text:
Inheritance and expression of the mouse metallothionein gene in tobacco
Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.
Maiti, I.B.; Wagner, G.J.; Yeargan, R.; Hunt, A.G. (Univ. of Kentucky, Lexington (USA))
Inheritance and Expression of the Mouse Metallothionein Gene in Tobacco
Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.
Maiti, Indu B.; Wagner, George J.; Yeargan, Ricky; Hunt, Arthur G.
Mouse hepatic metallothionein-I gene cleavage by micrococcal nuclease is enhanced after induction by cadmium.
Micrococcal nuclease has been shown to preferentially cleave chromatin in the region of genes actively engaged in transcription. We have used this preferential cleavage to show that the metallothionein (MT) gene in adult mouse liver, when induced to produce mRNA by injection of cadmium, becomes more susceptible to nuclease cleavage. However, the MT gene in uninduced liver, and the alphafoetal protein (AFP) gene in both induced and uninduced liver, remain relatively resistant to nuclease cleavage. The AFP gene is not normally expressed in cadmium induced or uninduced liver. Thus, susceptibility of genes to nuclease cleavage appears to rise with increasing transcription of the gene. Images
Koropatnick, J; Andrews, G; Duerksen, J D; Varshney, U; Gedamu, L
Expression of a mouse metallothionein-Escherichia coli. beta. -galactosidase fusion gene (MT-. beta. gal) in early mouse embryos
The authors have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli {beta}-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolyl {beta}-D-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. They observed staining indicative of exogenous {beta}-galactosidase activity in 5-17% of DNA-injected embryos assayed at preimplantation stages after 16-24 h treatment with ZnSO{sub 4}. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.
Stevens, M.E.; Meneses, J.J.; Pedersen, R.A. (Univ. of California, San Francisco (United States))
Human metallothionein genes are clustered on chromosome 16
The metallothioneins are a family of heavy-metal binding proteins of low molecular weight. They function in the regulation of trace metal metabolism and in the protection against toxic heavy metal ions. In man, the metallothioneins are encoded by at least 10-12 genes separated into two groups, MT-I and MT-II. To understand the genomic organization of these genes and their involvement in hereditary disorders of trace metal metabolism, we have determined their chromosomal location. Using human-mouse cell hybrids and hybridization probes derived from cloned and functional human MT1 and MT2 genes, we show that the functional human genes are clustered on human chromosome 16. Analysis of RNA from somatic cell hybrids indicated that hybrids that contained human chromosome 16 expressed both human MT1 and MT2 mRNA, and this expression is regulated by both heavy metal ions and glucocorticoid hormones. 27 references, 4 figures, 2 tables.
Karin, M.; Eddy, R.L.; Henry, W.M.; Haley, L.L.; Byers, M.G.; Shows, T.B.
Identification of a signal transducer and activator of transcription (STAT) binding site in the mouse metallothionein-I promoter involved in interleukin-6-induced gene expression.
Mechanisms of regulation of mouse metallothionein (MT)-I gene expression in response to bacterial endotoxin-lipopolysaccharide (LPS) were examined. Northern blot analysis of hepatic MT-I mRNA in interleukin (IL)-6 or tumour necrosis factor (TNF)-receptor type I knock-out mice demonstrated that IL-6, not TNF-alpha, is of central importance in mediating hepatic MT-I gene expression in vivo after LPS injection. In vivo genomic footprinting of the MT-I promoter demonstrated a rapid increase, after LPS injection, in the protection of several guanine residues in the -250 to -300 bp region of the MT-I promoter. The protected bases were within sequences which resemble binding sites for the signal transducers and activators of transcription (STAT) transcription factor family. Electrophoretic mobility-shift assays using oligonucleotides from footprinted MT-I promoter regions showed that injection of LPS resulted in a rapid increase in the specific, high-affinity, in vitro binding of STAT1 and STAT3 to a binding site at -297 bp (TTCTCGTAA). Western blotting of hepatic nuclear proteins showed that the time-course for changes of total nuclear STAT1 and STAT3 after LPS injection paralleled the increased complex formation in vitro using this oligonucleotide, and binding was specifically competed for by a functional STAT-binding site from the rat alpha2-macroglobulin promoter. Furthermore, the MT-I promoter -297 bp STAT-binding site conferred IL-6 responsiveness in the context of a minimal promoter in transient transfection assays using HepG2 cells. This study suggests that the effects of LPS on hepatic MT-I gene expression are mediated by IL-6 and involve the activation of STAT-binding to the proximal promoter.
Lee, D K; Carrasco, J; Hidalgo, J; Andrews, G K
Drosophila melanogaster metallothionein genes: Selection for duplications
The metallothionein genes of Drosophila melanogaster, Mtn and Mto, may play an important role in heavy-metal detoxification. In order to investigate the possibility of increased selection for duplications of these genes in natural populations exposed to high levels of heavy metals, I compared the frequencies of such duplications among flies collected from metal-contaminated and non-contaminated orchards in Pennsylvania, Tennessee, and Georgia. Contaminated of collection sites and of local flies was confirmed by atomic absorption spectrosphotometry. Six-nucleotide-recognizing restriction enzyme analysis was used to screen 1666 wild third chromosomes for Mtn duplications. A subset (327) of these lines was screened for Mto duplications: none were found. Cadmium tolerance test performed on F{sub 2} progeny of wild females failed to detect a difference in tolerance levels between flies from contaminated orchards and flies from control orchards. Estimates of sequence diversity among a subsample (92) of the chromosomes used in the duplication survey, including all 27 Mtn duplication chromosomes, were obtained using four-nucleotide-recognizing restriction enzyme analysis.
Lange, B.W.
Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium.
We inactivated the mouse metallothionein (MT)-I and MT-II genes in embryonic stem cells and generated mice homozygous for these mutant alleles. These mice were viable and reproduced normally when reared under normal laboratory conditions. They were, however, more susceptible to hepatic poisoning by cadmium. This proves that these widely expressed MTs are not essential for development but that they do protect against cadmium toxicity. These mice provide a means for testing other proposed functions of MT in vivo. Images Fig. 2 Fig. 4
Masters, B A; Kelly, E J; Quaife, C J; Brinster, R L; Palmiter, R D
Transfection of neonatal rat Schwann cells with SV40 large T antigen gene under control of the metallothionein promoter
Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T an- tigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phos- phate method for transfection and obtained a transfec- tion efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were car- ried in medium
Gihan I. Tennekoon; Jun Yoshino; Keith W. C. Peden; John Bigbee; J. Lynn Rutkowski; Yasuo Kishimoto; George H. DeVries; Guy M. McKhann
The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression.
We have described and cloned previously a factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF-1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment. Images
Heuchel, R; Radtke, F; Georgiev, O; Stark, G; Aguet, M; Schaffner, W
Down-Regulation of Metallothionein 1 and 2 after Exposure to Electromagnetic Field in Mouse Testis
Background: It is proved that testis is sensitive to electromagnetic field (EMF) and its damage results in infertility. Exposure to EMF induces reactive oxygen species production and affects on anti-oxidants defense mechanisms. Metallothionein (MT) is a name for a group of low molecular weight (6-7 kDa), sulfhydryl rich proteins. Expression of MT1 and MT2 genes in testis tissue after EMF exposure was aimed in this study. Methods: Male BALB/c mice (8 weeks old) were exposed to 3 MT EMF for 8 weeks, 4 hours/day. After 8 weeks, the mice were sacrificed and the testis tissue was removed. The testis pieces were stained with hematoxylin and eosin and analyzed under an optical microscope. Assessment of MT1 and MT2 genes and also protein expression was performed by real-time PCR and Western-blot, respectively. Results: In light microscopic observation, the number of primary spermatocytes was increased significantly in EMF group (P<0.01). In addition, in interstitial space, the number of leydig cells was increased significantly in EMF group (P<0.01) and basement membrane thickness was increased as well. MT1 and MT2 genes were down-regulated significantly in testis tissue of mice exposed to EMF both in mRNA and protein level compared to control. Conclusion: It is clear that MT is mediated in testis development and spermatogenesis. Down-regulation of MT1 and MT2 after EMF in mouse testis might be followed by some consequences that result in infertility.
