text:
Adenoviral-mediated gene transfer to rabbit synovium in vivo.
Roessler, B J; Allen, E D; Wilson, J M; Hartman, J W; Davidson, B L
Currently, treatment for rheumatoid arthritis and other inflammatory arthropathies is often ineffective in ameliorating the progression of the disease, particularly the invasive destruction of cartilage and bone by rheumatoid synovium. Multiple aspects of this inflammatory process are mediated by the synovial lining cells (synoviocytes). Genetic modification of these cells in vivo represents a potential method for the treatment of these conditions. In this report, we describe a novel technique for the genetic transduction of synovial lining cells in vivo using recombinant adenoviral vectors and intraarticular injection techniques. Purified high titer suspensions of a recombinant adenoviral vector containing the gene for Escherichia coli beta-galactosidase (AdCMVlacZ) were directly injected into the hind knees of New Zealand white rabbits. Synovial tissues were then examined for transgenic lacZ expression using a combination of in situ staining for beta-galactosidase activity, immunohistochemical staining, and transmission electron microscopy. High efficiency gene transfer and lacZ expression was observed in both type A and type B synoviocytes throughout the articular and periarticular synovium of the rabbit knee, with continued expression of transgenic lacZ detected for > or = 8 wk after infection. Images PMID:8349791
Adenoviral-mediated Gene Transfer into the Canine Brain In Vivo
Candolfi, Marianela; Kroeger, Kurt M.; Pluhar, G. Elizabeth; Bergeron, Josee; Puntel, Mariana; Curtin, James F.; McNiel, Elizabeth A.; Freese, Andrew B.; Ohlfest, John R.; Moore, Peter; Lowenstein, Pedro R.; Castro, Maria G.
OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding β-galactosidase (βGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding βGal (Ad-βGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, βGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and βGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm2 area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials. PMID:17228266
Restoration of β -Adrenergic Signaling in Failing Cardiac Ventricular Myocytes via Adenoviral-Mediated Gene Transfer
NASA Astrophysics Data System (ADS)
Akhter, Shahab A.; Skaer, Christine A.; Kypson, Alan P.; McDonald, Patricia H.; Peppel, Karsten C.; Glower, Donald D.; Lefkowitz, Robert J.; Koch, Walter J.
Cardiovascular gene therapy is a novel approach to the treatment of diseases such as congestive heart failure (CHF). Gene transfer to the heart would allow for the replacement of defective or missing cellular proteins that may improve cardiac performance. Our laboratory has been focusing on the feasibility of restoring β -adrenergic signaling deficiencies that are a characteristic of chronic CHF. We have now studied isolated ventricular myocytes from rabbits that have been chronically paced to produce hemodynamic failure. We document molecular β -adrenergic signaling defects including down-regulation of myocardial β -adrenergic receptors (β -ARs), functional β -AR uncoupling, and an upregulation of the β -AR kinase (β ARK1). Adenoviral-mediated gene transfer of the human β 2-AR or an inhibitor of β ARK1 to these failing myocytes led to the restoration of β -AR signaling. These results demonstrate that defects present in this critical myocardial signaling pathway can be corrected in vitro using genetic modification and raise the possibility of novel inotropic therapies for CHF including the inhibition of β ARK1 activity in the heart.
AMELIORATION OF ETHANOL-INDUCED DYSMORPHOGENESIS BY ADENOVIRAL-MEDIATED Cu,Zn-SOD AND Mn-SOD EXPRESSION IN NEURULATION STAGED MOUSE EMBRYOS IN VITRO. JB Smith1, PC Hartig3, MR Blanton3, KK Sulik1,2, and ES Hunter3. 1Department of Cell and Developmental Biology and 2Bowles Cente...
Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation
Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal
Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo. PMID:26808122
STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.
Yang, Xuechao; van der Kraan, Peter M; van den Dolder, Juliette; Walboomers, X Frank; Bian, Zhuan; Fan, Mingwen; Jansen, John A
Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium. PMID:17824831
Permissive environment in postnatal wounds induced by adenoviral-mediated overexpression of the anti-inflammatory cytokine interleukin-10 prevents scar formation.
Gordon, Ashley; Kozin, Elliott D; Keswani, Sundeep G; Vaikunth, Sachin S; Katz, Anna B; Zoltick, Philip W; Favata, Michele; Radu, Antoneta P; Soslowsky, Louis J; Herlyn, Meenhard; Crombleholme, Timothy M
Wound healing in the mid-gestation fetus is scarless with minimal inflammation and a unique extracellular matrix. We have previously documented the relative lack of inflammatory cytokines in this environment. We demonstrate that interleukin (IL)-10 is highly expressed in mid-gestation human fetal skin but is absent in postnatal human skin. We hypothesize that overexpression of IL-10 in postnatal skin may replicate a permissive environment for scarless healing. To study the mechanism underlying this process we performed immunohistochemistry for IL-10 in human mid-gestation fetal and postnatal skin. We also determined if adenoviral-mediated overexpression of IL-10 could allow for scarless wound healing in a murine incisional wound model. Wounds were analyzed at 1-90 days postwounding for effects on scar formation, inflammatory response, and biomechanical properties. Ad-IL-10 reconstitutes a permissive environment for scarless healing as shown by reconstitution of a normal dermal reticular collagen pattern and distribution of dermal elements. Compared with controls, Ad-IL-10 treated wounds showed reduced inflammatory response and no difference in biomechanical parameters. Therefore, overexpression of IL-10 in postnatal wounds results in a permissive environment for scarless wound repair, possibly by replicating a fetal wound environment. PMID:18086289
Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2
Neumann, Alexander J.; Gardner, Oliver F. W.; Williams, Rebecca; Alini, Mauro; Archer, Charles W.; Stoddart, Martin J.
Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a
Adenoviral-Mediated Glial Cell Line–Derived Neurotrophic Factor Gene Transfer Has a Protective Effect on Sciatic Nerve Following Constriction-Induced Spinal Cord Injury
Chou, An-Kuo; Yang, Ming-Chang; Tsai, Hung-Pei; Chai, Chee-Yin; Tai, Ming-Hong; Kwan, Aij-Li; Hong, Yi-Ren
Neuropathic pain due to peripheral nerve injury may be associated with abnormal central nerve activity. Glial cell-line-derived neurotrophic factor (GDNF) can help attenuate neuropathic pain in different animal models of nerve injury. However, whether GDNF can ameliorate neuropathic pain in the spinal cord dorsal horn (SCDH) in constriction-induced peripheral nerve injury remains unknown. We investigated the therapeutic effects of adenoviral-mediated GDNF on neuropathic pain behaviors, microglial activation, pro-inflammatory cytokine expression and programmed cell death in a chronic constriction injury (CCI) nerve injury animal model. In this study, neuropathic pain was produced by CCI on the ipsilateral SCDH. Mechanical allodynia was examined with von Frey filaments and thermal sensitivity was tested using a plantar test apparatus post-operatively. Target proteins GDNF-1, GDNFRa-1, MMP2, MMP9, p38, phospho-p38, ED1, IL6, IL1β, AIF, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, PARP, cleaved PARP, SPECTRIN, cleaved SPECTRIN, Beclin-1, PKCσ, PKCγ, iNOS, eNOS and nNOS were detected. Microglial activity was measured by observing changes in immunoreactivity with OX-42. NeuN and TUNEL staining were used to reveal whether apoptosis was attenuated by GDNF. Results showed that administrating GDNF began to attenuate both allodynia and thermal hyperalgesia at day 7. CCI-rats were found to have lower GDNF and GDNFRa-1 expression compared to controls, and GDNF re-activated their expression. Also, GDNF significantly down-regulated CCI-induced protein expression except for MMP2, eNOS and nNOS, indicating that the protective action of GDNF might be associated with anti-inflammation and prohibition of microglia activation. Immunocytochemistry staining showed that GDNF reduced CCI-induced neuronal apoptosis. In sum, GDNF enhanced the neurotrophic effect by inhibiting microglia activation and cytokine production via p38 and PKC signaling. GDNF could be a good
λ Recombination and Recombineering.
Murphy, Kenan C
The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821
Organ distribution of transgene expression following intranasal mucosal delivery of recombinant replication-defective adenovirus gene transfer vector
Damjanovic, Daniela; Zhang, Xizhong; Mu, Jingyu; Fe Medina, Maria; Xing, Zhou
It is believed that respiratory mucosal immunization triggers more effective immune protection than parenteral immunization against respiratory infection caused by viruses and intracellular bacteria. Such understanding has led to the successful implementation of intranasal immunization in humans with a live cold-adapted flu virus vaccine. Furthermore there has been an interest in developing effective mucosal-deliverable genetic vaccines against other infectious diseases. However, there is a concern that intranasally delivered recombinant viral-based vaccines may disseminate to the CNS via the olfactory tissue. Initial experimental evidence suggests that intranasally delivered recombinant adenoviral gene transfer vector may transport to the olfactory bulb. However, there is a lack of quantitative studies to compare the relative amounts of transgene products in the respiratory tract, lung, olfactory bulb and brain after intranasal mucosal delivery of viral gene transfer vector. To address this issue, we have used fluorescence macroscopic imaging, luciferase quantification and PCR approaches to compare the relative distribution of transgene products or adenoviral gene sequences in the respiratory tract, lung, draining lymph nodes, olfactory bulb, brain and spleen. Intranasal mucosal delivery of replication-defective recombinant adenoviral vector results in gene transfer predominantly in the respiratory system including the lung while it does lead to a moderate level of gene transfer in the olfactory bulb. However, intranasal inoculation of adenoviral vector leads to little or no viral dissemination to the major region of the CNS, the brain. These experimental findings support the efficaciousness of intranasal adenoviral-mediated gene transfer for the purpose of mucosal immunization and suggest that it may not be of significant safety concern. PMID:18261231
ERIC Educational Resources Information Center
Whitehouse, H. L. K.
Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)
NASA Astrophysics Data System (ADS)
Wong, Wan Yan
In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.
ERIC Educational Resources Information Center
Greenslade, Thomas B., Jr.
Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)
Recombinant AAV9-TLK1B Administration Ameliorates Fractionated Radiation-Induced Xerostomia
Shanmugam, Prakash Srinivasan Timiri; Dayton, Robert D.; Palaniyandi, Senthilnathan; Abreo, Fleurette; Caldito, Gloria; Klein, Ronald L.
Abstract Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviral-mediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (1011 vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline- and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; p<0.01). Salivary gland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection
Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein
Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.
Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme's ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665
Recombinant protein production technology
Technology Transfer Automated Retrieval System (TEKTRAN)
Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...
Therapeutic Recombinant Monoclonal Antibodies
ERIC Educational Resources Information Center
Bakhtiar, Ray
During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…
Recombineering homologous recombination constructs in Drosophila.
Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael
The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070
Photoionization and Recombination
NASA Technical Reports Server (NTRS)
Nahar, Sultana N.
Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.
NASA Technical Reports Server (NTRS)
Johnsen, Rainer
Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.
Recombination in electron coolers
NASA Astrophysics Data System (ADS)
Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.
An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.
Genetic recombination. [Escherichia coli
Stahl, F.W.
The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.
[Recombinant antibodies against bioweapons].
Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique
The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695
Activated recombinant adenovirus proteinases
Anderson, C.W.; Mangel, W.F.
This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.
Activated recombinant adenovirus proteinases
Anderson, Carl W.; Mangel, Walter F.
This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.
Regulation of Meiotic Recombination
Gregory p. Copenhaver
Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system
Meiotic recombination mechanisms.
Grelon, Mathilde
Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110
Adenoviral-Mediated Endothelial Precursor Cell Delivery of Soluble CD115 Suppresses Human Prostate Cancer Xenograft Growth in Mice
Lucas, Trevor; Abraham, Dietmar; Untergasser, Gerold; Zins, Karin; Hofer, Erhard; Gunsilius, Eberhard; Aharinejad, Seyedhossein
Prostate cancer tumor growth and neovascularization is promoted by an interplay between migratory tumor stromal cells such as specialized tumor-associated macrophages (TAMs) and circulating endothelial precursor cells (CEPs). As vehicles for tumor therapy, human CEPs are relatively easy to isolate from peripheral blood, are able to proliferate long-term in vitro, are amenable to viral manipulation, and preferentially home to regions of ischemia found in growing tumors. We show here that human peripheral blood CEPs expanded ex vivo migrate to prostate cancer cells in vitro and efficiently home to human prostate tumor xenografts in vivo. Infection of precursors ex vivo with an adenovirus constructed to secrete a soluble form of the colony-stimulating factor-1 receptor CD115 that inhibits macrophage viability and migration in vitro significantly decreases the number of TAMs in xenografts (p < .05), reduces proliferation (p < .01) and vascular density (p < .03), and suppresses the growth of xenografts (p < .03). These data show for the first time that targeting stromal cell processes with cellular therapy has the potential to retard prostate tumor growth. PMID:19522014
Recombinant renewable polyclonal antibodies
Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM
Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082
ERIC Educational Resources Information Center
Duvall, James G., III
A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…
Recombineering Pseudomonas syringae
Technology Transfer Automated Retrieval System (TEKTRAN)
Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...
Oligonucleotide recombination in bacteria
Technology Transfer Automated Retrieval System (TEKTRAN)
Today, there are more than 1,500 completed or draft bacterial genome sequences available for public access. To functionally analyze these genomes and to test the hypotheses that are generated from the sequence information we require new and generically useful tools. Recombineering (genetic engineer...
Genomic homologous recombination in planta.
Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B
A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150
Recombinant vaccines against leptospirosis.
Dellagostin, Odir A; Grassmann, André A; Hartwig, Daiane D; Félix, Samuel R; da Silva, Éverton F; McBride, Alan J A
Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine. PMID:22048111
Recombinant influenza vaccines.
Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L
This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377
Jurka, Jerzy W.
Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.
The recombination epoch revisited
NASA Technical Reports Server (NTRS)
Krolik, Julian H.
Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.
Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.
Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T
The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528
Dielectronic recombination theory
LaGattuta, K.J.
A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.
Recombinant Collagenlike Proteins
NASA Technical Reports Server (NTRS)
Fertala, Andzej
A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.
NASA Technical Reports Server (NTRS)
Bartlett, James G.; Stebbins, Albert
The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies.
Expression of Recombinant Antibodies
Frenzel, André; Hust, Michael; Schirrmann, Thomas
Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with "human-like" post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655
Aitken, Michael G
Human coagulation factor (F) VII is a single chain protease that circulates in the blood as a weakly active zymogen at concentrations of approximately 10 nmol/L. When converted to the active 2 chain form (FVIIa), it is a powerful initiator of haemostasis. Recombinant factor VIIa (rFVIIa, eptacog alfa, NovoSeven) is a genetically engineered product that was first introduced in 1988 for the treatment of patients with haemophilia A and B with high inhibitory antibody titres to factors VIII and IX. Recent reports in the form of case studies and series, and early trial data, have suggested a role for rFVIIa across a diverse range of indications including bleeding associated with trauma, surgery, thrombocytopaenia, liver disease and oral anticoagulant toxicity. This review describes the physiology of the coagulation pathway and in particular the role of recombinant factor VIIa. It will also focus on the emerging role of rFVIIa in both trauma and non-trauma bleeding and its potential use in the ED. PMID:15537408
Unraveling recombination rate evolution using ancestral recombination maps
Munch, Kasper; Schierup, Mikkel H; Mailund, Thomas
Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it. PMID:25043668
Algebraic theory of recombination spaces.
Stadler, P F; Wagner, G P
A new mathematical representation is proposed for the configuration space structure induced by recombination, which we call "P-structure." It consists of a mapping of pairs of objects to the power set of all objects in the search space. The mapping assigns to each pair of parental "genotypes" the set of all recombinant genotypes obtainable from the parental ones. It is shown that this construction allows a Fourier decomposition of fitness landscapes into a superposition of "elementary landscapes." This decomposition is analogous to the Fourier decomposition of fitness landscapes on mutation spaces. The elementary landscapes are obtained as eigenfunctions of a Laplacian operator defined for P-structures. For binary string recombination, the elementary landscapes are exactly the p-spin functions (Walsh functions), that is, the same as the elementary landscapes of the string point mutation spaces (i.e., the hypercube). This supports the notion of a strong homomorphism between string mutation and recombination spaces. However, the effective nearest neighbor correlations on these elementary landscapes differ between mutation and recombination and among different recombination operators. On average, the nearest neighbor correlation is higher for one-point recombination than for uniform recombination. For one-point recombination, the correlations are higher for elementary landscapes with fewer interacting sites as well as for sites that have closer linkage, confirming the qualitative predictions of the Schema Theorem. We conclude that the algebraic approach to fitness landscape analysis can be extended to recombination spaces and provides an effective way to analyze the relative hardness of a landscape for a given recombination operator. PMID:10021760
Recombinant Human Erythropoietin
Bartels, Claudia; Späte, Kira; Krampe, Henning
Treatment of multiple sclerosis (MS) is still unsatisfactory and essentially non-existing for the progressive course of the disease. Recombinant human erythropoietin (EPO) may be a promising neuroprotective/neuroregenerative treatment of MS. In the nervous system, EPO acts anti-apoptotic, antioxidative, anti-inflammatory, neurotrophic and plasticity-modulating. Beneficial effects have been shown in animal models of various neurological and psychiatric diseases, including different models of experimental autoimmune encephalomyelitis. EPO is also effective in human brain disease, as shown in double-blind placebo-controlled clinical studies on ischemic stroke and chronic schizophrenia. An exploratory study on chronic progressive MS yielded lasting improvement in motor and cognitive performance upon high-dose long-term EPO treatment. PMID:21180577
Recombinant glucose uptake system
Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico
Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.
The recombination of genetic material
Low, K.B.
Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.
Coalescent Simulation of Intracodon Recombination
Arenas, Miguel; Posada, David
The coalescent with recombination is a very useful tool in molecular population genetics. Under this framework, genealogies often represent the evolution of the substitution unit, and because of this, the few coalescent algorithms implemented for the simulation of coding sequences force recombination to occur only between codons. However, it is clear that recombination is expected to occur most often within codons. Here we have developed an algorithm that can evolve coding sequences under an ancestral recombination graph that represents the genealogies at each nucleotide site, thereby allowing for intracodon recombination. The algorithm is a modification of Hudson's coalescent in which, in addition to keeping track of events occurring in the ancestral material that reaches the sample, we need to keep track of events occurring in ancestral material that does not reach the sample but that is produced by intracodon recombination. We are able to show that at typical substitution rates the number of nonsynonymous changes induced by intracodon recombination is small and that intracodon recombination does not generally result in inflated estimates of the overall nonsynonymous/synonymous substitution ratio (ω). On the other hand, recombination can bias the estimation of ω at particular codons, resulting in apparent rate variation among sites and in the spurious identification of positively selected sites. Importantly, in this case, allowing for variable synonymous rates across sites greatly reduces the false-positive rate and recovers statistical power. Finally, coalescent simulations with intracodon recombination could be used to better represent the evolution of nuclear coding genes or fast-evolving pathogens such as HIV-1.We have implemented this algorithm in a computer program called NetRecodon, freely available at http://darwin.uvigo.es. PMID:19933876
Delayed recombination and standard rulers
De Bernardis, Francesco; Melchiorri, Alessandro; Bean, Rachel; Galli, Silvia; Silk, Joseph I.; Verde, Licia
Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.
Three Decades of Recombinant DNA.
ERIC Educational Resources Information Center
Palmer, Jackie
Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)
Controlled Release from Recombinant Polymers
Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza
Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486
Recombinant DNA means and method
Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.
This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.
Stable recombination hotspots in birds.
Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly
The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757
Recombination device for storage batteries
Kraft, H.; Ledjeff, K.
A recombination device including a gas-tight enclosure connected to receive the discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.
Recombination device for storage batteries
Kraft, Helmut; Ledjeff, Konstantin
A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.
[Antithrombotic recombinant antibodies].
Muzard, Julien; Loyau, Stéphane; Ajzenberg, Nadine; Billiald, Philippe; Jandrot-Perrus, Martine
Coronary syndromes, stroke and other ischaemic arterial diseases are the leading cause of death in the world and will probably remain it at least until 2020. Cardiovascular diseases kill 17 million people each year with an expected increase to 20 million in 2020 and 24 million in 2030. The global impact of recurrence and death during the 6 months following an acute coronary syndrome remains at 8-15% in the present state of medical practice. Acute ischaemic syndromes have a common aetiology that is the formation of a platelet-rich clot at the site of severe coronary stenosis and of eroded atherosclerotic plaques. Therapy consists of medical treatments associating thrombolysis, antiplatelet drugs, and the re-opening of the coronary artery by angioplasty. But these treatments do not prevent morbidity and mortality reaching 15% at 6 months. Finally the treatment of stroke is very limited. There is thus a real clinical need to improve existing treatments and to discover new molecules. Platelet activation is a critical step in ischaemic cardiovascular diseases. This is the reason why antiplatelet drugs are most often prescribed in these cases. Currently, only one recombinant antithrombotic antibody is used in therapy. This is a chimeric Fab, c7E3 or abciximab, which inhibits the final phase of platelet aggregation. Abciximab is prescribed in acute coronary syndromes treated by angioplasty. However, treatment by abciximab can induce severe complications, principally, hemorrages and thrombopenia. Other platelet receptors involved in the earlier steps of platelet activation, such as the phases of contact with and of activation by the subendothelium matrix, have been identified as potential targets for the development of antithrombotic antibodies and are described in this revue. PMID:17652972
Genetic recombination in Streptomyces griseus.
Parag, Y
Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described. PMID:415037
[Vaccine application of recombinant herpesviruses].
Yokoyama, N; Xuan, X; Mikami, T
Recently, genetic engineering using recombinant DNA techniques has been applied to design new viral vaccines in order to reduce some problems which the present viral vaccines have. Up to now, many viruses have been investigated for development of recombinant attenuated vaccines or live viral vectors for delivery of foreign genes coding immunogenic antigens. In this article, we introduced the new vaccine strategy using genetically engineered herpesviruses. PMID:10774221
Combinatorics in Recombinational Population Genomics
NASA Astrophysics Data System (ADS)
Parida, Laxmi
The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.
Delayed recombination and cosmic parameters
NASA Astrophysics Data System (ADS)
Galli, Silvia; Bean, Rachel; Melchiorri, Alessandro; Silk, Joseph
Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: γα<0.39 and γi<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.
Ethanol production by recombinant hosts
Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.
Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.
Ethanol production by recombinant hosts
Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie
Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.
Delayed recombination and cosmic parameters
Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph
Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n{sub s}, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z{sub *}=1078{+-}11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1{sigma} to R=1.734{+-}0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: {epsilon}{sub {alpha}}<0.39 and {epsilon}{sub i}<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.
Recombination Drives Vertebrate Genome Contraction
Nam, Kiwoong; Ellegren, Hans
Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process. PMID:22570634
Recombination processes in ionised plasmas
NASA Astrophysics Data System (ADS)
Bastin, Robert
The observational analysis of astrophysical plasmas relies on accurate calculations of the atomic processes involved. The recombination spectra of singly ionised oxygen (O il) and carbon (C il) present excellent tools for investigating regions such as planetary nebulae and H II regions. In this thesis, detailed treatments of the recombination processes of both O II and C II are presented. Using the R-matrix solution to the close coupling equations, I present the results of accurate photoionisation calculations. Bound state energy levels are determined and oscillator strengths calculated for both species. Recombination coefficients were evalu ated for low n and 1, for C II in LS-coupling, and 0 II in intermediate coupling, taking particular care to treat resonances effectively. Sample photoionisation cross-sections are presented for both species, and compared to previous work. A complete radiative-cascade model is treated for both species, in order to determine line emissivities under nebular conditions at a wide range of temperatures and densities. Collisional effects are treated for C II, along with, for the first time, the effects of high temperature dielectronic recombination, allowing the modelling of regions of much higher electron temperature than previous work. The O II calculations were performed under intermediate coupling for the first time, allowing the effects of non-statistical popula tions of the parent ion fine-structure levels and dielectronic recombination onto bound states within this fine-structure to be taken into account in line emissivities. Detailed comparison with previous theoretical work was made for both species. The application of the C II and 0 n recombination spectra to determining tempera ture and densities from the observed spectra of a number of ionised nebulae is considered. The potential for using the new recombination spectra as diagnostic tools to solve some of the key problems in the study of ionised nebulae is demonstrated.
Recombination at the DNA level. Abstracts
Not Available
Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)
Moriya, Takashi J.
Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.
Recombinant allergens for specific immunotherapy.
Cromwell, Oliver; Häfner, Dietrich; Nandy, Andreas
Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient's sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years. PMID:21377719
Stable recombination hotspots in birds
Singhal, Sonal; Leffler, Ellen M.; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M.; Strand, Alva I.; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N.; Griffith, Simon C.; McVean, Gil; Przeworski, Molly
The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species, the zebra finch Taeniopygia guttata and the long-tailed finch Poephila acuticauda. We find that both species have hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, the two species share most hotspots, with conservation seemingly extending over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757
Recombinant snake venom prothrombin activators.
Lövgren, Ann
Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318
The Dissociative Recombination of OH(+)
NASA Technical Reports Server (NTRS)
Guberman, Steven L.
Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.
Current Drive in Recombining Plasma
P.F. Schmit and N.J. Fisch
The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.
Selenium incorporation using recombinant techniques
Walden, Helen
An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.
Classifications and comparisons of multilocus recombination distributions
Karlin, Samuel; Liberman, Uri
Various classifications and representations of multilocus recombination structures are delineated based on generalized notions of linkage values and recombination rates. An important class of recombination distributions (called the count-location chiasma process) is parameterized by a distribution of the number of crossover events and, for each such crossover count, by a conditional distribution of crossover locations. A number of properties of this recombination structure are developed. A multilocus definition of a "natural" recombination range is set forth. Orderings among recombination distributions in the multilocus setting are also discussed. Comparisons are made in terms of complete linkage, free assortment and noninterference schemes serving as standards. PMID:16592601
Improving recombinant protein purification yield
Technology Transfer Automated Retrieval System (TEKTRAN)
Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It's technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...
CRMAGE: CRISPR Optimized MAGE Recombineering
Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.; Nielsen, Alex Toftgaard
A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering. PMID:26797514
Inhomogeneous recombinations during cosmic reionization
NASA Astrophysics Data System (ADS)
Sobacchi, Emanuele; Mesinger, Andrei
By depleting the ionizing photon budget available to expand cosmic H II regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a subgrid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parametrizing photoheating feedback on star formation, we present large-scale, seminumeric reionization simulations which self-consistently track the local (subgrid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large H II regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reionization. As a result, typical H II regions are smaller by factors of ˜2 to 3 throughout reionization. The large-scale (k ≲ 0.2 Mpc-1) ionization power spectrum is suppressed by factors of ≳2-3 in the second half of reionization. Therefore properly modelling recombinations is important in interpreting virtually all reionization observables, including upcoming interferometry with the redshifted 21cm line. Consistent with previous works, we find the clumping factor of ionized gas to be C H II ˜ 4 at the end of reionization.
Recombinant DNA: History of the Controversy.
ERIC Educational Resources Information Center
Vigue, Charles L.; Stanziale, William G.
The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)
Antibacterial activity of recombinant murine beta interferon.
Fujiki, T; Tanaka, A
Recombinant murine beta interferon was protective and therapeutic for mice against Listeria monocytogenes infection in vivo. The recombinant murine beta interferon caused enhanced H2O2 release by macrophages in vivo, but not in vitro. PMID:3343048
[Recombination in Drosophila in space flight].
Filatova, L P; Vaulina, E N; Lapteva, N Sh; Grozdova, T Ia
An experiment with Drosophila melanogaster males was performed aboard the Artificial Satellite "Kosmos-1667". Mutagenic effects of a 7-day space flight on intergene recombination in chromosome 2 were studied. The space flight factors decreased the frequency of recombination. A model experiment on a laboratory centrifuge demonstrated insignificant increase in recombination frequency caused by acceleration. PMID:3135244
Selection of Recombinant Human Antibodies.
Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André
Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551
Chemical kinetics of geminal recombination
Levin, P.P.; Khudyakov, I.V.; Brin, E.F.; Kuz'min, V.A.
The kinetics of geminal recombination of triplet radical pairs formed in photoreduction of benzophenone by p-cresol in glycerin solution was studied by pulsed laser photolysis. The experiments were conducted at several temperatures and in a constant magnetic field of H = 0.34 T. The parameters in six kinetic equations describing geminal recombination were determined with a computer. The values of the sums of the squares of the residual deviations of the approximation were obtained. It was found that the kinetics are best described by the functions proposed by Noyes and Shushin. It was shown that it is necessary to use the mutual diffusion coefficient of the radicals, which is significantly smaller than the sum of the estimations of the experimental values of the radical diffusion coefficients, for describing the kinetics due to the correlations of the molecular motions of the radicals in the cage.
Recombination Catalysts for Hypersonic Fuels
NASA Technical Reports Server (NTRS)
Chinitz, W.
The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.
Surface recombination statistics at traps
NASA Astrophysics Data System (ADS)
Landsberg, P. T.; Abrahams, M. S.
The Shockley-Read-Hall recombination statistics was recently generalised by Dhariwal, Kothari and Jain to include the effect of a finite time of relaxation before the captured carrier settles into its ground state, and by Landsberg to allow for Auger effects and so-called "extra" carriers supplied to the semiconductor from the outside. The combined result of these effects is studied here theoretically, together with the consideration of a simple distribution of trap states. It is found that the surface recombination velocity s has the usual minimum in the near intrinsic state and that s passes through a maximum as a function of excess electron concentration. Both extrema are enhanced if the trap states are distributed over an energy range. Experimental plots of s as a function of excess electron and hole concentrations should yield insight concerning the numerical importance of (a) Auger effects with the participation of traps and (b) relaxation times.
Recombinant Toxins for Cancer Treatment
NASA Astrophysics Data System (ADS)
Pastan, Ira; Fitzgerald, David
Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.
Introductory experiments in recombinant DNA.
Tait, R C
Nine practical exercises demonstrate the basic principles in recombinant DNA. The exercises explain the principles that DNA equals genes and that changes in DNA cause changes in genetic properties. The aim is to provide a teaching resource that can be used to illustrate the theory and applications of molecular biology to highschool students, undergraduate students, medics, dentists, doctors, nurses, life scientists, and anyone learning the basics of DNA technology. PMID:11471559
Recombinant vector and eukaryotic host transformed thereby
Sugden, W.M.
A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.
A coalescent model of recombination hotspots.
Wiuf, Carsten; Posada, David
Recent experimental findings suggest that the assumption of a homogeneous recombination rate along the human genome is too naive. These findings point to block-structured recombination rates; certain regions (called hotspots) are more prone than other regions to recombination. In this report a coalescent model incorporating hotspot or block-structured recombination is developed and investigated analytically as well as by simulation. Our main results can be summarized as follows: (1) The expected number of recombination events is much lower in a model with pure hotspot recombination than in a model with pure homogeneous recombination, (2) hotspots give rise to large variation in recombination rates along the genome as well as in the number of historical recombination events, and (3) the size of a (nonrecombining) block in the hotspot model is likely to be overestimated grossly when estimated from SNP data. The results are discussed with reference to the current debate about block-structured recombination and, in addition, the results are compared to genome-wide variation in recombination rates. A number of new analytical results about the model are derived. PMID:12750351
Nondisjunction of chromosome 15: Origin and recombination
Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. ); Langlois, S. ); Morris, M.A.; Malcolm, S.
Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.
Effect of gamma radiation on retroviral recombination.
Hu, W S; Temin, H M
To elucidate the mechanism(s) of retroviral recombination, we exposed virions to gamma radiation prior to infecting target cells. By using previously described spleen necrosis virus-based vectors containing multiple markers, recombinant proviruses were studied after a single round of retrovirus replication. The current models of retroviral recombination predict that breaking virion RNA should promote minus-strand recombination (forced copy-choice model), decrease or not affect plus-strand recombination (strand displacement/assimilation model), and shift plus-strand recombination towards the 3' end of the genome. However, we found that while gamma irradiation of virions reduced the amount of recoverable viral RNA, it did not primarily cause breaks. Thus, the frequency of selected recombinants was not significantly altered with greater doses of radiation. In spite of this, the irradiation did decrease the number of recombinants with only one internal template switch. As a result, the average number of additional internal template switches in the recombinant proviruses increased from 0.7 to 1.4 as infectivity decreased to 6%. The unselected internal template switches tended to be 5' of the selected crossover even in the recombinants from irradiated viruses, inconsistent with a plus-strand recombination mechanism. PMID:1602553
Adenoviral-mediated gene transfer of vascular endothelial growth factor in critical limb ischemia: safety results from a phase I trial.
Mohler, Emile R; Rajagopalan, Sanjay; Olin, Jeffrey W; Trachtenberg, Jeffrey D; Rasmussen, Henrik; Pak, Raphael; Crystal, Ronald G
Critical limb ischemia (CLI) is typified by rest pain and/or tissue necrosis secondary to advanced peripheral arterial disease (PAD) and is characterized by diminution in limb perfusion at rest. We tested the safety of an angiogenic strategy with CI-1023 (Ad(GV)VEGF121.10), a replication-deficient adenovirus encoding human vascular endothelial growth factor isoform 121 in patients with CLI as part of a phase I trial. Fifteen subjects >35 years of age with CLI and angiographic disease involving the infra-inguinal vessels underwent intramuscular injection of CI-1023 (4 x 10(8) to 4 x 10(10) particle units, n = 13) or placebo (n = 2). All of the patients tolerated the injection well and there were no serious complications related to the procedure. Transient edema was noted in one patient. A total of 79 adverse events were reported over the course of one year. One death (day 136) and one malignancy (day 332) occurred in the CI-1023 group. CI-1023 appears to be well tolerated and safe for single-dose administration in patients with critical limb ischemia due to PAD. Further studies are needed to determine the efficacy of this form of therapeutic angiogenesis. PMID:12866606
Isoform-Specific Modulation of Inflammation Induced by Adenoviral Mediated Delivery of Platelet-Derived Growth Factors in the Adult Mouse Heart
Ylä-Herttuala, Seppo; Betsholtz, Christer; Andrae, Johanna
Platelet-derived growth factors (PDGFs) are key regulators of mesenchymal cells in vertebrate development. To what extent PDGFs also exert beneficial homeostatic or reparative roles in adult organs, as opposed to adverse fibrogenic responses in pathology, are unclear. PDGF signaling plays critical roles during heart development, during which forced overexpression of PDGFs induces detrimental cardiac fibrosis; other studies have implicated PDGF signaling in post-infarct myocardial repair. Different PDGFs may exert different effects mediated through the two PDGF receptors (PDGFRα and PDGFRβ) in different cell types. Here, we assessed responses induced by five known PDGF isoforms in the adult mouse heart in the context of adenovirus vector-mediated inflammation. Our results show that different PDGFs have different, in some cases even opposing, effects. Strikingly, whereas the major PDGFRα agonists (PDGF-A and -C) decreased the amount of scar tissue and increased the numbers of PDGFRα-positive fibroblasts, PDGFRβ agonists either induced large scars with extensive inflammation (PDGF-B) or dampened the adenovirus-induced inflammation and produced a small and dense scar (PDGF-D). These results provide evidence for PDGF isoform-specific inflammation-modulating functions that may have therapeutic implications. They also illustrate a surprising complexity in the PDGF-mediated pathophysiological responses. PMID:27513343
Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference
CAO, Huibi; WANG, Anan; MARTIN, Bernard; KOEHLER, David R.; ZEITLIN, Pamela L.; TANAWELL, A. Keith; HU, Jim
Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr−/−; C38, Cftr-corrected) stimulated with TNF-α, IL-1β or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-κB, or on the protein levels of IκB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases. PMID:15740640
Osteogenic gene regulation and relative acceleration of healing by adenoviral-mediated transfer of human BMP-2 or -6 in equine osteotomy and ostectomy models.
Ishihara, Akikazu; Shields, Kathleen M; Litsky, Alan S; Mattoon, John S; Weisbrode, Steven E; Bartlett, Jeffrey S; Bertone, Alicia L
This study evaluated healing of equine metatarsal osteotomies and ostectomies in response to percutaneous injection of adenoviral (Ad) bone morphogenetic protein (BMP)-2, Ad-BMP-6, or beta-galactosidase protein vector control (Ad-LacZ) administered 14 days after surgery. Radiographic and quantitative computed tomographic assessment of bone formation indicated greater and earlier mineralized callus in both the osteotomies and ostectomies of the metatarsi injected with Ad-BMP-2 or Ad-BMP-6. Peak torque to failure and torsional stiffness were greater in osteotomies treated with Ad-BMP-2 than Ad-BMP-6, and both Ad-BMP-2- and Ad-BMP-6-treated osteotomies were greater than Ad-LacZ or untreated osteotomies. Gene expression of ostectomy mineralized callus 8 weeks after surgery indicated upregulation of genes related to osteogenesis compared to intact metatarsal bone. Expression of transforming growth factor beta-1, cathepsin H, and gelsolin-like capping protein were greater in Ad-BMP-2- and Ad-BMP-6-treated callus compared to Ad-LacZ-treated or untreated callus. Evidence of tissue biodistribution of adenovirus in distant organs was not identified by quantitative PCR, despite increased serum antiadenoviral vector antibody. This study demonstrated a greater relative potency of Ad-BMP-2 over Ad-BMP-6 in accelerating osteotomy healing when administered in this regimen, although both genes were effective at increasing bone at both osteotomy and ostectomy sites. PMID:18241059
Bacteriophage recombination systems and biotechnical applications.
Nafissi, Nafiseh; Slavcev, Roderick
Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504
Recombinant DNA technology in apple.
Gessler, Cesare; Patocchi, Andrea
This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available. PMID:17522823
Recombinant erythropoietin in clinical practice
Ng, T; Marx, G; Littlewood, T; Macdougall, I
The introduction of recombinant human erythropoietin (RHuEPO) has revolutionised the treatment of patients with anaemia of chronic renal disease. Clinical studies have demonstrated that RHuEPO is also useful in various non-uraemic conditions including haematological and oncological disorders, prematurity, HIV infection, and perioperative therapies. Besides highlighting both the historical and functional aspects of RHuEPO, this review discusses the applications of RHuEPO in clinical practice and the potential problems of RHuEPO treatment. PMID:12897214
Annealing Vs. Invasion in Phage λ Recombination
Stahl, M. M.; Thomason, L.; Poteete, A. R.; Tarkowski, T.; Kuzminov, A.; Stahl, F. W.
Genetic recombination catalyzed by λ's Red pathway was studied in rec(+) and recA mutant bacteria by examining both intracellular λ DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec(+) cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of λ catalyzes recombination primarily by annealing. PMID:9383045
Recombinant DNA production of spider silk proteins
Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L
Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078
Recombinant bacteria for mosquito control.
Federici, B A; Park, H-W; Bideshi, D K; Wirth, M C; Johnson, J J
Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria. PMID:14506223
Dissociative recombination in planetary ionospheres
NASA Technical Reports Server (NTRS)
Fox, J. L.
Ionization in planetary atmospheres can be produced by solar photoionization, photoelectron impact ionization, and, in auroral regions, by impact of precipitating particles. This ionization is lost mainly in dissociative recombination (DR) of molecular ions. Although atomic ions cannot undergo DR, they can be transformed locally through ion-molecule reactions into molecular ions, or they may be transported vertically or horizontally to regions of the atmosphere where such transformations are possible. Because DR reactions tend to be very exothermic, they can be an important source of kinetically or internally excited fragments. In interplanetary thermospheres, the neutral densities decrease exponentially with altitude. Below the homopause (or turbopause), the atmosphere is assumed to be throughly mixed by convection and/or turbulence. Above the homopause, diffusion is the major transport mechanism, and each species is distributed according to its mass, with the logarithmic derivative of the density with repect to altitude given approximately by -1/H, where H = kT/mg is the scale height. In this expression, T is the neutral temperature, g is the local acceleratiion of gravity, and m is the mass of the species. Thus lighter species become relatively more abundant, and heavier species less abundant, as the altitude increases. This variation of the neutral composition can lead to changes in the ion composition; furthermore, as the neutral densities decrease, dissociative recombination becomes more important relative to ion-neutral reactions as a loss mechanism for molecular ions.
Human Insulin from Recombinant DNA Technology
NASA Astrophysics Data System (ADS)
Johnson, Irving S.
Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.
Dielectronic recombination of tungsten ions
NASA Astrophysics Data System (ADS)
Li, Bowen; O'Sullivan, Gerry; Dong, Chenzhong; Chen, Ximeng
Ab initio calculations of dielectronic recombination rate coefficients of Ne-, Pd- and Ag-like tungsten have been performed. Energy levels, radiative transition probabilities and autoionization rates were calculated using the Flexible Atomic Code. The contributions from different channels to the total rate coefficients are discussed. The present calculated rate coefficients are compared with other calculations where available. Excellent agreement has been found for Ne-like W while a large discrepancy was found for Pd-like W, which implies that more ab initio calculations and experimental measurements are badly needed. Further calculations demonstrated that the influence of configuration interaction is small while nonresonant radiative stabilizing (NRS) contribution to doubly excited non-autoionizing states are vital. The data obtained are expected to be useful for modeling plasmas for fusion applications, especially for the ITER community, which makes experimental verification even more essential.
Steric effects on geminate recombinations
Traylor, T.G.; Taube, D.; Jongeward, K.A.; Magde, D. )
Steric effects on the binding of isonitrile ligands to iron(II) porphyrins were investigated by picosecond flash photolysis. Two different types of steric effects were distinguished and characterized: (1) steric restrictions to porphyrin planarity and (2) blocking of the pathway for ligand approach. Heme planarity was restricted by coordinating 1,2-dimethylimidazole trans to the ligand binding site being investigated. Blocking of the binding site was explored by using adamantane heme 6,6-cyclophane, in which the adamantane moiety forms a cap over the binding site. Results of picosecond kinetic measurements demonstrate that the first effect, heme nonplanarity or trans strain, influences the bond-making step, whereas the second effect, ligand blocking, involves a conformational preequilibrium prior to bond making. Relevance of these findings for contact pair recombination, in general, and for heme protein ligation, in particular, are discussed.
Modeling Interference in Genetic Recombination
McPeek, M. S.; Speed, T. P.
In analyzing genetic linkage data it is common to assume that the locations of crossovers along a chromosome follow a Poisson process, whereas it has long been known that this assumption does not fit the data. In many organisms it appears that the presence of a crossover inhibits the formation of another nearby, a phenomenon known as ``interference.'' We discuss several point process models for recombination that incorporate position interference but assume no chromatid interference. Using stochastic simulation, we are able to fit the models to a multilocus Drosophila dataset by the method of maximum likelihood. We find that some biologically inspired point process models incorporating one or two additional parameters provide a dramatically better fit to the data than the usual ``no-interference'' Poisson model. PMID:7713406
Deleterious background selection with recombination
Hudson, R.R.; Kaplan, N.L.