Ebrahimi-Kalan, Abbas; Habibi Roudkenar, Mehryar; Halabian, Raheleh; Broki Milan, Peiman; Zarrintan, Armin; Mohammadi Roush, Amaneh
Evaluation of a Heterologous Metallothionein Gene Promoter in Transfected Mosquito Cells
Mosquito cells from the C7-10 Aedes albopictus line were transfected with a recombinant plasmid containing the Escherichia coli galactokinase gene under control of the promoter from the Drosophila melanogaster metallothionein gene, Mtn. Consistent with what has been observed with heterologous metallothionein promoters in several vertebrate systems, treatment of transiently transfected mosquito cells with CuSO4 or CdCl2 induced a 2- to
Christina C. N Wu; Ann M Fallon
Expression of the Zinc Transporters Genes and Metallothionein in Obese Women
Research has investigated the participation of zinc transport proteins and metallothionein in the metabolism of this mineral.\\u000a However, studies about the genetic expression of these proteins in obese patients are scarce. The study determined the expression\\u000a of zinc transporter protein codifying genes (ZnT-1, Zip-1 and Zip-3) and of metallothionein in 55 obese women, aged between\\u000a 20 and 56�years. The assessment
Paula Beatriz Krebsky dos Santos Rocha; Amanda de Castro Amorim; Artemizia Francisca de Sousa; Sem�ramis Jamil Hadad do Monte; Luiz Claudio Demes da Mata Sousa; Nadir do Nascimento Nogueira; Jos� Machado Moita Neto; Dilina do Nascimento Marreiro
Structure and Function of the Human Metallothionein Gene Family: Final Technical Report.
National Technical Information Service (NTIS)
The full nucleotide sequence of two additional human metallothionein (hMT) genes has been determined. These genes, hMT-I/sub B/ and hMT-I/sub F/, are located within the MT-I gene cluster we have described originally. The hMT-I/sub F/ gene is the first hMT...
M. Karin
Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione
Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study, Mtf1 conditional knockout mice and control littermates were both mock- or cadmium-treated and liver-specific transcription was analyzed. Besides the well-characterized metallothionein genes, several new MTF-1 target genes with MRE motifs in the promoter region emerged. MTF-1 is required for the basal expression of selenoprotein W, muscle 1 gene (Sepw1) that encodes a glutathione-binding and putative antioxidant protein, supporting a role of MTF-1 in the oxidative stress response. Furthermore, MTF-1 mediates the cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1), which is induced by several stress conditions and is overexpressed in many cancers. MTF-1 is also involved in the cadmium response of cysteine- and glycine-rich protein 1 gene (Csrp1), which is implicated in cytoskeletal organization. In contrast, MTF-1 represses the basal expression of Slc39a10, a putative zinc transporter. In a pathway independent of MTF-1, cadmium also induced the transcription of genes involved in the synthesis and regeneration of glutathione, a cadmium-binding antioxidant. These data provide strong evidence for two major branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in selenoprotein W.
Wimmer, Ursula; Wang, Ying; Georgiev, Oleg; Schaffner, Walter
Glucose regulates free cytosolic Zn�? concentration, Slc39 (ZiP), and metallothionein gene expression in primary pancreatic islet ?-cells.
Zn�? is an important cofactor for insulin biosynthesis and storage in pancreatic ?-cells. Correspondingly, polymorphisms in the SLC30A8 gene, encoding the secretory granule Zn�? transporter ZnT8, are associated with type 2 diabetes risk. Using a genetically engineered (FRET)-based sensor (eCALWY-4), we show here that elevated glucose time-dependently increases free cytosolic Zn�? ([Zn�?](cyt)) in mouse pancreatic ?-cells. These changes become highly significant (853 � 96 pm versus 452 � 42 pm, p < 0.001) after 24 h and are associated with increased expression of the Zn�? importer family members Slc39a6, Slc39a7, and Slc39a8, and decreased expression of metallothionein 1 and 2. Arguing that altered expression of the above genes is not due to altered [Zn�?](cyt), elevation of extracellular (and intracellular) [Zn�?] failed to mimic the effects of high glucose. By contrast, increases in intracellular cAMP prompted by 3-isobutyl-1-methylxanthine and forskolin partially mimicked the effects of glucose on metallothionein, although not ZiP, gene expression. Modulation of intracellular Ca�? and insulin secretion with pharmacological agents (tolbutamide and diazoxide) suggested a possible role for changes in these parameters in the regulation of Slc39a6 and Slc39a7 but not Slc39a8, nor metallothionein expression. In summary, 1) glucose induces increases in [Zn�?](cyt), which are then likely to facilitate the processing and/or the storage of insulin and its cocrystallization with Zn�?, and 2) these increases are associated with elevated expression of zinc importers. Conversely, a chronic increase in [Zn�?](cyt) following sustained hyperglycemia may contribute to ?-cell dysfunction and death in some forms of diabetes. PMID:21613223
Bellomo, Elisa A; Meur, Gargi; Rutter, Guy A
The transcription factors MTF-1 and USF1 cooperate to regulate mouse metallothionein-I expression in response to the essential metal zinc in visceral endoderm cells during early development
During early development of the mouse embryo, expression of the metallothionein-I (MT-I) gene is heightened specifically in the endoderm cells of the visceral yolk sac. The mechanisms of regulation of this cell-specific pattern of expression of metallothionein-I are unknown. However, it has recently been shown that MTF-1, functioning as a metalloregulatory transcription factor, activates metallothionein genes in response to the essential metal zinc. In contrast with the metallothionein genes, MTF-1 is essential for development; null mutant embryos die due to liver degeneration. We report here that MTF-1 is absolutely essential for upregulation of MT-I gene expression in visceral endoderm cells and that optimal expression also involves interactions of the basic helix�loop�helix upstream stimulatory factor-1 (USF1) with an E-box1-containing sequence at �223 bp in the MT-I promoter. Expression of MT-I in visceral endoderm cells was dependent on maternal dietary zinc. Thus, the essential metal, zinc, apparently provides the signaling ligand that activates cell- specific MT-I expression in visceral endoderm cells.
Andrews, Glen K.; Lee, Dae Kee; Ravindra, Rudravajhala; Lichtlen, Peter; Sirito, Mario; Sawadogo, Michele; Schaffner, Walter
Evaluation of a heterologous metallothionein gene promoter in transfected mosquito cells.
Mosquito cells from the C7-10 Aedes albopictus line were transfected with a recombinant plasmid containing the Escherichia coli galactokinase gene under control of the promoter from the Drosophila melanogaster metallothionein gene, Mtn. Consistent with what has been observed with heterologous metallothionein promoters in several vertebrate systems, treatment of transiently transfected mosquito cells with CuSO4 or CdCl2 induced a 2- to 5-fold increase in galactokinase gene expression. Levels of enzyme activity were not increased in tests using stably transformed lines despite wide ranges in the number of transfected gene copies detected in Southern blots. The importance of comparative studies with gene constructs that may eventually be used to produce genetically modified mosquitoes is underscored by the apparent variability in activity of heterologous promoters from D. melanogaster in different mosquito cell lines. PMID:9114495
Wu, C C; Fallon, A M
Isolation of a Vicia faba metallothionein-like gene: expression in foliar trichomes
Animal metallothioneins (MTs) are cysteine-rich, low-molecular-weight proteins that bind to heavy metals and are believed to play a role in their metabolism and detoxification. Genes encoding MT-like proteins have been isolated in a number of plants although their function remains to be elucidated. We describe the isolation and characterization of a bean cDNA encoding an MT-like protein. The bean gene,
Rhonda C. Foley; Karam B. Singh
Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses.