An analytic expression for the expected nucleotide diversity is obtained for a neutral locus in a region with deleterious mutation and recombination. Our analytic results are used to predict levels of variation for the entire third chromosome of Drosophila melanogaster. The predictions are consistent with the low levels of variation that have been observed at loci near the centromeres of the third chromosome of D. melanogaster. However, the low levels of variation observed near the tips of this chromosome are not predicted using currently available estimates of the deleterious mutation rate and of selection coefficients. If considerably smaller selection coefficients are assumed, the low observed levels of variation at the tips of the third chromosome are consistent with the background selection model. 33 refs., 4 figs., 1 tab.
Recombinant prolylcarboxypeptidase activates plasma prekallikrein.
Shariat-Madar, Zia; Mahdi, Fakhri; Schmaier, Alvin H
The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. PMID:14996700
Fundamental Studies of Recombinant Hydrogenases
Adams, Michael W
This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a "minimal" hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first 'minimal' form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.
RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.
Liao, C L; Lai, M M
Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur
Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila
Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.
Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062
Oligo Recombination in Gram Negative Bacteria
Technology Transfer Automated Retrieval System (TEKTRAN)
Homologous recombination is important for bacterial survival because it simultaneously provides genomic stability as well as genomic plasticity. Of the mechanistic pathways for homologous recombination, those mediated by RecA are the most thoroughly characterized and are understood to be structural...
Dissociative recombination of highly symmetric polyatomic ions.
Douguet, Nicolas; Orel, Ann E; Greene, Chris H; Kokoouline, Viatcheslav
A general first-principles theory of dissociative recombination is developed for highly symmetric molecular ions and applied to H(3)O(+) and CH(3)(+), which play an important role in astrophysical, combustion, and laboratory plasma environments. The theoretical cross sections obtained for the dissociative recombination of the two ions are in good agreement with existing experimental data from storage ring experiments. PMID:22324682
High efficiency recombineering in lactic acid bacteria
van Pijkeren, Jan-Peter; Britton, Robert A.
The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to Gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold. By creating a single amino acid change in the d-Ala-d-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other Gram-positive bacteria. PMID:22328729
Oligonucleotide recombination in gram negative bacteria
Technology Transfer Automated Retrieval System (TEKTRAN)
This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...
Dielectronic recombination lines of C{sup +}
Sochi, Taha Storey, Peter J.
The present paper presents atomic data generated to investigate the recombination lines of C II in the spectra of planetary nebulae. These data include energies of bound and autoionizing states, oscillator strengths and radiative transition probabilities, autoionization probabilities, and recombination coefficients. The R-matrix method of electron scattering theory was used to describe the C{sup 2+} plus electron system.
Recombinant Vaccinia Virus: Immunization against Multiple Pathogens
NASA Astrophysics Data System (ADS)
Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo
The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.
Recombinant Swinepox Virus for Veterinary Vaccine Development.
Fan, Hong-Jie; Lin, Hui-Xing
Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses. PMID:26458836
Recombinant DNA encoding a desulfurization biocatalyst
Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.
This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.
Recombinant DNA encoding a desulfurization biocatalyst
Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.
This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.
V(D)J recombination deficiencies.
de Villartay, Jean-Pierre
V(D)J recombination not only comprises the molecular mechanism that insures diversity of the immune system but also constitutes a critical checkpoint in the developmental program of B- and T-lymphocytes. The analysis of human patients with Severe Combined Immune Deficiency (SCID) has contributed to the understanding of the biochemistry of the V(D)J recombination reaction. The molecular study V(D)J recombination settings in humans, mice and in cellular mutants has allowed to unravel the process of Non Homologous End Joining (NHEJ), one of the key pathway that insure proper repair of DNA double strand breaks (dsb), whether they occur during V(D)J recombination or secondary to other DNA injuries. Two NHEJ factors, Artemis and Cernunnos, were indeed discovered through the study of human V(D)J recombination defective human SCID patients. PMID:19731800
Biochemistry of Meiotic Recombination: Formation, Processing, and Resolution of Recombination Intermediates
Ehmsen, Kirk T.
Meiotic recombination ensures accurate chromosome segregation during the first meiotic division and provides a mechanism to increase genetic heterogeneity among the meiotic products. Unlike homologous recombination in somatic (vegetative) cells, where sister chromatid interactions prevail and crossover formation is avoided, meiotic recombination is targeted to involve homologs, resulting in crossovers to connect the homologs before anaphase of the first meiotic division. The mechanisms responsible for homolog choice and crossover control are poorly understood, but likely involve meiosis-specific recombination proteins, as well as meiosis-specific chromosome organization and architecture. Much progress has been made to identify and biochemically characterize many of the proteins acting during meiotic recombination. This review will focus on the proteins that generate and process heteroduplex DNA, as well as those that process DNA junctions during meiotic recombination, with particular attention to how recombination activities promote crossover resolution between homologs. PMID:20098639
Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...
Charge recombination and thermoluminescence in photosystem II.
Rappaport, Fabrice; Cuni, Aude; Xiong, Ling; Sayre, Richard; Lavergne, Jérôme
In the recombination process of Photosystem II (S(2)Q(A)(-)-->S(1)Q(A)) the limiting step is the electron transfer from the reduced primary acceptor pheophytin Ph(-) to the oxidized primary donor P(+) and the rate depends on the equilibrium constant between states S(2)PPhQ(A)(-) and S(1)P(+)Ph(-)Q(A). Accordingly, mutations that affect the midpoint potential of Ph or of P result in a modified recombination rate. A strong correlation is observed between the effects on the recombination rate and on thermoluminescence (TL, the light emission from S(2)Q(A)(-) during a warming ramp): a slower recombination corresponds to a large enhancement and higher temperature of the TL peak. The current theory of TL does not account for these effects, because it is based on the assumption that the rate-limiting step coincides with the radiative process. When implementing the known fact that the radiative pathway represents a minor leak, the modified TL theory readily accounts qualitatively for the observed behavior. However, the peak temperature is still lower than predicted from the temperature-dependence of recombination. We argue that this reflects the heterogeneity of the recombination process combined with the enhanced sensitivity of TL to slower components. The recombination kinetics are accurately fitted as a sum of two exponentials and we show that this is not due to a progressive stabilization of the charge-separated state, but to a pre-existing conformational heterogeneity. PMID:15653722
Electron Recombination in a Dense Hydrogen Plasma
Jana, M.R.; Johnstone, C.; Kobilarcik, T.; Koizumi, G.M.; Moretti, A.; Popovic, M.; Tollestrup, A.V.; Yonehara, K.; Leonova, M.A.; Schwarz, T.A.; Chung, M.; /Unlisted /IIT, Chicago /Fermilab /MUONS Inc., Batavia /Turin Polytechnic
A high pressure hydrogen gas filled RF cavity was subjected to an intense proton beam to study the evolution of the beam induced plasma inside the cavity. Varying beam intensities, gas pressures and electric fields were tested. Beam induced ionized electrons load the cavity, thereby decreasing the accelerating gradient. The extent and duration of this degradation has been measured. A model of the recombination between ionized electrons and ions is presented, with the intent of producing a baseline for the physics inside such a cavity used in a muon accelerator. Analysis of the data taken during the summer of 2011 shows that self recombination takes place in pure hydrogen gas. The decay of the number of electrons in the cavity once the beam is turned off indicates self recombination rather than attachment to electronegative dopants or impurities. The cross section of electron recombination grows for larger clusters of hydrogen and so at the equilibrium of electron production and recombination in the cavity, processes involving H{sub 5}{sup +} or larger clusters must be taking place. The measured recombination rates during this time match or exceed the analytic predicted values. The accelerating gradient in the cavity recovers fully in time for the next beam pulse of a muon collider. Exactly what the recombination rate is and how much the gradient degrades during the 60 ns muon collider beam pulse will be extrapolated from data taken during the spring of 2012.
Intraspecific variation of recombination rate in maize
Background In sexually reproducing organisms, meiotic crossovers ensure the proper segregation of chromosomes and contribute to genetic diversity by shuffling allelic combinations. Such genetic reassortment is exploited in breeding to combine favorable alleles, and in genetic research to identify genetic factors underlying traits of interest via linkage or association-based approaches. Crossover numbers and distributions along chromosomes vary between species, but little is known about their intraspecies variation. Results Here, we report on the variation of recombination rates between 22 European maize inbred lines that belong to the Dent and Flint gene pools. We genotype 23 doubled-haploid populations derived from crosses between these lines with a 50 k-SNP array and construct high-density genetic maps, showing good correspondence with the maize B73 genome sequence assembly. By aligning each genetic map to the B73 sequence, we obtain the recombination rates along chromosomes specific to each population. We identify significant differences in recombination rates at the genome-wide, chromosome, and intrachromosomal levels between populations, as well as significant variation for genome-wide recombination rates among maize lines. Crossover interference analysis using a two-pathway modeling framework reveals a negative association between recombination rate and interference strength. Conclusions To our knowledge, the present work provides the most comprehensive study on intraspecific variation of recombination rates and crossover interference strength in eukaryotes. Differences found in recombination rates will allow for selection of high or low recombining lines in crossing programs. Our methodology should pave the way for precise identification of genes controlling recombination rates in maize and other organisms. PMID:24050704
Advances in recombinant antibody manufacturing.
Kunert, Renate; Reinhart, David
Since the first use of Chinese hamster ovary (CHO) cells for recombinant protein expression, production processes have steadily improved through numerous advances. In this review, we have highlighted several key milestones that have contributed to the success of CHO cells from the beginning of their use for monoclonal antibody (mAb) expression until today. The main factors influencing the yield of a production process are the time to accumulate a desired amount of biomass, the process duration, and the specific productivity. By comparing maximum cell densities and specific growth rates of various expression systems, we have emphasized the limiting parameters of different cellular systems and comprehensively described scientific approaches and techniques to improve host cell lines. Besides the quantitative evaluation of current systems, the quality-determining properties of a host cell line, namely post-translational modifications, were analyzed and compared to naturally occurring polyclonal immunoglobulin fractions from human plasma. In summary, numerous different expression systems for mAbs are available and also under scientific investigation. However, CHO cells are the most frequently investigated cell lines and remain the workhorse for mAb production until today. PMID:26936774
Recombinant protein production and streptomycetes.
Anné, Jozef; Maldonado, Bárbara; Van Impe, Jan; Van Mellaert, Lieve; Bernaerts, Kristel
The biopharmaceutical market has come a long way since 1982, when the first biopharmaceutical product, recombinant human insulin, was launched. Just over 200 biopharma products have already gained approval. The global market for biopharmaceuticals which is currently valued at over US$99 billion has been growing at an impressive compound annual growth rate over the previous years. To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options--from yield to glycosylation, to proper folding, to economics of scale-up. No host cell from which all the proteins can be universally expressed in large quantities has been found so far. Therefore, it is important to provide a variety of host-vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell. In this overview, we focus on Streptomyces lividans, a Gram-positive bacterium with a proven excellence in secretion capacity, as host for heterologous protein production. We will discuss its advantages and disadvantages, and how with systems biology approaches strains can be developed to better producing cell factories. PMID:21777629
Dissociative Recombination without a Curve Crossing
NASA Technical Reports Server (NTRS)
Guberman, Steven L.
Ab initio calculations show that a curve crossing is not always needed for a high dissociative- recombination cross section. For HeH(+), in which no neutral states cross the ion potential curve, dissociative recombination is driven by the nuclear kinetic-energy operator on adiabatic potential curves. The kinetic-energy derivative operator allows for capture into repulsive curves that are outside of the classical turning points for the nuclear motion. The dominant dissociative route is the C (2)Sigma(+) state leading to H(n = 2) atoms. An analogous mechanism is proposed for the dissociative recombination of H3(+).
Recombination of N4(+) ions with electrons
NASA Technical Reports Server (NTRS)
Cao, Y. S.; Johnsen, R.
Using a modified high-pressure-afterglow/mass spectrometer apparatus similar to that described by Lee and Johnsen (1989), spectroscopic observations of afterglow helium plasmas, with N2 as a minor additive, were carried out in order to verify the mechanism suggested by Bates (1991) for dissociative recombination of electrons with N4(+) ions. It was found that dissociative recombination of electrons with N4(+) ions results in the formation of N2 molecules in the C 3Pi(u) (v = 0,1) state, with the recombination rate coefficient of (2.6 +/- 0.3) x 10 exp -6 cu cm/sec at 300 K.
Spacecraft thermal energy accommodation from atomic recombination
NASA Technical Reports Server (NTRS)
Carleton, Karen L.; Marinelli, William J.
Measurements of atomic recombination probabilities important in determining energy release to reusable spacecraft thermal protection surfaces during reentry are presented. An experimental apparatus constructed to examine recombination of atomic oxygen from thermal protection and reference materials at reentry temperatures is described. The materials are examined under ultrahigh vacuum conditions to develop and maintain well characterized surface conditions that are free of contamination. When compared with stagnation point heat transfer measurements performed in arc jet facilities, these measurements indicate that a significant fraction of the excess energy available from atom recombination is removed from the surface as metastable O2.
Bacterial genome remodeling through bacteriophage recombination.
Menouni, Rachid; Hutinet, Geoffrey; Petit, Marie-Agnès; Ansaldi, Mireille
Bacteriophages co-exist and co-evolve with their hosts in natural environments. Virulent phages lyse infected cells through lytic cycles, whereas temperate phages often remain dormant and can undergo lysogenic or lytic cycles. In their lysogenic state, prophages are actually part of the host genome and replicate passively in rhythm with host division. However, prophages are far from being passive residents: they can modify or bring new properties to their host. In this review, we focus on two important phage-encoded recombination mechanisms, i.e. site-specific recombination and homologous recombination, and how they remodel bacterial genomes. PMID:25790500
Mapping Recombination Initiation Sites Using Chromatin Immunoprecipitation.
He, Yan; Wang, Minghui; Sun, Qi; Pawlowski, Wojciech P
Genome-wide maps of recombination sites provide valuable information not only on the recombination pathway itself but also facilitate the understanding of genome dynamics and evolution. Here, we describe a chromatin immunoprecipitation (ChIP) protocol to map the sites of recombination initiation in plants with maize used as an example. ChIP is a method that allows identification of chromosomal sites occupied by specific proteins. Our protocol utilizes RAD51, a protein involved in repair of double-strand breaks (DSBs) that initiate meiotic recombination, to identify DSB formation hotspots. Chromatin is extracted from meiotic flowers, sheared and enriched in fragments bound to RAD51. Genomic location of the protein is then identified by next-generation sequencing. This protocol can also be used in other species of plants, animals, and fungi. PMID:27511175
Constraints from jet calculus on quark recombination
Jones, L.M.; Lassila, K.E.; Sukhatme, U.; Willen, D.
Within the quantum-chromodynamic jet-calculus formalism, we deduce an equation describing recombination of quarks and antiquarks into mesons within a quark or gluon jet. This equation relates the recombination function R(x/sub 1/,x/sub 2/,x) used in current literature to the fragmentation function for producing that same meson out of the parton initiating the jet. We submit currently used recombination functions to our consistency test, taking as input mainly the u-quark fragmentation ''data'' into ..pi../sup +/ mesons. The qq-bar..--> pi.. recombination functions popular in the literature are consistent with measured fragmentation functions, but they must be supplemented by other contributions to provide the full D..pi../sup +//sub u/. We also discuss the Q/sup 2/ dependence of the resulting fragmentation functions.
The Kinetics of Nitrogen Atom Recombination
ERIC Educational Resources Information Center
Brown, G. Ronald; Winkler, C. A.
Describes a study of the kinetics of the recombination of nitrogen atoms in which concentration-time relations are determined directly by utilizing visual observations of emissions to make gas phase titrations of N atoms with NO. (MLH)
Chemical recombination in an expansion tube
NASA Technical Reports Server (NTRS)
Bakos, Robert J.; Morgan, Richard G.
The note describes the theoretical basis of chemical recombination in an expansion tube which simulates energy, Reynolds number, and stream chemistry at near-orbital velocities. Expansion tubes can satisfy ground-based hypersonic propulsion and aerothermal testing requirements.
Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine
This page contains brief information about recombinant human papillomavirus (HPV) nonavalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.
Recombinant Human Papillomavirus (HPV) Bivalent Vaccine
This page contains brief information about recombinant human papillomavirus (HPV) bivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.
Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine
This page contains brief information about recombinant human papillomavirus (HPV) quadrivalent vaccine and a collection of links to more information about the use of this vaccine, research results, and ongoing clinical trials.
Fermentations with new recombinant organisms
Bothast, R.J.; Nichols, N.N.; Dien, B.S.
US fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharomyces cerevisiae and Zymomonas mobilis for pentose utilization. The authors have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coli K011, E. coli SL40, E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21--34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different industrially hardened strains may find separate applications in the fermentation of specific feedstocks.
Recombination walking: Genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination
Miller, A.M.; Savinelli, E.A.; Couture, S.M.; Hannigan, G.M.; Han, Z.; Selden, R.F.; Treco, D.A. )
Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unorderd) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. 29 refs., 4 figs., 1 tab.
Recombination-deficient mutant of Streptococcus faecalis
Yagi, Y.; Clewell, D.B.
An ultraviolet radiation-sensitive derivative of Streptococcus faecalis strain JH2-2 was isolated and found to be deficient in recombination, using a plasmid-plasmid recombination system. The strain was sensitive to chemical agents which interact with deoxyribonucleic acid and also underwent deoxyribonucleic acid degradation after ultraviolet irradiation. Thus, the mutant has properties similar to those of recA strains of Escherichia coli.
Tunnel surface recombination in optoelectronic device modeling
NASA Astrophysics Data System (ADS)
Ptashchenko, Alexander A.; Ptashchenko, Fedor A.
The rate of tunnel surface recombination (TSR) in a p-n structure has been calculated as a function of the excitation level and temperature in a semiclassical approximation under the assumption that the excess energy of a recombining electron is transferred to phonons or to a photon. The approximating analytical expressions obtained are applied in calculations of the effect of TSR on the characteristics of photodiodes, solar cells, light-emitting diodes and diode lasers.
Co-factor activated recombinant adenovirus proteinases
Anderson, Carl W.; Mangel, Walter F.
This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.
Co-factor activated recombinant adenovirus proteinases
Anderson, C.W.; Mangel, W.F.
This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.
Recombination-generation currents in degenerate semiconductors
NASA Technical Reports Server (NTRS)
Von Roos, O.
The classical Shockley-Read-Hall theory of free carrier recombination and generation via traps is extended to degenerate semiconductors. A concise and simple expression is found which avoids completely the concept of a Fermi level, a concept which is alien to nonequilibrium situations. Assumptions made in deriving the recombination generation current are carefully delineated and are found to be basically identical to those made in the original theory applicable to nondegenerate semiconductors.
Detecting the cosmological recombination signal from space
NASA Astrophysics Data System (ADS)
Desjacques, Vincent; Chluba, Jens; Silk, Joseph; de Bernardis, Francesco; Doré, Olivier
Spectral distortions of the cosmic microwave background (CMB) have recently experienced an increased interest. One of the inevitable distortion signals of our cosmological concordance model is created by the cosmological recombination process, just a little before photons last scatter at redshift z ≃ 1100. These cosmological recombination lines, emitted by the hydrogen and helium plasma, should still be observable as tiny deviation from the CMB blackbody spectrum in the cm-dm spectral bands. In this paper, we present a forecast for the detectability of the recombination signal with future satellite experiments. We argue that serious consideration for future CMB experiments in space should be given to probing spectral distortions and, in particular, the recombination line signals. The cosmological recombination radiation not only allows determination of standard cosmological parameters, but also provides a direct observational confirmation for one of the key ingredients of our cosmological model: the cosmological recombination history. We show that, with present technology, such experiments are futuristic but feasible. The potential rewards won by opening this new window to the very early universe could be considerable.
Recombination hotspots: Models and tools for detection.
Paul, Prosenjit; Nag, Debjyoti; Chakraborty, Supriyo
Recombination hotspots are the regions within the genome where the rate, and the frequency of recombination are optimum with a size varying from 1 to 2kb. The recombination event is mediated by the double-stranded break formation, guided by the combined enzymatic action of DNA topoisomerase and Spo 11 endonuclease. These regions are distributed non-uniformly throughout the human genome and cause distortions in the genetic map. Numerous lines of evidence suggest that the number of hotspots known in humans has increased manifold in recent years. A few facts about the hotspot evolutions were also put forward, indicating the differences in the hotspot position between chimpanzees and humans. In mice, recombination hot spots were found to be clustered within the major histocompatibility complex (MHC) region. Several models, that help explain meiotic recombination has been proposed. Moreover, scientists also developed some computational tools to locate the hotspot position and estimate their recombination rate in humans is of great interest to population and medical geneticists. Here we reviewed the molecular mechanisms, models and in silico prediction techniques of hot spot residues. PMID:26991854
Biochemistry of homologous recombination in Escherichia coli.
Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M
Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921
Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses
Kilpatrick, David R.; Ching, Karen; Iber, Jane; Campagnoli, Ray; Freeman, Christopher J.; Mishrik, Nada; Liu, Hong-Mei; Pallansch, Mark A.; Kew, Olen M.
The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR. PMID:15365031
Oligonucleotide recombination enabled site-specific mutagenesis in bacteria
Technology Transfer Automated Retrieval System (TEKTRAN)
Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligo recombination is one type of recombineering that uses ssDNA olig...
The Saccharomyces cerevisiae recombination enhancer biases recombination during interchromosomal mating-type switching but not in interchromosomal homologous recombination.
Houston, Peter; Simon, Peter J; Broach, James R
Haploid Saccharomyces can change mating type through HO-endonuclease cleavage of an expressor locus, MAT, followed by gene conversion using one of two repository loci, HML or HMR, as donor. The mating type of a cell dictates which repository locus is used as donor, with a cells using HML and alpha cells using HMR. This preference is established in part by RE, a locus on the left arm of chromosome III that activates the surrounding region, including HML, for recombination in a cells, an activity suppressed by alpha 2 protein in alpha cells. We have examined the ability of RE to stimulate different forms of interchromosomal recombination. We found that RE exerted an effect on interchromosomal mating-type switching and on intrachromosomal homologous recombination but not on interchromosomal homologous recombination. Also, even in the absence of RE, MAT alpha still influenced donor preference in interchromosomal mating-type switching, supporting a role of alpha 2 in donor preference independent of RE. These results suggest a model in which RE affects competition between productive and nonproductive recombination outcomes. In interchromosome gene conversion, RE enhances both productive and nonproductive pathways, whereas in intrachromosomal gene conversion and mating-type switching, RE enhances only the productive pathway. PMID:15082540
Technology Transfer Automated Retrieval System (TEKTRAN)
This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ph:C31 integrase, that can mediate recombination between th...
Perinatal induction of Cre recombination with tamoxifen.
Lizen, Benoit; Claus, Melissa; Jeannotte, Lucie; Rijli, Filippo M; Gofflot, Françoise
Temporal control of site-specific recombination is commonly achieved by using a tamoxifen-inducible form of Cre or Flp recombinases. Although powerful protocols of induction have been developed for gene inactivation at adult stages or during embryonic development, induction of recombination at late gestational or early postnatal stages is still difficult to achieve. In this context, using the ubiquitous CMV-CreER(T2) transgenic mice, we have tested and validated two procedures to achieve recombination just before and just after birth. The efficiency of recombination was evaluated in the brain, which is known to be more problematic to target. For the late gestation treatment with tamoxifen, different protocols of complementary administration of progesterone and estrogen were tested. However, delayed delivery and/or mortality of pups due to difficult delivery were always observed. To circumvent this problem, pups were collected from tamoxifen-treated pregnant dams by caesarian section at E18.5 and given to foster mothers. For postnatal treatment, different dosages of tamoxifen were administered by intragastric injection to the pups during 3 or 4 days after birth. The efficiency of these treatments was analyzed at P7 using a transgenic reporter line. They were also validated with the Hoxa5 conditional allele. In conclusion, we have developed efficient procedures that allow achieving efficient recombination of floxed alleles at perinatal stages. These protocols will allow investigating the late/adult functions of many developmental genes, whose characterization has been so far restricted to embryonic development. PMID:26395370
Human recombinant lysosomal enzymes produced in microorganisms.
Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A
Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627
Graded recombination layers for multijunction photovoltaics.
Koleilat, Ghada I; Wang, Xihua; Sargent, Edward H
Multijunction devices consist of a stack of semiconductor junctions having bandgaps tuned across a broad spectrum. In solar cells this concept is used to increase the efficiency of photovoltaic harvesting, while light emitters and detectors use it to achieve multicolor and spectrally tunable behavior. In series-connected current-matched multijunction devices, the recombination layers must allow the hole current from one cell to recombine, with high efficiency and low voltage loss, with the electron current from the next cell. We recently reported a tandem solar cell in which the recombination layer was implemented using a progression of n-type oxides whose doping densities and work functions serve to connect, with negligible resistive loss at solar current densities, the constituent cells. Here we present the generalized conditions for design of efficient graded recombination layer solar devices. We report the number of interlayers and the requirements on work function and doping of each interlayer, to bridge an work function difference as high as 1.6 eV. We also find solutions that minimize the doping required of the interlayers in order to minimize optical absorption due to free carriers in the graded recombination layer (GRL). We demonstrate a family of new GRL designs experimentally and highlight the benefits of the progression of dopings and work functions in the interlayers. PMID:22554234
Recombination in Eukaryotic Single Stranded DNA Viruses
Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind
Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803
Recombination and collisionally excited Balmer lines
NASA Astrophysics Data System (ADS)
Raga, A. C.; Castellanos-Ramírez, A.; Esquivel, A.; Rodríguez-González, A.; Velázquez, P. F.
We present a model for the statistical equilibrium of the levels of H, considering recombinations to excited levels, collisional excitations up from the ground state and spontaneous radiative transitions. This problem has a simple "cascade matrix" solution, describing a cascade of downwards spontaneous transitions fed by both recombinations and collisional excitations. The resulting predicted Balmer line ratios show a transition between a low temperature and a high temperature regime (dominated by recombinations and by collisional excitations, respectively), both with only a weak line ratio vs. temperature dependence. This clear characteristic allows a direct observational identification of regions in which the Balmer lines are either recombination or collisionally excited transitions. We find that for a gas in coronal ionization equilibrium the Halpha and Hbeta lines are collisionally excited for all temperatures. In order to have recombination Halpha and Hbeta it is necessary to have higher ionization fractions of H than the ones obtained from coronal equilibrium (e.g., such as the ones found in a photoionized gas).
Meiotic recombination and genome evolution in plants.
Melamed-Bessudo, Cathy; Shilo, Shay; Levy, Avraham A
Homologous recombination affects genome evolution through crossover, gene conversion and point mutations. Whole genome sequencing together with a detailed epigenome analysis have shed new light on our understanding of how meiotic recombination shapes plant genes and genome structure. Crossover events are associated with DNA sequence motifs, together with an open chromatin signature (hypomethylated CpGs, low nucleosome occupancy or specific histone modifications). The crossover landscape may differ between male and female meiocytes and between species. At the gene level, crossovers occur preferentially in promoter regions in Arabidopsis. In recent years, there is rising support suggesting that biased mismatch repair during meiotic recombination may increase GC content genome-wide and may be responsible for the GC content gradient found in many plant genes. PMID:26939088
Dielectronic recombination of xenonlike tungsten ions
Schippers, S.; Bernhardt, D.; Mueller, A.; Krantz, C.; Grieser, M.; Repnow, R.; Wolf, A.; Lestinsky, M.; Hahn, M.; Novotny, O.; Savin, D. W.
Dielectronic recombination (DR) of xenonlike W{sup 20+} forming W{sup 19+} has been studied experimentally at a heavy-ion storage ring. A merged-beams method has been employed for obtaining absolute rate coefficients for electron-ion recombination in the collision-energy range 0-140 eV. The measured rate coefficient is dominated by strong DR resonances even at the lowest experimental energies. At plasma temperatures where the fractional abundance of W{sup 20+} is expected to peak in a fusion plasma, the experimentally derived plasma recombination rate coefficient is over a factor of 4 larger than the theoretically calculated rate coefficient which is currently used in fusion plasma modeling. The largest part of this discrepancy stems most probably from the neglect in the theoretical calculations of DR associated with fine-structure excitations of the W{sup 20+}([Kr]4d{sup 10} 4f{sup 8}) ion core.
Transposon-specified site-specific recombination.
Kitts, P; Symington, L; Burke, M; Reed, R; Sherratt, D
Cointegrate DNA molecules containing two copies of a transposable element appear to be intermediates in the transposition process. These structures are resolved by site-specific recombination to yield the normal end products of transposition. The transposable element gamma delta (Tn1000) synthesizes a product interchangeable with the Tn1/3tnpR protein in promoting Tn1/3 site-specific recombination. These data support the hypothesis that cointegrates containing directly repeated copies of Tn1/3 are obligatory intermediates in interreplicon transposition of Tn1/3. In addition, we show here that the reaction is independent of the element-encoded tnpA gene product. Tn501, which specifies mercury resistance, also produces cointegrates as intermediates in interreplicon transposition. The appearance of Tn501-specified recombination activity that can act on these cointegrates requires growth of cells in the presence of Hg2+. Images PMID:6275390
Generation of active immunotoxins containing recombinant restrictocin.
Rathore, D; Batra, J K
Restrictocin, a toxin produced by the fungus Aspergillus restrictus, is a potent inhibitor of eukaryotic protein synthesis. Recombinant restrictocin was made in Escherichia coli and purified to homogeneity in large amounts. The recombinant protein was found to be poorly immunogenic in mice with low toxicity, when injected intraperitoneally. Two immunotoxins were constructed by coupling the recombinant restrictocin to an antibody to the human transferrin receptor, using a cleavable and a stable linkage. The immunotoxins so generated showed specific cytotoxic activity toward receptor bearing cells in tissue culture. Immunotoxin with a cleavable linkage, however, was more active than that containing a stable linkage. Restrictocin appears to be a promising candidate to be developed as a chimeric toxin for targeted therapy. PMID:8630074
Transcription and Recombination: When RNA Meets DNA
Aguilera, Andrés; Gaillard, Hélène
A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910
Recombination and DNA Repair in Helicobacter pylori
Dorer, Marion S.; Sessler, Tate H.; Salama, Nina R.
All organisms have pathways that repair the genome, ensuring their survival and that of their progeny. But these pathways also serve to diversify the genome, causing changes on the level of nucleotide, whole gene, and genome structure. Sequencing of bacteria has revealed wide allelic diversity and differences in gene content within the same species, highlighting the importance of understanding pathways of recombination and DNA repair. The human stomach pathogen Helicobacter pylori is an excellent model system for studying these pathways. H. pylori harbors major recombination and repair pathways and is naturally competent, facilitating its ability to diversify its genome. Elucidation of DNA recombination, repair, and diversification programs in this pathogen will reveal connections between these pathways and their importance to infection. PMID:21682641
Jet fragmentation via recombination of parton showers
NASA Astrophysics Data System (ADS)
Han, Kyong Chol; Fries, Rainer J.; Ko, Che Ming
We propose to model hadronization of parton showers in QCD jets through a hybrid approach involving quark recombination and string fragmentation. This is achieved by allowing gluons at the end of the perturbative shower evolution to undergo a nonperturbative splitting into quark and antiquark pairs, then applying a Monte Carlo version of instantaneous quark recombination, and finally subjecting remnant quarks (those which have not found a recombination partner) to Lund string fragmentation. When applied to parton showers from the pythia Monte Carlo event generator, the final hadron spectra from our calculation compare quite well to pythia jets that have been hadronized with the default Lund string fragmentation. Our new approach opens up the possibility to generalize hadronization to jets embedded in a quark gluon plasma.
CosmoRec: Cosmological Recombination code
NASA Astrophysics Data System (ADS)
Chluba, Jens; Thomas, Rajat Mani
CosmoRec solves the recombination problem including recombinations to highly excited states, corrections to the 2s-1s two-photon channel, HI Lyn-feedback, n>2 two-photon profile corrections, and n≥2 Raman-processes. The code can solve the radiative transfer equation of the Lyman-series photon field to obtain the required modifications to the rate equations of the resolved levels, and handles electron scattering, the effect of HeI intercombination transitions, and absorption of helium photons by hydrogen. It also allows accounting for dark matter annihilation and optionally includes detailed helium radiative transfer effects.
Graph Model of Coalescence with Recombinations
NASA Astrophysics Data System (ADS)
Parida, Laxmi
One of the primary genetic events shaping an autosomal chromosome is recombination. This is a process that occurs during meiosis, in eukaryotes, that results in the offsprings having different combinations of (homologous) genes, or chromosomal segments, of the two parents. The presence of these recombination events in the evolutionary history of each chromosome complicates the genetic landscape of a population, and understanding the manifestations of these genetic exchanges in the chromosome sequences has been a subject of intense curiosity (see [Hud83, Gri99, HSW05] and citations therein).
Recent Theoretical Studies On Excitation and Recombination
NASA Technical Reports Server (NTRS)
Pradhan, Anil K.
New advances in the theoretical treatment of atomic processes in plasmas are described. These enable not only an integrated, unified, and self-consistent treatment of important radiative and collisional processes, but also large-scale computation of atomic data with high accuracy. An extension of the R-matrix work, from excitation and photoionization to electron-ion recombination, includes a unified method that subsumes both the radiative and the di-electronic recombination processes in an ab initio manner. The extensive collisional calculations for iron and iron-peak elements under the Iron Project are also discussed.
Lectin glycoprofiling of recombinant therapeutic interleukin-7.
Landemarre, Ludovic; Duverger, Eric
Lectins array is a powerfull and complementary method of glycans analysis allowing fast identification of specific motifs on molecules or cells. This technology is of increased interest for the development of therapeutic recombinant glycoproteins and particularly relevant for a first study of lot-to-lot comparison, or detection of unwanted glycans. In this chapter, we describe a lectin array-type method specifically designed for the study of recombinant therapeutic interleukin-7 (rhIL-7). This specific method allows the analysis of the glycans motifs, the distribution of the glycoforms population, and the detection of potential immunogen glycans in rhIL-7 purified CHO-produced batches. PMID:23475723
The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss.
Chambers, S R; Hunter, N; Louis, E J; Borts, R H
Efficient genetic recombination requires near-perfect homology between participating molecules. Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. The effects of chromosomal divergence in diploids of Saccharomyces cerevisiae in which one copy of chromosome III is derived from a closely related species, Saccharomyces paradoxus, have been examined. Meiotic recombination between the diverged chromosomes is decreased by 25-fold. Spore viability is reduced with an observable increase in the number of tetrads with only two or three viable spores. Asci with only two viable spores are disomic for chromosome III, consistent with meiosis I nondisjunction of the homeologs. Asci with three viable spores are highly enriched for recombinants relative to tetrads with four viable spores. In 96% of the class with three viable spores, only one spore possesses a recombinant chromosome III, suggesting that the recombination process itself contributes to meiotic death. This phenomenon is dependent on the activities of the mismatch repair genes PMS1 and MSH2. A model of mismatch-stimulated chromosome loss is proposed to account for this observation. As expected, crossing over is increased in pms1 and msh2 mutants. Furthermore, genetic exchange in pms1 msh2 double mutants is affected to a greater extent than in either mutant alone, suggesting that the two proteins act independently to inhibit homeologous recombination. All mismatch repair-deficient strains exhibited reductions in the rate of chromosome III nondisjunction. PMID:8887641
Recombinant microorganisms for increased production of organic acids
Yi, Jian; Kleff, Susanne; Guettler, Michael V.
Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.
Recombinant microorganisms for increased production of organic acids
Yi, Jian; Kleff, Susanne; Guettler, Michael V
Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.
Recombinant Bovine Growth Hormone Criticism Grows.
ERIC Educational Resources Information Center
Gaard, Greta
Discusses concerns related to the use of recombinant bovine growth hormone in the United States and other countries. Analyses the issue from the perspectives of animal rights, human health, world hunger, concerns of small and organic farmers, costs to the taxpayer, and environmental questions. A sidebar discusses Canadian review of the hormone.…
Science: The Recombinant DNA Advisory Committee.
ERIC Educational Resources Information Center
Wright, Susan
Reports on the status of the Recombinant DNA Advisory Committee (RAC) and attempts to rationalize Suburban Highway Policy. Effective communication among members of the RAC is a current problem facing the committee. A federal transportation priority spending policy is suggested during these times of money and fuel shortages. (MA)
Vaccine development using recombinant DNA technology
Technology Transfer Automated Retrieval System (TEKTRAN)
Vaccines induce an immune response in the host that subsequently recognizes infectious agents and helps fight off the disease; vaccines must do this without causing the disease. This paper reviews the development of recombinant DNA technologies as a means of providing new ways for attenuating diseas...
Recombinant protein blends: silk beyond natural design.
Dinjaski, Nina; Kaplan, David L
Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. PMID:26686863
King, L.D.P.
A hydrogen-oxygen recombiner is described for use with water-boiler type reactors. The catalyst used is the wellknown platinized alumina, and the novelty lies in the structural arrangement used to prevent flashback through the gas input system. The recombiner is cylindrical, the gases at the input end being deflected by a baffle plate through a first flashback shield of steel shot into an annular passage adjacent to and extending the full length of the housing. Below the baffle plate the gases flow first through an outer annular array of alumina pellets which serve as a second flashback shield, a means of distributing the flowing gases evenly and as a means of reducing radiation losses to the walls. Thereafter the gases flow inio the centrally disposed catalyst bed where recombination is effected. The steam and uncombined gases flow into a centrally disposed cylindrical passage inside the catalyst bod and thereafter out through the exit port. A high rate of recombination is effected.
Gas recombination assembly for electrochemical cells
Levy, Isaac; Charkey, Allen
An assembly for recombining gases generated in electrochemical cells wherein a catalyst strip is enveloped within a hydrophobic, gas-porous film which, in turn, is encased between gas-porous, metallic layers. The sandwich construction of metallic layers and film is formed into a spiral with a tab for connection to the cell.
Recombinant DNA: Scientific and Social Perspectives.
ERIC Educational Resources Information Center
Vandegrift, Vaughn
This article is designed to inform chemical educators not engaged in this technology as to the nature and methods used in the technology, the reasons for scientific and social concern, and the attempts made to assuage concerns involving recombinant DNA research. (author/BB)
Recombinant therapeutic proteins: production platforms and challenges.
Dingermann, Theo
Since the approval of insulin in 1982, more than 120 recombinant drug substances have been approved and become available as extremely valuable therapeutic options. Exact copying of the most common human form is no longer a value per se, as challenges, primarily related to the pharmacokinetics of artificial recombinant drugs, can be overcome by diverging from the original. However, relatively minor changes in manufacturing or packaging may impact safety of therapeutic proteins. A major achievement is the development of recombinant proteins capable of entering a cell. Such drugs open up completely new opportunities by targeting intracellular mechanisms or by substituting intracellularly operating enzymes. Concerns that protein variants would cause an intolerable immune response turned out to be exaggerated. Although most recombinant drugs provoke some immune response, they are still well tolerated. This knowledge might result in a change in attitude towards antibody formation, i.e., neutralizing antibody activity (in vitro) may be overcome by dosing consistently on the basis of antibody titers and not only on body weight. As with other drugs, efficacy and safety of therapeutic proteins have to be demonstrated in clinical studies, and superiority over available products has to be proven instead of just claimed. PMID:18041103
Precise genotyping and recombination detection of Enterovirus
Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286
DNA Sequence Alignment during Homologous Recombination.
Greene, Eric C
Homologous recombination allows for the regulated exchange of genetic information between two different DNA molecules of identical or nearly identical sequence composition, and is a major pathway for the repair of double-stranded DNA breaks. A key facet of homologous recombination is the ability of recombination proteins to perfectly align the damaged DNA with homologous sequence located elsewhere in the genome. This reaction is referred to as the homology search and is akin to the target searches conducted by many different DNA-binding proteins. Here I briefly highlight early investigations into the homology search mechanism, and then describe more recent research. Based on these studies, I summarize a model that includes a combination of intersegmental transfer, short-distance one-dimensional sliding, and length-specific microhomology recognition to efficiently align DNA sequences during the homology search. I also suggest some future directions to help further our understanding of the homology search. Where appropriate, I direct the reader to other recent reviews describing various issues related to homologous recombination. PMID:27129270
Precise genotyping and recombination detection of Enterovirus.
Lin, Chieh-Hua; Wang, Yu-Bin; Chen, Shu-Hwa; Hsiung, Chao Agnes; Lin, Chung-Yen
Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution. PMID:26678286
Recombination phenomena in high efficiency silicon solar cells
NASA Technical Reports Server (NTRS)
Sah, C. T.
The dominant recombination phenomena which limit the highest efficiency attainable in silicon solar cells under terrestrial sunlight are reviewed. The ultimate achievable efficiency is limited by the two intrinsic recombination mechanisms, the interband Auger recombination and interband Radiative recombination, both of which occur in the entire cell body but principally in the base layer. It is suggested that an optimum (26%) cell design is one with lowly doped 50 to 100 micron thick base, a perfect BSF, and zero extrinsic recombination such as the thermal mechanism at recombination centers the Shockley-Read-Hall process (SRH) in the bulk, on the surface and at the interfaces. The importance of recombination at the interfaces of a high-efficiency cell is demonstrated by the ohmic contact on the back surface whose interface recombination velocity is infinite. The importance of surface and interface recombination is demonstrated by representing the auger and radiative recombination losses by effective recombination velocities. It is demonstrated that the three highest efficiency cells may all be limited by the SRH recombination losses at recombination centers in the base layer.
Radiative transfer effects in primordial hydrogen recombination
Ali-Haiemoud, Yacine; Hirata, Christopher M.; Grin, Daniel
The calculation of a highly accurate cosmological recombination history has been the object of particular attention recently, as it constitutes the major theoretical uncertainty when predicting the angular power spectrum of cosmic microwave background anisotropies. Lyman transitions, in particular the Lyman-{alpha} line, have long been recognized as one of the bottlenecks of recombination, due to their very low escape probabilities. The Sobolev approximation does not describe radiative transfer in the vicinity of Lyman lines to a sufficient degree of accuracy, and several corrections have already been computed in other works. In this paper, we compute the impact of some radiative transfer effects that were previously ignored, or for which previous treatments were incomplete. First, the effect of Thomson scattering in the vicinity of the Lyman-{alpha} line is evaluated, using a full redistribution kernel incorporated into a radiative transfer code. The effect of feedback of distortions generated by the optically thick deuterium Lyman-{alpha} line blueward of the hydrogen line is investigated with an analytic approximation. It is shown that both effects are negligible during cosmological hydrogen recombination. Second, the importance of high-lying, nonoverlapping Lyman transitions is assessed. It is shown that escape from lines above Ly{gamma} and frequency diffusion in Ly{beta} and higher lines can be neglected without loss of accuracy. Third, a formalism generalizing the Sobolev approximation is developed to account for the overlap of the high-lying Lyman lines, which is shown to lead to negligible changes to the recombination history. Finally, the possibility of a cosmological hydrogen recombination maser is investigated. It is shown that there is no such maser in the purely radiative treatment presented here.