Cicer microphyllum, a wild relative of cultivated chickpea, is a high altitude cold desert-adapted species distributed in western and trans-Himalayas. A complementary DNA (cDNA) encoding metallothionein-like protein has been identified from a cold-induced subtraction cDNA library from C. microphyllum. The sequence of the cloned metallothionein gene from C. microphyllum (GQ900702) contains 240-bp-long open reading frame and encodes predicted 79-amino acid protein of 7.9�kDa. Sequence analysis identified the motifs characteristic of type II metallothionein and designated as CmMet-2. Southern hybridization confirms a single copy of the CmMet-2 gene in C. microphyllum genome. In situ hybridization indicated spatial transcript regulation of CmMet-2 in root and aerial parts and also confirmed through real-time PCR-based quantitative transcript analysis. The data revealed a significantly low level of transcript in the aerial parts than the roots. Quantitative analysis using real-time PCR assay revealed induction of transcript in all parts of plants in response to cold stress at 4�C. The transcript abundance was found to increase exponentially with time course from 6 to 24�h after exposure. Further, regulation of transcript accumulation in response to abscisic acid application, polyethylene glycol (100�?M)-induced osmotic stress, or ZnSO(4) (1�?M) foliar spray indicated by Northern hybridization suggests the involvement of CmMet-2 in multiple stress response. PMID:21161305
Singh, Rupesh K; Anandhan, Sivalingam; Singh, Shweta; Patade, Vikas Y; Ahmed, Zakwan; Pande, Veena
Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): Gene structure, genomic organization, and mRNA expression analysis
Two metallothionein genes, MLMT-IA and MLMT-IB, were isolated and characterized from the mud loach Misgurnus mizolepis (Teleostei; Cypriniformes). For these MTs, we determined a tandem �tail-to-head� genomic organizational pattern, identified conserved genomic features, showed high sequence identities in the coding regions, and examined the closest phylogenetic affiliation, suggesting their divergence by a recent gene duplication event. However, the 5?-flanking upstream
Young Sun Cho; Sang Yoon Lee; Keun-Yong Kim; Yoon Kwon Nam
Mercury accumulation and its distribution to metallothionein in mouse brain after sub-chronic pulse exposure to mercury vapor.
Previously we found that exposure to mercury vapor effectively induced metallothionein (MT) biosynthesis in rat brain. Although the induction of not only MT-I/II but also MT-III was evident, the induction rate of the latter was much lower than that of the former. The brain of an MT-null mouse lacks MT-I/II, but has MT-III. Here we examined the effects of sub-chronic pulse exposure to mercury vapor on the brain MT in MT-null mice and their wild type controls. MT-null and wild type mice were preliminarily exposed to mercury vapor for 2 weeks at 0.1 mg Hg/m(3) for 1 h/day for 3 days a week, and then exposed for 11 weeks at 4.1 mg Hg/m(3) for 30 min/day for 3 days a week. This exposure caused no toxic signs such as abnormal behavior or loss of body weight gain in the mice of either strain throughout the experimental period. Twenty-four hours after the termination of the exposure, mice were sacrificed and brain samples were subjected to mercury analysis, MT assay, and pathological examination. The MT-null mice showed lower accumulation of mercury in the brain than the wild type mice. Mercury exposure resulted in a 70% increase of brain MT in the wild type mice, which was mostly accounted for by the increase in MT-I/II. On the other hand, the brain MT in the MT-null mice increased by 19%, suggesting less reactivity of the MT-III gene to mercury vapor. Although histochemical examination revealed silver-mercury grains in the cytoplasm of nerve cells and glial cells throughout the brains of both strains, no significant difference was observed between the two strains. PMID:15138662
Yasutake, A; Sawada, M; Shimada, A; Satoh, M; Tohyama, C
Cyclic AMP induces metallothionein gene expression in rat hepatocytes but not in rat kidney.
Cyclic AMP analogues induced expression of metallothionein-I (MT-I) mRNA in adult rat liver and in rat hepatocytes cultured in serum-free medium. This induction occurred via an increased rate of transcription of the MT-I gene. The effect of cyclic AMP analogues was tissue-specific since no induction of MT-I mRNA occurred in rat kidney. Induction of MT-I mRNA by a combination of cyclic AMP analogue and dexamethasone was additive in liver and cultured hepatocytes, indicating that induction occurred via independent regulatory pathways. Images Fig. 1. Fig. 2.
Nebes, V L; DeFranco, D; Morris, S M
Induction of metallothionein in mouse cerebellum and cerebrum with low-dose thimerosal injection.
Thimerosal, an ethyl mercury compound, is used worldwide as a vaccine preservative. We previously observed that the mercury concentration in mouse brains did not increase with the clinical dose of thimerosal injection, but the concentration increased in the brain after the injection of thimerosal with lipopolysaccharide, even if a low dose of thimerosal was administered. Thimerosal may penetrate the brain, but is undetectable when a clinical dose of thimerosal is injected; therefore, the induction of metallothionein (MT) messenger RNA (mRNA) and protein was observed in the cerebellum and cerebrum of mice after thimerosal injection, as MT is an inducible protein. MT-1 mRNA was expressed at 6 and 9 h in both the cerebrum and cerebellum, but MT-1 mRNA expression in the cerebellum was three times higher than that in the cerebrum after the injection of 12 microg/kg thimerosal. MT-2 mRNA was not expressed until 24 h in both organs. MT-3 mRNA was expressed in the cerebellum from 6 to 15 h after the injection, but not in the cerebrum until 24 h. MT-1 and MT-3 mRNAs were expressed in the cerebellum in a dose-dependent manner. Furthermore, MT-1 protein was detected from 6 to 72 h in the cerebellum after 12 microg/kg of thimerosal was injected and peaked at 10 h. MT-2 was detected in the cerebellum only at 10 h. In the cerebrum, little MT-1 protein was detected at 10 and 24 h, and there were no peaks of MT-2 protein in the cerebrum. In conclusion, MT-1 and MT-3 mRNAs but not MT-2 mRNA are easily expressed in the cerebellum rather than in the cerebrum by the injection of low-dose thimerosal. It is thought that the cerebellum is a sensitive organ against thimerosal. As a result of the present findings, in combination with the brain pathology observed in patients diagnosed with autism, the present study helps to support the possible biological plausibility for how low-dose exposure to mercury from thimerosal-containing vaccines may be associated with autism. PMID:19357975
Minami, Takeshi; Miyata, Eriko; Sakamoto, Yamato; Yamazaki, Hideo; Ichida, Seiji
Insertion of a yeast metallothionein gene into the model insect Drosophila melanogaster (Diptera: Drosophilidae) to assess the potential for its use in genetic improvement programs with natural enemies
The use of copper-containing fungicides, such as Kocide, negatively affects nontarget beneficial insects in Florida citrus. To determine if the insertion of the yeast metallothionein gene (CUP1) into the genome of beneficial insects could produce populations resistant to copper, the model insect Drosophila melanogaster Meigen (Diptera: Drosophilidae) was transformed with the Drosophila metallothionein promoter Mtn fused to the CUP1 open
Jennifer L. Meyer; Marjorie A. Hoy; Ayyamperumal Jeyaprakash
The Metallothionein Gene, TaMT3, from Tamarix androssowii Confers Cd2+ Tolerance in Tobacco.