Ancestries of a recombining diploid population.
Sainudiin, R; Thatte, B; Véber, A
We derive the exact one-step transition probabilities of the number of lineages that are ancestral to a random sample from the current generation of a bi-parental population that is evolving under the discrete Wright-Fisher model with n diploid individuals. Our model allows for a per-generation recombination probability of r . When r = 1, our model is equivalent to Chang's (Adv Appl Probab 31:1002-1038, 1999) model for the karyotic pedigree. When r = 0, our model is equivalent to Kingman's (Stoch Process Appl 13:235-248, 1982) discrete coalescent model for the cytoplasmic tree or sub-karyotic tree containing a DNA locus that is free of intra-locus recombination. When 0 < r < 1 our model can be thought to track a sub-karyotic ancestral graph containing a DNA sequence from an autosomal chromosome that has an intra-locus recombination probability r . Thus, our family of models indexed by r ∈ [0, 1] connects Kingman's discrete coalescent to Chang's pedigree in a continuous way as r goes from 0 to 1. For large populations, we also study three properties of the ancestral process corresponding to a given r ∈ (0, 1): the time Tn to a most recent common ancestor (MRCA) of the population, the time Un at which all individuals are either common ancestors of all present day individuals or ancestral to none of them, and the fraction of individuals that are common ancestors at time Un. These results generalize the three main results of Chang's (Adv Appl Probab 31:1002-1038, 1999). When we appropriately rescale time and recombination probability by the population size, our model leads to the continuous time Markov chain called the ancestral recombination graph of Hudson (Theor Popul Biol 23:183-201, 1983) and Griffiths (The two-locus ancestral graph, Institute of Mathematical Statistics 100-117, 1991). PMID:25925241
Extrachromosomal recombination substrates recapitulate beyond 12/23 restricted VDJ recombination in nonlymphoid cells.
Jung, David; Bassing, Craig H; Fugmann, Sebastian D; Cheng, Hwei-Ling; Schatz, David G; Alt, Frederick W
V(D)J recombination occurs efficiently only between gene segments flanked by recombination signals (RSs) containing 12 and 23 base pair spacers (the 12/23 rule). A further limitation "beyond the 12/23 rule" (B12/23) exists at the TCRbeta locus and ensures Dbeta usage. Herein, we show that extrachromosomal V(D)J recombination substrates recapitulate B12/23 restriction in nonlymphoid cells. We further demonstrate that the Vbeta coding flank, the 12-RS heptamer/nonamer, and the 23-RS spacer each can significantly influence B12/23 restriction. Finally, purified core RAG1 and RAG2 proteins (together with HMG2) also reproduce B12/23 restriction in a cell-free system. Our findings indicate that B12/23 restriction of V(D)J recombination is cemented at the level of interactions between the RAG proteins and TCRbeta RS sequences. PMID:12530976
Bacteriophage T4 DNA Topoisomerase Mediates Illegitimate Recombination in vitro
NASA Astrophysics Data System (ADS)
Ikeda, Hideo
We have found that purified T4 DNA topoisomerase promotes recombination between two phage λ DNA molecules in an in vitro system. In this cross, T4 DNA topoisomerase alone is able to catalyze the recombination and produce a linear monomer recombinant DNA that can be packaged in vitro. ATP is not required for this recombination, while oxolinic acid stimulates it. The recombinant DNA molecules contain duplications or deletions, and the crossovers take place between nonhomologous and nonspecific sequences of λ DNA. Therefore, the recombination mediated by the T4 DNA topoisomerase is an illegitimate recombination that is similar to that mediated by Escherichia coli DNA gyrase. A model was proposed previously in which DNA gyrase molecules that are bound to DNA associate with each other and lead to the exchange of DNA strands through the exchange of DNA gyrase subunits. This model is also applicable to the recombination mediated by T4 DNA topoisomerase.
Serial Recombination during Circulation of Type 1 Wild-Vaccine Recombinant Polioviruses in China
Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Kew, Olen M.; Pallansch, Mark A.
Type 1 wild-vaccine recombinant polioviruses sharing a 367-nucleotide (nt) block of Sabin 1-derived sequence spanning the VP1 and 2A genes circulated widely in China from 1991 to 1993. We surveyed the sequence relationships among 34 wild-vaccine recombinants by comparing six genomic intervals: the conserved 5′-untranslated region (5′-UTR) (nt 186 to 639), the hypervariable portion of the 5′-UTR (nt 640 to 742), the VP4 and partial VP2 genes (nt 743 to 1176), the VP1 gene (nt 2480 to 3385), the 2A gene (nt 3386 to 3832), and the partial 3D gene (nt 6011 to 6544). The 5′-UTR, capsid (VP4-VP2 and VP1), and 2A sequence intervals had similar phylogenies. By contrast, the partial 3D sequences could be distributed into five divergent genetic classes. Most (25 of 34) of the wild-vaccine recombinant isolates showed no evidence of additional recombination beyond the initial wild-Sabin recombination event. Eight isolates from 1992 to 1993, however, appear to be derived from three independent additional recombination events, and one 1993 isolate was derived from two consecutive events. Complete genomic sequences of a representative isolate for each 3D sequence class demonstrated that these exchanges had occurred in the 2B, 2C, and 3D genes. The 3D gene sequences were not closely related to those of the Sabin strains or 53 diverse contemporary wild poliovirus isolates from China, but all were related to the 3D genes of species C enteroviruses. The appearance within approximately 2.5 years of five recombinant classes derived from a single ancestral infection illustrates the rapid emergence of new recombinants among circulating wild polioviruses. PMID:14512548
Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus.
Johnson, T M; Pease, E A; Li, J K; Tien, M
Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification. PMID:1632652
An improved recombineering approach by adding RecA to lambda Red recombination.
Wang, Junping; Sarov, Mihail; Rientjes, Jeanette; Fu, Jun; Hollak, Heike; Kranz, Harald; Xie, Wei; Stewart, A Francis; Zhang, Youming
Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date. PMID:16382181
Molecular hydrogen in the cosmic recombination epoch
Alizadeh, Esfandiar; Hirata, Christopher M.
The advent of precise measurements of the CMB anisotropies has motivated correspondingly precise calculations of the cosmic recombination history. Cosmic recombination proceeds far out of equilibrium because of a ''bottleneck'' at the n=2 level of hydrogen: atoms can only reach the ground state via slow processes--two-photon decay or Lyman-{alpha} resonance escape. However, even a small primordial abundance of molecules could have a large effect on the interline opacity in the recombination epoch and lead to an additional route for hydrogen recombination. Therefore, this paper computes the abundance of the H{sub 2} molecule during the cosmic recombination epoch. Hydrogen molecules in the ground electronic levels X{sup 1}{Sigma}{sub g}{sup +} can either form from the excited H{sub 2} electronic levels B{sup 1}{Sigma}{sub u}{sup +} and C{sup 1}{Pi}{sub u} or through the charged particles H{sub 2}{sup +}, HeH{sup +}, and H{sup -}. We follow the transitions among all of these species, resolving the rotational and vibrational sublevels. Since the energies of the X{sup 1}{Sigma}{sub g}{sup +}-B{sup 1}{Sigma}{sub u}{sup +} (Lyman band) and X{sup 1}{Sigma}{sub g}{sup +}-C{sup 1}{Pi}{sub u} (Werner band) transitions are near the Lyman-{alpha} energy, the distortion of the CMB spectrum caused by escaped H Lyman-line photons accelerates both the formation and the destruction of H{sub 2} due to this channel relative to the thermal rates. This causes the populations of H{sub 2} molecules in X{sup 1}{Sigma}{sub g}{sup +} energy levels to deviate from their thermal equilibrium abundances. We find that the resulting H{sub 2} abundance is 10{sup -17} at z=1200 and 10{sup -13} at z=800, which is too small to have any significant influence on the recombination history.
The Unconventional Xer Recombination Machinery of Streptococci/Lactococci
Le Bourgeois, Pascal; Bugarel, Marie; Campo, Nathalie; Daveran-Mingot, Marie-Line; Labonté, Jessica; Lanfranchi, Daniel; Lautier, Thomas; Pagès, Carine; Ritzenthaler, Paul
Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (difSL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine difSL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when difSL sites are located on chromosome dimers. Moreover, the XerS/difSL recombination requires the streptococcal protein FtsKSL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs. PMID:17630835
Using Crossover Breakpoints in Recombinant Inbred Lines to Identify Quantitative Trait Loci Controlling the Global Recombination Frequency
Technology Transfer Automated Retrieval System (TEKTRAN)
Recombination is a crucial component of evolution and breeding, producing new genetic combinations on which selection can act. Rates of recombination vary tremendously, not only between species but also within species and for specific chromosomal segments. In this study, by examining recombination...
Recombination energy in double white dwarf formation
NASA Astrophysics Data System (ADS)
Nandez, J. L. A.; Ivanova, N.; Lombardi, J. C.
In this Letter, we investigate the role of recombination energy during a common envelope event. We confirm that taking this energy into account helps to avoid the formation of the circumbinary envelope commonly found in previous studies. For the first time, we can model a complete common envelope event, with a clean compact double white dwarf binary system formed at the end. The resulting binary orbit is almost perfectly circular. In addition to considering recombination energy, we also show that between 1/4 and 1/2 of the released orbital energy is taken away by the ejected material. We apply this new method to the case of the double white dwarf system WD 1101+364, and we find that the progenitor system at the start of the common envelope event consisted of an ˜1.5 M⊙ red giant star in an ˜30 d orbit with a white dwarf companion.
The evolution of sex dimorphism in recombination.
Lenormand, Thomas
Sex dimorphism in recombination is widespread on both sex chromosomes and autosomes. Various hypotheses have been proposed to explain these dimorphisms. Yet no theoretical model has been explored to determine how heterochiasmy--the autosomal dimorphism--could evolve. The model presented here shows three circumstances in which heterochiasmy is likely to evolve: (i) a male-female difference in haploid epistasis, (ii) a male-female difference in cis-epistasis minus trans-epistasis in diploids, or (iii) a difference in epistasis between combinations of genes inherited maternally or paternally. These results hold even if sources of linkage disequilibria besides epistasis, such as migration or Hill-Robertson interference, are considered and shed light on previous verbal models of sex dimorphism in recombination rates. Intriguingly, these results may also explain why imprinted regions on the autosomes of humans or sheep are particularly heterochiasmate. PMID:12618416
Recombinant laccase: I. Enzyme cloning and characterization.
Nicolini, Claudio; Bruzzese, Debora; Cambria, Maria Teresa; Bragazzi, Nicola Luigi; Pechkova, Eugenia
We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-β-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. PMID:22991171
Recombination clumping factor during cosmic reionization
Kaurov, Alexander A.; Gnedin, Nickolay Y. E-mail: email@example.com
We discuss the role of recombinations in the intergalactic medium, and the related concept of the clumping factor, during cosmic reionization. The clumping factor is, in general, a local quantity that depends on both the local overdensity and the scale below which the baryon density field can be assumed smooth. That scale, called the filtering scale, depends on over-density and local thermal history. We present a method for building a self-consistent analytical model of inhomogeneous reionization, assuming the linear growth rate of the density fluctuation, which simultaneously accounts for these effects. We show that taking into account the local clumping factor introduces significant corrections to the total recombination rate, compared to the model with a globally uniform clumping factor.
Recombinant organisms for production of industrial products
Adrio, Jose-Luis
A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding techniques and their modifications are contributing greatly to the development of improved industrial processes. In addition, functional genomics, proteomics and metabolomics are being exploited for the discovery of novel valuable small molecules for medicine as well as enzymes for catalysis. The sequencing of industrial microbal genomes is being carried out which bodes well for future process improvement and discovery of new industrial products. PMID:21326937
Recombineering BAC transgenes for protein tagging.
Ciotta, Giovanni; Hofemeister, Helmut; Maresca, Marcello; Fu, Jun; Sarov, Mihail; Anastassiadis, Konstantinos; Stewart, A Francis
Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression. PMID:20868752
Hα diagnostic in a recombining plasma
NASA Astrophysics Data System (ADS)
Wenzel, U.; Goto, M.
In fusion devices the hydrogen Balmer lines are used to measure the neutral flux from the walls into the plasma using the atomic physics factor S/XB. This is a standard diagnostic which can be applied in ionizing plasma using {{H}α} , {{H}β} or {{H}γ} without knowledge of the electron density. We will extend this method to a recombining plasma in front of a surface. {{H}α} can be used in an analogous way to measure the plasma flow to this surface which can be e.g. a divertor target. The other Balmer lines are not suitable because the corresponding atomic physics factor R/YB depends on density due to three-body recombination. An application of this diagnostic method is provided.
Dissipative Stern-Gerlach recombination experiment
Oliveira, Thiago R. de; Caldeira, A. O.
The possibility of obtaining the initial pure state in a usual Stern-Gerlach experiment through the recombination of the two emerging beams is investigated. We have extended the previous work of Englert, Schwinger, and Scully [Found Phys. 18, 1045 (1988)] including the fluctuations of the magnetic field generated by a properly chosen magnet. As a result we obtained an attenuation factor to the possible revival of coherence when the beams are perfectly recombined. When the source of the magnetic field is a superconducting quantum interference device (SQUID) the attenuation factor can be controlled by external circuits and the spin decoherence directly measured. For the proposed SQUID with dimensions in the scale of microns the attenuation factor has been shown unimportant when compared with the interaction time of the spin with the magnet.
Designing recombinant Pseudomonas strains to enhance biodesulfurization.
Gallardo, M E; Ferrández, A; De Lorenzo, V; García, J L; Díaz, E
The dsz biodesulfurization cluster from Rhodococcus erythropolis IGTS8 has been engineered under the control of heterologous broad-host-range regulatory signals to alleviate the mechanism of sulfur repression, and it was stably inserted into the chromosomes of different Pseudomonas strains. The recombinant bacteria were able to desulfurize dibenzothiophene more efficiently than the native host. Furthermore, these new biocatalysts combine relevant industrial and environmental traits, such as production of biosurfactants, with the enhanced biodesulfurization phenotype. PMID:9371464
Recombinant organisms capable of fermenting cellobiose
Ingram, Lonnie O.; Lai, Xiaokuang; Moniruzzaman, Mohammed; York, Sean W.
This invention relates to a recombinant microorganism which expresses pyruvate decarboxylase, alcohol dehydrogenase, Klebsiella phospho-.beta.-glucosidase and Klebsiella (phosphoenolpyruvate-dependent phosphotransferase system) cellobiose-utilizing Enzyme II, wherein said phospho-.beta.-glucosidase and said (phosphoenolpyruvate-dependent phosphotransferase) cellobiose-utilizing Enzyme II are heterologous to said microorganism and wherein said microorganism is capable of utilizing both hemicellulose and cellulose, including cellobiose, in the production of ethanol.
Nonradiative Auger recombination in semiconductor nanocrystals.
Vaxenburg, Roman; Rodina, Anna; Shabaev, Andrew; Lifshitz, Efrat; Efros, Alexander L
We calculate the rate of nonradiative Auger recombination in negatively charged CdSe nanocrystals (NCs). The rate is nonmonotonic, strongly oscillating with NC size, and sensitive to the NC surface. The oscillations result in nonexponential decay of carriers in NC ensembles. Using a standard single-exponential approximation of the decay dynamics, we determine the apparent size dependence of the Auger rate in an ensemble and derive CdSe surface parameters consistent with the experimental dependence on size. PMID:25693512
Mechanism and regulation of meiotic recombination initiation
Lam, Isabel; Keeney, Scott
Meiotic recombination involves the formation and repair of programmed DNA double-strand breaks (DSBs) catalyzed by the conserved Spo11 protein. This review summarizes recent studies pertaining to the formation of meiotic DSBs, including the mechanism of DNA cleavage by Spo11, proteins required for break formation, and mechanisms that control the location, timing, and number of DSBs. Where appropriate, findings in different organisms are discussed to highlight evolutionary conservation or divergence. PMID:25324213
Development of Mycoplasma hyopneumoniae Recombinant Vaccines.
Marchioro, Silvana Beutinger; Simionatto, Simone; Dellagostin, Odir
Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a disease that affects swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of the disease. Research using genome-based approach has the potential to elucidate the biology and pathogenesis of M. hyopneumoniae and contribute to the development of more effective vaccines. Here, we describe the protocol for developing M. hyopneumoniae recombinant vaccines using reverse vaccinology approaches. PMID:27076288
Cultivating Insect Cells To Produce Recombinant Proteins
NASA Technical Reports Server (NTRS)
Spaulding, Glenn; Goodwin, Thomas; Prewett, Tacey; Andrews, Angela; Francis, Karen; O'Connor, Kim
Method of producing recombinant proteins involves growth of insect cells in nutrient solution in cylindrical bioreactor rotating about cylindrical axis, oriented horizontally and infecting cells with viruses into which genes of selected type cloned. Genes in question those encoding production of desired proteins. Horizontal rotating bioreactor preferred for use in method, denoted by acronym "HARV", described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662).
Three-Body Recombination of Oxygen Atoms
NASA Astrophysics Data System (ADS)
Huestis, D. L.; Kalogerakis, K. S.
Dayside photodissociation of O2 and CO2 in the atmospheres of Earth, Venus, and Mars produces oxygen atoms that eventually undergo three-body recombination O + O + M -> O2* + M The variety of electronic states produced is observable as nightglow emissions, which have been the subject of many laboratory and interpretive investigations. Here we review the current understanding of the overall temperature-dependent rate coefficient for three-body recombination of oxygen atoms and describe a strategy for its measurement. The most recent measurement [1] is almost 30 years old. The most comprehensive review [2] is more than 25 years old and shows that the absolute rate coefficients for recombination and the reverse process, collision-induced dissociation, as well as the dependence on temperature and collider, are poorly determined, in spite of the relatively narrow error bars reported in the various studies. The most recent high-temperature dissociation study [3] actually increases the divergence. We plan experiments with a commercial F2 laser, providing roughly 50 mJ of 157 nm radiation in a 3-4 mm beam, to achieve greater than 80% dissociation of molecular oxygen, in the range from 0.5 to 5 torr. In a high-pressure N2 background (30-200 torr) the oxygen atoms will recombine in a time scale from 0.1 to 10 ms, as monitored by 845 nm fluorescence excited by two photons at 226 nm. [1] I. M. Campbell and C. N. Gray, Chem. Phys. Lett. 18, 607 (1973). [2] D. L. Baulch, D. D. Drysdale, J. Duxbury, and S. J. Grant, Evaluated Kinetic Data for High Temperature Reactions Vol. 3 ``Homogeneous Gas Phase Reactions of the O2--O3 System, the CO--O2--H2 System, and of Sulphur-Containing Species," (Butterworths, London, 1976). [3] V. Naudet, S. Abid, and C. E. Paillard, J. Chim. Phys. 96, 1123 (1999).
Ion recombination in aircraft exhaust plumes
NASA Astrophysics Data System (ADS)
Sorokin, A.; Mirabel, P.
In this article, a model which examines the evolution of ion concentrations in a hot aircraft exhaust plume on the ground is proposed. The model includes plume dilution and ion-ion recombination with coefficients which vary with temperature. A comparison of the model is made with the available ground-based experimental data obtained on the ATTAS research aircraft engines. From this comparison, an ion emission index of the order of 8 1016 ions/kg(fuel) inferred.
[Homologous recombination among bacterial genomes: the measurement and identification].
Xianwei, Yang; Ruifu, Yang; Yujun, Cui
Homologous recombination is one of important sources in shaping the bacterial population diversity, which disrupts the clonal relationship among different lineages through horizontal transferring of DNA-segments. As consequence of blurring the vertical inheritance signals, the homologous recombination raises difficulties in phylogenetic analysis and reconstruction of population structure. Here we discuss the impacts of homologous recombination in inferring phylogenetic relationship among bacterial isolates, and summarize the tools and models separately used in recombination measurement and identification. We also highlight the merits and drawbacks of various approaches, aiming to assist in the practical application for the analysis of homologous recombination in bacterial evolution research. PMID:26907777
RECOMBINATION RATE COEFFICIENTS OF Be-LIKE Si
Orban, I.; Boehm, S.; Schuch, R.; Loch, S. D.
Recombination of Be-like Si{sup 10+} over the 0-43 eV electron-ion energy range is measured at the CRYRING electron cooler. In addition to radiative and dielectronic recombination, the recombination spectrum also shows strong contributions from trielectronic recombination. Below 100 meV, several very strong resonances associated with a spin-flip of the excited electron dominate the spectrum and also dominate the recombination in the photoionized plasma. The resonant plasma rate coefficients corrected for the experimental field ionization are in good agreement with calculated results by Gu and with AUTOSTRUCTURE calculations. All other calculations significantly underestimate the plasma rate coefficients at low temperatures.
Radiative recombination of hot carriers in narrow-gap semiconductors
Pavlov, N. V.; Zegrya, G. G.
The mechanism of the radiative recombination of hot carriers in narrow-gap semiconductors is analyzed using the example of indium antimonide. It is shown that the CHCC Auger recombination process may lead to pronounced carrier heating at high excitation levels. The distribution functions and concentrations of hot carriers are determined. The radiative recombination rate of hot carriers and the radiation gain coefficient are calculated in terms of the Kane model. It is demonstrated that the radiative recombination of hot carriers will make a substantial contribution to the total radiative recombination rate at high carrier concentrations.
Recombination between linear double-stranded DNA substrates in vivo
Narayanan, Kumaran; Sim, Edmund Ui-Hang; Ravin, Nikolai V.; Lee, Choon-Weng
Recombineering technology in E. coli enables targeting of linear donor DNA to circular recipient DNA using short shared homology sequences. In this work, we demonstrate that recombineering is also able to support recombination between a pair of linear DNA substrates (linear/linear recombineering) in vivo in E. coli. Linear DNA up to 100 kb is accurately modified and remains intact without undergoing rearrangements after recombination. This system will be valuable for direct in vivo manipulation of large linear DNA including the N15 and PY54 prophages and linear animal viruses, and for assembly of linear constructs as artificial chromosome vectors. PMID:19454252
Recombinant Origin of the Retrovirus XMRV
Paprotka, Tobias; Delviks-Frankenberry, Krista A.; Cingöz, Oya; Martinez, Anthony; Kung, Hsing-Jien; Tepper, Clifford G.; Hu, Wei-Shau; Fivash, Matthew J.; Coffin, John M.; Pathak, Vinay K.
The retrovirus XMRV (xenotropic murine leukemia virus-related virus) has been detected in human prostate tumors and in blood samples from patients with chronic fatigue syndrome, but these findings have not been replicated. We hypothesized that an understanding of when and how XMRV first arose might help explain the discrepant results. We studied human prostate cancer cell lines CWR22Rv1 and CWR-R1, which produce XMRV virtually identical to the viruses recently found in patient samples, as well as their progenitor human prostate tumor xenograft (CWR22) that had been passaged in mice. We detected XMRV infection in the two cell lines and in the later passage xenografts, but not in the early passages. Importantly, we found that the host mice contained two proviruses, PreXMRV-1 and PreXMRV-2, which share 99.92% identity with XMRV over >3.2-kb stretches of their genomes. We conclude that XMRV was not present in the original CWR22 tumor but was generated by recombination of two proviruses during tumor passaging in mice. The probability that an identical recombinant was generated independently is negligible (~10-12); our results suggest that the association of XMRV with human disease is due to contamination of human samples with virus originating from this recombination event. PMID:21628392
Production of recombinant allergens in plants
A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed. PMID:21258627
Dissociative recombination of molecular ions with electrons
NASA Technical Reports Server (NTRS)
Johnsen, Rainer
An overview is presented for the present state of the art of laboratory measurements of the dissociative recombination of molecular ions with electrons. Most work has focussed on obtaining rates and their temperature dependence, as these are of primary interest for model calculations of ionospheres. A comparison of data obtained using the microwave afterglow method, the flowing afterglow technique, and the merged beam technique shows that generally the agreement is quite good, but there are some serious discrepancies, especially in the case of H(3+) recombination, that need to be resolved. Results of some earlier experimental work need to be reexamined in the light of more recent developments. Such cases are pointed out and a compilation of rate coefficients that have withstood scrutiny is presented. Recent advances in experimental methods, such as the use of laser-in-duced fluorescence, make it possible to identify some neutral products of dissociative recombination. What has been done so far and what results one might expect from future work are briefly reviewed.
Recombinant vaccine for canine parvovirus in dogs.
López de Turiso, J A; Cortés, E; Martínez, C; Ruiz de Ybáñez, R; Simarro, I; Vela, C; Casal, I
VP2 is the major component of canine parvovirus (CPV) capsids. The VP2-coding gene was engineered to be expressed by a recombinant baculovirus under the control of the polyhedrin promoter. A transfer vector that contains the lacZ gene under the control of the p10 promoter was used in order to facilitate the selection of recombinants. The expressed VP2 was found to be structurally and immunologically indistinguishable from authentic VP2. The recombinant VP2 shows also the capability to self-assemble, forming viruslike particles similar in size and appearance to CPV virions. These viruslike particles have been used to immunize dogs in different doses and combinations of adjuvants, and the anti-CPV responses have been measured by enzyme-linked immunosorbent assay, monolayer protection assays, and an assay for the inhibition of hemagglutination. A dose of ca. 10 micrograms of VP2 was able to elicit a good protective response, higher than that obtained with a commercially available, inactivated vaccine. The results indicate that these viruslike particles can be used to protect dogs from CPV infection. PMID:1313899
FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements
Jinks-Robertson, Sue
The 2001 meeting entitled ''Genetic Recombination and Genome Rearrangements'' was held July 21-26 in Snowmass, Colorado. The goal of the meeting was to bring together scientists using diverse approaches to study all aspects of genetic recombination. This goal was achieved by integrating talks covering the genetics, biochemistry and structural biology of homologous recombination, site-specific recombination, and nonhomologous recombination. The format of the meeting consisted of a keynote address on the opening evening, two formal plenary sessions on each of the four full meeting days, a single afternoon workshop consisting of short talks chosen from among submitted abstracts, and afternoon poster sessions on each of the four full meeting days. The eight plenary session were entitled: (1) Recombination Mechanisms, (2) Prokaryotic Recombination, (3) Repair and Recombination, (4) Site-specific Recombination and Transposition, (5) Eukaryotic Recombination I, (6) Genome Rearrangements, (7) Meiosis, and (8) Eukaryotic Recombination II. Each session included a mix of genetic, biochemical and structural talks; talks were limited to 20 minutes, followed by 10 minutes of very lively, general discussion. Much of the data presented in the plenary sessions was unpublished, thus providing attendees with the most up-to-date knowledge of this rapidly-moving field.
Recombination Pattern Reanalysis of Some HIV-1 Circulating Recombination Forms Suggest the Necessity and Difficulty of Revision
Jia, Lei; Li, Lin; Li, Hanping; Liu, Siyang; Wang, Xiaolin; Bao, Zuoyi; Li, Tianyi; Zhuang, Daomin; Liu, Yongjian; Li, Jingyun
Background Recombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs), like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results. Methods Representative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08), BG recombinants (CRF23 and CRF24), and BF recombinants (CRF38 and CRF44). They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3. Results The results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i) length of inserted fragments; and (ii) number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution. Conclusion Results indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also
Mechanisms and Factors that Influence High Frequency Retroviral Recombination
Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga; Mens, Helene; Pathak, Vinay K.; Hu, Wei-Shau
With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment, and vaccine development. PMID:21994801
Evolution of recombination in eutherian mammals: insights into mechanisms that affect recombination rates and crossover interference
Segura, Joana; Ferretti, Luca; Ramos-Onsins, Sebastián; Capilla, Laia; Farré, Marta; Reis, Fernanda; Oliver-Bonet, Maria; Fernández-Bellón, Hugo; Garcia, Francisca; Garcia-Caldés, Montserrat; Robinson, Terence J.; Ruiz-Herrera, Aurora
Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primates and Carnivora to better understand the dynamics of mammalian recombination. Our results suggest a phylogenetic component in recombination rates (RRs), which appears to be directional, strongly punctuated and subject to selection. Species that diversified earlier in the evolutionary tree have lower RRs than those from more derived phylogenetic branches. Furthermore, chromosome-specific recombination maps in distantly related taxa show that crossover interference is especially weak in the species with highest RRs detected thus far, the tiger. This is the first example of a mammalian species exhibiting such low levels of crossover interference, highlighting the uniqueness of this species and its relevance for the study of the mechanisms controlling crossover formation, distribution and resolution. PMID:24068360
Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use
Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.
Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.
The imperfect ancestral recombination graph reconstruction problem: upper bounds for recombination and homoplasy.
Lam, Fumei; Tarpine, Ryan; Istrail, Sorin
One of the central problems in computational biology is the reconstruction of evolutionary histories. While models incorporating recombination and homoplasy have been studied separately, a missing component in the theory is a robust and flexible unifying model which incorporates both of these major biological events shaping genetic diversity. In this article, we introduce the first such unifying model and develop algorithms to find the optimal ancestral recombination graph incorporating recombinations and homoplasy events. The power of our framework is the connection between our formulation and the Directed Steiner Arborescence Problem in combinatorial optimization. We implement linear programming techniques as well as heuristics for the Directed Steiner Arborescence Problem, and use our methods to construct evolutionary histories for both simulated and real data sets. PMID:20583925
Late replicating domains are highly recombining in females but have low male recombination rates: implications for isochore evolution.
Pink, Catherine J; Hurst, Laurence D
In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in analysis of GC
Same-period emission and recombination in nonsequential double-recombination high-order-harmonic generation
NASA Astrophysics Data System (ADS)
Hansen, Kenneth K.; Madsen, Lars Bojer
Nonsequential double-recombination (NSDR) high-order-harmonic generation (HHG) is studied in a molecular model system. We observe a unique molecular two-electron effect with a characteristic cutoff in the HHG spectrum at higher energies than what was previously seen for NSDR HHG in atoms. The effect is corroborated with a classical model where it is found that the effect is sensitive to the molecular potential and originates from same-period emission and recombination (SPEAR) of two electrons. The effect persists for intermediate nuclear distances of R ≳8.0 a.u.
Live-Cell Imaging of Vaccinia Virus Recombination
Paszkowski, Patrick; Noyce, Ryan S.; Evans, David H.
Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren't detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories. PMID:27525721
An introduction to recombination and linkage analysis
Mcpeek, M.S.
With a garden as his laboratory, Mendel was able to discern basic probabilistic laws of heredity. Although it first appeared as a baffling exception to one of Mendel`s principles, the phenomenon of variable linkage between characters was soon recognized to be a powerful tool in the process of chromosome mapping and location of genes of interest. In this introduction, we first describe Mendel`s work and the subsequent discovery of linkage. Next we describe the apparent cause of variable linkage, namely recombination, and we introduce linkage analysis. 33 refs., 1 fig., 2 tabs.
Cooling and recombination processes in cometary plasma
NASA Technical Reports Server (NTRS)
Wallis, M. K.; Ong, R. S. B.
The ion electron plasma in comets is examined for cooling processes which result from its interactions with the neutral coma. A cometary coma model is formulated that is composed predominantly of H2O and its decomposition products where electrons are cooled in a variety of processes at rates varying with energy. It is shown that solar plasma plus accumulated cometary ions and electrons is affected very strongly as it flows into the coma. The electrons are rapidly cooled and all but some 10% of the ions undergo charge exchange. Photodissociation of H2O is assumed where ion electron recombination is the dominant loss process.
Nanobodies and recombinant binders in cell biology
Helma, Jonas; Cardoso, M. Cristina; Muyldermans, Serge
Antibodies are key reagents to investigate cellular processes. The development of recombinant antibodies and binders derived from natural protein scaffolds has expanded traditional applications, such as immunofluorescence, binding arrays, and immunoprecipitation. In addition, their small size and high stability in ectopic environments have enabled their use in all areas of cell research, including structural biology, advanced microscopy, and intracellular expression. Understanding these novel reagents as genetic modules that can be integrated into cellular pathways opens up a broad experimental spectrum to monitor and manipulate cellular processes. PMID:26056137
Dissociation and recombination in an inhomogeneous gas
NASA Astrophysics Data System (ADS)
Kuščer, Ivan
The system of Boltzmann equations of Ludwig and Heil for a dissociating gas mixture is reviewed and reformulated in terms of differential cross sections. For the purpose of Monte Carlo simulations, collision models of the type of Borgnakke and Larsen are proposed. In case of short-lived transient flows of molecular gases and at not too high temperatures, simulation of scattering collisions is expected to suffice; the distribution function of the dissociated particles can be evaluated afterwards by integration, and recombination can be ignored.
Engineering thermoacidophilic archaea using linear DNA recombination.
Maezato, Yukari; Dana, Karl; Blum, Paul
Thermoacidophilic archaea comprise one of the major classes of extremophiles. Most belong to the family Sulfolobales within the phylum Crenarchaeota. They are of applied interest as sources of hyperstable enzymes, for biomining of base and precious metals, and for evolutionary studies because of their use of eukaryotic-like subcellular mechanisms. Genetic methods are available for several species particularly Sulfolobus solfataricus. This organism has a considerable number of methods available for the construction of novel cell lines with unique functions. This chapter presents recent developments in the use of homologous recombination and linear DNA for the engineering of site-specific changes in the genome of S. solfataricus. PMID:21815108
Hydrogen recombiner catalyst test supporting data
Britton, M.D.
This is a data package supporting the Hydrogen Recombiner Catalyst Performance and Carbon Monoxide Sorption Capacity Test Report, WHC-SD-WM-TRP-211, Rev 0. This report contains 10 appendices which consist of the following: Mass spectrometer analysis reports: HRC samples 93-001 through 93-157; Gas spectrometry analysis reports: HRC samples 93-141 through 93-658; Mass spectrometer procedure PNL-MA-299 ALO-284; Alternate analytical method for ammonia and water vapor; Sample log sheets; Job Safety analysis; Certificate of mixture analysis for feed gases; Flow controller calibration check; Westinghouse Standards Laboratory report on Bois flow calibrator; and Sorption capacity test data, tables, and graphs.
Population inversion in a stationary recombining plasma
Otsuka, M.
Population inversion, which occurs in a recombining plasma when a stationary He plasma is brought into contact with a neutral gas, is examined. With hydrogen as a contact gas, noticeable inversion between low-lying levels of H as been found. The overpopulation density is of the order of 10/sup 8/ cm/sup -3/, which is much higher then that (approx. =10/sup 5/ cm/sup -3/) obtained previously with He as a contact gas. Relations between these experimental results and the conditions for population inversion are discussed with the CR model.
Patterns of recombination on human chromosome 22
Schlumpf, K.S.; Kim, D.; Haines, J.L.
Virtually all genetic linkage maps generated to date are gross averages across individuals, ages, and (often) sexes. In addition, although some level of positive interference has been assumed, until recently little evidence to support this in humans has been available. The major stumbling block has been the quality of the data available, since even a few genotypic errors can have drastic effects on both the map length and the number of apparent recombinants. In addition, variation in recombination by factors other than sex have pretty much been ignored. To explore recombination in more detail, we have generated a microsatellite marker map of human chromosome 22. This map includes 32 markers genotyped through 46 sibships of the Venezuelan Reference Pedigree (VRP). Extensive error checking and regenotyping was performed to remove as many genotypic errors as possible, but no genotypes were removed simply because they created unlikely events. The following 1000:1 odds map has been obtained: cen--F8VWFP1--11--S264--3-S311--4--S257--2--TOP1P2--3--S156--1--CRYB2--1--S258--2--S310--6--S193--1--S275--3--S268--1--S280--4--S304--3--S283--2--LiR1--3--IL2RB--3--S299--1--S302--1--S537--2--S270--4--PDGF--8--S274--qter. The female map (91 cM) is twice as long as the male map (46 cM) and the log-likelihood difference in the maps (22.3) is highly significant (P=0.001, df=22) and appears constant across the chromosome. Analysis of recombination with age showed no particular trends for either males or females when chromosomes were grouped into three categories (20, 20-30, 30+) by parental age at birth of child. Positive interference was found in maternally derived chromosomes ({chi}{sup 2}=30.5 (4), p<0.005), but not in paternally derived chromosomes ({chi}{sup 2}=6.24 (3), P=0.10). This contrasts to data from chromosomes 9 and 21 where positive interference was found for both sexes. More detailed analyses are in progress.
Cutaneous allergy to human (recombinant DNA) insulin.
Grammer, L C; Metzger, B E; Patterson, R
p6 report two cases of cutaneous allergy to human (recombinant DNA) insulin. Each patient had a history of systemic allergic reactions to porcine insulin and was at least as reactive to human as to porcine insulin by end-point cutaneous titration. Both patients' insulin allergy was managed with animal insulins and both have done well. Our experience with these two patients indicates that human insulin (rDNA) should not be expected to be efficacious in all patients with systemic allergy to insulin. PMID:6366262
Metal binding proteins, recombinant host cells and methods
Summers, Anne O.; Caguiat, Jonathan J.
The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.
Recombinant DNA products: Insulin, interferon and growth hormone
Bollon, A.P.
This book provides the discussion of products of biotechnology of recombinant DNA. The contents include: Recombinant DNA techniques; isolation, cloning, and expression of genes; from somatostatin to human insulin; yeast; an alternative organism for foreign protein production; background in human interferon; preclinical assessment of biological properties of recombinant DNA derived human interferons; human clinical trials of bacteria-derived human ..cap alpha.. interferon.f large scale production of human alpha interferon from bacteria; direct expression of human growth hormone in escherichia coli with the lipoprotein promoter; biological actions in humans of recombinant DNA synthesized human growth hormone; NIH guidelines for research involving recombinant DNA molecules; appendix; viral vectors and the NHY guidelines; FDA's role in approval and regulation of recombinant DNA drugs; and index.
The recombination mediator RAD51D promotes geminiviral infection.
Richter, Kathrin S; Serra, Heϊdi; White, Charles I; Jeske, Holger
To study a possible role for homologous recombination in geminivirus replication, we challenged Arabidopsis recombination gene knockouts by Euphorbia yellow mosaic virus infection. Our results show that the RAD51 paralog RAD51D, rather than RAD51 itself, promotes viral replication at early stages of infection. Blot hybridization analyses of replicative intermediates using one- and two-dimensional gels and deep sequencing point to an unexpected facet of recombination-dependent replication, the repair by single-strand annealing (SSA) during complementary strand replication. A significant decrease of both intramolecular, yielding defective DNAs and intermolecular recombinant molecules between the two geminiviral DNA components (A, B) were observed in the absence of RAD51D. By contrast, DNA A and B reacted differentially with the generation of inversions. A model to implicate single-strand annealing recombination in geminiviral recombination-dependent replication is proposed. PMID:27018825
The Many Landscapes of Recombination in Drosophila melanogaster
Comeron, Josep M.; Ratnappan, Ramesh; Bailin, Samuel
Recombination is a fundamental biological process with profound evolutionary implications. Theory predicts that recombination increases the effectiveness of selection in natural populations. Yet, direct tests of this prediction have been restricted to qualitative trends due to the lack of detailed characterization of recombination rate variation across genomes and within species. The use of imprecise recombination rates can also skew population genetic analyses designed to assess the presence and mode of selection across genomes. Here we report the first integrated high-resolution description of genomic and population variation in recombination, which also distinguishes between the two outcomes of meiotic recombination: crossing over (CO) and gene conversion (GC). We characterized the products of 5,860 female meioses in Drosophila melanogaster by genotyping a total of 139 million informative SNPs and mapped 106,964 recombination events at a resolution down to 2 kilobases. This approach allowed us to generate whole-genome CO and GC maps as well as a detailed description of variation in recombination among individuals of this species. We describe many levels of variation in recombination rates. At a large-scale (100 kb), CO rates exhibit extreme and highly punctuated variation along chromosomes, with hot and coldspots. We also show extensive intra-specific variation in CO landscapes that is associated with hotspots at low frequency in our sample. GC rates are more uniformly distributed across the genome than CO rates and detectable in regions with reduced or absent CO. At a local scale, recombination events are associated with numerous sequence motifs and tend to occur within transcript regions, thus suggesting that chromatin accessibility favors double-strand breaks. All these non-independent layers of variation in recombination across genomes and among individuals need to be taken into account in order to obtain relevant estimates of recombination rates, and should
Dissociative recombination of N2H+
NASA Astrophysics Data System (ADS)
dos Santos, S. Fonseca; Ngassam, V.; Orel, A. E.; Larson, Å.
The direct and indirect mechanisms of dissociative recombination of N2H+ are theoretically studied. At low energies, the electron capture is found to be driven by recombination into bound Rydberg states, while at collision energies above 0.1 eV, the direct capture and dissociation along electronic resonant states becomes important. Electron-scattering calculations using the complex Kohn variational method are performed to obtain the scattering matrix as well as energy positions and autoionization widths of resonant states. Potential-energy surfaces of electronic bound states of N2H and N2H+ are computed using structure calculations with the multireference configuration interaction method. The cross section for the indirect mechanism is calculated using a vibrational frame transformation of the elements of the scattering matrix at energies just above the ionization threshold. Here vibrational excitations of the ionic core from v =0 to v =1 and v =2 for all three normal modes are considered and autoionization is neglected. The cross section for the direct dissociation along electronic resonant states is computed with wave-packet calculations using the multiconfiguration time-dependent Hartree method, where all three internal degrees of freedom are considered. The calculated cross sections are compared to measurements.
Photoexcited charge pair escape and recombination
Braun, C.L.
We report photocurrent transients arising from the pulsed laser excitation of the dipolar first excited singlet sate S{sub 1} of trans 4-dimethyl-amino-4{prime}-nitrostilbene (DMANS) in toluene solution. The currents arise from rotational reorientation of DMANS dipoles with respect to the axis of an applied electric field. The method appears to offer a simple and general approach to the measurement of the change in dipole moment upon electronic excitation of a molecule. In another experiment, durene (1,2,4,5-tetramethylbenzene) dissolved in n-hexane was photoionized by 35 psec pulses at 266 nm. Transient absorption at 1064 nm arising chiefly from geminate electrons was detected and used to monitor the recombination of the electron-cation pairs produced by two-photon ionization. An excellent fit to the recombination kinetics was obtained by assuming that the distribution of initial electron-cation separations was of the form r{sup 2}EXP = r{sup 2}/(2L{sup 3})exp({minus}r/L) with a mean radius 3L = 5.7 nm.
The landscape of recombination in African Americans.