Cadmium (Cd) is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress. PMID:24918294
Zhou, Boru; Yao, Wenjing; Wang, Shengji; Wang, Xinwang; Jiang, Tingbo
The Metallothionein Gene, TaMT3, from Tamarix androssowii Confers Cd2+ Tolerance in Tobacco
Cadmium (Cd) is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress.
Zhou, Boru; Yao, Wenjing; Wang, Shengji; Wang, Xinwang; Jiang, Tingbo
Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents.
Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions. Images
Kaina, B; Lohrer, H; Karin, M; Herrlich, P
Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents
Experiments were designed to detect survival advantages that cells gain by overexpressing metallothionein (MT). Chinese hamster ovary K1-2 cells and an x-ray-sensitive derivative were transfected with a bovine papillomavirus (BPV)-linked construct carrying the human metallothionein IIA (hMT-IIA) gene. Transfectants survived 40-fold higher levels of cadmium chloride, harbored at least 30 copies of hMT-IIA, and contained 25- to 166-fold more MT than the parent cells. Even under conditions of reduced glutathione synthesis, the transfectants were not more resistant to the lethal effects of ionizing radiation and bleomycin than the parent cells. Thus free radicals generated by these agents cannot be scavenged efficiently by MT in vivo. The hMT-IIA transfectants, however, but not control transfectants harboring a BPV-MT promoter-neo construct, tolerated significantly higher doses of the alkylating agents N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. Resistance and MT overexpression occurred irrespective of selection and cultivation in cadmium and zinc. There was no increase in resistance to methyl methanesulfonate and N-hydroxyethyl-N-chloroethylnitrosourea. MT did not affect the degree of overall DNA methylation after N-methyl-N-nitrosourea treatment nor the level of O6-methylguanine-DNA methyltransferase. The results suggest that MT participates as a cofactor or regulatory element in repair or tolerance of toxic alkylation lesions.
Kaina, B.; Lohrer, H.; Karin, M.; Herrlich, P. (Kernforschungszentrum Karlsruhe, Karlsruhe (Germany, F.R.))
Cadmium-induced teratogenicity in lizard embryos: correlation with metallothionein gene expression.
Cadmium teratogenic effects and metallothionein expression were studied in tissues of lizard embryos at different stages of development. Incubation of eggs in cadmium contaminated soil had no effect on embryo survival, but strongly affected cranial morphogenesis. Cytological analyses demonstrated abnormalities in the development of proencephalic vesicles, mesencephalon and eyes. No defects were observed in somite or limb development. Northern blot analysis demonstrated that MT expression was much stronger in embryos developed in cadmium contaminated soil. In situ hybridization showed an early induction of MT gene expression in developing liver and gut, whereas in brain and eyes the spatial and temporal localization of MT transcripts did not change. A possible correlation between inability to induce MT expression and abnormalities observed in the head region of lizard developing embryos is suggested. PMID:20888429
Simoniello, Palma; Motta, Chiara Maria; Scudiero, Rosaria; Trinchella, Francesca; Filosa, Silvana
Coordinate amplification of metallothionein I and II gene sequences in cadmium-resistant CHO variants
Cadmium-resistanc (Cd/sup r/) variants of the Chinese hamster cell line, CHO, have been derived by stepwise selection for growth in medium containing CdCl/sub 2/. These variants show coordinately increased production of both metallothionein (MT) I and II and were stably resistant to Cd/sup 2 +/ in the absence of continued selection. Genomic DNAs from these Cd/sup r/ sublines were analyzed for both MT gene copy number and MT gene organization, using cDNA sequence probes specific for each of the two Chinese hamster isometallothioneins. These analyses revealed coordinate amplification of MT I and II genes in all Cd/sup r/ variants which had increased copies of MT-encoding sequences. In situ hybridization of an MT-encoding probe to mitotic chromosomes of a Cd/sup r/ variant, which has amplified MT genes at least 14-fold, revealed a single chromosomal site of hybridization. These results suggest that the isoMTs constitute a functionally related gene cluster which amplifies coordinately in response to toxic metal stress.
Hildebrand, C.E.; Crawford, B.D.; Enger, M.D.
Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter
Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2- negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.
Metallothionein is a cysteine rich, low molecular weight, metal binding protein. Basal levels of endogenous metallothioneins (MT) have been reported in all eucaryotes. MT has been shown to play an essential role in regulating physiological requirements of essential metals such a...
Occurrence of metallothionein gene smtA in synechococcus Tx-20 and other blue-green algae
Blue-green algae are often abundant at Zn- and Cd-contaminated sites. In order to understand the mechanisms associated with Zn- and Cd-tolerance, we have isolated a metallothionein gene, designated smtA, in Synechococcus Tx-20 (- Pcc 6301 - Anacystis nidulans), a strain apparently obtained from an unpolluted site. The gene was cloned and sequenced, and its expression investigated in a range of heavy-metal-tolerant strains of the same organism obtained by stepwise adaptation. The polymerase chain reaction was used to probe for the possible presence of the homologous gene in a range of other strains (especially Synechococcus) isolated from sites without and with heavy metal contamination.
Robinson, N.J.; Gupta, A.; Huckle, J.W.; Jackson, P.; Whitton, B.A. (Univ. of Durham (England))
Pharmacokinetic evaluation of technetium-99-metallothionein-conjugated mouse monoclonal antibody B72. 3 in rhesus monkeys
These studies were conducted to determine the biodistribution and pharmacokinetics of ({sup 99m}Tc)metallothionein-conjugated B72.3 ((Tc)MT-B72.3) in Rhesus monkeys (Macaca mulatta) that were performed as part of the preclinical evaluation of (Tc)MT-B72.3. The B72.3-MT conjugate was studied at three doses of B72.3 ranging from 0.03 mg/kg to 1 mg/kg to determine whether a relationship existed between the dose of total antibody administered intravenously and the biodistribution and clearance of the radiolabeled protein. Results indicated that (Tc)MT-B72.3 distributes rapidly to central body cavity organs and that there was no difference in the rate of blood elimination for the three doses of B72.3 studied. The terminal phase of blood elimination was found to be 26.2 +/- 6.1 hr for the combined groups of monkeys. Approximately one-half of injected {sup 99m}Tc activity was recovered in the urine within 24 hr. A second purpose of these studies was to evaluate the overall immunogenicity of the mouse monoclonal B72.3 IgG1 antibody in Rhesus monkeys. These results demonstrated that a single i.v. exposure to mouse monoclonal B72.3 at doses of 0.3 mg/kg or greater elicited antibody production to B72.3 in Rhesus monkeys within 3 wk. Analysis of (Tc)MT-B72.3 biodistribution and clearance in monkeys with circulating levels of antibodies to B72.3 (immunized monkeys) revealed that the liver was the primary site of clearance of the presumed immune complex and that blood elimination was greatly accelerated.
Burchiel, S.W.; Hadjian, R.A.; Hladik, W.B.; Drozynski, C.A.; Tolman, G.L.; Haber, S.B.; Gallagher, B.M. (Univ. of New Mexico College of Pharmacy, Albuquerque (USA))
Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.).
In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible. PMID:21868142
Wang, Xiaojuan; Song, Yu; Ma, Yanhua; Zhuo, Renying; Jin, Liang
Metallothionein modulates lipopolysaccharide-stimulated tumour necrosis factor expression in mouse peritoneal macrophages.
Metallothionein (MT) is a low-molecular-mass, cysteine-rich metal binding protein thought to be involved in the detoxification of heavy metals and scavenging of free radicals. MT is directly induced not only by heavy metals, but also by hormones and cytokines. The present study, which uses mice with genetic deletions of the MT proteins (MT(-/-) mice), was designed to evaluate the effects of MT on the expression of pro-inflammatory cytokines in macrophages. We found that the production of tumour necrosis factor (TNF) induced by lipopolysaccharide (LPS) in peritoneal macrophages is up-regulated by MT via the modulation of nuclear factor kappaB (NF-kappaB) activity. This conclusion is supported by the following observations: (1) LPS stimulated the secretion of less TNF activity from MT(-/-) peritoneal exudate macrophages (PEMs) than from wild-type controls (MT(+/+) mice) without a difference in the pattern of kinetics; (2) LPS-stimulated expression of TNF-alpha mRNA was decreased in MT(-/-) PEMs; (3) LPS-stimulated activation of NF-kappaB was decreased in MT(-/-) PEMs; and (4) production of TNF in PEMs of MT(-/-) mice after LPS treatment in vivo was decreased (compared with MT(+/+) PEMs). Expression of other inflammatory cytokines, interleukin (IL)-1alpha and IL-6 mRNA, which were modulated by NF-kappaB, were also down-regulated in MT(-/-) PEMs. Thus MT plays a key role in the LPS-induced activation of PEMs via the modulation of NF-kappaB activity.
Kanekiyo, Masako; Itoh, Norio; Kawasaki, Atsuko; Matsuyama, Akiko; Matsuda, Kimihiro; Nakanishi, Tsuyoshi; Tanaka, Keiichi
Association of Estrogen Receptor-? Gene & Metallothionein-1 Gene Polymorphisms in Type 2 Diabetic Women of Andhra Pradesh.
Type 2 diabetes mellitus (DM) is a multifactorial disease where both genetic and environmental factors contribute to its pathogenesis. Estrogen plays an important role in type 2 DM pathogenesis. A number of polymorphisms have been reported in the estrogen receptor (ESR1), including the XbaI and PvuII restriction enzyme polymorphisms of ESR1,which may be involved in disease pathogenesis. Metallothioneins (MT) act as potent antioxidants against various oxidative damages. Very few studies have indicated the association between Estrogen Receptor-?, MT1 gene polymorphisms with type2 DM. A total of 100 type 2 diabetic women and 100 age, sex matched controls were recruited. Using the PCR based RFLP method, the PvuII and XbaI polymorphisms of ESR1 and in MT1A (rs8052394 and rs11076161) gene polymorphisms were analysed. The genotype distribution and frequency of mutated allele showed no significant differences between diabetic and non-diabetic groups in PvuII (?2�=�2.443; P�=�0.1181) or XbaI (?2�=�1.789; P�=�0.1812) and rs8052394 (?2�=�1.154; P�=�0.2840) or rs11076161 (?2�=�0.4141; P�=�0.5199), polymorphisms. This is the first Indian study to conclude that ESR1 and MT1 gene polymorphisms are not associated with increased susceptibility to type 2 diabetes in Indian women. PMID:23277715
Ganasyam, Shilpa Reddy; Rao, Talluri Bhaskar; Murthy, Y S R; Jyothy, Akka; Sujatha, Madireddy
Zinc rescue of Akt2 gene deletion-linked murine cardiac dysfunction and pathological changes is metallothionein-dependent.
We have demonstrated that zinc supplementation provides cardiac protection from diabetes in mice, but its underlying mechanism remains unclear. Since zinc mimics the function of insulin, it may provide benefit to the heart via stimulating Akt-mediated glucose metabolism. Akt2 plays an important role in cardiac glucose metabolism and mice with Akt2 gene deletion (Akt2-KO) exhibit a type 2 diabetes phenotype; therefore, we assumed that no cardiac protection by zinc supplementation from diabetes would be observed in Akt2-KO mice. Surprisingly, despite Akt2 gene deletion, zinc supplementation provided protection against cardiac dysfunction and other pathological changes in Akt2-KO mice, which were accompanied by significant decreases in Akt and GSK-3? phosphorylation. Correspondingly, glycogen synthase phosphorylation and hexokinase II and PGC-1? expression, all involved in the regulation of glucose metabolism, were significantly altered in diabetic hearts, along with a significantly increased expression of Akt negative regulators: PTEN, PTP1B, and TRB3. All these molecular, pathological, and functional changes were significantly prevented by 3-month zinc supplementation. Furthermore, the stimulation of Akt-mediated glucose metabolic kinases or enzymes by zinc treatment was metallothionein-dependent since it could not be observed in metallothionein-knockout mice. These results suggest that zinc preserves cardiac function and structure in Akt2-KO mice presumably due to its insulin mimetic effect on cardiac glucose-metabolism. The cardioprotective effects of zinc are metallothionein-dependent. This is very important since zinc supplementation may be required for patients with Akt2 gene deficiency or insulin resistance. PMID:24819347
Sun, Weixia; Miao, Xiao; Zhou, Shanshan; Zhang, Li; Epstein, Paul N; Mellen, Nicholas; Zheng, Yang; Fu, Yaowen; Wang, Yuehui; Cai, Lu
Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora
In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730 bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17 kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17 kDa protein matched with the regulatory {beta}-subunit of calcineurin (Ca{sup 2+}-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.
Kumar, Kalari Satish [Department of Biochemistry, Osmania University, Hyderabad 500007 (India); Ravi Kumar, B. [Discovery Research Laboratory, Dr. Reddy's Research Foundation, Miyapur, Hyderabad (India); Siddavattam, Dayananda [Department of Animal Sciences, University of Hyderabad, Hyderabad 500046 (India); Subramanyam, Chivukula [Department of Biochemistry, Osmania University, Hyderabad 500007 (India)]. E-mail: firstname.lastname@example.com
Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): gene structure, genomic organization, and mRNA expression analysis.
Two metallothionein genes, MLMT-IA and MLMT-IB, were isolated and characterized from the mud loach Misgurnus mizolepis (Teleostei; Cypriniformes). For these MTs, we determined a tandem "tail-to-head" genomic organizational pattern, identified conserved genomic features, showed high sequence identities in the coding regions, and examined the closest phylogenetic affiliation, suggesting their divergence by a recent gene duplication event. However, the 5'-flanking upstream regions in MLMT-IA and MLMT-IB exposed large differences in the composition and distribution patterns of various transcription factor binding motifs, especially regarding the organization of the metal response element clusters. Real-time RT-PCR assays showed that mRNA levels of both MLMT-IA and MLMT-IB isoforms were variable among tissues and the ratios between them were also variable across tissues, although the MLMT-IA was always predominant in every adult tissue tested. We also found that the MLMT-IA and MLMT-IB mRNA expression levels were regulated dynamically during embryonic and larval development stages, in which the basal expression level of MLMT-IA was also consistently higher than that of MLMT-IB. Upon acute in vivo metal exposure to cadmium, chromium, copper, iron, manganese, nickel, or zinc at 5 microM for 48 h, the transcriptional modulations of MLMT-IA and MLMT-IB were quite different from each other and the type of response was affected significantly by the kind of metals and tissues. PMID:19383548
Cho, Young Sun; Lee, Sang Yoon; Kim, Keun-Yong; Nam, Yoon Kwon
Copper Metallothionein of Yeast, Structure of the Gene, and Regulation of Expression
Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copper-binding protein by DNA sequence
Tauseef R. Butt; Edmund J. Sternberg; Jessica A. Gorman; Philip Clark; Dean Hamer; Martin Rosenberg; Stanley T. Crooke
Intracellular sequestration of zinc, cadmium and silver in Hebeloma mesophaeum and characterization of its metallothionein genes.