Hinch, Anjali G; Tandon, Arti; Patterson, Nick; Song, Yunli; Rohland, Nadin; Palmer, Cameron D; Chen, Gary K; Wang, Kai; Buxbaum, Sarah G; Akylbekova, Ermeg L; Aldrich, Melinda C; Ambrosone, Christine B; Amos, Christopher; Bandera, Elisa V; Berndt, Sonja I; Bernstein, Leslie; Blot, William J; Bock, Cathryn H; Boerwinkle, Eric; Cai, Qiuyin; Caporaso, Neil; Casey, Graham; Cupples, L Adrienne; Deming, Sandra L; Diver, W Ryan; Divers, Jasmin; Fornage, Myriam; Gillanders, Elizabeth M; Glessner, Joseph; Harris, Curtis C; Hu, Jennifer J; Ingles, Sue A; Isaacs, William; John, Esther M; Kao, W H Linda; Keating, Brendan; Kittles, Rick A; Kolonel, Laurence N; Larkin, Emma; Le Marchand, Loic; McNeill, Lorna H; Millikan, Robert C; Murphy, Adam; Musani, Solomon; Neslund-Dudas, Christine; Nyante, Sarah; Papanicolaou, George J; Press, Michael F; Psaty, Bruce M; Reiner, Alex P; Rich, Stephen S; Rodriguez-Gil, Jorge L; Rotter, Jerome I; Rybicki, Benjamin A; Schwartz, Ann G; Signorello, Lisa B; Spitz, Margaret; Strom, Sara S; Thun, Michael J; Tucker, Margaret A; Wang, Zhaoming; Wiencke, John K; Witte, John S; Wrensch, Margaret; Wu, Xifeng; Yamamura, Yuko; Zanetti, Krista A; Zheng, Wei; Ziegler, Regina G; Zhu, Xiaofeng; Redline, Susan; Hirschhorn, Joel N; Henderson, Brian E; Taylor, Herman A; Price, Alkes L; Hakonarson, Hakon; Chanock, Stephen J; Haiman, Christopher A; Wilson, James G; Reich, David; Myers, Simon R
Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P value < 10(-245)). We identify a 17-base-pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of PRDM9 alleles common in West Africans and rare in Europeans. Sites of this motif are predicted to be risk loci for disease-causing genomic rearrangements in individuals carrying these alleles. More generally, this map provides a resource for research in human genetic variation and evolution. PMID:21775986
Recombination-aware alignment of diploid individuals
Background Traditionally biological similarity search has been studied under the abstraction of a single string to represent each genome. The more realistic representation of diploid genomes, with two strings defining the genome, has so far been largely omitted in this context. With the development of sequencing techniques and better phasing routines through haplotype assembly algorithms, we are not far from the situation when individual diploid genomes could be represented in their full complexity with a pair-wise alignment defining the genome. Results We propose a generalization of global alignment that is designed to measure similarity between phased predictions of individual diploid genomes. This generalization takes into account that individual diploid genomes evolve through a mutation and recombination process, and that predictions may be erroneous in both dimensions. Even though our model is generic, we focus on the case where one wants to measure only the similarity of genome content allowing free recombination. This results into efficient algorithms for direct application in (i) evaluation of variation calling predictions and (ii) progressive multiple alignments based on labeled directed acyclic graphs (DAGs) to represent profiles. The latter may be of more general interest, in connection to covering alignment of DAGs. Extensions of our model and algorithms can be foreseen to have applications in evaluating phasing algorithms, as well as more fundamental role in phasing child genome based on parent genomes. PMID:25572943
Modified Fragmentation Function from Quark Recombination
Majumder, A.; Wang, Enke; Wang, Xin-Nian
Within the framework of the constituent quark model, it isshown that the single hadron fragmentation function of a parton can beexpressed as a convolution of shower diquark or triquark distributionfunction and quark recombination probability, if the interference betweenamplitudes of quark recombination with different momenta is neglected.Therecombination probability is determined by the hadron's wavefunction inthe constituent quark model. The shower diquark or triquark distributionfunctions of a fragmenting jet are defined in terms of overlappingmatrices of constituent quarks and parton field operators. They aresimilar in form to dihadron or trihadron fragmentation functions in termsof parton operator and hadron states. Extending the formalism to thefield theory at finite temperature, we automatically derive contributionsto the effective single hadron fragmentation function from therecombination of shower and thermal constituent quarks. Suchcontributions involve single or diquark distribution functions which inturn can be related to diquark or triquark distribution functions via sumrules. We also derive QCD evolution equations for quark distributionfunctions that in turn determine the evolution of the effective jetfragmentation functions in a thermal medium.
Lipopolysaccharide induced conversion of recombinant prion protein
Saleem, Fozia; Bjorndahl, Trent C; Ladner, Carol L; Perez-Pineiro, Rolando; Ametaj, Burim N; Wishart, David S
The conformational conversion of the cellular prion protein (PrPC) to the β-rich infectious isoform PrPSc is considered a critical and central feature in prion pathology. Although PrPSc is the critical component of the infectious agent, as proposed in the "protein-only" prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrPC to proteinase K resistant PrPSc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrPC conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrPC to PrPSc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232). PMID:24819168
Ethanol precipitation for purification of recombinant antibodies.
Tscheliessnig, Anne; Satzer, Peter; Hammerschmidt, Nikolaus; Schulz, Henk; Helk, Bernhard; Jungbauer, Alois
Currently, the golden standard for the purification of recombinant humanized antibodies (rhAbs) from CHO cell culture is protein A chromatography. However, due to increasing rhAbs titers alternative methods have come into focus. A new strategy for purification of recombinant human antibodies from CHO cell culture supernatant based on cold ethanol precipitation (CEP) and CaCl2 precipitation has been developed. This method is based on the cold ethanol precipitation, the process used for purification of antibodies and other components from blood plasma. We proof the applicability of the developed process for four different antibodies resulting in similar yield and purity as a protein A chromatography based process. This process can be further improved using an anion-exchange chromatography in flowthrough mode e.g. a monolith as last step so that residual host cell protein is reduced to a minimum. Beside the ethanol based process, our data also suggest that ethanol could be replaced with methanol or isopropanol. The process is suited for continuous operation. PMID:25087738
Recombinant synthesis of hyaluronan by Agrobacterium sp.
Mao, Zichao; Chen, Rachel Ruizhen
Hyaluronan (HA) is a sugar polymer of a repeating disaccharide, beta1-3 D-N-acetylglucosamine (GlcNAc) beta1-4 D-glucuronic acid (GlcA). It finds applications in numerous biomedical procedures such as ophthalmic surgery and osteoarthritis treatment. Until recently, the only commercial sources were extraction of rooster combs and from fermentation of pathogenic Streptococcus. In this work, we demonstrate that metabolic engineering strategies enable the recombinant synthesis of hyaluronan in a safe microorganism. Agrobacterium sp. ATCC 31749 is a commercial production strain for a food polymer, Curdlan. A broad host range expression vector was successfully developed to express the 3 kb HA synthase gene from Pasteurella multocida, along with a kfiD gene encoding UDP-glucose dehydrogenase from Escherichia coli K5 strain. Coexpression of these two heterologous enzymes enables Agrobacterium to produce HA. Hyaluronan was accumulated up to 0.3 g/L in shaker flask cultivation. The molecular weight of the polymer from various Agrobacterium strains is in the range of 0.7-2 MD. To our knowledge, this is the first successful recombinant hyaluronan synthesis in a Gram-negative bacterium that naturally produces a food product. The ease of genetic modifications provides future opportunities to tailor properties of polymers for specific applications. PMID:17705506
Recombinant viral vaccines for enzootic bovine leucosis.
Daniel, R C; Gatei, M H; Good, M F; Boyle, D B; Lavin, M F
Recently published studies on the development and use of recombinant vaccinia virus (VV) vaccines incorporating either the complete envelope (env) gene or only a fragment of the env gene consisting of the coding sequence for the env glycoprotein 51 (gp51) and part of gp30 of the bovine leukaemia virus (BLV) are described. It has been reported that vaccination of sheep with recombinant VV vaccines containing the complete env gene appears to protect sheep against challenge infection with BLV. The evidence for this protection is based on the lack of persistence of high titres of anti-gp51 antibodies compared with unvaccinated BLV infected controls, on the enhanced CD4 proliferative responses to specific BLV gp51 synthetic peptides in the vaccinated sheep, and on the inability to detect BLV pro-virus by polymerase chain reaction in the vaccinated sheep after 4 months following challenge infection compared with continual detection in unvaccinated sheep over a 16 month trial period. It has been suggested that cell-mediated immune responses may be an important aspect of protective immunity against BLV infection and it has been reported that large tracts of amino acid sequences within the env and pol genes are highly conserved in different isolates from different countries which is of importance in designing peptide derived vaccines. PMID:8270269
Recombinant protein scaffolds for tissue engineering.
Werkmeister, Jerome A; Ramshaw, John A M
New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. PMID:22262725
Geminate recombination of O2 and hemoglobin.
Chernoff, D A; Hochstrasser, R M; Steele, A W
The photolysis of HbO2 and HbCO has been studied by measuring transient absorption spectra in the Soret region after excitation with picosecond pulses at 530 nm. Dissociation occurred promptly in both cases, followed (for HbO2) by geminate recombination of ca. 40% of the photodissociated O2 with a lifetime of 200 +/- 70 psec (25 degrees C). No recombination of Hb + CO was observed up to 1200 psec after photolysis. The HbO2 and HbCO photoproduct spectra were broader, weaker, and red-shifted in comparison to the spectrum of stable Hb and Gibson's fast-reacting form, Hb. For HbO2 the spectrum was initially much broader to longer wavelengths but relaxed to a constant shape within 90 psec, whereas for HbCO there was no spectral evolution. The photophysics is analyzed by considering the effect of spin constraints as well as spin--orbit coupling and orbital correlation among the various electronic states of liganded and deoxy hemoglobins. The small quantum yield of HbO2 dissociation is not primarily due to rebinding but rather to electronic relaxation to nonreactive states. PMID:6932659
[Telomere Recombination in Normal Mammalian Cells].
Zhdanova, N S; Rubtsov, N B
Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15-20% of tumors lacking active telomerase and, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the "gold standard" of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed,and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed. PMID:27183789
The landscape of recombination in African Americans
Hinch, Anjali G.; Tandon, Arti; Patterson, Nick; Song, Yunli; Rohland, Nadin; Palmer, Cameron D.; Chen, Gary K.; Wang, Kai; Buxbaum, Sarah G.; Akylbekova, Meggie; Aldrich, Melinda C.; Ambrosone, Christine B.; Amos, Christopher; Bandera, Elisa V.; Berndt, Sonja I.; Bernstein, Leslie; Blot, William J.; Bock, Cathryn H.; Boerwinkle, Eric; Cai, Qiuyin; Caporaso, Neil; Casey, Graham; Cupples, L. Adrienne; Deming, Sandra L.; Diver, W. Ryan; Divers, Jasmin; Fornage, Myriam; Gillanders, Elizabeth M.; Glessner, Joseph; Harris, Curtis C.; Hu, Jennifer J.; Ingles, Sue A.; Isaacs, Williams; John, Esther M.; Kao, W. H. Linda; Keating, Brendan; Kittles, Rick A.; Kolonel, Laurence N.; Larkin, Emma; Le Marchand, Loic; McNeill, Lorna H.; Millikan, Robert C.; Murphy, Adam; Musani, Solomon; Neslund-Dudas, Christine; Nyante, Sarah; Papanicolaou, George J.; Press, Michael F.; Psaty, Bruce M.; Reiner, Alex P.; Rich, Stephen S.; Rodriguez-Gil, Jorge L.; Rotter, Jerome I.; Rybicki, Benjamin A.; Schwartz, Ann G.; Signorello, Lisa B.; Spitz, Margaret; Strom, Sara S.; Thun, Michael J.; Tucker, Margaret A.; Wang, Zhaoming; Wiencke, John K.; Witte, John S.; Wrensch, Margaret; Wu, Xifeng; Yamamura, Yuko; Zanetti, Krista A.; Zheng, Wei; Ziegler, Regina G.; Zhu, Xiaofeng; Redline, Susan; Hirschhorn, Joel N.; Henderson, Brian E.; Taylor, Herman A.; Price, Alkes L.; Hakonarson, Hakon; Chanock, Stephen J.; Haiman, Christopher A.; Wilson, James G.; Reich, David; Myers, Simon R.
Recombination, together with mutation, is the ultimate source of genetic variation in populations. We leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing-over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P<10−245). We identify a 17 base pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of African-enriched alleles of PRDM9. PMID:21775986
Monitoring recombinant human erythropoietin abuse among athletes.
Citartan, Marimuthu; Gopinath, Subash C B; Chen, Yeng; Lakshmipriya, Thangavel; Tang, Thean-Hock
The illegal administration of recombinant human erythropoietin (rHuEPO) among athletes is largely preferred over blood doping to enhance stamina. The advent of recombinant DNA technology allowed the expression of EPO-encoding genes in several eukaryotic hosts to produce rHuEPO, and today these performance-enhancing drugs are readily available. As a mimetic of endogenous EPO (eEPO), rHuEPO augments the oxygen carrying capacity of blood. Thus, monitoring the illicit use of rHuEPO among athletes is crucial in ensuring an even playing field and maintaining the welfare of athletes. A number of rHuEPO detection methods currently exist, including measurement of hematologic parameters, gene-based detection methods, glycomics, use of peptide markers, electrophoresis, isoelectric focusing (IEF)-double immunoblotting, aptamer/antibody-based methods, and lateral flow tests. This review gleans these different strategies and highlights the leading molecular recognition elements that have potential roles in rHuEPO doping detection. PMID:25058943
Kinetics of radiative recombination in quantum wells
NASA Astrophysics Data System (ADS)
Ridley, B. K.
A theory of radiative-recombination kinetics which treats free carriers, excitons, and photon recycling in a quantum-well system is presented. An expression for the temporal decay of excess carriers which encompasses large- and small-signal regimes is derived. When excitons are present the decay can be approximated by two exponentials in general, and in the large-signal regime the photoluminescence time constant is half as long as that associated with photoconductivity. Explicit expressions for the recombination coefficients are given and their magnitudes discussed for nondegenerate and degenerate populations in GaAs. Excitons are shown to enhance the temperature dependence. A simple model of exciton screening is used to illustrate the dependence of radiative time constants on background carrier density, which deviates significantly from the conventional free-carrier dependence. The magnitudes of radiative time constants in real systems depend, in addition to material characteristics, upon the details of exciton screening, the overlap of the electron and hole wave functions in the quantum well, and the probability of photon reabsorption, all of which are specimen specific. It is pointed out that the transition from a degenerate to a nondegenerate population may be misinterpreted in terms of Auger processes.
Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.
Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan
A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells. PMID:17581705
The evolution of recombination in a heterogeneous environment.
Lenormand, T; Otto, S P
Most models describing the evolution of recombination have focused on the case of a single population, implicitly assuming that all individuals are equally likely to mate and that spatial heterogeneity in selection is absent. In these models, the evolution of recombination is driven by linkage disequilibria generated either by epistatic selection or drift. Models based on epistatic selection show that recombination can be favored if epistasis is negative and weak compared to directional selection and if the recombination modifier locus is tightly linked to the selected loci. In this article, we examine the joint effects of spatial heterogeneity in selection and epistasis on the evolution of recombination. In a model with two patches, each subject to different selection regimes, we consider the cases of mutation-selection and migration-selection balance as well as the spread of beneficial alleles. We find that including spatial heterogeneity extends the range of epistasis over which recombination can be favored. Indeed, recombination can be favored without epistasis, with negative and even with positive epistasis depending on environmental circumstances. The selection pressure acting on recombination-modifier loci is often much stronger with spatial heterogeneity, and even loosely linked modifiers and free linkage may evolve. In each case, predicting whether recombination is favored requires knowledge of both the type of environmental heterogeneity and epistasis, as none of these factors alone is sufficient to predict the outcome. PMID:10978305
Charge recombination in disordered neat polymer films under imbalanced excitation conditions studied using the recombination time of flight technique
NASA Astrophysics Data System (ADS)
Ravia, Israel; Tessler, Nir
It has recently been suggested that the charge recombination rate in amorphous polymers could be affected by the energy distribution of electrons and holes as well as that of the resulting excitons. To test this hypothesis, we developed a new method for measuring charge recombination under highly imbalanced conditions. We find that if the electron density is higher than that of the holes, increasing the electron density further results in reduction of the recombination coefficient. We attribute this to the very different energy distribution between low and high carrier densities, which is not accounted for in the Langevin recombination model.
Photoionization and electron-ion recombination of Ti I
NASA Astrophysics Data System (ADS)
Nahar, Sultana N.
Study of the inverse processes of photoionization and electron-ion recombination of (Ti I + h ν ⇋ Ti II + e) using the unified method is reported. The method, based on close coupling (CC) approximation and R-matrix method, subsumes both the radiative recombination (RR) and dielectronic recombination (DR) in a unified manner and provides state-specific and total electron-ion recombination rate coefficients which are self-consistent with the state-specific photoionization cross sections. The present results include state-specific electron-ion recombination rates (αRC(i))and partial photoionization cross sections (σPI(i)) leaving the ion in the ground state of 813 bound states with n ≤ 10 and l ≤ 9 of Ti I. Various features of state-specific and total electron-ion recombination with temperature, and the corresponding photoionization cross sections with energies are discussed with illustrations. Due to closely lying excited states near the ground state of the core, photoionization cross sections show presence of narrow Rydberg resonances in low energy region near the ionization threshold. Many excited states also show broad and enhanced Seaton resonances due to PEC (photo-excitation-of-core) which contribute to the high temperature recombination. The total recombination rate coefficient is found to show a low hump around temperature 280 K and a high dielectronic recombination peak at temperature 25,000 K. Total spectrum of recombination cross sections and rates with photoelectron energy are also presented for experimental observation. Calculations were carried out using a CC wave function expansion of 36 states of the core ion Ti II. The large set of data for recombination rates and partial photoionization cross sections with resonances should provide a complete and accurate modelings of plasmas.
Recombination of Globally Circulating Varicella-Zoster Virus
Depledge, Daniel P.; Kundu, Samit; Atkinson, Claire; Brown, Julianne; Haque, Tanzina; Hussaini, Yusuf; MacMahon, Eithne; Molyneaux, Pamela; Papaevangelou, Vassiliki; Sengupta, Nitu; Koay, Evelyn S. C.; Tang, Julian W.; Underhill, Gillian S.; Grahn, Anna; Studahl, Marie; Breuer, Judith; Bergström, Tomas
ABSTRACT Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo, creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the
Visible charge exchange recombination spectroscopy on TFTR
Stratton, B.C.; Fonck, R.J.; Jaehnig, K.P.; Schechtman, N.; Synakowski, E.J.
Visible charge exchange recombination spectroscopy is routinely used to measure the time evolution of the ion temperature (T{sub i}) and toroidal rotation velocity (v{sub {phi}}) profiles on TFTR. These measurements are made with the CHERS diagnostic, a fiber-optically coupled spectrometer equipped with a two-dimensional photodiode array detector which provides both spectral and spatial resolution. The instrumentation, data analysis techniques, and examples of T{sub i} and v{sub {phi}} measurements are described. Recently, CHERS has been used to perform impurity transport experiments: radial profiles of diffusivities and convective velocities for helium and iron have been deduced from measurements of the time evolutions of He{sup 2+} and Fe{sup 24+} profiles following impurity injection. Examples of these measurements are given. 12 refs., 8 figs.
Recombinant hepatitis B triple antigen vaccine: Hepacare.
Zuckerman, Jane N; Zuckerman, Arie J
Infection with hepatitis B virus is a public health problem throughout the world. Hepatitis B vaccines are now included in national immunization programmes of infants and/or adolescents in 129 countries. Current single antigen vaccines, that are plasma-derived or produced by recombinant DNA technology are highly effective, but between 5-10% or more of healthy immunocompetent subjects do not mount an antihepatitis B surface antibody protective response and others respond poorly (hyporesponders). The inclusion of pre-S1 and -S2 components of hepatitis B surface antigen in addition to the single antigen (triple antigen) in a novel vaccine, Hepacare, Medeva Pharma Plc, Speke, UK, overcomes nonresponsiveness and hyporesponsiveness in a significant number of individuals. The triple antigen is indicated for vaccination of nonresponders (and hyporesponders) to the current single antigen vaccines and for persons who require rapid protection against hepatitis B infection. PMID:12901552
Recombining overlapping BACs into single large BACs.
Kotzamanis, George; Kotsinas, Athanassios
BAC clones containing the entire genomic region of a gene including the long-range regulatory elements are very useful for gene functional analysis. However, large genes often span more than the insert of a BAC clone, and single BACs covering the entire region of interest are not available. Here, we describe a general system for linking two or more overlapping BACs into a single clone. Two rounds of homologous recombination are used. In the first, the BAC inserts are subcloned into the pBACLink vectors. In the second, the two BACs are combined together. Multiple BACs in a contig can be combined by alternating use of the pBACLInk vectors, resulting in several BAC clones containing as much of the genomic region of a gene as required. Such BACs can then be used in gene expression studies and/or gene therapy applications. PMID:25239744
Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses.
Su, Shuo; Wong, Gary; Shi, Weifeng; Liu, Jun; Lai, Alexander C K; Zhou, Jiyong; Liu, Wenjun; Bi, Yuhai; Gao, George F
Human coronaviruses (HCoVs) were first described in the 1960s for patients with the common cold. Since then, more HCoVs have been discovered, including those that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), two pathogens that, upon infection, can cause fatal respiratory disease in humans. It was recently discovered that dromedary camels in Saudi Arabia harbor three different HCoV species, including a dominant MERS HCoV lineage that was responsible for the outbreaks in the Middle East and South Korea during 2015. In this review we aim to compare and contrast the different HCoVs with regard to epidemiology and pathogenesis, in addition to the virus evolution and recombination events which have, on occasion, resulted in outbreaks amongst humans. PMID:27012512
Integrative mobile elements exploiting Xer recombination.
Das, Bhabatosh; Martínez, Eriel; Midonet, Caroline; Barre, François-Xavier
Integrative mobile genetic elements directly participate in the rapid response of bacteria to environmental challenges. They generally encode their own dedicated recombination machineries. CTXφ, a filamentous bacteriophage that harbors the genes encoding cholera toxin in Vibrio cholerae provided the first notable exception to this rule: it hijacks XerC and XerD, two chromosome-encoded tyrosine recombinases for lysogenic conversion. XerC and XerD are highly conserved in bacteria because of their role in the topological maintenance of circular chromosomes and, with the advent of high throughput sequencing, numerous other integrative mobile elements exploiting them have been discovered. Here, we review our understanding of the molecular mechanisms of integration of the different integrative mobile elements exploiting Xer (IMEXs) so far described. PMID:23127381
Charge exchange recombination spectroscopy on fusion devices
Duval, B. P.
For fusion, obtaining reliable measurements of basic plasma parameters like ion and electron densities and temperatures is a primary goal. For theory, measurements are needed as a function of time and space to understand plasma transport and confinement with the ultimate goal of achieving economic nuclear fusion power. Electron profile measurements and plasma spectroscopy for the plasma ions are introduced. With the advent of Neutral Beam auxiliary plasma heating, Charge Exchange Recombination Spectroscopy provides accurate and time resolved measurements of the ions in large volume fusion devices. In acknowledgement of Nicol Peacock's role in the development of these techniques, still at the forefront of plasma fusion research, this paper describes the evolution of this diagnostic method.
Dissociative Recombination Dynamics of the Ozone Cation
Zhaunerchyk, Vitali; Geppert, W.; sterdahl, F.; Larsson, Mats; Thomas, R. D.; Bahati Musafiri, Eric; Bannister, Mark E; Fogle, Mark R.; Vane, C Randy
The dissociative recombination of the ozone cation has been studied at the heavy-ion storage ring CRYRING. The total cross section and branching fractions have been measured. The cross section from {approx}0eV to 0.2 eV follows a nearly E{sup -1} dependence, which was theoretically predicted to be a characteristic of the direct dissociative recombination mechanism. The thermal rate coefficient has been deduced from the cross section to be 7.37x10{sup -7}(T/300){sup -0.55}cm{sup 3}s{sup -1}. The branching fraction analysis carried out at {approx}0eV interaction energy has shown a strong propensity (94%) to dissociate through the three-body channel. Due to the overwhelming dominance of this channel it has been investigated in more detail. Of the six energetically available three-body pathways only three are significantly populated, such that the production of O(S1) is highly unfavored and all atomic oxygen fragments are predominantly formed in P3 and D1 states. Analysis of the breakup geometries has been performed by means of the Dalitz plot. It is observed that the molecules dissociating through the O(P3)+O(P3)+O(P3) and O(P3)+O(P3)+O(D1) channels have an open linear geometry where the cleavage of two valence bonds occurs preferentially in unison, while the O(P3)+O(D1)+O(D1) breakup might proceed partly through a sequential mechanism.
Functional, Responsive Materials Assembled from Recombinant Oleosin
NASA Astrophysics Data System (ADS)
Hammer, Daniel
Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..
The Landscape of Realized Homologous Recombination in Pathogenic Bacteria
Yahara, Koji; Didelot, Xavier; Jolley, Keith A.; Kobayashi, Ichizo; Maiden, Martin C.J.; Sheppard, Samuel K.; Falush, Daniel
Recombination enhances the adaptive potential of organisms by allowing genetic variants to be tested on multiple genomic backgrounds. Its distribution in the genome can provide insight into the evolutionary forces that underlie traits, such as the emergence of pathogenicity. Here, we examined landscapes of realized homologous recombination of 500 genomes from ten bacterial species and found all species have "hot" regions with elevated rates relative to the genome average. We examined the size, gene content, and chromosomal features associated with these regions and the correlations between closely related species. The recombination landscape is variable and evolves rapidly. For example in Salmonella, only short regions of around 1 kb in length are hot whereas in the closely related species Escherichia coli, some hot regions exceed 100 kb, spanning many genes. Only Streptococcus pyogenes shows evidence for the positive correlation between GC content and recombination that has been reported for several eukaryotes. Genes with function related to the cell surface/membrane are often found in recombination hot regions but E. coli is the only species where genes annotated as "virulence associated" are consistently hotter. There is also evidence that some genes with "housekeeping" functions tend to be overrepresented in cold regions. For example, ribosomal proteins showed low recombination in all of the species. Among specific genes, transferrin-binding proteins are recombination hot in all three of the species in which they were found, and are subject to interspecies recombination. PMID:26516092
Radiative recombination and excited-state photoionization of lithium
Lahiri, J. ); Manson, S.T. )
The radiative-recombination rate coefficients for electrons impinging on Li[sup +], along with the associated excited-state photoionization cross sections for Li, are calculated in the low-energy region. In addition to the totals, the contribution of the recombination of individual excited states to the total is discussed.
Inference of Ancestral Recombination Graphs through Topological Data Analysis
Cámara, Pablo G.; Levine, Arnold J.; Rabadán, Raúl
The recent explosion of genomic data has underscored the need for interpretable and comprehensive analyses that can capture complex phylogenetic relationships within and across species. Recombination, reassortment and horizontal gene transfer constitute examples of pervasive biological phenomena that cannot be captured by tree-like representations. Starting from hundreds of genomes, we are interested in the reconstruction of potential evolutionary histories leading to the observed data. Ancestral recombination graphs represent potential histories that explicitly accommodate recombination and mutation events across orthologous genomes. However, they are computationally costly to reconstruct, usually being infeasible for more than few tens of genomes. Recently, Topological Data Analysis (TDA) methods have been proposed as robust and scalable methods that can capture the genetic scale and frequency of recombination. We build upon previous TDA developments for detecting and quantifying recombination, and present a novel framework that can be applied to hundreds of genomes and can be interpreted in terms of minimal histories of mutation and recombination events, quantifying the scales and identifying the genomic locations of recombinations. We implement this framework in a software package, called TARGet, and apply it to several examples, including small migration between different populations, human recombination, and horizontal evolution in finches inhabiting the Galápagos Islands. PMID:27532298
Not so fast on recombination analysis of Newcastle disease virus
Technology Transfer Automated Retrieval System (TEKTRAN)
Regarding the letter published in the Journal of Virology Vol. 82 No. 13 p. 6782 indicating that "powerful evidence" of recombination is a call for caution in the use of Newcastle Disease Virus (NDV) based vaccines, I would like to suggest that evidence for recombination is still weak. The authors ...
Termolecular ionic recombination at high ambient gas density
NASA Technical Reports Server (NTRS)
Bates, D. R.
It is shown that the importance of diffusion relative to drift in ion-ion recombination is a decreasing function of the separation between the ions (contrary to a common supposition). Langevin's formula for the recombination coefficient is obtained very simply using a physical model which allows for both diffusion and drift.
Experimental studies of the dissociative recombination of H3 +
NASA Astrophysics Data System (ADS)
Larsson, M.
Despite many experimental efforts over several decades, no consensus has bee n reached concerning the rate of dissociative recombination of H3+. We review the current status concerning different experimental appro aches to the measurement of the cross-section, rate coefficient and branching r atios in electron recombination of H3+.
Recombination in viruses: mechanisms, methods of study, and evolutionary consequences.
Pérez-Losada, Marcos; Arenas, Miguel; Galán, Juan Carlos; Palero, Ferran; González-Candelas, Fernando
Recombination is a pervasive process generating diversity in most viruses. It joins variants that arise independently within the same molecule, creating new opportunities for viruses to overcome selective pressures and to adapt to new environments and hosts. Consequently, the analysis of viral recombination attracts the interest of clinicians, epidemiologists, molecular biologists and evolutionary biologists. In this review we present an overview of three major areas related to viral recombination: (i) the molecular mechanisms that underlie recombination in model viruses, including DNA-viruses (Herpesvirus) and RNA-viruses (Human Influenza Virus and Human Immunodeficiency Virus), (ii) the analytical procedures to detect recombination in viral sequences and to determine the recombination breakpoints, along with the conceptual and methodological tools currently used and a brief overview of the impact of new sequencing technologies on the detection of recombination, and (iii) the major areas in the evolutionary analysis of viral populations on which recombination has an impact. These include the evaluation of selective pressures acting on viral populations, the application of evolutionary reconstructions in the characterization of centralized genes for vaccine design, and the evaluation of linkage disequilibrium and population structure. PMID:25541518
Recombinant HT.sub.m4 gene, protein and assays
Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel
The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.
Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus
Thor, Sharmi W.; Hilt, Deborah A.; Kissinger, Jessica C.; Paterson, Andrew H.; Jackwood, Mark W.
Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV) isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus. PMID:21994806
Oxygen recombination in the sealed nickel-cadmium cell
NASA Technical Reports Server (NTRS)
Zimmerman, Al H.; Barrera, Tom P.
The purpose of this study was to determine what parameters are most critical for controlling overcharge pressure in a sealed nickel cadmium cell. Topics covered in viewgraph format are: parameters examined; oxygen evolution and recombination; experimental test description; and effect of parameters on recombination rate.
Short communication: A comparative analysis of recombinant chymosins.
Vallejo, J A; Ageitos, J M; Poza, M; Villa, T G
The first step in cheesemaking is the milk clotting process, in which κ-caseinolytic enzymes contribute to micelle precipitation. The best enzyme for this purpose is chymosin because of its high degree of specificity toward κ-casein. Although recombinant bovine chymosin is the most frequently used chymosin in the industry, new sources of recombinant chymosin, such as goat, camel, or buffalo, are now available. The present work represents a comparative study of 4 different recombinant chymosins (goat and buffalo chymosins expressed in Pichia pastoris, and bovine and camel chymosin expressed in Aspergillus niger). Recombinant goat chymosin exhibited the best catalytic efficiency compared with the buffalo, bovine, or camel recombinant enzymes. Moreover, recombinant goat chymosin exhibited the best specific proteolytic activity, a wider pH range of action, and a lower glycosylation degree than the other 3 enzymes. In conclusion, we propose that recombinant goat chymosin represents a serious alternative to recombinant bovine chymosin for use in the cheesemaking industry. PMID:22281325
Heterogeneity in rates of recombination across the mouse genome
Nachman, M.W.; Churchill, G.A.
If loci are randomly distributed on a physical map, the density of markers on a genetic map will be inversely proportional to recombination rate. First proposed by Mary Lyon, we have used this idea to estimate recombination rates from the Drosophila melanogaster linkage map. These results were compared with results of two other studies that estimated regional recombination rates in D. melanogaster using both physical and genetic maps. The three methods were largely concordant in identifying large-scale genomic patterns of recombination. The marker density method was then applied to the Mus musculus microsatellite linkage map. The distribution of microsatellites provided evidence for heterogeneity in recombination rates. Centromeric regions for several mouse chromosomes had significantly greater numbers of markers than expected, suggesting that recombination rates were lower in these regions. In contrast, most telomeric regions contained significantly fewer markers than expected. This indicates that recombination rates are elevated at the telomeres of many mouse chromosomes and is consistent with a comparison of the genetic and cytogenetic maps in these regions. The density of markers on a genetic map may provide a generally useful way to estimate regional recombination rates in species for which genetic, but not physical, maps are available. 44 refs., 5 figs., 4 tabs.
A Heritable Recombination system for synthetic Darwinian evolution in yeast.
Romanini, Dante W; Peralta-Yahya, Pamela; Mondol, Vanessa; Cornish, Virginia W
Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology. PMID:23412545
Procedures for monitoring recombinant erythropoietin and analogs in doping.
Lamon, Séverine; Robinson, Neil; Saugy, Martial
Hemoglobin concentration is one of the principal factors of aerobic power and, consequently, of performance in many types of physical activities. The use of recombinant human erythropoietin is, therefore, particularly powerful for improving the physical performances of patients, and, more generally, improving their quality of life. This article discusses procedures for monitoring recombinant erythropoietin and its analogues in doping for athletic performance. PMID:20122455
Inference of Ancestral Recombination Graphs through Topological Data Analysis.
Cámara, Pablo G; Levine, Arnold J; Rabadán, Raúl
The recent explosion of genomic data has underscored the need for interpretable and comprehensive analyses that can capture complex phylogenetic relationships within and across species. Recombination, reassortment and horizontal gene transfer constitute examples of pervasive biological phenomena that cannot be captured by tree-like representations. Starting from hundreds of genomes, we are interested in the reconstruction of potential evolutionary histories leading to the observed data. Ancestral recombination graphs represent potential histories that explicitly accommodate recombination and mutation events across orthologous genomes. However, they are computationally costly to reconstruct, usually being infeasible for more than few tens of genomes. Recently, Topological Data Analysis (TDA) methods have been proposed as robust and scalable methods that can capture the genetic scale and frequency of recombination. We build upon previous TDA developments for detecting and quantifying recombination, and present a novel framework that can be applied to hundreds of genomes and can be interpreted in terms of minimal histories of mutation and recombination events, quantifying the scales and identifying the genomic locations of recombinations. We implement this framework in a software package, called TARGet, and apply it to several examples, including small migration between different populations, human recombination, and horizontal evolution in finches inhabiting the Galápagos Islands. PMID:27532298
Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity
Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.
We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906
Applications of recombinant DNA technology in the production of glycosylated recombinant human granulocyte colony stimulating factor.
Holloway, C J
Lenograstim has been developed by recombinant DNA technology and is expressed in large-scale mammalian cell culture. It has been shown that lenograstim is indistinguishable in its physicochemical, structural and biological properties with respect to native granulocyte colony stimulating factor isolated from a human cell line. In particular, both the recombinant and natural proteins have identical amino acid sequences, contain the same intra-polypeptide chain disulphide bridges and exhibit the same posttranslational carbohydrate structures. Lenograstim is manufactured by expanding inoculum from vials of the Manufacturer's Working Cell Bank (from molecular cloning) followed by culture in a large bioreactor. Purification of lenograstim involves a four-step chromatographic process. The active ingredient is monitored by in-process controls at all stages of manufacture and routinely as purified bulk. The finished product is formulated into excipients reflecting conditions close to the natural environment of the protein with respect to pH, osmolarity and the presence of human serum albumin. PMID:7535067
Modulating Mek1 kinase alters outcomes of meiotic recombination and the stringency of the recombination checkpoint response
Hsin-Yen, Wu; Hsuan-Chung, Ho; Burgess, Sean M.
Summary Background During meiosis, recombination between homologous chromosomes promotes their proper segregation. In budding yeast, programmed double-strand breaks (DSBs) promote recombination between homologs versus sister chromatids by dimerizing and activating Mek1, a chromosome axis-associated kinase. Mek1 is also a proposed effector kinase in the recombination checkpoint that arrests exit from pachytene in response to aberrant DNA/axis structures. Elucidating a role for Mek1 in the recombination checkpoint has been difficult since in mek1 loss-of-function mutants DSBs are rapidly repaired using a sister chromatid thereby bypassing formation of checkpoint-activating lesions. Here we tested the hypothesis that a MEK1 gain-of-function allele would enhance interhomolog bias and the recombination checkpoint response. Results When Mek1 activation was artificially maintained through GST-mediated dimerization, there was an enhanced skew toward interhomolog recombination and reduction of intersister events including multi-chromatid joint molecules. Increased interhomolog events were specifically repaired as noncrossovers rather than crossovers. Ectopic Mek1 dimerization was also sufficient to impose interhomolog bias in the absence of recombination checkpoint functions, thereby uncoupling these two processes. Finally, the stringency of the recombination checkpoint was enhanced in weak meiotic recombination mutants by blocking prophase exit in a subset of cells where arrest is not absolute. Conclusions We propose that Mek1 plays dual roles during meiotic prophase I by phosphorylating targets directly involved in the recombination checkpoint as well as targets involved in sister chromatid recombination. We discuss how regulation of pachytene exit by Mek1 or similar kinases could influence checkpoint stringency, which may differ among species and between sexes. PMID:20888230
Recombination in liquid xenon for low-energy recoils
NASA Astrophysics Data System (ADS)
Wang, Lu; Mei, Dongming; Cubed Collaboration
Detector response to low-energy recoils in sub-keV region is critical to detection of low-mass dark matter particles-WIMPS (Weakly interacting massive particles). The role of electron-ion recombination is important to the interpretation of the relation between ionization yield and scintillation yield, which are in general anti-correlated. Recent experimental results show that ionization yield increases down to keV range. This phenomenon contradicts general understanding for low energy recoils in the keV range in which direct excitation dominates. The explanation is that recombination becomes much less efficient when the track length is smaller than the thermalization distance of electrons. However, recombination rate is also proportional to ionization density, which is very high for keV recoils. To understand how recombination rate behaves for keV recoils, we calculated both initial recombination rate and volume recombination rate for keV recoils in liquid xenon. In this paper, we show the results of the calculated recombination rate as a function of recoil energy for both electronic recoils and nuclear recoils. Detector response to low-energy recoils in sub-keV region is critical to detection of low-mass dark matter particles-WIMPS (Weakly interacting massive particles). The role of electron-ion recombination is important to the interpretation of the relation between ionization yield and scintillation yield, which are in general anti-correlated. Recent experimental results show that ionization yield increases down to keV range. This phenomenon contradicts general understanding for low energy recoils in the keV range in which direct excitation dominates. The explanation is that recombination becomes much less efficient when the track length is smaller than the thermalization distance of electrons. However, recombination rate is also proportional to ionization density, which is very high for keV recoils. To understand how recombination rate behaves for keV recoils
Two dimensional Langevin recombination in regioregular poly(3-hexylthiophene)
NASA Astrophysics Data System (ADS)
Juška, Gytis; Genevičius, Kristijonas; Nekrašas, Nerijus; Sliaužys, Gytis; Österbacka, Ronald
In this work, it is shown that recombination in regioregular poly(3-hexylthiophene):[6,6]-phenyl-C61-butyric acid methyl ester (RRP3HT:PCBM) bulk-heterojunction solar cells is caused by the two dimensional (2D) Langevin recombination in the lamellar structures of RRP3HT, which are formed after annealing process. Due to 2D Langevin process, bimolecular recombination coefficient is reduced in comparison with three dimensional Langevin case, and bimolecular recombination coefficient depends on the density of charge carriers n1/2. Data obtained from the different experimental techniques (charge extraction with linearly increasing voltage, integral time of flight, double injection current transients and transient absorption spectroscopy) confirms 2D Langevin recombination in RR3PHT.
Charge carrier recombination dynamics in perovskite and polymer solar cells
NASA Astrophysics Data System (ADS)
Paulke, Andreas; Stranks, Samuel D.; Kniepert, Juliane; Kurpiers, Jona; Wolff, Christian M.; Schön, Natalie; Snaith, Henry J.; Brenner, Thomas J. K.; Neher, Dieter
Time-delayed collection field experiments are applied to planar organometal halide perovskite (CH3NH3PbI3) based solar cells to investigate charge carrier recombination in a fully working solar cell at the nanosecond to microsecond time scale. Recombination of mobile (extractable) charges is shown to follow second-order recombination dynamics for all fluences and time scales tested. Most importantly, the bimolecular recombination coefficient is found to be time-dependent, with an initial value of ca. 10-9 cm3/s and a progressive reduction within the first tens of nanoseconds. Comparison to the prototypical organic bulk heterojunction device PTB7:PC71BM yields important differences with regard to the mechanism and time scale of free carrier recombination.
Recombinant human bone morphogenetic protein induces bone formation.
Wang, E A; Rosen, V; D'Alessandro, J S; Bauduy, M; Cordes, P; Harada, T; Israel, D I; Hewick, R M; Kerns, K M; LaPan, P
We have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 micrograms of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans. Images PMID:2315314
Sex recombination, and reproductive fitness: an experimental study using Paramecium
Nyberg, D.
The effect of sex and recombination on reproductive fitness are measured using five wild stocks of Paramecium primaurelia. Among the wild stocks there were highly significant differences in growth rates. No hybrid had as low a fitness as the least fit parental stock. Recombination produced genotypes of higher fitness than those of either parent only in the cross between the two stocks of lowest fitness. The increase in variance of fitness as a result of recombination was almost exclusively attributable to the generation lines with low fitness. The fitness consequences of sexuality and mate choice were stock specific; some individuals leaving the most descendants by inbreeding, others by outcrossing. For most crosses the short-term advantage of sex, if any, accrue from the fusion of different gametes (hybrid vigor) and not from recombination. Since the homozygous genotype with the highest fitnes left the most progeny by inbreeding (no recombination), the persistence of conjugation in P. primaurelia is paradoxical. (JMT)
Utilization of Site-Specific Recombination in Biopharmaceutical Production
Ahmadi, Maryam; Damavandi, Narges; Akbari, Mohammad Reza; Davami, Fatemeh
Mammalian expression systems, due to their capacity in post-translational modification, are preferred systems for biopharmaceutical protein production. Several recombinant protein systems have been introduced to the market, most of which are under clinical development. In spite of significant improvements such as cell line engineering, introducing novel expression methods, gene silencing and process development, expression level is unpredictable and unstable because of the random location of integration in the genome. Site-specific recombination techniques are capable of producing stable and high producer clonal cells; therefore, they are gaining more importance in the biopharmaceutical production. Site-specific recombination methods increase the recombinant protein production by specifically inserting a vector at a locus with specific expression trait. The present review focused on the latest developments in site-specific recombination techniques, their specific features and comparisons. PMID:26602035
Production and secretion of recombinant proteins in Dictyostelium discoideum.