Sequestration of intracellular heavy metals in eukaryotes involves compartmentalization and binding with cytosolic, cysteine-rich metallothionein (MT) peptides. We examined the roles of these processes in handling of zinc (Zn), cadmium (Cd) and silver (Ag) in sporocarps and a metal-exposed extraradical mycelium of Hebeloma mesophaeum, the Zn-accumulating ectomycorrhizal (EM) species frequently associated with metal disturbed sites. Size exclusion chromatography revealed that the majority of Zn and Cd in the sporocarps and mycelium was contained in a low molecular mass fraction attributable to compartmentalized metal. The staining of hyphal cells with the Zn-specific Zinquin and Cd-specific Leadmium fluorescent tracers labeled Zn and Cd in small, punctuated vesicles and vacuoles, respectively. By contrast, the sporocarp and mycelium Ag was associated with cysteine-rich, 5-kDa peptides. The peptides of the same size were also identified in minor Zn and Cd complexes from the metal-exposed mycelium. We have further isolated and characterized HmMT1, HmMT2 and HmMT3 genes coding for different 5-kDa MTs of H. mesophaeum collected at a lead smelter site. Heterologous complementation assays in metal-sensitive yeast mutants indicated that HmMTs encode functional, metal-specific peptides: only HmMT1 was able to complement sensitivity to Zn; HmMT1 conferred higher tolerance to Cd and Cu than HmMT2 or HmMT3; and both HmMT2 and HmMT3, but not HmMT1, conferred increased tolerance to Ag. The presence of HmMT1 and HmMT3, but not HmMT2, was also confirmed in a H. mesophaeum isolate from an unpolluted site. Gene expression analysis in the extraradical mycelium of this isolate revealed that the transcription of HmMT1 was preferentially induced in the presence of Zn and Cd, while Ag was a stronger inducer of HmMT3. Altogether, these results improve our understanding of the handling of intracellular Zn, Cd and Ag in Hebeloma and represent the first evidence suggesting involvement of MTs in sequestration of Zn in EM fungi. PMID:24674773
S�ck�, Jan; Leonhardt, Tereza; Borovi?ka, Jan; Gryndler, Milan; Briks�, Ale�; Kotrba, Pavel
Two major branches of anti-cadmium defense in the mouse: MTF1\\/metallothioneins and glutathione
Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer\\/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study,
Ursula Wimmer; Ying Wang; Oleg Georgiev; Walter Schaffner
Regulation of metallothionein gene expression by oxidative stress and metal ions
The metallothioneins (MT) are small, cysteine-rich heavy metal-binding proteins which participate in an array of protective stress responses. Although a single essential function of MT has not been demonstrated, MT of higher eukaryotes evolved as a mechanism to regulate zinc levels and distribution within cells and organisms. These proteins can also protect against some toxic metals and oxidative stress-inducing agents.
Glen K Andrews
Cancer gene discovery in mouse and man
The elucidation of the human and mouse genome sequence and developments in high-throughput genome analysis, and in computational tools, have made it possible to profile entire cancer genomes. In parallel with these advances mouse models of cancer have evolved into a powerful tool for cancer gene discovery. Here we discuss the approaches that may be used for cancer gene identification in both human and mouse and discuss how a cross-species �oncogenomics� approach to cancer gene discovery represents a powerful strategy for finding genes that drive tumourigenesis.
Mattison, Jenny; van der Weyden, Louise; Hubbard, Tim; Adams, David J.
Comparative Gene Prediction in Human and Mouse
The completion of the sequencing of the mouse genome promises to help predict human genes with greater accuracy. While current ab initio gene prediction programs are remarkably sensitive (i.e., they predict at least a fragment of most genes), their specificity is often low, predicting a large number of false-positive genes in the human genome. Sequence conservation at the protein level
Genis Parra; Pankaj Agarwal; Josep F. Abril; Thomas Wiehe; James W. Fickett; Roderic Guigo
Comparative Gene Prediction in Human and Mouse
The completion of the sequencing of the mouse genome promises to help predict human genes with greater accuracy. While current ab initio gene prediction programs are remarkably sensitive (i.e., they predict at least a fragment of most genes), their specificity is often low, predicting a large number of false-positive genes in the human genome. Sequence conservation at the protein level with the mouse genome can help eliminate some of those false positives. Here we describe SGP2, a gene prediction program that combines ab initio gene prediction with TBLASTX searches between two genome sequences to provide both sensitive and specific gene predictions. The accuracy of SGP2 when used to predict genes by comparing the human and mouse genomes is assessed on a number of data sets, including single-gene data sets, the highly curated human chromosome 22 predictions, and entire genome predictions from ENSEMBL. Results indicate that SGP2 outperforms purely ab initio gene prediction methods. Results also indicate that SGP2 works about as well with 3x shotgun data as it does with fully assembled genomes. SGP2 provides a high enough specificity that its predictions can be experimentally verified at a reasonable cost. SGP2 was used to generate a complete set of gene predictions on both the human and mouse by comparing the genomes of these two species. Our results suggest that another few thousand human and mouse genes currently not in ENSEMBL are worth verifying experimentally.
Parra, Genis; Agarwal, Pankaj; Abril, Josep F.; Wiehe, Thomas; Fickett, James W.; Guigo, Roderic
Characterization of the mouse Prox1 gene
Prox1, a vertebrate homologue of Drosophila prospero, encodes a divergent homeodomain protein. We have isolated and characterized full length mouse Prox1 cDNA and genomic clones. Mouse Prox1 gene mapped to position 106.3 cM from the centromere of Chromosome 1, which is very close to the retinal degeneration mutation, rd3. Although the coding sequence and exon-intron junctions of the Prox1 genes
S I Tomarev; R D Zinovieva; B Chang; N L Hawes
Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.
The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression. Images
Scanlon, K J; Jiao, L; Funato, T; Wang, W; Tone, T; Rossi, J J; Kashani-Sabet, M
Expression analysis of buckwheat (Fagopyrum esculentum Moench) metallothionein-like gene (MT3) under different stress and physiological conditions.
The buckwheat metallothionein-like (MT3) gene expression was studied throughout seed and leaf development, as well as under the influence of different external stimuli. MT3 mRNAs were detected from the early stage of seed development to the end of maturation, reaching the highest level during the mid-maturation stage. High MT3 mRNA level was noticed for both green and senescent leaves. The influence of raising Cu ion concentrations on MT3 gene expression was studied only in leaves, while the effect of Zn ions was analyzed through seed development as well. It was found that Cu and Zn ions had stimulatory effects on expression in leaves. MT3 expression was significantly enhanced in the early stage of seed development in response to Zn ions, while after this stage, influence of Zn ions was not detected. After H2O2/NaCl treatment, MT3 mRNA level was decreased in green leaves, contrary to senescent leaves where expression levels remained unchanged. H2O2 treatment caused the increase of MT3 mRNA levels in the mid-maturation stage of seed development. NaCl had no effect on expression levels in seeds. According to obtained results, proposed functions in different plant organs regarding oxidative stress and metal homeostasis are discussed. PMID:15266722
Brkljaci?, Jelena M; Samardzi?, Jelena T; Timotijevi?, Gordana S; Maksimovi?, Vesna R
Spatiotemporal changes in metallothionein gene expression during embryogenesis in the wall lizard Podarcis sicula.