Dittrich, W; Williams, K L; Slade, M B
We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection. PMID:7764951
CRISPR-directed mitotic recombination enables genetic mapping without crosses.
Sadhu, Meru J; Bloom, Joshua S; Day, Laura; Kruglyak, Leonid
Linkage and association studies have mapped thousands of genomic regions that contribute to phenotypic variation, but narrowing these regions to the underlying causal genes and variants has proven much more challenging. Resolution of genetic mapping is limited by the recombination rate. We developed a method that uses CRISPR (clustered, regularly interspaced, short palindromic repeats) to build mapping panels with targeted recombination events. We tested the method by generating a panel with recombination events spaced along a yeast chromosome arm, mapping trait variation, and then targeting a high density of recombination events to the region of interest. Using this approach, we fine-mapped manganese sensitivity to a single polymorphism in the transporter Pmr1. Targeting recombination events to regions of interest allows us to rapidly and systematically identify causal variants underlying trait differences. PMID:27230379
High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production.
Zhou, Ying; Ma, Xue; Hou, Zheng; Xue, Xiaoyan; Meng, Jingru; Li, Mingkai; Jia, Min; Luo, Xiaoxing
Glucagon-like peptide-1 (GLP-1)(2) has been attracting increasing interest on account of its prominent benefits in type 2 diabetes. However, its clinical applications are limited by the short half-life in vivo. To overcome this limitation, a new polymer of GLP-1 was developed by prodrug strategy. In this study a recombinant protein, rhGLPs, was successfully constructed, cloned into plasmid pET30a (+) and expressed in Escherichia coli ArcticExpress(DE3)RP in the form of inclusion body. The recombinant fusion protein productivity could be enhanced by high cell density culture of the recombinant strain. As a result, about 40 g wet weight cells per liter were obtained. The protein was purified by size-exclusion chromatography on a Superdex 75 column and refolded using reverse dilution and dialysis methods. SDS-PAGE, HPLC and MALDI-TOF mass spectrometry were undertaken to determine the purity and molecular weight of rhGLPs. Bioactivity assay revealed that it had glucose-lowering and insulin-releasing action in vivo. PMID:22771632
Recombinational Landscape and Population Genomics of Caenorhabditis elegans
Rockman, Matthew V.; Kruglyak, Leonid
Recombination rate and linkage disequilibrium, the latter a function of population genomic processes, are the critical parameters for mapping by linkage and association, and their patterns in Caenorhabditis elegans are poorly understood. We performed high-density SNP genotyping on a large panel of recombinant inbred advanced intercross lines (RIAILs) of C. elegans to characterize the landscape of recombination and, on a panel of wild strains, to characterize population genomic patterns. We confirmed that C. elegans autosomes exhibit discrete domains of nearly constant recombination rate, and we show, for the first time, that the pattern holds for the X chromosome as well. The terminal domains of each chromosome, spanning about 7% of the genome, exhibit effectively no recombination. The RIAILs exhibit a 5.3-fold expansion of the genetic map. With median marker spacing of 61 kb, they are a powerful resource for mapping quantitative trait loci in C. elegans. Among 125 wild isolates, we identified only 41 distinct haplotypes. The patterns of genotypic similarity suggest that some presumed wild strains are laboratory contaminants. The Hawaiian strain, CB4856, exhibits genetic isolation from the remainder of the global population, whose members exhibit ample evidence of intercrossing and recombining. The population effective recombination rate, estimated from the pattern of linkage disequilibrium, is correlated with the estimated meiotic recombination rate, but its magnitude implies that the effective rate of outcrossing is extremely low, corroborating reports of selection against recombinant genotypes. Despite the low population, effective recombination rate and extensive linkage disequilibrium among chromosomes, which are techniques that account for background levels of genomic similarity, permit association mapping in wild C. elegans strains. PMID:19283065
Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.
Kolodner, R; Fishel, R A; Howard, M
Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product. PMID:2993230
Genome Sequence of Complex HIV-1 Unique Recombinant Forms Sharing a Common Recombination Breakpoint Identified in Malaysia
Cheong, Hui Ting; Ng, Kim Tien; Ong, Lai Yee; Takebe, Yutaka; Chan, Kok Gan; Koh, Clayton; Al-Darraji, Haider Abdulrazzaq Abed; Kamarulzaman, Adeeba
Three strains of HIV-1 unique recombinant forms (URFs) descended from subtypes B, B′, and CRF01_AE were identified among people who inject drugs in Kuala Lumpur, Malaysia. These three URFs shared a common recombination breakpoint in the reverse transcriptase region, indicating frequent linkage within the drug-injecting networks in Malaysia. PMID:26543107
75 FR 42114 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH...
Federal Register 2010, 2011, 2012, 2013, 2014
... HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Proposed Action Under the NIH Guidelines for Research Involving Recombinant DNA Molecules (NIH... transgenic rodents by recombinant DNA technology must be registered with the Institutional...
Effect of sex, age, and breed on genetic recombination features in cattle
Technology Transfer Automated Retrieval System (TEKTRAN)
Meiotic recombination is a fundamental biological process which generates genetic diversity, affects fertility, and influences evolvability. Here we investigate the roles of sex, age, and breed in cattle recombination features, including recombination rate, location and crossover interference. Usin...
Auger recombination as the dominant recombination process in indium nitride at low temperatures during steady-state photoluminescence
Seetoh, I. P.; Soh, C. B.; Fitzgerald, E. A.; Chua, S. J.
Auger recombination in InN films grown by metal-organic chemical vapor deposition was studied by steady-state photoluminescence at different laser excitation powers and sample temperatures. It was dominant over radiative recombination and Shockley-Read-Hall recombination at low temperatures, contributing to the sub-linear relationship between the integrated photoluminescence intensity and laser excitation power. Auger recombination rates increased gradually with temperature with an activation energy of 10-17 meV, in good agreement with values from transient photoluminescence reported in literature. As the Auger recombination rates were independent of material quality, they may form an upper limit to the luminous efficiency of InN.
Auger recombination as the dominant recombination process in indium nitride at low temperatures during steady-state photoluminescence
NASA Astrophysics Data System (ADS)
Seetoh, I. P.; Soh, C. B.; Fitzgerald, E. A.; Chua, S. J.
Auger recombination in InN films grown by metal-organic chemical vapor deposition was studied by steady-state photoluminescence at different laser excitation powers and sample temperatures. It was dominant over radiative recombination and Shockley-Read-Hall recombination at low temperatures, contributing to the sub-linear relationship between the integrated photoluminescence intensity and laser excitation power. Auger recombination rates increased gradually with temperature with an activation energy of 10-17 meV, in good agreement with values from transient photoluminescence reported in literature. As the Auger recombination rates were independent of material quality, they may form an upper limit to the luminous efficiency of InN.
Ethanol produces dysmorphogenesis in offspring when pregnant animals are exposed in vivo (Chaudhuri 2000; Hannigan and Armant, 2000) and by direct exposure of neurulation staged embryos to ethanol in vitro (Kotch et al., 1995; Hunter et al., 1994 ; Thompson and Folb, 1982). The a...
Regulation of amino acid transporters by adenoviral-mediated human insulin-like growth factor-1 in a mouse model of placental insufficiency in vivo and the human trophoblast line BeWo in vitro
Jones, H.; Crombleholme, T.; Habli, M.
Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor-11 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined amino acid transporter expression and localization in both a mouse model of placental Insufficiency (PI) and a model of human trophoblast, the BeWo Choriocarcinoma cell line. For in vitro human studies, BeWo Choriocarcinoma cells were maintained in F12 complete medium + 10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 h. MOIs of 10:1 and 100:1 were utilized. In BeWo, transfection efficiency was 100% at an MOI of 100:1 and Ad-IGF-1 significantly increased IGF-1 secretion, proliferation and invasion but reduced apoptosis compared to controls. In vitro, amino acid uptake was increased following Ad-IGF-1 treatment and associated with significantly increased RNA expression of SNAT1, 2, LAT1 and 4F2hc. Only SNAT2 protein expression was increased but LAT1 showed relocalization from a perinuclear location to the cytoplasm and cell membrane. For in vivo studies, timed-pregnant animals were divided into four groups on day 18; sham-operated controls, uterine artery branch ligation (UABL), UABL + Ad-hIGF-1 (108 PFU), UABL + Ad-LacZ (108 PFU). At gestational day 20, pups and placentas were harvested by C-section. Only LAT1 mRNA expression changed, showing that a reduced expression of the transporter levels in the PI model could be partially rectified with Ad-hIGF1 treatment. At the protein level, System L was reduced in PI but remained at control levels following Ad-hIGF1. The System A isoforms were differentially regulated with SNAT2 expression diminished but SNAT1 increased in PI and Ad-hIGF1 groups. Enhanced amino acid isoform transporter expression and relocalization to the membrane may be an important mechanism contributing to Ad-hIGF-1 mediated correction of placental insufficiency. PMID:24360522
Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination
Rawson, Jonathan M.O.; Mansky, Louis M.
Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved. PMID:25254386
Calculations of effective recombination coefficients for nebular astrophysics
NASA Astrophysics Data System (ADS)
Fang, Xuan; Liu, Xiaowei; Storey, Pete J.
In the seemingly well established field of nebular astrophysics, there has been a long-standing dichotomy in plasma diagnostics and heavy elemental abundance determinations using the traditional method based on collisionally excited lines on the one hand, and optical recombination lines/continua, on the other. Several mechanisms have been proposed to explain this fundamental problem. Deep spectroscopy and recombination line analysis of emission line nebulae (planetary nebulae and H II regions) in the past decade have pointed to the existence of another previously unknown component of cold, H-deficient material as the culprit. Better constraints are needed on the physical conditions (electron temperature and density), chemical composition, mass, and spatial distribution of the postulated H-deficient inclusions in order to unravel their astrophysical origins. This requires knowledge of the relevant atomic parameters, most importantly the effective recombination coefficients of abundant heavy element ions, such as C II, O II, N II, and Ne II, appropriate for the physical conditions prevailing in those cold inclusions (e.g., electron temperature Te < 1000 K).In this contribution, I will introduce the creation of new effective recombination coefficients for the heavy element optical recombination lines, and review the recent progress in nebular astrophysics since the availability of new and high-quality atomic data. I will also present our new calculations of the effective recombination coefficients for the Ne II recombination line spectrum.
Phylogenetic and Recombination Analysis of Tomato Spotted Wilt Virus
Yu, Jisuk; Kim, Mi-Kyeong; Choi, Hong-Soo; Kim, Kook-Hyung
Tomato spotted wilt virus (TSWV) severely damages and reduces the yield of many economically important plants worldwide. In this study, we determined the whole-genome sequences of 10 TSWV isolates recently identified from various regions and hosts in Korea. Phylogenetic analysis of these 10 isolates as well as the three previously sequenced isolates indicated that the 13 Korean TSWV isolates could be divided into two groups reflecting either two different origins or divergences of Korean TSWV isolates. In addition, the complete nucleotide sequences for the 13 Korean TSWV isolates along with previously sequenced TSWV RNA segments from Korea and other countries were subjected to phylogenetic and recombination analysis. The phylogenetic analysis indicated that both the RNA L and RNA M segments of most Korean isolates might have originated in Western Europe and North America but that the RNA S segments for all Korean isolates might have originated in China and Japan. Recombination analysis identified a total of 12 recombination events among all isolates and segments and five recombination events among the 13 Korea isolates; among the five recombinants from Korea, three contained the whole RNA L segment, suggesting reassortment rather than recombination. Our analyses provide evidence that both recombination and reassortment have contributed to the molecular diversity of TSWV. PMID:23696821
The influence of recombination on human genetic diversity.
Spencer, Chris C A; Deloukas, Panos; Hunt, Sarah; Mullikin, Jim; Myers, Simon; Silverman, Bernard; Donnelly, Peter; Bentley, David; McVean, Gil
In humans, the rate of recombination, as measured on the megabase scale, is positively associated with the level of genetic variation, as measured at the genic scale. Despite considerable debate, it is not clear whether these factors are causally linked or, if they are, whether this is driven by the repeated action of adaptive evolution or molecular processes such as double-strand break formation and mismatch repair. We introduce three innovations to the analysis of recombination and diversity: fine-scale genetic maps estimated from genotype experiments that identify recombination hotspots at the kilobase scale, analysis of an entire human chromosome, and the use of wavelet techniques to identify correlations acting at different scales. We show that recombination influences genetic diversity only at the level of recombination hotspots. Hotspots are also associated with local increases in GC content and the relative frequency of GC-increasing mutations but have no effect on substitution rates. Broad-scale association between recombination and diversity is explained through covariance of both factors with base composition. To our knowledge, these results are the first evidence of a direct and local influence of recombination hotspots on genetic variation and the fate of individual mutations. However, that hotspots have no influence on substitution rates suggests that they are too ephemeral on an evolutionary time scale to have a strong influence on broader scale patterns of base composition and long-term molecular evolution. PMID:17044736
Mechanics and Single-Molecule Interrogation of DNA Recombination.
Bell, Jason C; Kowalczykowski, Stephen C
The repair of DNA by homologous recombination is an essential, efficient, and high-fidelity process that mends DNA lesions formed during cellular metabolism; these lesions include double-stranded DNA breaks, daughter-strand gaps, and DNA cross-links. Genetic defects in the homologous recombination pathway undermine genomic integrity and cause the accumulation of gross chromosomal abnormalities-including rearrangements, deletions, and aneuploidy-that contribute to cancer formation. Recombination proceeds through the formation of joint DNA molecules-homologously paired but metastable DNA intermediates that are processed by several alternative subpathways-making recombination a versatile and robust mechanism to repair damaged chromosomes. Modern biophysical methods make it possible to visualize, probe, and manipulate the individual molecules participating in the intermediate steps of recombination, revealing new details about the mechanics of genetic recombination. We review and discuss the individual stages of homologous recombination, focusing on common pathways in bacteria, yeast, and humans, and place particular emphasis on the molecular mechanisms illuminated by single-molecule methods. PMID:27088880
Identification and Manipulation of the Molecular Determinants Influencing Poliovirus Recombination
Runckel, Charles; Westesson, Oscar; Andino, Raul; DeRisi, Joseph L.
The control and prevention of communicable disease is directly impacted by the genetic mutability of the underlying etiological agents. In the case of RNA viruses, genetic recombination may impact public health by facilitating the generation of new viral strains with altered phenotypes and by compromising the genetic stability of live attenuated vaccines. The landscape of homologous recombination within a given RNA viral genome is thought to be influenced by several factors; however, a complete understanding of the genetic determinants of recombination is lacking. Here, we utilize gene synthesis and deep sequencing to create a detailed recombination map of the poliovirus 1 coding region. We identified over 50 thousand breakpoints throughout the genome, and we show the majority of breakpoints to be concentrated in a small number of specific "hotspots," including those associated with known or predicted RNA secondary structures. Nucleotide base composition was also found to be associated with recombination frequency, suggesting that recombination is modulated across the genome by predictable and alterable motifs. We tested the predictive utility of the nucleotide base composition association by generating an artificial hotspot in the poliovirus genome. Our results imply that modification of these motifs could be extended to whole genome re-designs for the development of recombination-deficient, genetically stable live vaccine strains. PMID:23408891
A Glance at Recombination Hotspots in the Domestic Cat.
Alhaddad, Hasan; Zhang, Chi; Rannala, Bruce; Lyons, Leslie A
Recombination has essential roles in increasing genetic variability within a population and in ensuring successful meiotic events. The objective of this study is to (i) infer the population-scaled recombination rate (ρ), and (ii) identify and characterize regions of increased recombination rate for the domestic cat, Felis silvestris catus. SNPs (n = 701) were genotyped in twenty-two East Asian feral cats (random bred). The SNPs covered ten different chromosomal regions (A1, A2, B3, C2, D1, D2, D4, E2, F2, X) with an average region size of 850 Kb and an average SNP density of 70 SNPs/region. The Bayesian method in the program inferRho was used to infer regional population recombination rates and hotspots localities. The regions exhibited variable population recombination rates and four decisive recombination hotspots were identified on cat chromosome A2, D1, and E2 regions. As a description of the identified hotspots, no correlation was detected between the GC content and the locality of recombination spots, and the hotspots enclosed L2 LINE elements and MIR and tRNA-Lys SINE elements. PMID:26859385
Monte Carlo Simulations of the Inside Intron Recombination
NASA Astrophysics Data System (ADS)
Cebrat, Stanisław; PȨKALSKI, Andrzej; Scharf, Fabian
Biological genomes are divided into coding and non-coding regions. Introns are non-coding parts within genes, while the remaining non-coding parts are intergenic sequences. To study evolutionary significance of the inside intron recombination we have used two models based on the Monte Carlo method. In our computer simulations we have implemented the internal structure of genes by declaring the probability of recombination between exons. One situation when inside intron recombination is advantageous is recovering functional genes by combining proper exons dispersed in the genetic pool of the population after a long period without selection for the function of the gene. Populations have to pass through the bottleneck, then. These events are rather rare and we have expected that there should be other phenomena giving profits from the inside intron recombination. In fact we have found that inside intron recombination is advantageous only in the case when after recombination, besides the recombinant forms, parental haplotypes are available and selection is set already on gametes.
Data Mining for Expressivity of Recombinant Protein Expression
NASA Astrophysics Data System (ADS)
Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki
We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.
Recombination-mediated genetic engineering of large genomic DNA transgenes.
Ejsmont, Radoslaw Kamil; Ahlfeld, Peter; Pozniakovsky, Andrei; Stewart, A Francis; Tomancak, Pavel; Sarov, Mihail
Faithful gene activity reporters are a useful tool for evo-devo studies enabling selective introduction of specific loci between species and assaying the activity of large gene regulatory sequences. The use of large genomic constructs such as BACs and fosmids provides an efficient platform for exploration of gene function under endogenous regulatory control. Despite their large size they can be easily engineered using in vivo homologous recombination in Escherichia coli (recombineering). We have previously demonstrated that the efficiency and fidelity of recombineering are sufficient to allow high-throughput transgene engineering in liquid culture, and have successfully applied this approach in several model systems. Here, we present a detailed protocol for recombineering of BAC/fosmid transgenes for expression of fluorescent or affinity tagged proteins in Drosophila under endogenous in vivo regulatory control. The tag coding sequence is seamlessly recombineered into the genomic region contained in the BAC/fosmid clone, which is then integrated into the fly genome using ϕC31 recombination. This protocol can be easily adapted to other recombineering projects. PMID:22065454
A Glance at Recombination Hotspots in the Domestic Cat
Alhaddad, Hasan; Zhang, Chi; Rannala, Bruce; Lyons, Leslie A.
Recombination has essential roles in increasing genetic variability within a population and in ensuring successful meiotic events. The objective of this study is to (i) infer the population-scaled recombination rate (ρ), and (ii) identify and characterize regions of increased recombination rate for the domestic cat, Felis silvestris catus. SNPs (n = 701) were genotyped in twenty-two East Asian feral cats (random bred). The SNPs covered ten different chromosomal regions (A1, A2, B3, C2, D1, D2, D4, E2, F2, X) with an average region size of 850 Kb and an average SNP density of 70 SNPs/region. The Bayesian method in the program inferRho was used to infer regional population recombination rates and hotspots localities. The regions exhibited variable population recombination rates and four decisive recombination hotspots were identified on cat chromosome A2, D1, and E2 regions. As a description of the identified hotspots, no correlation was detected between the GC content and the locality of recombination spots, and the hotspots enclosed L2 LINE elements and MIR and tRNA-Lys SINE elements. PMID:26859385
The Contribution of Genetic Recombination to CRISPR Array Evolution
Kupczok, Anne; Landan, Giddy; Dagan, Tal
CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and
Recombinant phage probes for Listeria monocytogenes
NASA Astrophysics Data System (ADS)
Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.
Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.
Recombinant albumin monolayers on latex particles.
Sofińska, Kamila; Adamczyk, Zbigniew; Kujda, Marta; Nattich-Rak, Małgorzata
The adsorption of recombinant human serum albumin (rHSA) on negatively charged polystyrene latex micro-particles was studied at pH 3.5 and the NaCl concentration range of 10(-3) to 0.15 M. The electrophoretic mobility of latex monotonically increased with the albumin concentration in the suspension. The coverage of adsorbed albumin was quantitatively determined using the depletion method, where the residual protein concentration was determined by electrokinetic measurements and AFM imaging. It was shown that albumin adsorption was irreversible. Its maximum coverage on latex varied between 0.7 mg m(-2) for 10(-3) M NaCl to 1.3 mg m(-2) for 0.15 M NaCl. The latter value matches the maximum coverage previously determined for human serum albumin on mica using the streaming potential method. The increase in the maximum coverage was interpreted in terms of reduced electrostatic repulsion among adsorbed molecules. These facts confirm that albumin adsorption at pH 3.5 is governed by electrostatic interactions and proceeds analogously to colloid particle deposition. The stability of albumin monolayers was measured in additional experiments where changes in the latex electrophoretic mobility and the concentration of free albumin in solutions were monitored over prolonged time periods. Based on these experimental data, a robust procedure of preparing albumin monolayers on latex particles of well-controlled coverage and molecule distribution was proposed. PMID:24354916
Developing recombinant antibodies for biomarker detection
Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.; Miller, Keith D.; Kagen, Jacob; Srivastava, Sudhir; Rodland, Karin D.
Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.
Multivalent Recombinant Protein Vaccine against Coccidioidomycosis
Tarcha, Eric J.; Basrur, Venkatesha; Hung, Chiung-Yu; Gardner, Malcolm J.; Cole, Garry T.
Coccidioidomycosis is a human respiratory disease that is endemic to the southwestern United States and is caused by inhalation of the spores of a desert soilborne fungus. Efforts to develop a vaccine against this disease have focused on identification of T-cell-reactive antigens derived from the parasitic cell wall which can stimulate protective immunity against Coccidioides posadasii infection in mice. We previously described a productive immunoproteomic/bioinformatic approach to the discovery of vaccine candidates which makes use of the translated genome of C. posadasii and a computer-based method of scanning deduced sequences of seroreactive proteins for epitopes that are predicted to bind to human major histocompatibility (MHC) class II-restricted molecules. In this study we identified a set of putative cell wall proteins predicted to contain multiple, promiscuous MHC II binding epitopes. Three of these were expressed by Escherichia coli, combined in a vaccine, and tested for protective efficacy in C57BL/6 mice. Approximately 90% of the mice survived beyond 90 days after intranasal challenge, and the majority cleared the pathogen. We suggest that the multicomponent vaccine stimulates a broader range of T-cell clones than the single recombinant protein vaccines and thereby may be capable of inducing protection in an immunologically heterogeneous human population. PMID:16988258
Dielectronic Recombination In Active Galactic Nuclei
NASA Technical Reports Server (NTRS)
Lukic, D. V.; Schnell, M.; Savin, D. W.; Altun, Z.; Badnell, N.; Brandau, C.; Schmidt, E. W.; Mueller, A.; Schippers, S.; Sprenger, F.; Lestinsky, M.; Wolf, A.
XMM-Newton and Chandra observations of active galactic nuclei (AGN) show rich spectra of X-ray absorption lines. These observations have detected a broad unresolved transition array (UTA) between approx. 15-17 A. This is attributed to inner-shell photoexcitation of M-shell iron ions. Modeling these UTA features is currently limited by uncertainties in the low-temperature dielectronic recombination (DR) data for M-shell iron. In order to resolve this issue, and to provide reliable iron M-shell DR data for plasma modeling, we are carrying out a series of laboratory measurements using the heavy-ion Test Storage Ring (TSR) at the Max-Plank-Institute for Nuclear Physics in Heidelberg, Germany. Currently, laboratory measurements of low temperature DR can only be performed at storage rings. We use the DR data obtained at TSR, to calculate rate coefficients for plasma modeling and to benchmark theoretical DR calculations. Here we report our recent experimental results for DR of Fe XIV forming Fe XIII.
Development of recombinant vaccines for botulinum neurotoxin.
Smith, L A
Synthetic genes encoding non-toxic, carboxyl-terminal regions (approximately 50 kDa) of botulinum neurotoxin (BoNT) serotypes A and B (referred to as fragment C or HC) were constructed and cloned into the methylotropic yeast, Pichia pastoris. Genes specifying BoNTA(HC) and BoNTB(HC) were expressed as both intracellular and secreted products. Recombinants, expressed intracellularly, yielded products with the expected molecular weight as judged by SDS PAGE and Western blot (immunoblot) analysis, while secreted products were larger due to glycosylation. Gene products were used to vaccinate mice and evaluated for their ability to elicit protective antibody titers in vivo. Mice given three intramuscular vaccinations with yeast supernatant containing glycosylated BoNTA(HC) were protected against an intraperitoneal challenge of 10(6) 50% mouse lethal doses (MLD50) of serotype A neurotoxin, a result not duplicated by its BoNTB(HC) counterpart. Vaccinating mice with cytoplasmically produced BoNTA(HC) and BoNTB(HC) protected animals from a challenge of 10(6) MLD50 of serotype A and B toxins, respectively. Because of the glycosylation encountered with secreted BoNT(HC), our efforts focused on the production and purification of products from intracellular expression. PMID:9792170
HSV Recombinant Vectors for Gene Therapy
Manservigi, Roberto; Argnani, Rafaela; Marconi, Peggy
The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges. PMID:20835362
Epigenetic Codes Programing Class Switch Recombination
Vaidyanathan, Bharat; Chaudhuri, Jayanta
Class switch recombination imparts B cells with a fitness-associated adaptive advantage during a humoral immune response by using a precision-tailored DNA excision and ligation process to swap the default constant region gene of the antibody with a new one that has unique effector functions. This secondary diversification of the antibody repertoire is a hallmark of the adaptability of B cells when confronted with environmental and pathogenic challenges. Given that the nucleotide sequence of genes during class switching remains unchanged (genetic constraints), it is logical and necessary therefore, to integrate the adaptability of B cells to an epigenetic state, which is dynamic and can be heritably modulated before, after, or even during an antibody-dependent immune response. Epigenetic regulation encompasses heritable changes that affect function (phenotype) without altering the sequence information embedded in a gene, and include histone, DNA and RNA modifications. Here, we review current literature on how B cells use an epigenetic code language as a means to ensure antibody plasticity in light of pathogenic insults. PMID:26441954
Therapeutic Use of Native and Recombinant Enteroviruses.
Ylä-Pelto, Jani; Tripathi, Lav; Susi, Petri
Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these "viral" receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy. PMID:26907330
Recombinant Canine Coronaviruses in Dogs, Europe
Mari, Viviana; Elia, Gabriella; Addie, Diane D.; Camero, Michele; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio
Coronaviruses of potential recombinant origin with porcine transmissible gastroenteritis virus (TGEV), referred to as a new subtype (IIb) of canine coronavirus (CCoV), were recently identified in dogs in Europe. To assess the distribution of the TGEV-like CCoV subtype, during 2001–2008 we tested fecal samples from dogs with gastroenteritis. Of 1,172 samples, 493 (42.06%) were positive for CCoV. CCoV-II was found in 218 samples, and CCoV-I and CCoV-II genotypes were found in 182. Approximately 20% of the samples with CCoV-II had the TGEV-like subtype; detection rates varied according to geographic origin. The highest and lowest rates of prevalence for CCoV-II infection were found in samples from Hungary and Greece (96.87% and 3.45%, respectively). Sequence and phylogenetic analyses showed that the CCoV-IIb strains were related to prototype TGEV-like strains in the 5′ and the 3′ ends of the spike protein gene. PMID:20031041
Therapeutic Use of Native and Recombinant Enteroviruses
Ylä-Pelto, Jani; Tripathi, Lav; Susi, Petri
Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these "viral" receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy. PMID:26907330
Recombinant protein vaccines produced in insect cells.
Cox, Manon M J
The baculovirus-insect cell expression system is a well known tool for the production of complex proteins. The technology is also used for commercial manufacture of various veterinary and human vaccines. This review paper provides an overview of how this technology can be applied to produce a multitude of vaccine candidates. The key advantage of this recombinant protein manufacturing platform is that a universal "plug and play" process may be used for producing a broad range of protein-based prophylactic and therapeutic vaccines for both human and veterinary use while offering the potential for low manufacturing costs. Large scale mammalian cell culture facilities previously established for the manufacturing of monoclonal antibodies that have now become obsolete due to yield improvement could be deployed for the manufacturing of these vaccines. Alternatively, manufacturing capacity could be established in geographic regions that do not have any vaccine production capability. Dependent on health care priorities, different vaccines could be manufactured while maintaining the ability to rapidly convert to producing pandemic influenza vaccine when the need arises. PMID:22265860
Homologous recombination deficiency and ovarian cancer.
Ledermann, Jonathan A; Drew, Yvette; Kristeleit, Rebecca S
The discovery that PARP inhibitors block an essential pathway of DNA repair in cells harbouring a BRCA mutation has opened up a new therapeutic avenue for high-grade ovarian cancers. BRCA1 and BRCA2 proteins are essential for high-fidelity repair of double-strand breaks of DNA through the homologous recombination repair (HRR) pathway. Deficiency in HRR (HRD) is a target for PARP inhibitors. The first PARP inhibitor, olaparib, has now been licensed for BRCA-mutated ovarian cancers. While mutated BRCA genes are individually most commonly associated with HRD other essential HRR proteins may be mutated or functionally deficient potentially widening the therapeutic opportunities for PARP inhibitors. HRD is the first phenotypically defined predictive marker for therapy with PARP inhibitors in ovarian cancer. Several different PARP inhibitors are being trialled in ovarian cancer and this class of drugs has been shown to be a new selective therapy for high-grade ovarian cancer. Around 20% of high-grade serous ovarian cancers harbour germline or somatic BRCA mutations and testing for BRCA mutations should be incorporated into routine clinical practice. The expanded use of PARP inhibitors in HRD deficient (non-BRCA mutant) tumours using a signature of HRD in clinical practice requires validation. PMID:27065456
Recombinant Human Elastase Treatment of Cephalic Veins
Wong, Marco D; Bingham, Karen; Moss, Emma; Warn, J Donald; Smirnov, Igor; Bland, Kimberly S; Starcher, Barry; Franano, F Nicholas; Burke, Steven K
Background Vessel injury at the time of Arteriovenous Fistula (AVF) creation may lead to neointimal hyperplasia that impairs AVF maturation. Vonapanitase, a recombinant human chymotrypsin-like elastase family member 1, is an investigational drug under development to improve AVF maturation and patency. The current studies were designed to document vonapanitase effects in human cephalic veins that are used in AVF creation. Methods Human cephalic veins were mounted on a perfusion myograph. Vonapanitase 1.2, 4, 13.2, and 40 μg/ml or saline was applied drop wise on the vein followed by saline rinse. Vein segments were cut into rings for elastin content determination by desmosine radioimmunoassay and histology. Fluorescently-labelled vonapanitase was applied to veins and adventitial imaging was performed using laser scanning confocal microscopy. In vivo time course experiments were performed by treating rabbit jugular veins and harvesting 1 h and 4 h after vonapanitase treatment. Results / Conclusion Vonapanitase reduced desmosine content in a dose-related manner. Histology also confirmed a dose-related reduction in elastic fiber staining. Fluorescently-labelled vonapanitase persistently localized to elastic fibers in the vein adventitia. In vivo experiments showed a reduction in desmosine content in jugular veins from 1 h to 4 h following treatment. These data suggest that vonapanitase targets elastin in elastic fibers in a dose related manner and that elastase remains in the vessel wall and has catalytic activity for at least 1 h.
Overview of the Purification of Recombinant Proteins
Wingfield, Paul T.
When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302
Dissociative recombination of rotationally cold H+3
NASA Astrophysics Data System (ADS)
McCall, B. J.; Huneycutt, A. J.; Saykally, R. J.; Djuric, N.; Dunn, G. H.; Semaniak, J.; Novotny, O.; Al-Khalili, A.; Ehlerding, A.; Hellberg, F.; Kalhori, S.; Neau, A.; Thomas, R. D.; Paal, A.; Österdahl, F.; Larsson, M.
This paper presents the first dissociative recombination (DR) measurement of electrons with rotationally and vibrationally cold H+3 ions. A dc discharge pinhole supersonic jet source was developed and characterized using infrared cavity ringdown spectroscopy before installation on the CRYRING ion storage ring for the DR measurements. Rotational state distributions ( Trot ˜30 K ) produced using the source were comparable to those in the diffuse interstellar medium. Our measurement of the electron energy dependence of the DR cross section showed resonances not clearly seen in experiments using rotationally hot ions, and allowed calculation of the thermal DR rate coefficient for ions at interstellar temperatures, αDR (23 K)=2.6× 10-7 cm3 s-1 . This value is in general agreement with recent theoretical predictions by
Dielectronic recombination studies based on EBIT
Xiao Jun; Han Chuan; Yao Ke; Shen Yang; Yang Yang; Wei Baoren; Fu Yunqing; Lu Di; Hutton, Roger; Zou Yaming
Dielectronic recombination (DR) process plays an important role in high temperature plasmas, where DR can affect charge balance and level populations significantly, and can cause radiative energy loss. Resolvable DR sourced satellite lines are often used for plasma temperature diagnostics, while the un-resolvable ones disturb determining spectral line shape, line intensity, and line position. Data of DR resonant strength is vital for accurate modeling of high temperature plasmas. DR studies are also important for testing atomic structure and atomic collision theories, since they carry information on quantum electrodynamics, relativistic effects, electron correlations and so on. Electron beam ion trap (EBIT) is an accelerator type device, which is capable of acting as both ion sources and light sources. EBIT can produce a special sort of plasma, in which electron energy is tunable and has a very narrow distribution. This made it possible for disentanglement studies on electron ion collision processes in plasmas. In this paper, experimental studies of DR processes based on electron beam ion traps (EBIT) will be discussed.
Dissociative Recombination of Molecular Ions for Astrochemistry
NASA Astrophysics Data System (ADS)
Savin, Daniel W.; Novotný, O.; Becker, A.; Buhr, H.; Geppert, W.; Hamberg, M.; Krantz, C.; Kreckel, H.; Schwalm, D.; Spruck, K.; Stützel, J.; Wolf, A.; Yang, B.
Dissociative recombination (DR) of molecular ions is a key chemical process in the cold interstellar medium (ISM). DR affects the composition, charge state, and energy balance of such environments. Astrochemical models of the ISM require reliable total DR cross sections as well as knowledge of the chemical composition and excitation states of the neutral DR products. Theory cannot reliably provide these data. We have systematically measured DR for many astrophysically relevant molecular ions utilizing the TSR storage ring at the Max-Planck-Institute for Nuclear Physics in Heidelberg, Germany. We used the merged ion-electron beam technique combined with an energy- and position-sensitive imaging detector and are able to study DR down to plasma temperatures of 10 K. The DR count rate is used to obtain absolute DR rate coefficient. Additionally we determine the masses of the DR products by measuring their kinetic energy. This allows us to assign particular DR fragmentation channels and to obtain their branching ratios. Moreover, the distribution of detected fragment distances provides information on the kinetic energy released in DR and thus also on the internal excitation of the DR products. All this information is particularly important for understanding DR of heteronuclear polyatomic ions. We will present DR results for several ions recently investigated at TSR. This work is supported in part by NASA and the NSF.
Functional divergence of gene duplicates through ectopic recombination
Christiaens, Joaquin F; Van Mulders, Sebastiaan E; Duitama, Jorge; Brown, Chris A; Ghequire, Maarten G; De Meester, Luc; Michiels, Jan; Wenseleers, Tom; Voordeckers, Karin; Verstrepen, Kevin J
Gene duplication stimulates evolutionary innovation as the resulting paralogs acquire mutations that lead to sub- or neofunctionalization. A comprehensive in silico analysis of paralogs in Saccharomyces cerevisiae reveals that duplicates of cell-surface and subtelomeric genes also undergo ectopic recombination, which leads to new chimaeric alleles. Mimicking such intergenic recombination events in the FLO (flocculation) family of cell-surface genes shows that chimaeric FLO alleles confer different adhesion phenotypes than the parental genes. Our results indicate that intergenic recombination between paralogs can generate a large set of new alleles, thereby providing the raw material for evolutionary adaptation and innovation. PMID:23070367
Resonant phenomena in laser-assisted radiative attachment or recombination
NASA Astrophysics Data System (ADS)
Zheltukhin, A. N.; Flegel, A. V.; Frolov, M. V.; Manakov, N. L.; Starace, Anthony F.
Resonant enhancements are predicted in cross sections σn for laser-assisted radiative attachment or electron-ion recombination accompanied by absorption of n laser photons. These enhancements occur for incoming electron energies at which the electron can be attached or recombined by emitting μ laser photons followed by emission of a spontaneous photon upon absorbing n + μ laser photons. The close similarity between rescattering plateaus in spectra of resonant attachment/recombination and of high-order harmonic generation is shown based on a general parametrization for σn and on numerical results for e - H attachment.
General features of the dissociative recombination of polyatomic molecules
Pratt, S. T.; Jungen, Ch.; Schneider, I. F.; Dulieu, O.; Robert, J.
We discuss some aspects of a simple expression for the low-energy dissociative recombination cross section that applies when the recombination process is dominated by the indirect mechanism. In most previous applications, this expression has been applied to capture into vibrationally excited Rydberg states with the assumption that capture is always followed by prompt dissociation. Here we consider the dissociative recombination of larger polyatomic ions and electrons. More specifically, we consider capture into electronically core-excited Rydberg states, and begin to assess its potential importance for larger systems.
Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus
NASA Astrophysics Data System (ADS)
Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard
The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.
Dynamics of carrier recombination in a semiconductor laser structure
Dzhioev, R. I. Kavokin, K. V.; Kusrayev, Yu. G.; Poletaev, N. K.
Carrier-recombination dynamics is studied by the method of optical orientation at room temperature in the active layer of a laser diode structure. The dependence of the degree of electron-spin orientation on the excitation density is attributed to saturation of the nonradiative-recombination channel. The time of electron capture at recombination centers is determined to be τ{sub e} = 5 × 10{sup –9} s. The temperature of nonequilibrium electrons heated by a He–Ne laser is estimated.
Minimizing recombinations in consensus networks for phylogeographic studies
Parida, Laxmi; Javed, Asif; Melé, Marta; Calafell, Francesc; Bertranpetit, Jaume
Background We address the problem of studying recombinational variations in (human) populations. In this paper, our focus is on one computational aspect of the general task: Given two networks G1 and G2, with both mutation and recombination events, defined on overlapping sets of extant units the objective is to compute a consensus network G3 with minimum number of additional recombinations. We describe a polynomial time algorithm with a guarantee that the number of computed new recombination events is within ϵ = sz(G1, G2) (function sz is a well-behaved function of the sizes and topologies of G1 and G2) of the optimal number of recombinations. To date, this is the best known result for a network consensus problem. Results Although the network consensus problem can be applied to a variety of domains, here we focus on structure of human populations. With our preliminary analysis on a segment of the human Chromosome X data we are able to infer ancient recombinations, population-specific recombinations and more, which also support the widely accepted 'Out of Africa' model. These results have been verified independently using traditional manual procedures. To the best of our knowledge, this is the first recombinations-based characterization of human populations. Conclusion We show that our mathematical model identifies recombination spots in the individual haplotypes; the aggregate of these spots over a set of haplotypes defines a recombinational landscape that has enough signal to detect continental as well as population divide based on a short segment of Chromosome X. In particular, we are able to infer ancient recombinations, population-specific recombinations and more, which also support the widely accepted 'Out of Africa' model. The agreement with mutation-based analysis can be viewed as an indirect validation of our results and the model. Since the model in principle gives us more information embedded in the networks, in our future work, we plan to investigate
Determination of surface recombination velocity in heavily doped silicon
NASA Technical Reports Server (NTRS)
Watanabe, M.; Gatos, H. C.; Actor, G.
A method was developed and successfully tested for the determination of the effective surface recombination velocity of silicon layers doped by diffusion of phosphorus to a level of 10 to the 19th to 10 to the 21st per cu cm. The effective recombination velocity was obtained from the dependence of the electron-beam-induced current on the penetration of the electron beam of a scanning electron microscope. A special silicon diode was constructed which permitted the collection at the p-n junction of the carriers excited by the electron beam. This diode also permitted the study of the effects of surface preparation on the effective surface recombination velocity.
Further corrections to the theory of cosmological recombination
NASA Technical Reports Server (NTRS)
Krolik, Julian H.
Krolik (1989) pointed out that frequency redistribution due to scattering is more important than cosmological expansion in determining the Ly-alpha frequency profile during cosmological recombination, and that its effects substantially modify the rate of recombination. Although the first statement is true, the second statement is not: a basic symmetry of photon scattering leads to identical cancellations which almost completely erase the effects of both coherent and incoherent scattering. Only a small correction due to atomic recoil alters the line profile from the prediction of pure cosmological expansion, so that the pace of cosmological recombination can be well approximated by ignoring Ly-alpha scattering.
Clinical experience with recombinant molecules for allergy vaccination.
Cromwell, Oliver; Niederberger, Verena; Horak, Friedrich; Fiebig, Helmut
Numerous allergens have been cloned and produced by the use of recombinant DNA technology. In several cases recombinant variants with reduced IgE-reactivity have also been developed as candidates for allergen specific immunotherapy. Only very few of these proteins have as yet been tested in the clinic, and the major focus has been on birch and grass pollen, two of the most common causes of IgE-mediated allergic disease. This article serves to justify the rational for using recombinant products and reviews the progress that has been made to date with their clinical assessment. PMID:21562972
Characterization and biodistribution of recombinant and recombinant/chimeric constructs of monoclonal antibody B72. 3
Colcher, D.; Milenic, D.; Roselli, M.; Raubitschek, A.; Yarranton, G.; King, D.; Adair, J.; Whittle, N.; Bodmer, M.; Schlom, J.
Radiolabeled B72.3 has been administered both i.v. and i.p. in patients with colorectal and ovarian cancer as well as other carcinomas and has been shown to selectively bind to approximately 70-80% of metastatic lesions. Greater than 50% of the patients that have been treated with B72.3 have developed an immunological response to murine IgG after a single injection. In an attempt to minimize the immune response of these patients to the administered murine monoclonal antibody, we developed a recombinant form of the murine B72.3 as well as a recombinant/chimeric antibody, using the variable regions of the murine B72.3 and human heavy chain (gamma 4) and light chain (kappa) constant regions. We report here that both the recombinant B72.3 (rB72.3) and the recombinant/chimeric B72.3 (cB72.3(gamma 4)) IgGs maintain the tissue binding and idiotypic specificity of the native murine IgG. The native B72.3, rB72.3, and cB72.3(gamma 4) IgGs were radiolabeled and the biodistribution of these IgGs was studied in athymic mice bearing human colon carcinoma xenografts (LS-174T). Differences were observed between the cB72.3(gamma 4) and the native B72.3 in the percentage of injected dose/g that localized in the tumor. The somewhat lower absolute amounts of the cB72.3(gamma 4) in the tumor are mostly likely due to the observed more rapid clearance from the blood and body of the mouse as compared to the native B72.3 and rB72.3. All three forms (native B72.3, rB72.3, and cB72.3(gamma 4)) of the IgG, however, were able to localize the colon tumor with similar radiolocalization indices (percentage of injected dose/g in tumor divided by the percentage of injected dose/g in normal tissue).
Rate coefficients for N2(+)(v) dissociative recombination
NASA Astrophysics Data System (ADS)
Bates, D. R.; Mitchell, J. B. A.
The data of Zipf (1980) on N2(+)(v) dissociative recombination are analyzed taking into account the fact that there is coupling due to reversible symmetrical resonance charge transfer, N2(+)(v) + N2(0) yields N2(+)(0) + N2(v). The vibrational deactivation in N2(+)(v)-Ne collisions is also considered. A reported experimental value of the vibrational deactivation coefficient is found to be much higher than can be reconciled with the results of Zipf and it is therefore rejected. The analysis shows that the recombination coefficient for N2(+)(0) is about 2.6 x 10 exp-7 cu cm/s at 300 K and that recombination coefficients for N2(+)(1) and N2(+)(2) are substantially smaller. It is concluded that these coefficients conflict with the dissociative recombination cross section vs energy curve obtained by the merged beam method.
[Processing and Modification of Recombinant Spider Silk Proteins].
Liu, Bin; Wang, Tao; Liu, Xiaobing; Luo, Yongen
Due to its special sequence structure, spider silk protein has unique physical and chemical properties, mechanical properties and excellent biological properties. With the expansion of the application value of spider silk in many fields as a functional material, progress has been made in the studies on the expression of recombinant spider silk proteins through many host systems by gene recombinant techniques. Recombinant spider silk proteins can be processed into high performance fibers, and a wide range of nonfibrous morphologies. Moreover, for their excellent biocompatibility and low immune response they are ideal for biomedical applications. Here we review the process and mechanism of preparation in vitro, chemistry and genetic engineering modification on recombinant spider silk protein. PMID:26710473
Experimental study of columnar recombination in fission chambers
NASA Astrophysics Data System (ADS)
Filliatre, P.; Lamirand, V.; Geslot, B.; Jammes, C.
In this paper, we present experimental saturation curves of a small gap miniature fission chamber obtained in the MINERVE reactor. The chamber is filled with argon at various pressures, and the fissile material can be coated on the anode, cathode, or both. For analyzing the recombination regime, we consider a model of columnar recombination and discuss its applicability to our chamber. By applying this model to the data, it is possible to estimate the ratio between the recombination coefficient k and an effective column radius b, appearing in the model, to be k / b =(2.5 ± 0.9) ×10-6m2 / s for argon. From these results, a routine measurement of the recombination regime is proposed in order to detect gas leakage. This online diagnosis would be beneficial in terms of lifetime and reliability of the neutron instrumentation of nuclear reactors.
Interchromosomal recombination is suppressed in mammalian somatic cells.
Shulman, M J; Collins, C; Connor, A; Read, L R; Baker, M D
Homologous recombination occurs intrachromosomally as well as interchromosomally, both in mitotic (somatic) cells as well as meiotically in the germline. These different processes can serve very different purposes in maintaining the integrity of the organism and in enhancing diversity in the species. As shown here, comparison of the frequencies of intra- and interchromosomal recombination in meiotic and mitotic cells of both mouse and yeast argues that interchromosomal recombination is particularly low in mitotic cells of metazoan organisms. This result in turn suggests that the recombination machinery of metazoa might be organized to avoid the deleterious effects of homozygotization in somatic cells while still deriving the benefits of species diversification and of DNA repair. Images PMID:7664750
DNA damage tolerance by recombination: Molecular pathways and DNA structures.
Branzei, Dana; Szakal, Barnabas
Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research. PMID:27236213
Auger recombination in sodium-iodide scintillators from first principles
McAllister, Andrew; Åberg, Daniel; Schleife, André; Kioupakis, Emmanouil
Scintillator radiation detectors suffer from low energy resolution that has been attributed to non-linear light yield response to the energy of the incident gamma rays. Auger recombination is a key non-radiative recombination channel that scales with the third power of the excitation density and may play a role in the non-proportionality problem of scintillators. In this work, we study direct and phonon-assisted Auger recombination in NaI using first-principles calculations. Our results show that phonon-assisted Auger recombination, mediated primarily by short-range phonon scattering, dominates at room temperature. We discuss our findings in light of the much larger values obtained by numerical fits to z-scan experiments.
Nonradiative recombination of excitons in semimagnetic quantum dots
Chernenko, A. V.
The mechanisms of the nonradiative recombination of excitons in neutral and charged quantum dots based on II–VI semimagnetic semiconductors are investigated. It is shown that, along with the dipole–dipole and direct-exchange mechanisms, there is one more mechanism referred to as the indirect-exchange mechanism and related to sp–d mixing. The selection rules for nonradiative recombination by exchange mechanisms are subsequently derived. The dependence of the efficiency of all recombination mechanisms on the quantum-dot size is studied. The experimentally observed growth in the intracenter photoluminescence intensity with decreasing size of dots and nanocrystals is accounted for. Methods for experimental determination of the contributions of different mechanisms to nonradiative recombination are discussed.
New strategies for genetic engineering Pseudomonas syringae using recombination
Technology Transfer Automated Retrieval System (TEKTRAN)
Here we report that DNA oligonucleotides (oligos) introduced directly into bacteria by electroporation can recombine with the bacterial chromosome. This phenomenon was identified in Pseudomonas syringae and we subsequently found that Escherichia coli, Salmonella typhimurium and Shigella flexneri are...
Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes
Throop, Andrea L.; LaBaer, Joshua
The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088
Paracentric inversions do not normally generate monocentric recombinant chromosomes
Sutherland, G.R.; Callen, D.F.; Gardner, R.J.M.
Dr. Pettenati et al. recently reported a review of paracentric inversions in humans in which they concluded that carriers of these have a 3.8% risk of viable offspring with recombinant chromosomes. We are of the view that there are serious problems with this estimate which should be much closer to zero. The only recombinant chromosomes which can be generated by a paracentric inversion undergoing a normal meiotic division are dicentrics and acentric fragments. Only two such cases were found by Pettenati et al. Several of the alleged monocentric recombinants were originally reported as arising from parental insertions (3-break rearrangements) and it is not legitimate to include them in any analysis of paracentric inversions. Any monocentric recombinant chromosome can only arise from a paracentric inversion by some abnormal process which must involve chromatid breakage and reunion. 4 refs.
Meiotic recombination in normal and cloned bulls and their offspring
Technology Transfer Automated Retrieval System (TEKTRAN)
Homologous chromosome pairing and recombination are essential components of meiosis and sexual reproduction. The reshuffling of genetic material through breakage and reunion of chromatids ensure proper segregation of homologous chromosomes in reduction division and genetic diversity in the progeny....
Effect Of Auger Recombination In An Ion Track
NASA Technical Reports Server (NTRS)
Edmonds, Larry D.
Report presents theoretical calculations of contribution of Auger recombination to depletion of charge carriers from ionization track left by passage of energetic heavy ion through silicon-based electronic device.
Iron ionization and recombination rates and ionization equilibrium
NASA Technical Reports Server (NTRS)
Arnaud, M.; Raymond, J.
In the past few years important progress has been made on the knowledge of ionization and recombination rates of iron, an astrophysically abundant heavy element and a major impurity in laboratory fusion devices. We make a critical review of the existing data on ionization and dielectronic recombination and present new computations of radiative recombination rate coefficients of Fe(+14) through Fe(+25) using the photoionization cross sections of Clark et al. (1986). We provide analytical fits to the recommended data (direct ionization and excitation-autoionization cross sections; radiative and dielectronic recombination rate coefficients). Finally we determine the iron ionic fractions at ionization equilibrium and compare them with previous computations as well as with observational data.
Evidence for recombination in scorpion mitochondrial DNA (Scorpiones: Buthidae)
Gantenbein, Benjamin; Fet, Victor; Gantenbein-Ritter, Iris A; Balloux, François
There has been very little undisputed evidence for recombination in animal mitochondrial DNA (mtDNA) provided so far. Previous unpublished results suggestive of mtDNA recombination in the scorpion family Buthidae, together with cytological evidence for a unique mechanism of mitochondrial fusion in that family, prompted us to investigate this group in more details. First, we sequenced the complete mtDNA genome of Mesobuthus gibbosus, and chose two genes opposing each other (16S and coxI). We then sequenced 150 individuals from the natural populations of four species of Buthidae (Old World genera Buthus and Mesobuthus). We observed strong evidence for widespread recombination through highly significant negative correlations between linkage disequilibrium and physical distance in three out of four species. The evidence is further confirmed when using five other tests for recombination and by the presence of a high amount of homoplasy in phylogenetic trees. PMID:15870032
Products of Dissociative Recombination in the Ionosphere
NASA Technical Reports Server (NTRS)
Cosby, Philip
SRI International undertook a novel experimental measurement of the product states formed by dissociative recombination (DR) of O2(+), NO(+), and N2(+) as a function of both electron energy and reactant ion vibrational level. For these measurements we used a recently developed experimental technique for measuring dissociation product distributions that allows both the branching ratios to be accurately determined and the electronic and rovibrational state composition of the reactant ions to be specified. DR is the dominant electron loss mechanism in all regions of the ionosphere. In this process, electron attachment to the molecular ion produces an unstable neutral molecule that rapidly dissociates. For a molecular ion such as O2(+), the dissociation recombination reaction is (1) O2(+) + e yields O + O + W. The atomic products of this reaction, in this case two oxygen atoms, can be produced in a variety of excited states and with a variety of kinetic energies, as represented by W in Eq. (1). These atoms are not only active in the neutral chemistry of the ionosphere, but are also especially important because their optical emissions are often used to infer in situ concentrations of the parent molecular ion and ambient electron densities. Many laboratory measurements have been made of DR reaction rates under a wide range of electron temperatures, but very little is known about the actual distributions among the final states of the atomic products. This lack of knowledge seriously limits the validity and effectiveness of efforts to model both natural and man-made ionospheric disturbances. Bates recently identified major deficiencies in the currently accepted branching ratios for O2(+) as they relate to blue and green line emission measurements in the nocturnal F-region. During our two-year effort, we partially satisfied our ambitious goals. We constructed and operated a variable pressure, electron-impact ion source and a high pressure, hollow-cathode discharge ion
Therapeutic use of recombinant methionyl human leptin.
Vatier, Camille; Gautier, Jean-François; Vigouroux, Corinne
Recombinant methionyl human leptin (r-metHuLeptin) was first used as a replacement therapy in patients bearing inactivating mutations in the leptin gene. In this indication, it was shown since 1999 to be very efficient in inducing a dramatic weight loss in rare children and adults with severe obesity due to the lack of leptin. These first clinical trials clearly showed that r-metHuLeptin acted centrally to reduce food intake, inducing loss of fat mass, and to correct metabolic alterations, immune and neuroendocrine defects. A few years later, r-metHuLeptin was also shown to reverse the metabolic complications associated with lipodystrophic syndromes, due to primary defects in fat storage, which induce leptin deficiency. The beneficial effects, which could be mediated by central and/or peripheral mechanisms, are thought to mainly involve the lowering effects of leptin on ectopic lipid storage, in particular in liver and muscles, reducing insulin resistance. Interestingly, r-metHuLeptin therapy also reversed the hypothalamic-pituitary-gonadal axis dysfunctions associated with hypothalamic amenorrhea. However, if r-metHuLeptin treatment has been shown to be dramatically efficient in leptin-deficient states, its very limited effect in inducing weight loss in common obese patients revealed that, in patients with adequate leptin secretion, mechanisms of leptin resistance and leptin tolerance prevent r-metHuLeptin from inducing any additional effects. This review will present the current data about the effects of r-metHuLeptin therapy in humans, and discuss the recent perspectives of this therapy in new indications. PMID:22464954
Integrated continuous production of recombinant therapeutic proteins.
Warikoo, Veena; Godawat, Rahul; Brower, Kevin; Jain, Sujit; Cummings, Daniel; Simons, Elizabeth; Johnson, Timothy; Walther, Jason; Yu, Marcella; Wright, Benjamin; McLarty, Jean; Karey, Kenneth P; Hwang, Chris; Zhou, Weichang; Riske, Frank; Konstantinov, Konstantin
In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins. PMID:22729761
Dissociative Recombination of Molecular Ions for Astrochemistry
NASA Astrophysics Data System (ADS)
Novotny, Oldrich; Becker, A.; Buhr, H.; Fleischmann, Andreas; Gamer, Lisa; Geppert, W.; Krantz, C.; Kreckel, H.; Schwalm, D.; Spruck, K.; Wolf, A.; Savin, Daniel Wolf
Dissociative recombination (DR) of molecular ions is a key chemical process in the cold interstellar medium (ISM). DR affects the composition, charge state, and energy balance of such environments. Astrochemical models of the ISM require reliable total DR cross sections as well as knowledge of the chemical composition of the neutral DR products. We have systematically measured DR for many astrophysically relevant molecular ions utilizing the TSR storage ring at the Max-Planck-Institute for Nuclear Physics (MPIK) in Heidelberg, Germany. We used the merged ion-electron beam technique combined with an energy- and position-sensitive imaging detector and are able to study DR down to plasma temperatures as low as 10 K. The DR count rate is used to obtain an absolute merged beams DR rate coefficient from which we can derive a thermal rate coefficient needed for plasma models. Additionally we determine the masses of the DR products by measuring their kinetic energy in the laboratory reference frame. This allows us to assign particular DR fragmentation channels and to obtain their branching ratios. All this information is particularly important for understanding DR of heteronuclear polyatomic ions. We will present DR results for several ions recently investigated at TSR. A new Cryogenic Storage Ring (CSR) is currently being commissioned at MPIK. With the chamber cooled down to ~10 K and a base pressure better than 10-13 mbar, this setup will allow internal cooling of the stored ions down to their rotational ground states, thus opening a new era in DR experiments. New technological challenges arise due to the ultracold, ultra-high vacuum environment of the CSR and thus the detection techniques used at TSR cannot be easily transferred to CSR. We will present new approaches for DR fragment detection in cryogenic environment. This work is supported in part by NASA and the NSF.
The Time Scale of Recombination Rate Evolution in Great Apes.
Stevison, Laurie S; Woerner, August E; Kidd, Jeffrey M; Kelley, Joanna L; Veeramah, Krishna R; McManus, Kimberly F; Bustamante, Carlos D; Hammer, Michael F; Wall, Jeffrey D
We present three linkage-disequilibrium (LD)-based recombination maps generated using whole-genome sequence data from 10 Nigerian chimpanzees, 13 bonobos, and 15 western gorillas, collected as part of the Great Ape Genome Project (Prado-Martinez J, et al. 2013. Great ape genetic diversity and population history. Nature 499:471-475). We also identified species-specific recombination hotspots in each group using a modified LDhot framework, which greatly improves statistical power to detect hotspots at varying strengths. We show that fewer hotspots are shared among chimpanzee subspecies than within human populations, further narrowing the time scale of complete hotspot turnover. Further, using species-specific PRDM9 sequences to predict potential binding sites (PBS), we show higher predicted PRDM9 binding in recombination hotspots as compared to matched cold spot regions in multiple great ape species, including at least one chimpanzee subspecies. We found that correlations between broad-scale recombination rates decline more rapidly than nucleotide divergence between species. We also compared the skew of recombination rates at centromeres and telomeres between species and show a skew from chromosome means extending as far as 10-15 Mb from chromosome ends. Further, we examined broad-scale recombination rate changes near a translocation in gorillas and found minimal differences as compared to other great ape species perhaps because the coordinates relative to the chromosome ends were unaffected. Finally, on the basis of multiple linear regression analysis, we found that various correlates of recombination rate persist throughout the African great apes including repeats, diversity, and divergence. Our study is the first to analyze within- and between-species genome-wide recombination rate variation in several close relatives. PMID:26671457
Fixation of Emerging Interviral Recombinants in Cucumber Mosaic Virus Populations
Pita, Justin S.
Interstrain recombinants were observed in the progenies of the Cucumber mosaic virus (CMV) reassortant L1L2F3 containing RNAs 1 and 2 from LS-CMV and RNA 3 from Fny-CMV. We characterized these recombinants, and we found that their fixation was controlled by the nature of the replicating RNAs 1 and 2. We demonstrate that the 2b gene partially affects this fixation process, but only in the context of homologous RNAs 1 and 2. PMID:23115282
Strongly Enhanced Recombination via Two-Center Electronic Correlations
Mueller, C.; Voitkiv, A. B.; Lopez-Urrutia, J. R. Crespo; Harman, Z.
In the presence of a neighboring atom, electron-ion recombination can proceed resonantly via excitation of an electron in the atom, with subsequent relaxation through radiative decay. It is shown that this two-center dielectronic process can largely dominate over single-center radiative recombination at internuclear distances as large as several nanometers. The relevance of the predicted process is demonstrated by using examples of water-dissolved alkali cations and warm dense matter.
Negative-continuum dielectronic recombination for heavy ions
Artemyev, A.N.; Yerokhin, V.A.; Beier, T.; Kozhuharov, C.; Eichler, J.; Klasnikov, A.E.; Shabaev, V.M.; Stoehlker, T.
The process of recombination of an electron with a bare heavy nucleus via the creation of a free-positron-bound-electron pair is considered. This process is denoted as 'negative-continuum dielectronic recombination' because it results in the capture of an incident electron into a bound state accompanied by a transition of a negative-continuum electron into a bound state. The calculations are performed for a wide range of incident electron energies for Z=82 and 92.
Recombinant Protein Production and Insect Cell Culture and Process
NASA Technical Reports Server (NTRS)
Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)
A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.
Recombinant protein production and insect cell culture and process
NASA Technical Reports Server (NTRS)
Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)
A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.
Recombinant HT{sub m4} gene, protein and assays
Lim, B.; Adra, C.N.; Lelias, J.M.
The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.
Green factory: plants as bioproduction platforms for recombinant proteins.
Xu, Jianfeng; Dolan, Maureen C; Medrano, Giuliana; Cramer, Carole L; Weathers, Pamela J
Molecular farming, long considered a promising strategy to produce valuable recombinant proteins not only for human and veterinary medicine, but also for agriculture and industry, now has some commercially available products. Various plant-based production platforms including whole-plants, aquatic plants, plant cell suspensions, and plant tissues (hairy roots) have been compared in terms of their advantages and limits. Effective recombinant strategies are summarized along with descriptions of scalable culture systems and examples of commercial progress and success. PMID:21924345
Unified Theoretical Approach to Electron-Ion Recombination
NASA Astrophysics Data System (ADS)
Pradhan, Anil
Electron-ion recombination occurs via the electron continuum and embedded resonances that are coupled to each other and thus unified in nature. However, theoretical methods generally separate the two as ``radiative recombination" (RR), referring to recombination through the non-resonant continuum, and ``di-electronic recombination" (DR) through the autoionizing resonances. A computationally unified scheme(S.N. Nahar and A.K. Pradhan, Phys.Rev.Lett.) 68,1488(1992); H.L. Zhang and A.K. Pradhan, Phys.Rev.Lett. 78,195(1997) is described that employs the coupled channel R-matrix method, and detailed balance (the Milne relation), to obtain photo-recombination cross sections including both the continuum and resonant recombination in an ab intio manner. In the energy region corresponding to high-n Rydberg resonances, where the background recombination (RR) is negligibly small, a precise theory of DR (R.H. Bell and M.J. Seaton, J.Phys.B) 18,1589(1985) yields DR collision strengths consistent with their threshold behaviour leading to the cross section for electron impact excitation. For highly charged ions the relativistic fine structure and the effect of radiation damping of resonances are considered. Theoretical cross sections agree well with recent experiments on ion stograge rings and the electron-beam-ion-trap (EBIT). Total electron-ion recombination rates can be obtained for practical applications. For many complex atomic systems, such as the important Iron-peak elements, the new results differ considerably from those hitherto available.
Fine-scale recombination and adaptive radiation could be linked.
Bodilis, Josselin
The difficult reconstruction of the evolutionary history of the major surface protein gene oprF highlighted an adaptive radiation in the Pseudomonas fluorescens group. The recent work of Hao (2013) showed that partial recombination events in oprF gene occurred specifically in a P. fluorescens lineage under ecological niche segregation. So, I suggest that identification of lineage-specific fine-scale recombination may be a way to detect putative adaptive radiation in bacteria. PMID:23774687
Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.
Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P
Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF. PMID:22808513
Recombinant TCR ligand reverses clinical signs and CNS damage of EAE induced by recombinant human MOG.
Sinha, Sushmita; Subramanian, Sandhya; Emerson-Webber, Ashley; Lindner, Maren; Burrows, Gregory G; Grafe, Marjorie; Linington, Christopher; Vandenbark, Arthur A; Bernard, Claude C A; Offner, Halina
Increasing evidence suggests that in addition to T cell-dependent effector mechanisms, autoantibodies are also involved in the pathogenesis of MS, including demyelinating antibodies specific for myelin oligodendrocyte glycoprotein (MOG). Our previous studies have demonstrated that recombinant T cell receptor ligands (RTLs) are very effective for treating T cell-mediated experimental autoimmune encephalomyelitis (EAE). In order to expand the scope of RTL therapy in MS patients, it was of interest to study RTL treatment of EAE involving a demyelinating antibody component. Therefore, we evaluated the therapeutic effects of RTL551, specific for T cells reactive to mouse (m)MOG-35-55 peptide, on EAE induced with recombinant human (rh)MOG in C57BL/6 mice. We report that RTL551 therapy can reverse disease progression and reduce demyelination and axonal damage induced by rhMOG without suppressing the anti-MOG antibody response. This result suggests that T cell-mediated inflammation and associated blood-brain barrier dysfunction are the central contributors to EAE pathogenesis and that successful regulation of these key players restricts potential damage by demyelinating antibodies. The results of our study lend support for the use of RTL therapy for treatment of MS subjects whose disease includes inflammatory T cells as well as those with an additional antibody component. PMID:19789980
Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination
Martín, M. Cruz; Alonso, Juan C.; Suárez, Juan E.; Alvarez, Miguel A.
The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation. PMID:10831443
Posttranslational modifications and activity of natural and recombinant tissue factor
Butenas, Saulius; Krudysz-Amblo, Jolanta; Mann, Kenneth G
Tissue factor is a membrane protein, which in a complex with factor VIIa initiates in vivo blood coagulation. Due to the scarcity of natural tissue factor protein, most studies have relied upon recombinant tissue factor forms. However, there have been only cursory experimental comparisons of natural and recombinant tissue factor proteins. Our preliminary data suggested that placental tissue factor in a complex with factor VIIa was more efficient activator of factor X than the recombinant protein. After deglycosylation, both forms of tissue factor showed almost an identical activity in the extrinsic factor Xase. Analyses using tryptic digestion and mass-spectrometry revealed that the levels of glycosylation and the composition of carbohydrates present in natural placental tissue factor were different than those in its recombinant counterpart. These data indicate that natural and recombinant tissue factor proteins differ in their posttranslational modifications and that these differences translate into different cofactor activity. Thus the use of recombinant tissue factor proteins for the quantitation of natural tissue factor is misleading. PMID:20138335
Recombination Suppression by Heterozygous Robertsonian Chromosomes in the Mouse
Davisson, M. T.; Akeson, E. C.
Robertsonian chromosomes are metacentric chromosomes formed by the joining of two telocentric chromosomes at their centromere ends. Many Robertsonian chromosomes of the mouse suppress genetic recombination near the centromere when heterozygous. We have analyzed genetic recombination and meiotic pairing in mice heterozygous for Robertsonian chromosomes and genetic markers to determine (1) the reason for this recombination suppression and (2) whether there are any consistent rules to predict which Robertsonian chromosomes will suppress recombination. Meiotic pairing was analyzed using synaptonemal complex preparations. Our data provide evidence that the underlying mechanism of recombination suppression is mechanical interference in meiotic pairing between Robertsonian chromosomes and their telocentric partners. The fact that recombination suppression is specific to individual Robertsonian chromosomes suggests that the pairing delay is caused by minor structural differences between the Robertsonian chromosomes and their telocentric homologs and that these differences arise during Robertsonian formation. Further understanding of this pairing delay is important for mouse mapping studies. In 10 mouse chromosomes (3, 4, 5, 6, 8, 9, 10, 11, 15 and 19) the distances from the centromeres to first markers may still be underestimated because they have been determined using only Robertsonian chromosomes. Our control linkage studies using C-band (heterochromatin) markers for the centromeric region provide improved estimates for the centromere-to-first-locus distance in mouse chromosomes 1, 2 and 16. PMID:8454207
RPA homologs and ssDNA processing during meiotic recombination.
Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle
Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair. PMID:26520106
Recombinant medaka (Oryzias melastigmus) pro-hepcidin: Multifunctional characterization.
Cai, Ling; Cai, Jing-Jing; Liu, Hai-Peng; Fan, Dan-Qing; Peng, Hui; Wang, Ke-Jian
Recently, two hepcidin variant genes (Om-hep1 and Om-hep2) were identified in a model fish marine medaka and both were highly induced in vivo with bacterial challenge, suggesting that the medaka hepcidin may have a similar function to other reported teleostean hepcidins. In the present study, the antibacterial, antiviral and antitumor activities of Om-hep1 were determined using its synthetic and recombinant pro-peptides. The recombinant pro-hepcidin1 was expressed in Escherichia coli and an effective method to produce recombinant Pro-Omhep1 was developed in order to obtain a right folded structure. The results showed that both the synthetic mature peptide and recombinant pro-peptide had similar antibacterial activity against Gram-positive and negative bacteria. In particular, both the synthetic mature Om-hep1 and recombinant Pro-Omhep1 inhibited the viral replication of white spot syndrome virus in the hematopoietic tissue cells of the crayfish Cherax quadricarinatus. Om-hep1 also presented antitumor activity on the cultured human hepatocellular carcinoma cells. In addition, the antimicrobial mechanism of Om-hep1 was measured and it was found that Om-hep1 was likely to be non-membranolytic. The recombinant Pro-Omhep1 performed better biological activity compared to the synthetic mature Om-hep1. This study suggested that Om-hep1 was likely to be an important multifunction protein involved in various resistance actions in the marine medaka immune system. PMID:22051539
Charge relaxation and recombination in photo-excited Mott insulators
NASA Astrophysics Data System (ADS)
Prelovšek, P.; Lenarčič, Z.
Recent femtosecond pump-probe experiments on Mott insulators reveal charge recombination, which is in picosecond range, i.e., much faster than in clean bandgap semiconductors although excitation gaps in Mott insulators are even larger. The charge response in photo-excited insulators can be generally divided in femtosecond transient relaxation of charge excitations, which are holons and doublons, and a second slower, but still very fast, holon-doublon (HD) recombination. We present a theory of the recombination rate of the excited HD pairs, based on the two-dimensional (2D) model relevant for cuprates, which shows that such fast processes can be explained even quantitatively with the multi-magnon emission. We show that the condition for the exponential decay as observed in the experiment is the existence of the exciton, i.e., the bound HD pair. Its recombination rate is exponentially dependent on the charge gap and on the magnon energy, while the ultrafast process can be traced back to strong charge-spin coupling. We comment also fast recombination times in the one-dimensional (1D) Mott insulators, as e.g., organic salts. The recombination rate in the latter cases can be explained with the stronger coupling with phonon excitations.
Genetic diversity and recombination analysis of sweepoviruses from Brazil
Background Monopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect sweet potato (Ipomoea batatas) around the world are known as sweepoviruses. Because sweet potato plants are vegetatively propagated, the accumulation of viruses can become a major constraint for root production. Mixed infections of sweepovirus species and strains can lead to recombination, which may contribute to the generation of new recombinant sweepoviruses. Results This study reports the full genome sequence of 34 sweepoviruses sampled from a sweet potato germplasm bank and commercial fields in Brazil. These sequences were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic diversity and patterns of genetic exchange in sweepoviruses isolated from Brazil, as well as to review the classification and nomenclature of sweepoviruses in accordance with the current guidelines proposed by the Geminiviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV). Co-infections and extensive recombination events were identified in Brazilian sweepoviruses. Analysis of the recombination breakpoints detected within the sweepovirus dataset revealed that most recombination events occurred in the intergenic region (IR) and in the middle of the C1 open reading frame (ORF). Conclusions The genetic diversity of sweepoviruses was considerably greater than previously described in Brazil. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of sweepovirus species and strains and provided valuable new information for understanding the diversity and evolution of sweepoviruses. PMID:23082767
Recombination, chromosome number and eusociality in the Hymenoptera
Ross, L; Blackmon, H; Lorite, P; Gokhman, V E; Hardy, N B
Extraordinarily high rates of recombination have been observed in some eusocial species. The most popular explanation is that increased recombination increases genetic variation among workers, which in turn increases colony performance, for example by increasing parasite resistance. However, support for the generality of higher recombination rates among eusocial organisms remains weak, due to low sample size and a lack of phylogenetic independence of observations. Recombination rate, although difficult to measure directly, is correlated with chromosome number. As predicted, several authors have noted that chromosome numbers are higher among the eusocial species of Hymenoptera (ants, bees and wasps). Here, we present a formal comparative analysis of karyotype data from 1567 species of Hymenoptera. Contrary to earlier studies, we find no evidence for an absolute difference between chromosome number in eusocial and solitary species of Hymenoptera. However, we find support for an increased rate of chromosome number change in eusocial taxa. We show that among eusocial taxa colony size is able to explain some of the variation in chromosome number: intermediate-sized colonies have more chromosomes than those that are either very small or very large. However, we were unable to detect effects of a number of other colony characteristics predicted to affect recombination rate – including colony relatedness and caste number. Taken together, our results support the view that a eusocial lifestyle has led to variable selection pressure for increased recombination rates, but that identifying the factors contributing to this variable selection will require further theoretical and empirical effort. PMID:25382409
Meiotic recombination analysis in female ducks (Anas platyrhynchos).
Pigozzi, M I; Del Priore, L
Meiotic recombination in female ducks was directly studied by immunolocalization of MLH1 protein, a mismatch repair protein of mature recombination nodules. In total, 6820 crossovers were scored along the autosomal synaptonemal complexes in 122 meiotic nuclei. From this analysis we predict that the female map length of the duck is 2845 cM, with a genome wide recombination rate of 2 cM/Mb. MLH1-focus mapping along the six largest bivalents shows regional variations of recombination frequencies that can be linked to differences in chromosome morphology. From this MLH1 mapping it can be inferred that distally located markers will appear more separated in genetic maps than physically equidistant markers located near the centromeres on bivalents 1 and 2. Instead, markers at interstitial positions on the acrocentric bivalents 3-6 will appear more tightly linked than expected on the basis of their physical distance because recombination is comparatively lower at the mid region of these chromosomes. The present results provide useful information to complement linkage mapping in ducks and extend previous knowledge about the variation of recombination rates among domestic Galloanserae. PMID:27115519
Immunoglobulin/Myc recombinations in murine Peyer's patch follicles.
Müller, J R; Mushinski, E B; Williams, J A; Hausner, P F
Immunoglobulin heavy chain (Igh)/Myc recombinations are a hallmark of pristane-induced mouse plasmacytomas but are also frequently found in non-tumorous tissues. Here we describe for the first time a PCR-based technique for detecting fusions between Igh mu or Igh alpha and Myc in situ. Igh/Myc recombinations were found in transplanted and primary plasmacytomas. In addition, the gut-associated lymphoid tissues of plasmacytoma-free BALB/c mice were investigated for the presence of Igh/Myc fusions. Igh/Myc rearrangements were detected in Peyer's patch follicles and in the intestinal lamina propria both in normal mice and in mice shortly after pristane treatment. The sequence analysis showed that i) three to five different Igh/Myc hybrid sequences were present in individual follicles, ii) Igh/Myc recombinations can be subjected to additional switch recombinations as shown by related sequences in neighboring cells, and iii) cells harboring these rearrangements migrate into the adjacent lamina propria. The results indicate that Peyer's patches are a hyper-recombinogenic tissue. Myc recombination-positive cells are present in at least 100-fold more frequently than expected if recombinations were random, which suggests that this kind of trans-chromosomal rearrangement may be targeted. PMID:9290947
Recombination Coefficient Measurements of O and N radicals
NASA Astrophysics Data System (ADS)
Singh, Harmeet; Coburn, John; Graves, David
Surface recombination of radicals in low-pressure high-density plasmas has direct influence on the neutral and ionic composition of the plasma. While, electron impact dissociation of molecules is the dominant mechanism for creation of radicals, the surface recombination of radicals is often expected to be the dominant loss mechanism. We have a combination of measurements and a model to determine the recombination coefficients of O and N, to O2 and N2, respectively on the stainless steel walls of our inductively coupled plasma chamber. The radial variation of the electron energy distribution function (EEDF) is measured using a tuned, cylindrical Langmuir probe. The number density of the molecular species is measured using line-of-sight modulated beam mass spectrometry. The mass spectrometer is differentially pumped in three stages to ensure a good beam to background signal ratio. The radical absolute number density is measured using appearance potential mass spectrometry with the aforementioned mass spectrometer. The recombination coefficient is calculated using a balance of the volume-generation and surface-loss rates of the radicals in the plasma. The generation rate of the radicals is calculated using the number density measurements of the parent molecule and the spatially resolved EEDFs. At approximately 330 K on stainless steel, the recombination coefficient for O is 0.16, and recombination coefficient for N is 0.07.
Seroreactive recombinant herpes simplex virus type 2-specific glycoprotein G.
Parkes, D L; Smith, C M; Rose, J M; Brandis, J; Coates, S R
The herpes simplex virus type 2 (HSV-2) genome codes for an envelope protein, glycoprotein G (gG), which contains predominantly type 2-specific epitopes. A portion of this gG gene has been expressed as a fusion protein in Escherichia coli. Expression was regulated by a lambda phage pL promoter. The 60,000-molecular-weight recombinant protein was purified by ion-exchange chromatography. Amino acid sequence analysis confirmed the N terminus of the purified protein. Mice immunized with recombinant gG developed antibodies reactive with native HSV-2 protein, but not with HSV-1 protein, in an indirect immunofluorescence assay. The serological activity of this purified recombinant gG protein was evaluated by immunoblot assay. This protein was reactive with an HSV-2 gG monoclonal antibody. It was also reactive with HSV-2 rabbit antiserum but not with HSV-1 rabbit antiserum. Of 15 patient serum samples known to have antibody to HSV-2, 14 were reactive with this recombinant type 2-specific gG protein, and none of 15 HSV antibody-negative patient serum samples showed reactivity. In agreement with the expected prevalence of HSV-2 infection, 27.6% of 134 serum samples from random normal individuals had antibodies reactive with recombinant gG. This recombinant gG protein may be of value in detecting HSV-2-specific antibody responses in patients infected with HSV-2. Images PMID:1653787
Electron-ion recombination of FetII
NASA Astrophysics Data System (ADS)
Nahar, Sultana N.
A complete treatment of electron-ion recombination of e+Fe III-->Fe II employing a unified method is presented. The treatment incorporates both the radiative and dielectronic recombinations in a self-consistent manner. Total recombination rate coefficients are obtained from photoionization cross sections, and from collision strengths for dielectronic recombination calculated using the precise theory of Bell and Seaton [J. Phys. B 18, 1589 (1985)]. Large-scale computations for both of these quantities are carried out in the close coupling approximation using the R-matrix method with an eigenfunction expansion that includes 83 states of Fe III dominated by the ground 3d6, and the excited 3d54s and 3d54p configurations. Both the total and state-specific recombination rate coefficients are obtained. Comparison of the present results with the previous ones shows considerable difference for most of the temperature regions. The present results provide accurate and self-consistent recombination rates, in the temperature range of practical applications (T<105 K), for ionization balance in photoionization models employing the detailed photoionization cross sections from the Opacity Project.
Electron-ion recombination of FeII
Nahar, S.N.
A complete treatment of electron-ion recombination of e+FeIII{r_arrow}FeII employing a unified method is presented. The treatment incorporates both the radiative and dielectronic recombinations in a self-consistent manner. Total recombination rate coefficients are obtained from photoionization cross sections, and from collision strengths for dielectronic recombination calculated using the precise theory of Bell and Seaton [J. Phys. B {bold 18}, 1589 (1985)]. Large-scale computations for both of these quantities are carried out in the close coupling approximation using the R-matrix method with an eigenfunction expansion that includes 83 states of FeIII dominated by the ground 3d{sup 6}, and the excited 3d{sup 5}4s and 3d{sup 5}4p configurations. Both the total and state-specific recombination rate coefficients are obtained. Comparison of the present results with the previous ones shows considerable difference for most of the temperature regions. The present results provide accurate and self-consistent recombination rates, in the temperature range of practical applications (T{lt}10{sup 5} K), for ionization balance in photoionization models employing the detailed photoionization cross sections from the Opacity Project. {copyright} {ital 1997} {ital The American Physical Society}
Distribution of Recombination Crossovers and the Origin of Haplotype Blocks: The Interplay of Population History, Recombination, and Mutation
Wang, Ning; Akey, Joshua M.; Zhang, Kun; Chakraborty, Ranajit; Jin, Li
Recent studies suggest that haplotypes are arranged into discrete blocklike structures throughout the human genome. Here, we present an alternative haplotype block definition that assumes no recombination within each block but allows for recombination between blocks, and we use it to study the combined effects of demographic history and various population genetic parameters on haplotype block characteristics. Through extensive coalescent simulations and analysis of published haplotype data on chromosome 21, we find that (1) the combined effects of population demographic history, recombination, and mutation dictate haplotype block characteristics and (2) haplotype blocks can arise in the absence of recombination hot spots. Finally, we provide practical guidelines for designing and interpreting studies investigating haplotype block structure. PMID:12384857
"Genome-wide recombination and chromosome segregation in human oocytes and embryos reveal selection for maternal recombination rates"
Natesan, Senthilkumar A.; Joshi, Hrishikesh A.; Cimadomo, Danilo; Griffin, Darren K.; Sage, Karen; Summers, Michael C.; Thornhill, Alan R.; Housworth, Elizabeth; Herbert, Alex D.; Rienzi, Laura; Ubaldi, Filippo M.; Handyside, Alan H.; Hoffmann, Eva R.
Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here, we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping > 4 million informative single-nucleotide polymorphisms (SNPs) from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a novel reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germline by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings reveal that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II. PMID:25985139
Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates.
Ottolini, Christian S; Newnham, Louise J; Capalbo, Antonio; Natesan, Senthilkumar A; Joshi, Hrishikesh A; Cimadomo, Danilo; Griffin, Darren K; Sage, Karen; Summers, Michael C; Thornhill, Alan R; Housworth, Elizabeth; Herbert, Alex D; Rienzi, Laura; Ubaldi, Filippo M; Handyside, Alan H; Hoffmann, Eva R
Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping >4 million informative SNPs from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a new reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germ line by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings show that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II. PMID:25985139
Urinary hMG (Meropur) versus recombinant FSH plus recombinant LH (Pergoveris) in IVF: a multicenter, prospective, randomized controlled trial.
Pacchiarotti, Alessandro; Sbracia, Marco; Frega, Antonio; Selman, Helmy; Rinaldi, Leonardo; Pacchiarotti, Arianna
To compare IVF outcome in ovarian stimulation protocols with recombinant FSH plus recombinant LH versus hMG, 122 patients were randomized into two study groups: group A, patients treated with urinary hMG, and group B, patients treated with rFSH plus rLH. The two groups proved to be comparable to the main IVF outcome (pregnancy rate, implantation rate, oocytes, and embryos quality), with an increasing risk of ovarian hyperstimulation in the Pergoveris group. PMID:20537626
Differential Requirements of Singleplex and Multiplex Recombineering of Large DNA Constructs
Reddy, Thimma R.; Kelsall, Emma J.; Fevat, Léna M. S.; Munson, Sarah E.; Cowley, Shaun M.
Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency. PMID:25954970
Recombinant thrombomodulin for secondary thrombotic thrombocytopenic purpura.
Nakamura, Kensuke; Inokuchi, Ryota; Hiruma, Takahiro; Ohshima, Kazuma; Sonoo, Tomohiro; Tokunaga, Kurato; Doi, Kent; Nakajima, Susumu
In the pathogenesis of thrombotic thrombocytopenic purpura (TTP), reductions in the enzyme activity of ADAMTS13, which cuts ultralarge von Willebrand multimers, generates shear stress on the microvascular endothelium, leading to platelet aggregation and the formation of a thrombus. ADAMTS13 activity is markedly decreased in typical TTP, but is only mildly reduced in secondary TTP, which concomitantly develops with primary disease. The latter develops with septic disseminated intravascular coagulation (DIC) and often causes organ failure. Recombinant thrombomodulin (rTM) is a drug that is used to treat DIC and may also remit TTP because it improves vascular endothelial dysfunction. Therefore, we herein investigated the efficacy of rTM in patients treated for the pathology of secondary TTP. Patients who were admitted to the Emergency and Critical Care Center of our hospital and met the following conditions were extracted and retrospectively analyzed: hemolytic anemia accompanied by fragmented red blood cells (Hb < 12 g/dL or lower); thrombocytopenia (<100 × 10/μL); and ADAMTS13 activity <50%. Sixteen patients were included and accompanied by Kidney Disease: Improving Global Outcomes (KDIGO) stage 2 or more severe nephropathy and DIC. Eleven and 5 patients treated with and without rTM (the rTM and non-rTM treatment groups, respectively) were compared, and no significant difference was noted in their basic characteristics, such as background disease and severity. No significant difference was observed in survival rates; however, the platelet count, which is an important outcome of treatments for TTP, significantly increased in the rTM treatment group: 3.3 ± 2.6→11.3 ± 14.6 versus 3.5 ± 3.7→5.7 ± 3.9 (×1000/μL) (P = 0.034). Thrombotic thrombocytopenic purpura originally requires invasive treatments and its prognosis is not favorable. Blood thrombomodulin levels also markedly increase due to vascular endothelial dysfunction
Transforming the treatment for hemophilia B patients: update on the clinical development of recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP).