Lizard embryos are nutritionally independent from their environment. During the early phases of oogenesis, the egg prepares for development by storing reserve organelles, proteins, and RNAs sufficient to allow the zygote to transform into a juvenile. This preparation also includes the storage of metallothionein (MT) transcripts. This study investigated the localization of these transcripts by in situ hybridization throughout Podarcis sicula developmental stages. Our data show that MT expression undergoes shifts in both regional and cellular localization. MT transcripts were detected early in the central nervous system, later in tissues implicated in metabolic processes. Results are discussed highlighting differences in lizard embryonic spatial and temporal MT expression compared with piscine, amphibian, and mammalian embryos. We hypothesize that, under natural conditions, the nutritionally closed system represented by the lizard egg protects the developing embryo from an unwanted excess of metals. This mechanism would make MT expression and accumulation in detoxifying organs in developing animals unnecessary until hatching and food intake begins. Conversely, the presence of MT transcripts during brain development may ensure the correct final architecture of this organ. PMID:20623798
Simoniello, Palma; Motta, Chiara Maria; Scudiero, Rosaria; Trinchella, Francesca; Filosa, Silvana
Effect of cadmium on glutathione S-transferase and metallothionein gene expression in coho salmon liver, gill and olfactory tissues
The glutathione S-transferases (GSTs) are a multifunctional family of phase II enzymes that detoxify a variety of environmental chemicals, reactive intermediates, and secondary products of oxidative damage. GST mRNA expression and catalytic activity have been used as biomarkers of exposure to environmental chemicals. However, factors such as species differences in induction, partial analyses of multiple GST isoforms, and lack of understanding of fish GST gene regulation, have confounded the use of GST as markers of pollutant exposure. In the present study, we examined the effect of exposure to cadmium (Cd), a prototypical environmental contaminant and inducer of mammalian GST, on GST mRNA expression in coho salmon (Oncorhynchus kisutch) liver, gill, and olfactory tissues. GST expression data were compared to those for metallothionein (MT), a prototypical biomarker of metal exposure. Data mining of genomic databases led to the development of quantitative real-time PCR (qPCR) assays for salmon GST isoforms encompassing 9 subfamilies, including alpha, mu, pi, theta, omega, kappa, rho, zeta and microsomal GST. In vivo acute (8-48 hr) exposures to low (3.7 ppb) and high (347 ppb) levels of Cd relevant to environmental scenarios elicited a variety of transient, albeit minor changes (<2.5-fold) in tissue GST profiles, including some reductions in GST mRNA expression. In general, olfactory GSTs were the earliest to respond to cadmium, whereas, more pronounced effects in olfactory and gill GST expression were observed at 48 hr relative to earlier time points. Although evaluation of GSTs reflected a cadmium-associated oxidative stress response, there was no clear GST isoform in any tissue that could serve as a reliable biomarker of acute cadmium exposure. By contrast, metallothionein (MT) mRNA was consistently and markedly induced in all three tissues by cadmium, and among the tissues examined, olfactory MT was the most sensitive marker of cadmium exposures. In summary, coho salmon exhibit a complex GST tissue profile consisting of at least 9 isoforms, all of which are present in the peripheral olfactory system. Short-term exposure to environmental levels of Cd causes transient changes in salmon GST consistent with oxidative stress, and in some cases, includes a loss of GST. In a biomarker context, however, monitoring of tissue MT mRNA expression, especially in the peripheral olfactory system, may be of greater utility for assessing short-term environmental exposures to cadmium.
Espinoza, Herbert M.; Williams, Chase R.; Gallagher, Evan P.
Differential Expression of a Metallothionein Gene during the Presymbiotic versus the Symbiotic Phase of an Arbuscular Mycorrhizal Fungus1
A full-length cDNA encoding a metallothionein (MT)-like polypeptide, designated GmarMT1, was identified in an expressed sequence tag collection from germinated spores of the arbuscular mycorrhizal fungus Gigaspora margarita (BEG34). The GmarMT1 gene is composed of two exons separated by an 81-bp intron. It codes for a 65-amino acid polypeptide comprising a plant type 1 MT-like N-terminal domain and a C-terminal domain that is most closely related to an as-yet-uncharacterized fungal MT. As revealed by heterologous complementation assays in yeast, GmarMT1 encodes a functional polypeptide capable of conferring increased tolerance against Cd and Cu. The GmarMT1 RNA is expressed in both presymbiotic spores and symbiotic mycelia, even in the absence of metal exposure, but is significantly less abundant in the latter stage. An opposite pattern was observed upon Cu exposure, which up-regulated GmarMT1 expression in symbiotic mycelia but not in germinated spores. Together, these data provide the first evidence, to our knowledge, for the occurrence in an arbuscular mycorrhizal fungus of a structurally novel MT that is modulated in a metal and life cycle stage-dependent manner and may afford protection against heavy metals (and other types of stress) to both partners of the endomycorrhizal symbiosis.
Lanfranco, Luisa; Bolchi, Angelo; Ros, Emanuele Cesale; Ottonello, Simone; Bonfante, Paola
Gene expression variation between mouse inbred strains
Background In this study, we investigated the effect of genetic background on expression profiles. We analysed the transcriptome of mouse hindlimb muscle of five frequently used mouse inbred strains using spotted oligonucleotide microarrays. Results Through ANOVA analysis with a false discovery rate of 10%, we show that 1.4% of the analysed genes is significantly differentially expressed between these mouse strains. Differential expression of several of these genes has been confirmed by quantitative RT-PCR. The number of genes affected by genetic background is approximately ten-fold lower than the number of differentially expressed genes caused by a dystrophic genetic defect. Conclusions We conclude that evaluation of the effect of background on gene expression profiles in the tissue under study is an effective and sensible approach when comparing expression patterns in animal models with heterogeneous genetic backgrounds. Genes affected by the genetic background can be excluded in subsequent analyses of the disease-related changes in expression profiles. This is often a more effective strategy than backcrossing and inbreeding to obtain isogenic backgrounds.
Turk, Rolf; 't Hoen, Peter AC; Sterrenburg, Ellen; de Menezes, Renee X; de Meijer, Emile J; Boer, Judith M; van Ommen, Gert-Jan B; den Dunnen, Johan T
Expression Patterns of the Human and Mouse IFGP Family Genes
The IFGP gene family has recently been found in human and mouse cells and is structurally related to the leukocytic Fc receptor genes. Expression of six human and four mouse IFGP genes was studied. Apart from mouse IFGP2, the genes of the family are expressed predominantly in hematopoietic cells. Expression of human IFGP1-IFGP5 and mouse IFGP3 is restricted to B
S. A. Ershova; A. M. Najakshin; L. V. Mechetina; M. M. Peklo; A. Ya. Shevelev; T. N. Vlasik; N. A. Chikaev; A. V. Taranin
Promoter architecture of mouse olfactory receptor genes.
Odorous chemicals are detected by the mouse main olfactory epithelium (MOE) by about 1100 types of olfactory receptors (OR) expressed by olfactory sensory neurons (OSNs). Each mature OSN is thought to express only one allele of a single OR gene. Major impediments to understand the transcriptional control of OR gene expression are the lack of a proper characterization of OR transcription start sites (TSSs) and promoters, and of regulatory transcripts at OR loci. We have applied the nanoCAGE technology to profile the transcriptome and the active promoters in the MOE. nanoCAGE analysis revealed the map and architecture of promoters for 87.5% of the mouse OR genes, as well as the expression of many novel noncoding RNAs including antisense transcripts. We identified candidate transcription factors for OR gene expression and among them confirmed by chromatin immunoprecipitation the binding of TBP, EBF1 (OLF1), and MEF2A to OR promoters. Finally, we showed that a short genomic fragment flanking the major TSS of the OR gene Olfr160 (M72) can drive OSN-specific expression in transgenic mice. PMID:22194471
Plessy, Charles; Pascarella, Giovanni; Bertin, Nicolas; Akalin, Altuna; Carrieri, Claudia; Vassalli, Anne; Lazarevic, Dejan; Severin, Jessica; Vlachouli, Christina; Simone, Roberto; Faulkner, Geoffrey J; Kawai, Jun; Daub, Carsten O; Zucchelli, Silvia; Hayashizaki, Yoshihide; Mombaerts, Peter; Lenhard, Boris; Gustincich, Stefano; Carninci, Piero
Characterization and expression of a metallothionein gene in the aquatic fern Azolla filiculoides under heavy metal stress
A cDNA encoding a type 2 metallothionein (MT) was isolated from Azolla filiculoides, termed AzMT2, accession no. AF482470. The AzMT2 transcript was expressed in sterile A. filiculoides that were free of the cyanobiont Anabaena azollae after erythromycin treatment, proving that AzMT2 is encoded by the fern genome. AzMT2 RNA expression was enhanced by the addition of Cd+2, Cu+2, Zn+2 and
Tamar Schor-Fumbarov; Peter B. Goldsbrough; Zach Adam; Elisha Tel-Or
Synteny of the genes for thymidine kinase and galactokinase in the mouse and their assignment to mouse chromosome 11
A bstract We have studied the expression of mouse galactokinase in human-mouse somatic cell hybrids segregating mouse chromosomes. Since concordant segregation of the expression of mouse galactokinase and the presence of mouse chromosome 11 were observed in the hybrid clones, we conclude that the gene for mouse galactokinase is located on mouse chromosome 11. We have also investigated the expression
P. McBreen; K. G. Orkwiszewski; C. J. Chern; W. J. Mellman; C. M. Croce
Production of a bifunctional hybrid molecule B72.3/metallothionein-1 by protein engineering.