Santagostino, Elena
Recombinant fusion protein linking recombinant coagulation factor IX with recombinant albumin (rIX-FP; Idelvion®(†)) is an innovative new treatment designed to extend the half-life of factor IX (FIX) and ease the burden of care for hemophilia B patients. The rIX-FP clinical development program - PROLONG-9FP - is in its advanced phases, with pivotal studies in previously treated adults, adolescents, and pediatrics now completed. Across all age groups studied, rIX-FP has demonstrated a markedly improved pharmacokinetic profile compared with plasma-derived and recombinant FIX treatments, with a 30-40% higher incremental recovery, an approximately 5-fold longer half-life, a lower clearance, and a greater area under the curve. rIX-FP has been very well tolerated with an excellent safety profile. In the pivotal studies, there have been no reports of FIX inhibitors or antidrug antibodies, and few treatment-related adverse events have been observed. Prophylactic regimens of rIX-FP administered once weekly to once every 14 days have been highly effective. When used for surgical prophylaxis, a single infusion of rIX-FP has been sufficient to maintain hemostasis, even during major orthopedic surgery. An ongoing study is now enrolling previously untreated patients and evaluating the possibility of extending the dosing interval to every 21 days. There is little doubt that rIX-FP will transform the treatment of hemophilia B. PMID:27288064
Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins.
Demasi, Marcos A; de S Molina, Erika; Bowman-Colin, Christian; Lojudice, Fernando H; Muras, Angelita; Sogayar, Mari C
Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg(1648) and Glu(1649). By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg(1648) FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg(1648) FVIII processing site by PCSK6. PMID:27126696
Genome-wide recombination dynamics are associated with phenotypic variation in maize.
Pan, Qingchun; Li, Lin; Yang, Xiaohong; Tong, Hao; Xu, Shutu; Li, Zhigang; Li, Weiya; Muehlbauer, Gary J; Li, Jiansheng; Yan, Jianbing
Meiotic recombination is a major driver of genetic diversity, species evolution, and agricultural improvement. Thus, an understanding of the genetic recombination landscape across the maize (Zea mays) genome will provide insight and tools for further study of maize evolution and improvement. Here, we used c. 50 000 single nucleotide polymorphisms to precisely map recombination events in 12 artificial maize segregating populations. We observed substantial variation in the recombination frequency and distribution along the ten maize chromosomes among the 12 populations and identified 143 recombination hot regions. Recombination breakpoints were partitioned into intragenic and intergenic events. Interestingly, an increase in the number of genes containing recombination events was accompanied by a decrease in the number of recombination events per gene. This kept the overall number of intragenic recombination events nearly invariable in a given population, suggesting that the recombination variation observed among populations was largely attributed to intergenic recombination. However, significant associations between intragenic recombination events and variation in gene expression and agronomic traits were observed, suggesting potential roles for intragenic recombination in plant phenotypic diversity. Our results provide a comprehensive view of the maize recombination landscape, and show an association between recombination, gene expression and phenotypic variation, which may enhance crop genetic improvement. PMID:26720856
CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.
Oh, Jee-Hwan; van Pijkeren, Jan-Peter
Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379
Synonymous and nonsynonymous distances help untangle convergent evolution and recombination.
Chi, Peter B; Chattopadhyay, Sujay; Lemey, Philippe; Sokurenko, Evgeni V; Minin, Vladimir N
When estimating a phylogeny from a multiple sequence alignment, researchers often assume the absence of recombination. However, if recombination is present, then tree estimation and all downstream analyses will be impacted, because different segments of the sequence alignment support different phylogenies. Similarly, convergent selective pressures at the molecular level can also lead to phylogenetic tree incongruence across the sequence alignment. Current methods for detection of phylogenetic incongruence are not equipped to distinguish between these two different mechanisms and assume that the incongruence is a result of recombination or other horizontal transfer of genetic information. We propose a new recombination detection method that can make this distinction, based on synonymous codon substitution distances. Although some power is lost by discarding the information contained in the nonsynonymous substitutions, our new method has lower false positive probabilities than the comparable recombination detection method when the phylogenetic incongruence signal is due to convergent evolution. We apply our method to three empirical examples, where we analyze: (1) sequences from a transmission network of the human immunodeficiency virus, (2) tlpB gene sequences from a geographically diverse set of 38 Helicobacter pylori strains, and (3) hepatitis C virus sequences sampled longitudinally from one patient. PMID:26061623
Electron-ion Recombination and Photoionization of P II
NASA Astrophysics Data System (ADS)
Nahar, Sultana
Study of the inverse processes of photoionization and electron-ion recombination of P II will be reported. It is a highly reactive ion and has been difficult to detect without detailed information of its interactions. Although a low charged ion, present study shows features in photoionization resulting from relativistic fine structure couplings at low energy region near the ionization threshold of many levels. Unified method under the framework of close coupling approximation and R-matrix method and an extension of Bell and Seaton theory has been used to study the inverse processes. The method gives the level-specific as well as the total recombination rate coefficients which include both the radiative recombination (RR) and dielectronic recombination (DR) in a precise manner. The present results include level specific rates and photoionization cross sections of 475 fine structure levels with n <= 10. Preliminary results on the total recombination rates show considerable interference of RR and DR around 4000 K and a DR peak around 105 K. NSF,DOE,OSC.
Suppression of auger recombination in ""giant"" core/shell nanocrystals
Garcia Santamaria, Florencio; Vela, Javier; Schaller, Richard D; Hollingsworth, Jennifer A; Klimov, Victor I; Chen, Yongfen
Many potential applications of semiconductor nanocrystals are hindered by nonradiative Auger recombination wherein the electron-hole (exciton) recombination energy is transferred to a third charge carrier. This process severely limits the lifetime and bandwidth of optical gain, leads to large nonradiative losses in light emitting diodes and photovoltaic cells, and is believed to be responsible for intermittency ('blinking') of emission from single nanocrystals. The development of nanostructures in which Auger recombination is suppressed has been a longstanding goal in colloidal nanocrystal research. Here, we demonstrate that such suppression is possible using so-called 'giant' nanocrystals that consist of a small CdSe core and a thick CdS shell. These nanostructures exhibit a very long biexciton lifetime ({approx}10 ns) that is likely dominated by radiative decay instead of non-radiative Auger recombination. As a result of suppressed Auger recombination, even high-order multiexcitons exhibit high emission efficiencies, which allows us to demonstrate optical amplification with an extraordinarily large bandwidth (>500 me V) and record low excitation thresholds.
Epistasis and the selective advantage of sex and recombination
NASA Astrophysics Data System (ADS)
de Oliveira, Viviane M.; da Silva, Juliana K.; Campos, Paulo R. A.
The understanding of the central mechanisms favoring sex and recombination in real populations is one of the fundamental issues in evolutionary biology. Based on a previous stochastic formulation for the study of sex, here we aim to investigate the conditions under which epistasis favors the fixation of the sexual mode of reproduction in a given population. In addition, we try to identify the evolutionary forces which contribute to this process. One considers a finite population model which assumes the existence of a recombination modifier allele that can activate the recombination mechanism. We have found that sex is very little favored in a scenario of antagonistic epistasis, and this advantage only occurs in a narrow range of values of the selection coefficient sd . On the other hand, synergistic epistasis favors recombination in a very broad domain. However, the major mechanism contributing to the spreading of the modifier allele depends on the range of values of sd . At large sd , background selection favors recombination since it increases the efficacy of selection, while at low sd Muller's ratchet is the leading mechanism.
Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses
Saleem, Huma; Nahid, Nazia; Shakir, Sara; Ijaz, Sehrish; Murtaza, Ghulam; Khan, Asif Ali; Mubin, Muhammad; Nawaz-ul-Rehman, Muhammad Shah
The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent. PMID:26963635
Recombination and the maintenance of plant organelle genome stability.
Maréchal, Alexandre; Brisson, Normand
Like their nuclear counterpart, the plastid and mitochondrial genomes of plants have to be faithfully replicated and repaired to ensure the normal functioning of the plant. Inability to maintain organelle genome stability results in plastid and/or mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly, deregulation of recombination in organelles results in genomic instability, often accompanied by adverse consequences for plant fitness. The recent identification of four families of proteins that prevent aberrant recombination of organelle DNA sheds much needed mechanistic light on this important process. What comes out of these investigations is a partial portrait of the recombination surveillance machinery in which plants have co-opted some proteins of prokaryotic origin but have also evolved whole new factors to keep their organelle genomes intact. These new features presumably optimized the protection of plastid and mitochondrial genomes against the particular genotoxic stresses they face. PMID:20180912
Application of internal standard method in recombinant luminescent bacteria test.
Wang, Yong-Zhi; Li, Dan; He, Miao
Mercury and its organic compounds have been of severe concern worldwide due to their damage to the ecosystem and human health. The development of effective and affordable technology to monitor and signal the presence of bioavailable mercury is an urgent need. The Mer gene is a mercury-responsive resistant gene, and a mercury-sensing recombinant luminescent bacterium using the Mer gene was constructed in this study. The mer operon from marine Pseudomonas putida strain SP1 was amplified and fused with prompterless luxCDABE in the pUCD615 plasmid within Escherichia coli cells, resulting in pTHE30-E. coli. The recombinant strain showed high sensitivity and specificity. The detection limit of Hg(2+) was 5nmol/L, and distinct luminescence could be detected in 30min. Cd(2+), Cu(2+), Zn(2+), Ca(2+), Pb(2+), Mg(2+), Mn(2+), and Al(3+) did not interfere with the detection over a range of 10(-5)-1mM. Application of recombinant luminescent bacteria testing in environmental samples has been a controversial issue: especially for metal-sensing recombinant strains, false negatives caused by high cytotoxicity are one of the most important issues when applying recombinant luminescent bacteria in biomonitoring of heavy metals. In this study, by establishing an internal standard approach, the false negative problem was overcome; furthermore, the method can also help to estimate the suspected mercury concentration, which ensures high detection sensitivity of bioavailable Hg(2+). PMID:26354701
Recombination spot identification Based on gapped k-mers
Wang, Rong; Xu, Yong; Liu, Bin
Recombination is crucial for biological evolution, which provides many new combinations of genetic diversity. Accurate identification of recombination spots is useful for DNA function study. To improve the prediction accuracy, researchers have proposed several computational methods for recombination spot identification. The k-mer feature is one of the most useful features for modeling the properties and function of DNA sequences. However, it suffers from the inherent limitation. If the value of word length k is large, the occurrences of k-mers are closed to a binary variable, with a few k-mers present once and most k-mers are absent. This usually causes the sparse problem and reduces the classification accuracy. To solve this problem, we add gaps into k-mer and introduce a new feature called gapped k-mer (GKM) for identification of recombination spots. By using this feature, we present a new predictor called SVM-GKM, which combines the gapped k-mers and Support Vector Machine (SVM) for recombination spot identification. Experimental results on a widely used benchmark dataset show that SVM-GKM outperforms other highly related predictors. Therefore, SVM-GKM would be a powerful predictor for computational genomics. PMID:27030570
Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies
Chuang, Katherine; Nguyen, Eileen; Sergeev, Yuri; Badea, Tudor C.
Site-specific recombinases (SSRs) such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants. PMID:26715092
Recombination spot identification Based on gapped k-mers.
Wang, Rong; Xu, Yong; Liu, Bin
Recombination is crucial for biological evolution, which provides many new combinations of genetic diversity. Accurate identification of recombination spots is useful for DNA function study. To improve the prediction accuracy, researchers have proposed several computational methods for recombination spot identification. The k-mer feature is one of the most useful features for modeling the properties and function of DNA sequences. However, it suffers from the inherent limitation. If the value of word length k is large, the occurrences of k-mers are closed to a binary variable, with a few k-mers present once and most k-mers are absent. This usually causes the sparse problem and reduces the classification accuracy. To solve this problem, we add gaps into k-mer and introduce a new feature called gapped k-mer (GKM) for identification of recombination spots. By using this feature, we present a new predictor called SVM-GKM, which combines the gapped k-mers and Support Vector Machine (SVM) for recombination spot identification. Experimental results on a widely used benchmark dataset show that SVM-GKM outperforms other highly related predictors. Therefore, SVM-GKM would be a powerful predictor for computational genomics. PMID:27030570
Recombination coefficients of O and N radicals on stainless steel
NASA Astrophysics Data System (ADS)
Singh, Harmeet; Coburn, J. W.; Graves, David B.
Surface recombination coefficients of O and N radicals in pure O2 and N2 plasmas, respectively, have been estimated on the stainless steel walls of a low-pressure inductively coupled plasma reactor. The recombination coefficients are estimated using a steady state plasma model describing the balance between the volume generation of the radicals from electron-impact dissociation of the parent molecules, and the loss of the radicals due to surface recombination. The model uses radical and parent molecule number densities and the electron energy distribution function (EEDF) as input parameters. We have measured the radical number density using appearance potential mass spectrometry. The parent neutral number density is measured using mass spectrometry. The EEDF is measured using a Langmuir probe. The recombination coefficient of O radicals on stainless steel walls at approximately 330 K is estimated to be 0.17±0.02, and agrees well with previous measurements. The recombination coefficient of N radicals is estimated to be 0.07±0.02 on stainless steel at 330 K.
Recombinant host cells and media for ethanol production
Wood, Brent E; Ingram, Lonnie O; Yomano, Lorraine P; York, Sean W
Disclosed are recombinant host cells suitable for degrading an oligosaccharide that have been optimized for growth and production of high yields of ethanol, and methods of making and using these cells. The invention further provides minimal media comprising urea-like compounds for economical production of ethanol by recombinant microorganisms. Recombinant host cells in accordance with the invention are modified by gene mutation to eliminate genes responsible for the production of unwanted products other than ethanol, thereby increasing the yield of ethanol produced from the oligosaccharides, relative to unmutated parent strains. The new and improved strains of recombinant bacteria are capable of superior ethanol productivity and yield when grown under conditions suitable for fermentation in minimal growth media containing inexpensive reagents. Systems optimized for ethanol production combine a selected optimized minimal medium with a recombinant host cell optimized for use in the selected medium. Preferred systems are suitable for efficient ethanol production by simultaneous saccharification and fermentation (SSF) using lignocellulose as an oligosaccharide source. The invention also provides novel isolated polynucleotide sequences, polypeptide sequences, vectors and antibodies.
Enhancing radiotherapy through a greater understanding of homologous recombination
Barker, Christopher A.; Powell, Simon N.
Radiotherapy for the treatment of cancer can cause a wide range of cellular effects, the most biologically potent of which is the double strand break in DNA. The process of repairing DNA double strand breaks involves one of two major mechanisms: non-homologous end-joining or homologous recombination. In this review, we review the molecular mechanisms of homologous recombination, in particular as it relates to the repair of DNA damage from ionizing radiation. We also present specific situations where homologous recombination may be dysfunctional in human cancers, and how this functional abnormality can be recognized. We also discuss the therapeutic opportunities that can be exploited based on deficiencies in homologous recombination at various steps in the DNA repair pathway. Side-by-side with these potential therapeutic opportunities, we review the contemporary clinical trials in which strategies to exploit these defects in homologous recombination can be enhanced by the use of radiotherapy in conjunction with biologically-targeted agents. We conclude that the field of radiation oncology has only scratched the surface of a potentially highly efficacious therapeutic strategy. PMID:20832019
Directed Ig class switch recombination in activated murine B cells.
Winter, E; Krawinkel, U; Radbruch, A
Immunoglobulin class switch recombination occurs at frequencies of up to 10%/cell/generation in activated murine B-lymphocytes. We analysed cH gene rearrangements and switch recombinations from active and inactive IgH loci of B-cells activated in various ways and immortalized by cell fusion. Although about half of the IgM+ cells show rearrangement of c mu genes, the deletion of c mu is a rare event. Half of the IgG3+ and IgG1+ cells show rearrangement of c mu genes on the inactive IgH locus and the other half of the IgG+ cells have deleted c mu from both IgH loci by switch recombination. This recombination is directed to the same switch regions on both IgH loci in 60-80% of all cases. Interleukin 4 may play a critical role in programming murine B-lymphocytes for specific switch recombination. Images Fig. 1. Fig. 2. Fig. 6. PMID:3038529
Auger recombination in InN from first principles
NASA Astrophysics Data System (ADS)
McAllister, Andrew; Kioupakis, Emmanouil
Group-III Nitride materials are used in numerous electronic and optoelectronic devices including solid-state lighting, energy conversion, sensor technologies, and high-power electronics. Indium nitride in particular is interesting for fast electronics and optoelectronics in the infrared. Auger recombination is a non-radiative carrier recombination process that would limit the efficiency of these devices. The small band gap (0.7 eV) and the high intrinsic free-electron concentrations in InN possibly make Auger recombination particularly important in this material. We used first-principles computational methods to determine the Auger recombination rates in InN. Our results suggest that direct Auger recombination is dominant in this material and that phonon-assisted Auger processes are not as important as in wider-gap nitrides such as GaN. This research was supported by the National Science Foundation CAREER award through Grant No. DMR-1254314. Computational resources were provided by the DOE NERSC facility.
Replication-Associated Recombinational Repair: Lessons from Budding Yeast
Bonner, Jaclyn N.; Zhao, Xiaolan
Recombinational repair processes multiple types of DNA lesions. Though best understood in the repair of DNA breaks, recombinational repair is intimately linked to other situations encountered during replication. As DNA strands are decorated with many types of blocks that impede the replication machinery, a great number of genomic regions cannot be duplicated without the help of recombinational repair. This replication-associated recombinational repair employs both the core recombination proteins used for DNA break repair and the specialized factors that couple replication with repair. Studies from multiple organisms have provided insights into the roles of these specialized factors, with the findings in budding yeast being advanced through use of powerful genetics and methods for detecting DNA replication and repair intermediates. In this review, we summarize recent progress made in this organism, ranging from our understanding of the classical template switch mechanisms to gap filling and replication fork regression pathways. As many of the protein factors and biological principles uncovered in budding yeast are conserved in higher eukaryotes, these findings are crucial for stimulating studies in more complex organisms. PMID:27548223
Recombination shapes the structure of an environmental Vibrio cholerae population.
Keymer, Daniel P; Boehm, Alexandria B
Vibrio cholerae consists of pathogenic strains that cause sporadic gastrointestinal illness or epidemic cholera disease and nonpathogenic strains that grow and persist in coastal aquatic ecosystems. Previous studies of disease-causing strains have shown V. cholerae to be a primarily clonal bacterial species, but isolates analyzed have been strongly biased toward pathogenic genotypes, while representing only a small sample of the vast diversity in environmental strains. In this study, we characterized homologous recombination and structure among 152 environmental V. cholerae isolates and 13 other putative Vibrio isolates from coastal waters and sediments in central California, as well as four clinical V. cholerae isolates, using multilocus sequence analysis of seven housekeeping genes. Recombinant regions were identified by at least three detection methods in 72% of our V. cholerae isolates. Despite frequent recombination, significant linkage disequilibrium was still detected among the V. cholerae sequence types. Incongruent but nonrandom associations were observed for maximum likelihood topologies from the individual loci. Overall, our estimated recombination rate in V. cholerae of 6.5 times the mutation rate is similar to those of other sexual bacteria and appears frequently enough to restrict selection from purging much of the neutral intraspecies diversity. These data suggest that frequent recombination among V. cholerae may hinder the identification of ecotypes in this bacterioplankton population. PMID:21075874
Systemic delivery of recombinant proteins by genetically modified myoblasts
Barr, E.; Leiden, J.M. )
The ability to stably deliver recombinant proteins to the systemic circulation would facilitate the treatment of a variety of acquired and inherited diseases. To explore the feasibility of the use of genetically engineered myoblasts as a recombinant protein delivery system, stable transfectants of the murine C2C12 myoblast cell line were produced that synthesize and secrete high levels of human growth hormone (hGH) in vitro. Mice injected with hGH-transfected myoblasts had significant levels of hGH in both muscle and serum that were stable for at least 3 weeks after injection. Histological examination of muscles injected with {beta}-galactosidase-expressing C2C12 myoblasts demonstrated that many of the injected cells had fused to form multinucleated myotubes. Thus, genetically engineered myoblasts can be used for the stable delivery of recombinant proteins into the circulation.
Interspecific plastidial recombination in the diatom genus Pseudo-nitzschia.
D'Alelio, Domenico; Ruggiero, Maria Valeria
Plastids are usually uni-parentally inherited and genetic recombination between these organelles is seldom observed. The genus Pseudo-nitzschia, a globally relevant marine diatom, features bi-parental plastid inheritance in the course of sexual reproduction. This observation inspired the recombination detection we pursued in this paper over a ~1,400-nucleotide-long region of the plastidial rbcL, a marker used in both molecular taxonomy and phylogenetic studies in diatoms. Among all the rbcL-sequences available in web-databases for Pseudo-nitzschia, 42 haplotypes were identified and grouped in five clusters by Bayesian phylogeny. Signs of hybridization were evident in four of five clusters, at both intra- and interspecific levels, suggesting that, in diatoms, (i) plastidial recombination is not absent and (ii) hybridization can play a role in speciation of Pseudo-nitzschia spp. PMID:26986997
Tailoring Charge Recombination in Photoelectrodes Using Oxide Nanostructures.
Iandolo, Beniamino; Wickman, Björn; Svensson, Elin; Paulsson, Daniel; Hellman, Anders
Optimizing semiconductor devices for solar energy conversion requires an explicit control of the recombination of photogenerated electron-hole pairs. Here we show how the recombination of charge carriers can be controlled in semiconductor thin films by surface patterning with oxide nanodisks. The control mechanism relies on the formation of dipole-like electric fields at the interface that, depending on the field direction, attract or repel minority carriers from underneath the disks. The charge recombination rate can be controlled through the choice of oxide material and the surface coverage of nanodisks. We provide proof-of-principle demonstration of this approach by patterning the surface of Fe2O3, one of the most studied semiconductors for light-driven water splitting, with TiO2 and Cu2O nanodisks. We expect this method to be generally applicable to a range of semiconductor-based solar energy conversion devices. PMID:26978576
Self-assembly of tunable protein suprastructures from recombinant oleosin
Vargo, Kevin B.; Parthasarathy, Ranganath; Hammer, Daniel A.
Using recombinant amphiphilic proteins to self-assemble suprastructures would allow precise control over surfactant chemistry and the facile incorporation of biological functionality. We used cryo-TEM to confirm self-assembled structures from recombinantly produced mutants of the naturally occurring sunflower protein, oleosin. We studied the phase behavior of protein self-assembly as a function of solution ionic strength and protein hydrophilic fraction, observing nanometric fibers, sheets, and vesicles. Vesicle membrane thickness correlated with increasing hydrophilic fraction for a fixed hydrophobic domain length. The existence of a bilayer membrane was corroborated in giant vesicles through the localized encapsulation of hydrophobic Nile red and hydrophilic calcein. Circular dichroism revealed that changes in nanostructural morphology in this family of mutants was unrelated to changes in secondary structure. Ultimately, we envision the use of recombinant techniques to introduce novel functionality into these materials for biological applications. PMID:22753512
Meiotic recombination and the crossover assurance checkpoint in Caenorhabditis elegans.
Yu, Zhouliang; Kim, Yumi; Dernburg, Abby F
During meiotic prophase, chromosomes pair and synapse with their homologs and undergo programmed DNA double-strand break (DSB) formation to initiate meiotic recombination. These DSBs are processed to generate a limited number of crossover recombination products on each chromosome, which are essential to ensure faithful segregation of homologous chromosomes. The nematode Caenorhabditis elegans has served as an excellent model organism to investigate the mechanisms that drive and coordinate these chromosome dynamics during meiosis. Here we focus on our current understanding of the regulation of DSB induction in C. elegans. We also review evidence that feedback regulation of crossover formation prolongs the early stages of meiotic prophase, and discuss evidence that this can alter the recombination pattern, most likely by shifting the genome-wide distribution of DSBs. PMID:27013114
RECOMBINATION RATE COEFFICIENTS OF BORON-LIKE Ne
Mahmood, S.; Orban, I.; Ali, S.; Schuch, R.; Glans, P.; Bleda, E. A.; Altun, Z.
Recombination of Ne{sup 5+} was measured in a merged-beam type experiment at the heavy-ion storage ring CRYRING. In the collision energy range 0-110 eV resonances due to 2s {sup 2}2p {yields} 2s2p {sup 2} ({Delta}n = 0) and 2s {sup 2}2p {yields} 2s {sup 2}3l ({Delta}n = 1), core excitations were observed. The experimentally derived rate coefficients agree well with the calculations obtained using AUTOSTRUCTURE. At low energies, recombination is dominated by resonances belonging to the spin-forbidden 2s2p {sup 2}({sup 4} P{sub J})nl series. The energy-dependent rate coefficients were convoluted with a Maxwell-Boltzmann electron energy distribution to obtain plasma recombination rate coefficients. The data from the literature deviate from the measured results at low temperature.
Cre-mediated site-specific recombination in zebrafish embryos.
Thummel, Ryan; Burket, Christopher T; Brewer, Jeffrey L; Sarras, Michael P; Li, Li; Perry, Martin; McDermott, Jeffrey P; Sauer, Brian; Hyde, David R; Godwin, Alan R
Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish. PMID:15977183
Historical Perspectives Pertaining to the NIH Recombinant DNA Advisory Committee
Abstract Science is host to a constantly emerging series of new paradigms, and it is this characteristic that makes science both interesting and dynamic. As a part of this continuum, it became possible to create recombinant DNA molecules. Immediately it was recognized that there was a potential for serious adverse events associated with this new technology. Following two scientific conferences at Asilomar, California, the National Institutes of Health moved quickly to create the Recombinant DNA Advisory Committee (RAC). For approximately 38 years the RAC has served as an open forum for review of various recombinant DNA experiments, and for the last 23 years it has played a pivotal role in the oversight of human gene therapy. The RAC's existence obviated the need for more restrictive governmental legislation and has supported the development of genetic interventions that are leading to actual human therapies. PMID:24444182
Matter-wave recombiners for trapped Bose-Einstein condensates
NASA Astrophysics Data System (ADS)
Berrada, T.; van Frank, S.; Bücker, R.; Schumm, T.; Schaff, J.-F.; Schmiedmayer, J.; Julía-Díaz, B.; Polls, A.
Interferometry with trapped atomic Bose-Einstein condensates (BECs) requires the development of techniques to recombine the two paths of the interferometer and map the accumulated phase difference to a measurable atom number difference. We have implemented and compared two recombining procedures in a double-well-based BEC interferometer. The first procedure utilizes the bosonic Josephson effect and controlled tunneling of atoms through the potential barrier, similar to laser light in an optical fiber coupler. The second one relies on the interference of the reflected and transmitted parts of the BEC wave function when impinging on the potential barrier, analogous to light impinging on a half-silvered mirror. Both schemes were implemented successfully, yielding an interferometric contrast of ˜20 % and 42% respectively. Building efficient matter-wave recombiners represents an important step towards the coherent manipulation of external quantum superposition states of BECs.
Mechanisms underlying allergy vaccination with recombinant hypoallergenic allergen derivatives
Linhart, Birgit; Valenta, Rudolf
Hundred years ago therapeutic vaccination with allergen-containing extracts has been introduced as a clinically effective, disease-modifying, allergen-specific and long-lasting form of therapy for allergy, a hypersensitivity disease affecting more than 25% of the population. Today, the structures of most of the disease-causing allergens have been elucidated and recombinant hypoallergenic allergen derivatives with reduced allergenic activity have been engineered to reduce side effects during allergen-specific immunotherapy (SIT). These recombinant hypoallergens have been characterized in vitro, in experimental animal models and in clinical trials in allergic patients. This review provides a summary of the molecular, immunological and preclinical evaluation criteria applied for this new generation of allergy vaccines. Furthermore, we summarize the mechanisms underlying SIT with recombinant hypoallergens which are thought to be responsible for their therapeutic effect. PMID:22100888
A recombineering-based gene tagging system for Arabidopsis.
Alonso, Jose M; Stepanova, Anna N
Many of the experimental approaches aimed at studying gene function heavily rely on the ability to make precise modifications in the gene's DNA sequence. Homologous recombination (HR)-based strategies provide a convenient way to create such types of modifications. HR-based DNA sequence manipulations can be enormously facilitated by expressing in E. coli a small set of bacteriophage proteins that make the exchange of DNA between a linear donor and the target DNA molecules extremely efficient. These in vivo recombineering techniques have been incorporated as essential components of the molecular toolbox in many model organisms. In this chapter, we describe the experimental procedures involved in recombineering-based tagging of an Arabidopsis gene contained in a plant transformation-ready bacterial artificial chromosome (TAC). PMID:25239749
Optimising yeast as a host for recombinant protein production (review).
Bonander, Nicklas; Bill, Roslyn M
Having access to suitably stable, functional recombinant protein samples underpins diverse academic and industrial research efforts to understand the workings of the cell in health and disease. Synthesising a protein in recombinant host cells typically allows the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to the native human source cells of many proteins of interest, while also being quick, easy, and cheap to grow and process. Even in these cells the production of some proteins can be plagued by low functional yields. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast cell factories. In this chapter, we provide an overview of the opportunities available to improve yeast as a host system for recombinant protein production. PMID:22454109
Attenuated vaccines can recombine to form virulent field viruses.
Lee, Sang-Won; Markham, Philip F; Coppo, Mauricio J C; Legione, Alistair R; Markham, John F; Noormohammadi, Amir H; Browning, Glenn F; Ficorilli, Nino; Hartley, Carol A; Devlin, Joanne M
Recombination between herpesviruses has been seen in vitro and in vivo under experimental conditions. This has raised safety concerns about using attenuated herpesvirus vaccines in human and veterinary medicine and adds to other known concerns associated with their use, including reversion to virulence and disease arising from recurrent reactivation of lifelong chronic infection. We used high-throughput sequencing to investigate relationships between emergent field strains and vaccine strains of infectious laryngotracheitis virus (ILTV, gallid herpesvirus 1). We show that independent recombination events between distinct attenuated vaccine strains resulted in virulent recombinant viruses that became the dominant strains responsible for widespread disease in Australian commercial poultry flocks. These findings highlight the risks of using multiple different attenuated herpesvirus vaccines, or vectors, in the same populations. PMID:22798607
Production of biologically active recombinant human lactoferrin in Aspergillus oryzae.
Ward, P P; Lo, J Y; Duke, M; May, G S; Headon, D R; Conneely, O M
We report the production of recombinant human lactoferrin in Aspergillus oryzae. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae alpha-amylase promoter and the 3' flanking region of the A. niger glucoamylase gene. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron-binding capacity. The recombinant protein appears to be appropriately N-linked glycosylated and correctly processed at the N-terminus by the A. oryzae secretory apparatus. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date. Hence, this expression system appears suitable for the large-scale production and secretion of biologically active mammalian glycoproteins. PMID:1368268
Recombinant lentivector as a genetic immunization vehicle for antitumor immunity
He, Yukai; Munn, David; Falo, Louis D
Summary Encouraged by remarkable successes in preventing infectious diseases and by the well established potential of immune system for controlling tumor growth, active therapeutic immunization approaches hold great promise for treating malignant tumors. In recent years, engineered recombinant viral vectors have been carefully examined as genetic immunization vehicles and have been demonstrated to induce potent T cell mediated immune responses that can control tumor growth. Very recent efforts suggest that lentivectors possess important advantages over other candidate recombinant viral vectors for genetic immunization. Here we review the development of recombinant lentivectors and the characteristics of T cell immune responses elicited by lentivector immunization, including the mechanism of T cell priming with a focus on the role of skin dendritic cells (DC) and potential applications for tumor immunotherapy. PMID:18377355
An Overview of the Molecular Mechanisms of Recombinational DNA Repair.
Kowalczykowski, Stephen C
Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148
Recombination and collisional X-UV lasers at ORSAY
Klisnick, A.; Carillon, A.; Dhez, P.; Goedtkindt, P.; Guennou, H.; Jamelot, G.; Jaegle, P.; Sureau, A.; Rus, B.; Zeitoun, P. ); Chenais-Popovics, C.; Renaudin, P.; Rancu, O.; Gauthier, J.C. ); Back, C.A. )
In this paper we describe the progress achieved recently in our laboratory in the field of X-ray lasers. Both collisional excitation and recombination pumped systems are under investigation. We show that the 5g-4f transition in lithium-like ions could bring out a significant increase of the gain-length accessible with recombination X-ray lasers. We present preliminary results on an absorption spectroscopy experiment designed to probe the ionization state of recombination X-ray laser plasmas. Finally we report on the observation of a strong amplified signal at 212 A, the wavelength of a 3p-3s (J=0--1) in neon-like zinc. [copyright] 1995 [ital American] [ital Institute] [ital of] [ital Physics
Plant cell cultures for the production of recombinant proteins.
Hellwig, Stephan; Drossard, Jürgen; Twyman, Richard M; Fischer, Rainer
The use of whole plants for the synthesis of recombinant proteins has received a great deal of attention recently because of advantages in economy, scalability and safety compared with traditional microbial and mammalian production systems. However, production systems that use whole plants lack several of the intrinsic benefits of cultured cells, including the precise control over growth conditions, batch-to-batch product consistency, a high level of containment and the ability to produce recombinant proteins in compliance with good manufacturing practice. Plant cell cultures combine the merits of whole-plant systems with those of microbial and animal cell cultures, and already have an established track record for the production of valuable therapeutic secondary metabolites. Although no recombinant proteins have yet been produced commercially using plant cell cultures, there have been many proof-of-principle studies and several companies are investigating the commercial feasibility of such production systems. PMID:15529167
Replication Origin Selection Regulates the Distribution of Meiotic Recombination
Wu, Pei-Yun Jenny; Nurse, Paul
Summary The program of DNA replication, defined by the temporal and spatial pattern of origin activation, is altered during development and in cancers. However, whether changes in origin usage play a role in regulating specific biological processes remains unknown. We investigated the consequences of modifying origin selection on meiosis in fission yeast. Genome-wide changes in the replication program of premeiotic S phase do not affect meiotic progression, indicating that meiosis neither activates nor requires a particular origin pattern. In contrast, local changes in origin efficiencies between different replication programs lead to changes in Rad51 recombination factor binding and recombination frequencies in these domains. We observed similar results for Rad51 when changes in efficiencies were generated by directly targeting expression of the Cdc45 replication factor. We conclude that origin selection is a key determinant for organizing meiotic recombination, providing evidence that genome-wide modifications in replication program can modulate cellular physiology. PMID:24560273
Challenges and opportunities in the purification of recombinant tagged proteins.
Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A
The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. PMID:24334194
Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination.
Yang, W; Summers, J
Linear hepadnavirus DNA in primary hepatocyte cultures efficiently participates in intra- and intermolecular nonhomologous recombination at its ends. The products of this recombination are (i) monomeric covalently closed circular DNAs (cccDNAs) with deletions and insertions around the site of joining and (ii) oligomeric forms in which monomers are joined near the ends in random orientation. A fraction of monomeric cccDNAs can serve as intermediates in further DNA replication through at least five generations of nonhomologous recombination in a process we call illegitimate replication. We suggest that the monomeric and oligomeric linear DNAs produced by illegitimate replication may be precursors of the integrated and other high-molecular-weight hepadnaviral DNA forms seen in chronic infection. PMID:7769660
Unraveling a Region-Specific Hyper-Recombination Phenomenon: Genetic Control and Modalities of Terminal Recombination in Escherichia Coli
Corre, J.; Cornet, F.; Patte, J.; Louarn, J. M.
The propensity of the terminus of the Escherichia coli chromosome for recombination has been further explored, using a test based on the selectable loss of a λ prophage inserted between repeated sequences from Tn10. Terminal recombination appears region-specific and unrelated to replication termination in a strain harboring a major chromosomal rearrangement. It requires RecBC(D) activity and must therefore occur between sister chromosomes, to conserve genomic integrity in spite of DNA degradation by RecBCD. Terminal recombination is maximal in the dif region and its intensity on either side of this recombination site depends on the orientation of the repeated sequences, probably because of the single χ site present in each repeat. Additional observations support the model that the crossover is initiated by single-strand invasion between sister chromosomes followed by RecBCD action as a consequence of DNA breakage due to the initial invasion event. Crossover location within repeats inserted at dif position supports the possibility that sister chromosomes are tightly paired in the centre of the terminal recombination zone. These data reinforce the model that postreplicative reconstruction of nucleoid organization creates a localized synapsis between the termini of sister chromosomes. PMID:9383046
A Network Approach to Analyzing Highly Recombinant Malaria Parasite Genes
Larremore, Daniel B.; Clauset, Aaron; Buckee, Caroline O.
The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs), and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα) domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences. PMID:24130474
Yeast meiotic mutants proficient for the induction of ectopic recombination.
Engebrecht, J; Masse, S; Davis, L; Rose, K; Kessel, T
A screen was designed to identify Saccharomyces cerevisiae mutants that were defective in meiosis yet proficient for meiotic ectopic recombination in the return-to-growth protocol. Seven mutants alleles were isolated; two are important for chromosome synapsis (RED1, MEK1) and five function independently of recombination (SPO14, GSG1, SPOT8/MUM2, 3, 4). Similar to the spoT8-1 mutant, mum2 deletion strains do not undergo premeiotic DNA synthesis, arrest prior to the first meiotic division and fail to sporulate. Surprisingly, although DNA replication does not occur, mum2 mutants are induced for high levels of ectopic recombination. gsg1 diploids are reduced in their ability to complete premeiotic DNA synthesis and the meiotic divisions, and a small percentage of cells produce spores. mum3 mutants sporulate poorly and the spores produced are inviable. Finally, mum4-1 mutants produce inviable spores. The meiotic/sporulation defects of gsg1, mum2, and mum3 are not relieved by spo11 or spo13 mutations, indicating that the mutant defects are not dependent on the initiation of recombination or completion of both meiotic divisions. In contrast, the spore inviability of the mum4-1 mutant is rescued by the spo13 mutation. The mum4-1 spo13 mutant undergoes a single, predominantly equational division, suggesting that MUM4 functions at or prior to the first meiotic division. Although recombination is variably affected in the gsg1 and mum mutants, we hypothesize that these mutants define genes important for aspects of meiosis not directly related to recombination. PMID:9504908
Recombinant production of mecasermin in E. coli expression system.
Jafari, S; Babaeipour, V; Seyedi, H A Eslampanah; Rahaie, M; Mofid, M R; Haddad, L; Namvaran, M M; Fallah, J
Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260
Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes.
Smith, Shavannor M; Steinau, Martin; Trick, Harold N; Hulbert, Scot H
Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant. PMID:20443026
Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.
Baker, Christopher L; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M; Paigen, Kenneth
Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape. PMID:26368021
Adaptive evolution of a lactose-consuming Saccharomyces cerevisiae recombinant.
Guimarães, Pedro M R; François, Jean; Parrou, Jean Luc; Teixeira, José A; Domingues, Lucília
The construction of Saccharomyces cerevisiae strains that ferment lactose has biotechnological interest, particularly for cheese whey fermentation. A flocculent lactose-consuming S. cerevisiae recombinant expressing the LAC12 (lactose permease) and LAC4 (beta-galactosidase) genes of Kluyveromyces lactis was constructed previously but showed poor efficiency in lactose fermentation. This strain was therefore subjected to an evolutionary engineering process (serial transfer and dilution in lactose medium), which yielded an evolved recombinant strain that consumed lactose twofold faster, producing 30% more ethanol than the original recombinant. We identified two molecular events that targeted the LAC construct in the evolved strain: a 1,593-bp deletion in the intergenic region (promoter) between LAC4 and LAC12 and a decrease of the plasmid copy number by about 10-fold compared to that in the original recombinant. The results suggest that the intact promoter was unable to mediate the induction of the transcription of LAC4 and LAC12 by lactose in the original recombinant and that the deletion established the transcriptional induction of both genes in the evolved strain. We propose that the tuning of the expression of the heterologous LAC genes in the evolved recombinant was accomplished by the interplay between the decreased copy number of both genes and the different levels of transcriptional induction for LAC4 and LAC12 resulting from the changed promoter structure. Nevertheless, our results do not exclude other possible mutations that may have contributed to the improved lactose fermentation phenotype. This study illustrates the usefulness of simple evolutionary engineering approaches in strain improvement. The evolved strain efficiently fermented threefold-concentrated cheese whey, providing an attractive alternative for the fermentation of lactose-based media. PMID:18245248
Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris
Weidner, Maria; Taupp, Marcus; Hallam, Steven J.
Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system. As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1]. As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3]. Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4]. The vector used here (pPICZαA) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the α-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector. PMID:20186119
Recombinant production of mecasermin in E. coli expression system
Jafari, S.; Babaeipour, V.; Seyedi, H.A. Eslampanah; Rahaie, M.; Mofid, M.R.; Haddad, L.; Namvaran, M.M.; Fallah, J.
Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260
Oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.
Krylov, Alexander A; Kolontaevsky, Egor E; Mashko, Sergey V
Brevibacterium lactofermentum and Corynebacterium glutamicum are important biotechnology species of the genus Corynebacterium. The single-strand DNA annealing protein (SSAP)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains B. lactofermentum AJ1511 and C. glutamicum ATCC13032. When the rpsL gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies per 10(8) viable cells for the corresponding strains. We tested the influence of several parameters that are known to enhance the efficiency of oligonucleotide-mediated recombination in other bacterial species. Among them, increasing the concentration of oligonucleotides and targeting the lagging strand of the chromosome have proven to have positive effects on both of the tested species. No difference in the efficiency of recombination was observed between the oligonucleotides phosphorothiorated at the 5' ends and the unmodified oligonucleotides or between the oligonucleotides with four mutated nucleotides and those with one mutated nucleotide. The described approach demonstrates that during the adaptation of the recombineering technique, testing SSAP-independent oligonucleotide-mediated recombination could be a good starting point. Such testing could decrease the probability of an incorrect interpretation of the effect of exogenous protein factors (such as SSAP and/or corresponding exonucleases) due to non-optimal experimental conditions. In addition, SSAP-independent recombination itself could be useful in combination with suitable selection/enrichment methods. PMID:25087479
Evidence and Consequence of Porcine Endogenous Retrovirus Recombination
Bartosch, Birke; Stefanidis, Dimitrios; Myers, Richard; Weiss, Robin; Patience, Clive; Takeuchi, Yasuhiro
The genetic nature and biological effects of recombination between porcine endogenous retroviruses (PERV) were studied. An infectious molecular clone was generated from a high-titer, human-tropic PERV isolate, PERV-A 14/220 (B. A. Oldmixon, et al. J. Virol. 76:3045-3048, 2002; T. A. Ericsson et al. Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). To analyze this sequence and 15 available full-length PERV nucleotide sequences, we developed a sequence comparison program, LOHATM to calculate local sequence homology between two sequences. This analysis determined that PERV-A 14/220 arose by homologous recombination of a PERV-C genome replacing an 850-bp region around the pol-env junction with that of a PERV-A sequence. This 850-bp PERV-A sequence encompasses the env receptor binding domain, thereby conferring a wide host range including human cells. In addition, we determined that multiple regions derived from PERV-C are responsible for the increased infectious titer of PERV-A 14/220. Thus, a single recombination event may be a fast and effective way to generate high-titer, potentially harmful PERV. Further, local homology and phylogenetic analyses between 16 full-length sequences revealed evidence for other recombination events in the past that give rise to other PERV genomes that possess the PERV-A, but not the PERV-B, env gene. These results indicate that PERV-A env is more prone to recombination with heterogeneous backbone genomes than PERV-B env. Such recombination events that generate more active PERV-A appear to occur in pigs rather frequently, which increases the potential risk of zoonotic PERV transmission. In this context, pigs lacking non-human-tropic PERV-C would be more suitable as donor animals for clinical xenotransplantation. PMID:15564496
Phylogenetic Mapping of Recombination Hotspots in Human Immunodeficiency Virus via Spatially Smoothed Change-Point Processes
Minin, Vladimir N.; Dorman, Karin S.; Fang, Fang; Suchard, Marc A.
We present a Bayesian framework for inferring spatial preferences of recombination from multiple putative recombinant nucleotide sequences. Phylogenetic recombination detection has been an active area of research for the last 15 years. However, only recently attempts to summarize information from several instances of recombination have been made. We propose a hierarchical model that allows for simultaneous inference of recombination breakpoint locations and spatial variation in recombination frequency. The dual multiple change-point model for phylogenetic recombination detection resides at the lowest level of our hierarchy under the umbrella of a common prior on breakpoint locations. The hierarchical prior allows for information about spatial preferences of recombination to be shared among individual data sets. To overcome the sparseness of breakpoint data, dictated by the modest number of available recombinant sequences, we a priori impose a biologically relevant correlation structure on recombination location log odds via a Gaussian Markov random field hyperprior. To examine the capabilities of our model to recover spatial variation in recombination frequency, we simulate recombination from a predefined distribution of breakpoint locations. We then proceed with the analysis of 42 human immunodeficiency virus (HIV) intersubtype gag recombinants and identify a putative recombination hotspot. PMID:17194781
Charge recombination in the muon collider cooling channel
Fernow, R. C.; Palmer, R. B.
The final stage of the ionization cooling channel for the muon collider must transversely recombine the positively and negatively charged bunches into a single beam before the muons can be accelerated. It is particularly important to minimize any emittance growth in this system since no further cooling takes place before the bunches are collided. We have found that emittance growth could be minimized by using symmetric pairs of bent solenoids and careful matching. We show that a practical design can be found that has transmission {approx}99%, emittance growth less than 0.1%, and minimal dispersion in the recombined bunches.
Mechanism for radiative recombination in ZnCdO alloys
Buyanova, I. A.; Bergman, J. P.; Pozina, G.; Chen, W. M.; Rawal, S.; Norton, D. P.; Pearton, S. J.; Osinsky, A.; Dong, J. W.
Temperature dependent cw- and time-resolved photoluminescence combined with absorption measurements are employed to evaluate the origin of radiative recombination in ZnCdO alloys grown by molecular-beam epitaxy. The near-band-edge emission is attributed to recombination of excitons localized within band tail states likely caused by nonuniformity in Cd distribution. Energy transfer between the tail states is argued to occur via tunneling of localized excitons. The transfer is shown to be facilitated by increasing Cd content due to a reduction of the exciton binding energy and, therefore, an increase of the exciton Bohr radius in the alloys with a high Cd content.
Recombinant chromosome 18 resulting from a maternal pericentric inversion
Ayukawa, Hiroshi; Tsukahara, Masato; Fukuda, Masamichi; Kondoh, Osamu
We report on a newborn girl with duplication of 18q12.2{yields}18 qter and deficiency of 18p11.2{yields}18pter which resulted from meiotic recombination of the maternal pericentric inversion, inv(18)(p11.2q12.2). Her clinical manifestations were compatible with those of partial trisomy 18q syndrome. We review the previously reported 9 cases in 8 families of rec(18) resulting from recombination of a parental pericentric inversion. 8 refs., 3 figs., 1 tab.
Prominent Higher-Order Contributions to Electronic Recombination
Beilmann, C.; Mokler, P. H.; Bernitt, S.; Keitel, C. H.; Ullrich, J.; Lopez-Urrutia, J. R. Crespo; Harman, Z.
Intershell higher-order (HO) electronic recombination is reported for highly charged Ar, Fe, and Kr ions, where simultaneous excitation of one K-shell electron and one or two additional L-shell electrons occurs upon resonant capture of a free electron. For the mid-Z region, HO resonance strengths grow unexpectedly strong with decreasing atomic number Z ({proportional_to}Z{sup -4}), such that, for Ar ions the 2nd-order overwhelms the 1st-order resonant recombination considerably. The experimental findings are confirmed by multiconfiguration Dirac-Fock calculations including hitherto neglected excitation pathways.
Modeling of recombinant yeast cells: reduction of phase space.
Birol, G; Birol, I; Kirdar, B; Onsan, Z I
The mechanism of starch fermentation by recombinant Saccharomyces cerevisiae in batch reactor is studied. Experiments were carried in the presence and absence of oxygen, with different initial starch concentrations. A variety of data concerning biotic and abiotic phases are collected. Nonlinear data analysis techniques are used to determine the block diagram of the system under study. Data analysis and processing reported here, are believed to form a basis in further work in structured modeling of biological systems, recombinant yeast cultures in particular. PMID:9603032
Enteric Immunization of Mice Against Influenza with Recombinant Vaccinia
NASA Astrophysics Data System (ADS)
Meitin, Catherine A.; Bender, Bradley S.; Small, Parker A., Jr.
Intrajejunal administration to mice of a recombinant vaccinia virus containing the influenza virus hemagglutinin gene induced IgA antibody in nasal, gut, and vaginal secretions. It also induced IgG antibody in serum and cell-mediated immunity. The immunization provided significant protection against an influenza virus challenge. This work suggests that enteric-coated recombinant vaccinia could be an orally administered, inexpensive, multivalent, temperature-stable, safe, and effective vaccine for children that could be particularly useful in developing nations, where multiple injections are not easily administered. Oral administration of vaccines should also reduce children's fear of shots at the doctor's office.
Recombinant protein expression in Escherichia coli: advances and challenges
Rosano, Germán L.; Ceccarelli, Eduardo A.
Escherichia coli is one of the organisms of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of heterologous proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field. PMID:24860555
A ring-shaped recombination chamber for hadron therapy dosimetry.
Jakubowska, E; Zielczyński, M; Golnik, N; Gryziński, M A; Krzemiński, Ł
An innovative recombination chamber has been designed for estimation of stray radiation doses and quality factors in hadron therapy. The chamber allows for determination of absorbed dose and recombination index of radiation quality in phantoms at small distances from simulated organs. The chamber body and electrodes are ring shaped, so the beam may be directed through the empty centre of the ring. The ionisation of the filling gas is caused by secondary or scattered radiation and can be related to the dose absorbed in the tissues close to the irradiated target volume. PMID:24430949
Recombination of H(3+) and D(3+) ions with electrons
NASA Technical Reports Server (NTRS)
Johnsen, R.; Gougousi, T.; Golde, M. F.
Flowing-afterglow measurements in decaying H3(+) or D3(+) plasmas suggest that de-ionization does not occur by simple binary recombination of a single ion species. We find that vibrational excitation of the ions fails to provide an explanation for the effect, contrary to an earlier suggestion. Instead, we suggest that collisional stabilization of H3** Rydberg molecules by ambient electrons introduces an additional dependence on electron density. The proposed mechanism would permit plasma de-ionization to occur without the need for dissociative recombination by the mechanism of potential-surface crossings.
Acclimation to temperature and irradiance modulates PSII charge recombination.
Ivanov, A G; Sane, P V; Krol, M; Gray, G R; Balseris, A; Savitch, L V; Oquist, G; Hüner, N P A
Acclimation of wild type and the chlorina F2 mutant of barley to either high light or low temperature results in a 2- to 3-fold increase in non-photochemical quenching which occurred independently of either energy-dependent quenching (qE), xanthophyll cycle-mediated antenna quenching or state transitions. Results of in vivo thermoluminescence measurements used to address this conundrum indicated that excitation pressure regulates the temperature gap for S(2)Q(B)(-) and S(2)Q(A)(-) charge recombinations within photosystem II reaction centers. This is discussed in terms of photoprotection through non-radiative charge recombination. PMID:16674953
Radiative recombination and photon recycling in gallium arsenide solar cells
NASA Astrophysics Data System (ADS)
Lundstrom, M. S.; Melloch, M. R.; Lush, G. B.; Patkar, M. P.; Young, M.; Durbin, S. M.; Gray, J. L.; MacMillan, H. F.; Keyes, B. M.; Levi, D. H.; Ahrenkiel, R. K.
This talk reviews experimental work to develop a detailed understanding of radiative recombination in n-GaAs. Photoluminescence decay studies of minority carrier lifetimes versus doping in n-GaAs are presented. We show that when the substrate is removed by etching, photon recycling is enhanced, and lifetimes increase by nearly a factor of 10. The doping-dependent absorption coefficient is measured, and detailed balance arguments are used to relate absorption and recombination. Modeling surfaces, verified by comparison with experiments, are used to examine the effects of recycling in conventional solar cells and to explore new design options.
A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency
Lee, Yu Nee; Frugoni, Francesco; Dobbs, Kerry; Walter, Jolan E.; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Haddad, Elie; LeDeist, Francoise; Bleesing, Jack H.; Henderson, Lauren A.; Pai, Sung-Yun; Nelson, Robert P.; El-Ghoneimy, Dalia H.; El-Feky, Reem A.; Reda, Shereen M.; Hossny, Elham; Soler-Palacin, Pere; Fuleihan, Ramsay L.; Patel, Niraj C.; Massaad, Michel J.; Geha, Raif S.; Puck, Jennifer M.; Palma, Paolo; Cancrini, Caterina; Chen, Karin; Vihinen, Mauno; Alt, Frederick W.; Notarangelo, Luigi D.
Background The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T−B− severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes. Objective We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations. Methods We have developed a flow cytometry–based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1−/− pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology. Results Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity. Conclusions Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process. PMID:24290284
RecQ promotes toxic recombination in cells lacking recombination intermediate-removal proteins.
Magner, Daniel B; Blankschien, Matthew D; Lee, Jennifer A; Pennington, Jeanine M; Lupski, James R; Rosenberg, Susan M
The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs. PMID:17466628
RecQ Promotes Toxic Recombination in Cells Lacking Recombination-Intermediate-Removal Proteins
Magner, Daniel B.; Blankschien, Matthew D.; Lee, Jennifer A.; Pennington, Jeanine M.; Lupski, James R.; Rosenberg, Susan M.
Summary The RecQ-helicase family is widespread, highly conserved, and includes human orthologues that suppress genomic instability and cancer. In vivo, some RecQ homologues promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand-exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promote this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in-vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologues. PMID:17466628
High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast
Dominska, Margaret; Moriel-Carretero, María; Herrera-Moyano, Emilia; Aguilera, Andrés; Petes, Thomas D.
The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister
Recombinational landscape of porcine X chromosome and individual variation in female meiotic recombination associated with haplotypes of Chinese pigs
Background Variations in recombination fraction (θ) among chromosomal regions, individuals and families have been observed and have an important impact on quantitative trait loci (QTL) mapping studies. Such variations on porcine chromosome X (SSC-X) and on other mammalian chromosome X are rarely explored. The emerging assembly of pig sequence provides exact physical location of many markers, facilitating the study of a fine-scale recombination landscape of the pig genome by comparing a clone-based physical map to a genetic map. Using large offspring of F1 females from two large-scale resource populations (Large White ♂ × Chinese Meishan ♀, and White Duroc ♂ × Chinese Erhualian ♀), we were able to evaluate the heterogeneity in θ for a specific interval among individual F1 females. Results Alignments between the cytogenetic map, radiation hybrid (RH) map, genetic maps and clone map of SSC-X with the physical map of human chromosome X (HSA-X) are presented. The most likely order of 60 markers on SSC-X is inferred. The average recombination rate across SSC-X is of ~1.27 cM/Mb. However, almost no recombination occurred in a large region of ~31 Mb extending from the centromere to Xq21, whereas in the surrounding regions and in the Xq telomeric region a recombination rate of 2.8-3.3 cM/Mb was observed, more than twice the chromosome-wide average rate. Significant differences in θ among F1 females within each population were observed for several chromosomal intervals. The largest variation was observed in both populations in the interval UMNP71-SW1943, or more precisely in the subinterval UMNP891-UMNP93. The individual variation in θ over this subinterval was found associated with F1 females' maternal haplotypes (Chinese pig haplotypes) and independent of paternal haplotype (European pig haplotypes). The θ between UMNP891 and UMNP93 for haplotype 1122 and 4311 differed by more than fourteen-fold (10.3% vs. 0.7%). Conclusions This study reveals marked
Primordial helium recombination. II. Two-photon processes
Hirata, Christopher M.; Switzer, Eric R.
Interpretation of precision measurements of the cosmic microwave background (CMB) will require a detailed understanding of the recombination era, which determines such quantities as the acoustic oscillation scale and the Silk damping scale. This paper is the second in a series devoted to the subject of helium recombination, with a focus on two-photon processes in He i. The standard treatment of these processes includes only the spontaneous two-photon decay from the 2{sup 1}S level. We extend this treatment by including five additional effects, some of which have been suggested in recent papers but whose impact on He i recombination has not been fully quantified. These are: (i) stimulated two-photon decays; (ii) two-photon absorption of redshifted He i line radiation; (iii) two-photon decays from highly excited levels in He i (n{sup 1}S and n{sup 1}D, with n{>=}3); (iv) Raman scattering; and (v) the finite width of the 2{sup 1}P{sup o} resonance. We find that effect (iii) is highly suppressed when one takes into account destructive interference between different intermediate states contributing to the two-photon decay amplitude. Overall, these effects are found to be insignificant: they modify the recombination history at the level of several parts in 10{sup 4}.
From gateway to multisite gateway in one recombination event
Technology Transfer Automated Retrieval System (TEKTRAN)
We created a construct, pDONR-R4-R3, to easily convert any available Gateway destination vector to a MultiSite Gateway vector in a single recombination reaction. In addition, we designed pDONR-R4-R3 so that DNA fragments already cloned upstream or downstream of the Gateway cassette in the original d...
Phylogenomic evidence for recombination of adenoviruses in wild gorillas.
Hoppe, Eileen; Pauly, Maude; Robbins, Martha; Gray, Maryke; Kujirakwinja, Deo; Nishuli, Radar; Boji Mungu-Akonkwa, Dieu-Donné; Leendertz, Fabian H; Ehlers, Bernhard
Human adenoviruses (HAdVs) of species Human mastadenovirus B (HAdV-B) are genetically highly diverse and comprise several pathogenic types. AdVs closely related to members of HAdV-B infect African great apes and the evolutionary origin of HAdV-B has recently been determined in ancient gorillas. Genetic evidence for intra- and inter-species recombination has been obtained for AdVs of humans and captive great apes, but evidence from wild great apes is lacking. In this study, potential HAdV-B members of wild Eastern gorillas were analysed for evidence of recombination. One near-complete genome was amplified from primary sample material and sequenced, and from another six individuals genome fragments were obtained. In phylogenomic analysis, their penton base, pVII-pVI, hexon and fiber genes were compared with those of all publicly available HAdV-B full-genome sequences of humans and captive great apes. Evidence for intra-species recombination between different HAdV-B members of wild gorillas as well as between HAdV-B members of chimpanzees and gorillas was obtained. Since zoonotic AdVs have been reported to cause respiratory outbreaks in both humans and monkeys, and humans in West and Central Africa frequently hunt and butcher primates thereby increasing the chance of zoonotic transmission, such HAdV-B recombinants might widen the pool of potential human pathogens. PMID:26219820
Spin-dependent recombination probed through the dielectric polarizability
Bayliss, Sam L.; Greenham, Neil C.; Friend, Richard H.; Bouchiat, Hélène; Chepelianskii, Alexei D
Despite residing in an energetically and structurally disordered landscape, the spin degree of freedom remains a robust quantity in organic semiconductor materials due to the weak coupling of spin and orbital states. This enforces spin-selectivity in recombination processes which plays a crucial role in optoelectronic devices, for example, in the spin-dependent recombination of weakly bound electron-hole pairs, or charge-transfer states, which form in a photovoltaic blend. Here, we implement a detection scheme to probe the spin-selective recombination of these states through changes in their dielectric polarizability under magnetic resonance. Using this technique, we access a regime in which the usual mixing of spin-singlet and spin-triplet states due to hyperfine fields is suppressed by microwave driving. We present a quantitative model for this behaviour which allows us to estimate the spin-dependent recombination rate, and draw parallels with the Majorana–Brossel resonances observed in atomic physics experiments. PMID:26439933
Doping in the recombinant era: strategies and counterstrategies.
Azzazy, Hassan M E; Mansour, Mai M H; Christenson, Robert H
Advances in recombinant DNA technology have created one of the most powerful weapons in the current doping arsenal: recombinant proteins [Sweeney HL. Gene doping. Sci Am 2004;291:62-9; Unal M, Ozer Unal D. Gene doping in sports. Sports Med 2004;34:357-62]. Recombinant erythropoietin (EPO) and human growth hormone (hGH) are currently being abused but are fortunately detectable either directly by employing isoelectric focusing and immunoassays or indirectly by assessing changes in selected hematopoietic parameters. The detection is technically demanding due to the extent of similarity between the recombinant proteins and their endogenous counterparts. Another issue facing detection efforts is the speed and conditions at which blood samples are collected and analyzed in a sports setting. Recently, gene doping, which stemmed out of legitimate gene therapy trials, has emerged as the next level of doping. Erythropoietin (EPO), human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), peroxisome proliferator-activated receptor-delta (PPAR delta), and myostatin inhibitor genes have been identified as primary targets for doping. Sports clinical scientists today are racing against the clock because assuring the continued integrity of sports competition depends on their ability to outpace the efforts of dopers by developing new detection strategies. PMID:16286094
Detecting Recombination Hotspots from Patterns of Linkage Disequilibrium
Wall, Jeffrey D.; Stevison, Laurie S.
With recent advances in DNA sequencing technologies, it has become increasingly easy to use whole-genome sequencing of unrelated individuals to assay patterns of linkage disequilibrium (LD) across the genome. One type of analysis that is commonly performed is to estimate local recombination rates and identify recombination hotspots from patterns of LD. One method for detecting recombination hotspots, LDhot, has been used in a handful of species to further our understanding of the basic biology of recombination. For the most part, the effectiveness of this method (e.g., power and false positive rate) is unknown. In this study, we run extensive simulations to compare the effectiveness of three different implementations of LDhot. We find large differences in the power and false positive rates of these different approaches, as well as a strong sensitivity to the window size used (with smaller window sizes leading to more accurate estimation of hotspot locations). We also compared our LDhot simulation results with comparable simulation results obtained from a Bayesian maximum-likelihood approach for identifying hotspots. Surprisingly, we found that the latter computationally intensive approach had substantially lower power over the parameter values considered in our simulations. PMID:27226166
Meiotic Segregation and Male Recombination in Interspecific Hybrids of Drosophila
Coyne, Jerry A.
Male hybrids between three pairs of Drosophila species show no substantial distortion of Mendelian segregation and no appreciable male recombination. These results do not support the theories that meiotic drive alleles of large effect are often fixed within species and that transposable genetic elements cause speciation. PMID:3021573
Identification of Recombination and Positively Selected Genes in Brucella.
Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash
Brucella is a facultative intracellular bacterium belongs to the class alpha proteobacteria. It causes zoonotic disease brucellosis to wide range of animals. Brucella species are highly conserved in nucleotide level. Here, we employed a comparative genomics approach to examine the role of homologous recombination and positive selection in the evolution of Brucella. For the analysis, we have selected 19 complete genomes from 8 species of Brucella. Among the 1599 core genome predicted, 24 genes were showing signals of recombination but no significant breakpoint was found. The analysis revealed that recombination events are less frequent and the impact of recombination occurred is negligible on the evolution of Brucella. This leads to the view that Brucella is clonally evolved. On other hand, 56 genes (3.5 % of core genome) were showing signals of positive selection. Results suggest that natural selection plays an important role in the evolution of Brucella. Some of the genes that are responsible for the pathogenesis of Brucella were found positively selected, presumably due to their role in avoidance of the host immune system. PMID:26543263
Gene Delivery into Plant Cells for Recombinant Protein Production
Chen, Qiang
Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications. PMID:26075275
Inference on recombination and block structure using unphased data.
Wiuf, Carsten
In this study compatibility with a tree for unphased genotype data is discussed. If the data are compatible with a tree, the data are consistent with an assumption of no recombination in its evolutionary history. Further, it is said that there is a solution to the perfect phylogeny problem; i.e., for each individual a pair of haplotypes can be defined and the set of all haplotypes can be explained without invoking recombination. A new algorithm to decide whether or not a sample is compatible with a tree is derived. The new algorithm relies on an equivalence relation between sites that mutually determine the phase of each other. (The previous algorithm was based on advanced graph theoretical tools.) The equivalence relation is used to derive the number of solutions to the perfect phylogeny problem. Further, a series of statistics, R ( j ) ( M ), j >or= 2, are defined. These can be used to detect recombination events in the sample's history and to divide the sample into regions that are compatible with a tree. The new statistics are applied to real data from human genes. The results from this application are discussed with reference to recent suggestions that recombination in the human genome is highly heterogeneous. PMID:15020442
Spin-dependent recombination probed through the dielectric polarizability.
Bayliss, Sam L; Greenham, Neil C; Friend, Richard H; Bouchiat, Hélène; Chepelianskii, Alexei D
Despite residing in an energetically and structurally disordered landscape, the spin degree of freedom remains a robust quantity in organic semiconductor materials due to the weak coupling of spin and orbital states. This enforces spin-selectivity in recombination processes which plays a crucial role in optoelectronic devices, for example, in the spin-dependent recombination of weakly bound electron-hole pairs, or charge-transfer states, which form in a photovoltaic blend. Here, we implement a detection scheme to probe the spin-selective recombination of these states through changes in their dielectric polarizability under magnetic resonance. Using this technique, we access a regime in which the usual mixing of spin-singlet and spin-triplet states due to hyperfine fields is suppressed by microwave driving. We present a quantitative model for this behaviour which allows us to estimate the spin-dependent recombination rate, and draw parallels with the Majorana-Brossel resonances observed in atomic physics experiments. PMID:26439933
Progress toward the development of diploid recombinant inbred lines
Technology Transfer Automated Retrieval System (TEKTRAN)
Recombinant inbred lines (RILs) offer new opportunities for mapping traits in potato. They also provide an opportunity to evaluate responses to inbreeding in interspecific hybrids. We are developing a set of six RILs, which will comprise a nested association mapping population. The common parent is ...
Recombinative Generalization of Subword Units Using Matching to Sample
ERIC Educational Resources Information Center
Mahon, Catherine; Lyddy, Fiona; Barnes-Holmes, Dermot
The purpose of the current study was to develop and test a computerized matching-to-sample (MTS) protocol to facilitate recombinative generalization of subword units (onsets and rimes) and recognition of novel onset-rime and onset-rime-rime words. In addition, we sought to isolate the key training components necessary for recombinative…
Dense genomic sampling identifies highways of pneumococcal recombination
Chewapreecha, Claire; Harris, Simon R; Croucher, Nicholas J; Turner, Claudia; Marttinen, Pekka; Cheng, Lu; Pessia, Alberto; Aanensen, David M; Mather, Alison E; Page, Andrew J; Salter, Susannah J.; Harris, David; Nosten, Francois; Goldblatt, David; Corander, Jukka; Parkhill, Julian
Evasion of clinical interventions by Streptococcus pneumoniae occurs through selection of non-susceptible genomic variants. Here we use genome sequencing of 3,085 pneumococcal carriage isolates from a 2.4 km2 refugee camp to enable unprecedented resolution of the process of recombination, and highlight its impact on population evolution. Genomic recombination hotspots show remarkable consistency between lineages, indicating common selective pressures acting at certain loci, particularly those associated with antibiotic resistance. Temporal changes in antibiotic consumption are reflected in changes in recombination trends demonstrating rapid spread of resistance when selective pressure is high. The highest frequencies of receipt and donation of recombined DNA fragments were observed in non-encapsulated lineages, implying that this largely overlooked pneumococcal group, which is beyond the reach of current vaccines, may play a major role in genetic exchange and adaptation of the species as a whole. These findings advance our understanding of pneumococcal population dynamics and provide important information for the design of future intervention strategies. PMID:24509479
Tetrasomic Recombination Is Surprisingly Frequent in Allotetraploid Arachis
Leal-Bertioli, Soraya; Shirasawa, Kenta; Abernathy, Brian; Moretzsohn, Marcio; Chavarro, Carolina; Clevenger, Josh; Ozias-Akins, Peggy; Jackson, Scott; Bertioli, David
Arachis hypogaea L. (cultivated peanut) is an allotetraploid (2n = 4x = 40) with an AABB genome type. Based on cytogenetic studies it has been assumed that peanut and wild-derived induced AABB allotetraploids have classic allotetraploid genetic behavior with diploid-like disomic recombination only between homologous chromosomes, at the exclusion of recombination between homeologous chromosomes. Using this assumption, numerous linkage map and quantitative trait loci studies have been carried out. Here, with a systematic analysis of genotyping and gene expression data, we show that this assumption is not entirely valid. In fact, autotetraploid-like tetrasomic recombination is surprisingly frequent in recombinant inbred lines generated from a cross of cultivated peanut and an induced allotetraploid derived from peanut's most probable ancestral species. We suggest that a better, more predictive genetic model for peanut is that of a "segmental allotetraploid" with partly disomic, partly tetrasomic genetic behavior. This intermediate genetic behavior has probably had a previously overseen, but significant, impact on the genome and genetics of cultivated peanut. PMID:25701284
Recombinant pharmaceuticals from microbial cells: a 2015 update.
Sanchez-Garcia, Laura; Martín, Lucas; Mangues, Ramon; Ferrer-Miralles, Neus; Vázquez, Esther; Villaverde, Antonio
Diabetes, growth or clotting disorders are among the spectrum of human diseases related to protein absence or malfunction. Since these pathologies cannot be yet regularly treated by gene therapy, the administration of functional proteins produced ex vivo is required. As both protein extraction from natural producers and chemical synthesis undergo inherent constraints that limit regular large-scale production, recombinant DNA technologies have rapidly become a choice for therapeutic protein production. The spectrum of organisms exploited as recombinant cell factories has expanded from the early predominating Escherichia coli to alternative bacteria, yeasts, insect cells and especially mammalian cells, which benefit from metabolic and protein processing pathways similar to those in human cells. Up to date, around 650 protein drugs have been worldwide approved, among which about 400 are obtained by recombinant technologies. Other 1300 recombinant pharmaceuticals are under development, with a clear tendency towards engineered versions with improved performance and new functionalities regarding the conventional, plain protein species. This trend is exemplified by the examination of the contemporary protein-based drugs developed for cancer treatment. PMID:26861699
Recombination Difference between Sexes: A Role for Haploid Selection
Why the autosomal recombination rate differs between female and male meiosis in most species has been a genetic enigma since the early study of meiosis. Some hypotheses have been put forward to explain this widespread phenomenon and, up to now, only one fact has emerged clearly: In species in which meiosis is achiasmate in one sex, it is the heterogametic one. This pattern, known as the Haldane-Huxley rule, is thought to be a side effect, on autosomes, of the suppression of recombination between the sex chromosomes. However, this rule does not hold for heterochiasmate species (i.e., species in which recombination is present in both sexes but varies quantitatively between sexes) and does not apply to species lacking sex chromosomes, such as hermaphroditic plants. In this paper, we show that in plants, heterochiasmy is due to a male-female difference in gametic selection and is not influenced by the presence of heteromorphic sex chromosomes. This finding provides strong empirical support in favour of a population genetic explanation for the evolution of heterochiasmy and, more broadly, for the evolution of sex and recombination. PMID:15736976
Genetically encoded optical activation of DNA recombination in human cells.
Luo, J; Arbely, E; Zhang, J; Chou, C; Uprety, R; Chin, J W; Deiters, A
We developed two tightly regulated, light-activated Cre recombinase enzymes through site-specific incorporation of two genetically-encoded photocaged amino acids in human cells. Excellent optical off to on switching of DNA recombination was achieved. Furthermore, we demonstrated precise spatial control of Cre recombinase through patterned illumination. PMID:27277957
Recombination patterns in aphthoviruses mirror those found in other picornaviruses.
Heath, Livio; van der Walt, Eric; Varsani, Arvind; Martin, Darren P
Foot-and-mouth disease virus (FMDV) is thought to evolve largely through genetic drift driven by the inherently error-prone nature of its RNA polymerase. There is, however, increasing evidence that recombination is an important mechanism in the evolution of these and other related picornoviruses. Here, we use an extensive set of recombination detection methods to identify 86 unique potential recombination events among 125 publicly available FMDV complete genome sequences. The large number of events detected between members of different serotypes suggests that horizontal flow of sequences among the serotypes is relatively common and does not incur severe fitness costs. Interestingly, the distribution of recombination breakpoints was found to be largely nonrandom. Whereas there are clear breakpoint cold spots within the structural genes, two statistically significant hot spots precisely separate these from the nonstructural genes. Very similar breakpoint distributions were found for other picornovirus species in the genera Enterovirus and Teschovirus. Our results suggest that genome regions encoding the structural proteins of both FMDV and other picornaviruses are functionally interchangeable modules, supporting recent proposals that the structural and nonstructural coding regions of the picornaviruses are evolving largely independently of one another. PMID:16971423
Dense genomic sampling identifies highways of pneumococcal recombination.
Chewapreecha, Claire; Harris, Simon R; Croucher, Nicholas J; Turner, Claudia; Marttinen, Pekka; Cheng, Lu; Pessia, Alberto; Aanensen, David M; Mather, Alison E; Page, Andrew J; Salter, Susannah J; Harris, David; Nosten, Francois; Goldblatt, David; Corander, Jukka; Parkhill, Julian; Turner, Paul; Bentley, Stephen D
Evasion of clinical interventions by Streptococcus pneumoniae occurs through selection of non-susceptible genomic variants. We report whole-genome sequencing of 3,085 pneumococcal carriage isolates from a 2.4-km(2) refugee camp. This sequencing provides unprecedented resolution of the process of recombination and its impact on population evolution. Genomic recombination hotspots show remarkable consistency between lineages, indicating common selective pressures acting at certain loci, particularly those associated with antibiotic resistance. Temporal changes in antibiotic consumption are reflected in changes in recombination trends, demonstrating rapid spread of resistance when selective pressure is high. The highest frequencies of receipt and donation of recombined DNA fragments were observed in non-encapsulated lineages, implying that this largely overlooked pneumococcal group, which is beyond the reach of current vaccines, may have a major role in genetic exchange and the adaptation of the species as a whole. These findings advance understanding of pneumococcal population dynamics and provide information for the design of future intervention strategies. PMID:24509479
Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract
Not Available
Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)
Production of recombinant proteins in microalgae at pilot greenhouse scale.
Gimpel, Javier A; Hyun, James S; Schoepp, Nathan G; Mayfield, Stephen P
Recombinant protein production in microalgae chloroplasts can provide correctly folded proteins in significant quantities and potentially inexpensive costs compared to other heterologous protein production platforms. The best results have been achieved by using the psbA promoter and 5' untranslated region (UTR) to drive the expression of heterologous genes in a psbA-deficient, non-photosynthetic, algal host. Unfortunately, using such a strategy makes the system unviable for large scale cultivation using natural sunlight for photosynthetic growth. In this study we characterized eight different combinations of 5' regulatory regions and psbA coding sequences for their ability to restore photosynthesis in a psbA-deficient Chlamydomonas reinhardtii, while maintaining robust accumulation of a commercially viable recombinant protein driven by the psbA promoter/5'UTR. The recombinant protein corresponded to bovine Milk Amyloid A (MAA), which is present in milk colostrum and could be used to prevent infectious diarrhea in mammals. This approach allowed us to identify photosynthetic strains that achieved constitutive production of MAA when grown photosynthetically in 100 L bags in a greenhouse. Under these conditions, the maximum MAA expression achieved was 1.86% of total protein, which corresponded to 3.28 mg/L of culture medium. Within our knowledge, this is the first report of a recombinant protein being produced this way in microalgae. PMID:25116083
The kinetochore prevents centromere-proximal crossover recombination during meiosis.
Vincenten, Nadine; Kuhl, Lisa-Marie; Lam, Isabel; Oke, Ashwini; Kerr, Alastair Rw; Hochwagen, Andreas; Fung, Jennifer; Keeney, Scott; Vader, Gerben; Marston, Adèle L
During meiosis, crossover recombination is essential to link homologous chromosomes and drive faithful chromosome segregation. Crossover recombination is non-random across the genome, and centromere-proximal crossovers are associated with an increased risk of aneuploidy, including Trisomy 21 in humans. Here, we identify the conserved Ctf19/CCAN kinetochore sub-complex as a major factor that minimizes potentially deleterious centromere-proximal crossovers in budding yeast. We uncover multi-layered suppression of pericentromeric recombination by the Ctf19 complex, operating across distinct chromosomal distances. The Ctf19 complex prevents meiotic DNA break formation, the initiating event of recombination, proximal to the centromere. The Ctf19 complex independently drives the enrichment of cohesin throughout the broader pericentromere to suppress crossovers, but not DNA breaks. This non-canonical role of the kinetochore in defining a chromosome domain that is refractory to crossovers adds a new layer of functionality by which the kinetochore prevents the incidence of chromosome segregation errors that generate aneuploid gametes. PMID:26653857
Recombinational repair and restart of damaged replication forks.
McGlynn, Peter; Lloyd, Robert G
Genome duplication necessarily involves the replication of imperfect DNA templates and, if left to their own devices, replication complexes regularly run into problems. The details of how cells overcome these replicative 'hiccups' are beginning to emerge, revealing a complex interplay between DNA replication, recombination and repair that ensures faithful passage of the genetic material from one generation to the next. PMID:12415303
Production of recombinant antibody fragments in Bacillus megaterium
Jordan, Eva; Hust, Michael; Roth, Andreas; Biedendieck, Rebekka; Schirrmann, Thomas; Jahn, Dieter; Dübel, Stefan
Background Recombinant antibodies are essential reagents for research, diagnostics and therapy. The well established production host Escherichia coli relies on the secretion into the periplasmic space for antibody synthesis. Due to the outer membrane of Gram-negative bacteria, only a fraction of this material reaches the medium. Recently, the Gram-positive bacterium Bacillus megaterium was shown to efficiently secrete recombinant proteins into the growth medium. Here we evaluated B. megaterium for the recombinant production of antibody fragments. Results The lysozyme specific single chain Fv (scFv) fragment D1.3 was succesfully produced using B. megaterium. The impact of culture medium composition, gene expression time and culture temperatures on the production of functional scFv protein was systematically analyzed. A production and secretion at 41°C for 24 h using TB medium was optimal for this individual scFv. Interestingly, these parameters were very different to the optimal conditions for the expression of other proteins in B. megaterium. Per L culture supernatant, more than 400 μg of recombinant His6-tagged antibody fragment were purified by one step affinity chromatography. The material produced by B. megaterium showed an increased specific activity compared to material produced in E. coli. Conclusion High yields of functional scFv antibody fragments can be produced and secreted into the culture medium by B. megaterium, making this production system a reasonable alternative to E. coli. PMID:17224052
Recombination facilitates neofunctionalization of duplicate genes via originalization
Background Recently originalization was proposed to be an effective way of duplicate-gene preservation, in which recombination provokes the high frequency of original (or wild-type) allele on both duplicated loci. Because the high frequency of wild-type allele might drive the arising and accumulating of advantageous mutation, it is hypothesized that recombination might enlarge the probability of neofunctionalization (Pneo) of duplicate genes. In this article this hypothesis has been tested theoretically. Results Results show that through originalization recombination might not only shorten mean time to neofunctionalizaiton, but also enlarge Pneo. Conclusions Therefore, recombination might facilitate neofunctionalization via originalization. Several extensive applications of these results on genomic evolution have been discussed: 1. Time to nonfunctionalization can be much longer than a few million generations expected before; 2. Homogenization on duplicated loci results from not only gene conversion, but also originalization; 3. Although the rate of advantageous mutation is much small compared with that of degenerative mutation, Pneo cannot be expected to be small. PMID:20534125
The insecticidal activity of recombinant garlic lectins towards aphids.
Fitches, Elaine; Wiles, Duncan; Douglas, Angela E; Hinchliffe, Gareth; Audsley, Neil; Gatehouse, John A
The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at < or =48 h exposure; similar timescale to starvation) was only apparent at the highest lectin concentrations tested (2.0 mg ml(-)1), but dose-dependent chronic toxicity (mortality at >3d exposure) was observed over the concentration range 0.125-2.0 mg ml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding. PMID:18707000
Detecting Recombination Hotspots from Patterns of Linkage Disequilibrium.
Wall, Jeffrey D; Stevison, Laurie S
With recent advances in DNA sequencing technologies, it has become increasingly easy to use whole-genome sequencing of unrelated individuals to assay patterns of linkage disequilibrium (LD) across the genome. One type of analysis that is commonly performed is to estimate local recombination rates and identify recombination hotspots from patterns of LD. One method for detecting recombination hotspots, LDhot, has been used in a handful of species to further our understanding of the basic biology of recombination. For the most part, the effectiveness of this method (e.g., power and false positive rate) is unknown. In this study, we run extensive simulations to compare the effectiveness of three different implementations of LDhot. We find large differences in the power and false positive rates of these different approaches, as well as a strong sensitivity to the window size used (with smaller window sizes leading to more accurate estimation of hotspot locations). We also compared our LDhot simulation results with comparable simulation results obtained from a Bayesian maximum-likelihood approach for identifying hotspots. Surprisingly, we found that the latter computationally intensive approach had substantially lower power over the parameter values considered in our simulations. PMID:27226166
Recombinant inbred lines derived from potato interspecific hybrids
Technology Transfer Automated Retrieval System (TEKTRAN)
Recombinant inbred lines (RILs) offer new opportunities for mapping traits of interest to potato breeders. We are developing a set of six RILs, which will comprise a nested association mapping population. The common parent is M6, an inbred line of the diploid wild relative Solanum chacoense. Other g...
Recombinant Temporal Aberration Detection Algorithms for Enhanced Biosurveillance
Murphy, Sean Patrick; Burkom, Howard
Objective Broadly, this research aims to improve the outbreak detection performance and, therefore, the cost effectiveness of automated syndromic surveillance systems by building novel, recombinant temporal aberration detection algorithms from components of previously developed detectors. Methods This study decomposes existing temporal aberration detection algorithms into two sequential stages and investigates the individual impact of each stage on outbreak detection performance. The data forecasting stage (Stage 1) generates predictions of time series values a certain number of time steps in the future based on historical data. The anomaly measure stage (Stage 2) compares features of this prediction to corresponding features of the actual time series to compute a statistical anomaly measure. A Monte Carlo simulation procedure is then used to examine the recombinant algorithms' ability to detect synthetic aberrations injected into authentic syndromic time series. Results New methods obtained with procedural components of published, sometimes widely used, algorithms were compared to the known methods using authentic datasets with plausible stochastic injected signals. Performance improvements were found for some of the recombinant methods, and these improvements were consistent over a range of data types, outbreak types, and outbreak sizes. For gradual outbreaks, the WEWD MovAvg7+WEWD Z-Score recombinant algorithm performed best; for sudden outbreaks, the HW+WEWD Z-Score performed best. Conclusion This decomposition was found not only to yield valuable insight into the effects of the aberration detection algorithms but also to produce novel combinations of data forecasters and anomaly measures with enhanced detection performance. PMID:17947614
Recombineering using RecET from Pseudomonas syringae
Technology Transfer Automated Retrieval System (TEKTRAN)
Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...
Genetic manipulation of poxviruses using bacterial artificial chromosome recombineering.
Cottingham, Matthew G
Traditional methods for genetic manipulation of poxviruses rely on low-frequency natural recombination in virus-infected cells. Although these powerful systems represent the technical foundation of current knowledge and applications of poxviruses, they require long (≥ 500 bp) flanking sequences for homologous recombination, an efficient viral selection method, and burdensome, time-consuming plaque purification. The beginning of the twenty-first century has seen the application of bacterial artificial chromosome (BAC) technology to poxviruses as an alternative method for their genetic manipulation, following the invention of a long-sought-after method for deriving a BAC clone of vaccinia virus (VAC-BAC) by Arban Domi and Bernard Moss. The key advantages of the BAC system are the ease and versatility of performing genetic manipulation using bacteriophage λ Red recombination (recombineering), which requires only ∼50 bp homology arms that can be easily created by PCR, and which allows seamless mutations lacking any marker gene without having to perform transient-dominant selection. On the other hand, there are disadvantages, including the significant setup time, the risk of contamination of the cloned genome with bacterial insertion sequences, and the nontrivial issue of removal of the BAC cassette from derived viruses. These must be carefully weighed to decide whether the use of BACs will be advantageous for a particular application, making pox-BAC systems likely to complement, rather than supplant, traditional methods in most laboratories. PMID:22688760
A new recombineering system for Photorhabdus and Xenorhabdus.
Yin, Jia; Zhu, Hongbo; Xia, Liqiu; Ding, Xuezhi; Hoffmann, Thomas; Hoffmann, Michael; Bian, Xiaoying; Müller, Rolf; Fu, Jun; Stewart, A Francis; Zhang, Youming
Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed 'recombineering', in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5'-3' exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes. PMID:25539914
Strand invasion promoted by recombination protein of coliphage
NASA Astrophysics Data System (ADS)
Rybalchenko, Nataliya; Golub, Efim I.; Bi, Baoyuan; Radding, Charles M.
Studies of phage in vivo have indicated that its own recombination enzymes, protein and exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that protein has annealing activity, and that exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA. genetic recombination | phage λ
Male recombination in Brazilian populations of Drosophila ananassae.
Goñi, Beatriz; Matsuda, Muneo; Tobari, Yoshiko N
With few exceptions, spontaneous crossing over does not normally occur in male Drosophila. Drosophila ananassae males show considerable amounts of crossing over. In wild males of D. ananassae from Asian (2008) and Brazilian populations (1986 and 2007) variable frequencies of meiotic crossing over, estimated from chiasmata counts, suggested the existence of factors controlling male crossing over in these populations. To corroborate for such prediction, we present data on spontaneous recombination in F1 males of D. ananassae heterozygous for chromosomes of the same Brazilian populations (1986) and marker chromosomes using three testers stocks. Mean recombination value was low, although high variability existed between individual frequencies. Recombination frequencies between lines in each tester stock were not significantly different, excepting when the 3ple-px and 3ple-cy testers were compared (p < 0.05). These two testers differ in respect to the regional distribution of crossovers. The occurrence of recombination in chromosomes 2 and 3 in F1 males tested with e(65) se; bri ru was not related, suggesting they are under independent genetic control. Our data are consistent with proposed genetic factors controlling male crossing over in the tester stocks and to the presence of enhancers and suppressors of male crossing over segregating in the Brazilian populations (1986). PMID:27314475