A hybrid anti-tumour B72.3 antibody/metallothionein protein B72.3MT-1 was produced by the construction of the expression vector mpSV2neo-EP1-B72.3MT-1. This vector contained the neo gene as a selection marker, the murine immunoglobulin promoter and enhancer, and the hybrid B72.3 heavy chain gene fragment with mouse metallothionein-1 cDNA gene ligated into its CH2 domain. The expression vector was transfected to the heavy chain loss mutant B72.3Mut(K) cell line. The hybrid protein B72.3MT-1 was purified from transfectant supernates using a Protein G column. We showed that the hybrid protein retained the binding reactivity for the TAG72 antigen as the original B72.3 antibody, and the metal-binding capacity of the native metallothionein molecule. Therefore, the bifunctional hybrid protein B72.3MT-1 may be very useful in cancer imaging when labelled with radionuclides such as 99mTc. Images Figure 3 Figure 6 Figure 7
Xiang, J; Koropatnick, J; Qi, Y; Luo, X; Moyana, T; Li, K; Chen, Y
The biology of novel animal genes: Mouse APEX gene knockout
This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.
MacInnes, M.; Altherr, M.R.; Ludwig, D. [Los Alamos National Lab., NM (United States); Pedersen, R.; Mold, C. [Univ. of California, San Francisco, CA (United States)
Target gene search for the metal-responsive transcription factor MTF1
Activation of genes by heavy metals, notably zinc, cadmium and copper, depends on MTF-1, a unique zinc finger transcription factor conserved from insects to human. Knockout of MTF-1 in the mouse results in embryonic lethality due to liver decay, while knockout of its best characterized target genes, the stress-inducible metallothionein genes I and II, is viable, suggesting additional target genes
P. Lichtlen; Y. Wang; T. Belser; O. Georgiev; U. Certa; R. Sack; W. Schaffner
The Mouse Genome Database (MGD): from genes to mice--a community resource for mouse biology.
The Mouse Genome Database (MGD) forms the core of the Mouse Genome Informatics (MGI) system (http://www.informatics.jax.org), a model organism database resource for the laboratory mouse. MGD provides essential integration of experimental knowledge for the mouse system with information annotated from both literature and online sources. MGD curates and presents consensus and experimental data representations of genotype (sequence) through phenotype information, including highly detailed reports about genes and gene products. Primary foci of integration are through representations of relationships among genes, sequences and phenotypes. MGD collaborates with other bioinformatics groups to curate a definitive set of information about the laboratory mouse and to build and implement the data and semantic standards that are essential for comparative genome analysis. Recent improvements in MGD discussed here include the enhancement of phenotype resources, the re-development of the International Mouse Strain Resource, IMSR, the update of mammalian orthology datasets and the electronic publication of classic books in mouse genetics. PMID:15608240
Eppig, Janan T; Bult, Carol J; Kadin, James A; Richardson, Joel E; Blake, Judith A; Anagnostopoulos, A; Baldarelli, R M; Baya, M; Beal, J S; Bello, S M; Boddy, W J; Bradt, D W; Burkart, D L; Butler, N E; Campbell, J; Cassell, M A; Corbani, L E; Cousins, S L; Dahmen, D J; Dene, H; Diehl, A D; Drabkin, H J; Frazer, K S; Frost, P; Glass, L H; Goldsmith, C W; Grant, P L; Lennon-Pierce, M; Lewis, J; Lu, I; Maltais, L J; McAndrews-Hill, M; McClellan, L; Miers, D B; Miller, L A; Ni, L; Ormsby, J E; Qi, D; Reddy, T B K; Reed, D J; Richards-Smith, B; Shaw, D R; Sinclair, R; Smith, C L; Szauter, P; Walker, M B; Walton, D O; Washburn, L L; Witham, I T; Zhu, Y
Gene Expression Profile Analysis of Type 2 Diabetic Mouse Liver
Liver plays a key role in glucose metabolism and homeostasis, and impaired hepatic glucose metabolism contributes to the development of type 2 diabetes. However, the precise gene expression profile of diabetic liver and its association with diabetes and related diseases are yet to be further elucidated. In this study, we detected the gene expression profile by high-throughput sequencing in 9-week-old normal and type 2 diabetic db/db mouse liver. Totally 12132 genes were detected, and 2627 genes were significantly changed in diabetic mouse liver. Biological process analysis showed that the upregulated genes in diabetic mouse liver were mainly enriched in metabolic processes. Surprisingly, the downregulated genes in diabetic mouse liver were mainly enriched in immune-related processes, although all the altered genes were still mainly enriched in metabolic processes. Similarly, KEGG pathway analysis showed that metabolic pathways were the major pathways altered in diabetic mouse liver, and downregulated genes were enriched in immune and cancer pathways. Analysis of the key enzyme genes in fatty acid and glucose metabolism showed that some key enzyme genes were significantly increased and none of the detected key enzyme genes were decreased. In addition, FunDo analysis showed that liver cancer and hepatitis were most likely to be associated with diabetes. Taken together, this study provides the digital gene expression profile of diabetic mouse liver, and demonstrates the main diabetes-associated hepatic biological processes, pathways, key enzyme genes in fatty acid and glucose metabolism and potential hepatic diseases.
Zhang, Fang; Xu, Xiang; Zhang, Yi; Zhou, Ben; He, Zhishui; Zhai, Qiwei
ScMT2-1-3, a Metallothionein Gene of Sugarcane, Plays an Important Role in the Regulation of Heavy Metal Tolerance/Accumulation
Plant metallothioneins (MTs), which are cysteine-rich, low-molecular-weight, and metal-binding proteins, play important roles in detoxification, metal ion homeostasis, and metal transport adjustment. In this study, a novel metallothionein gene, designated as ScMT2-1-3 (GenBank Accession number JQ627644), was identified from sugarcane. ScMT2-1-3 was 700?bp long, including a 240?bp open reading frame (ORF) encoding 79 amino acid residues. A His-tagged ScMT2-1-3 protein was successfully expressed in Escherichia coli system which had increased the host cell's tolerance to Cd2+, Cu2+, PEG, and NaCl. The expression of ScMT2-1-3 was upregulated under Cu2+ stress but downregulated under Cd2+ stress. Real-time qPCR demonstrated that the expression levels of ScMT2-1-3 in bud and root were over 14 times higher than those in stem and leaf, respectively. Thus, both the E. coli assay and sugarcane plantlets assay suggested that ScMT2-1-3 is significantly involved in the copper detoxification and storage in the cell, but its functional mechanism in cadmium detoxification and storage in sugarcane cells needs more testification though its expressed protein could obviously increase the host E. coli cell's tolerance to Cd2+. ScMT2-1-3 constitutes thus a new interesting candidate for elucidating the molecular mechanisms of MTs-implied plant heavy metal tolerance/accumulation and for developing sugarcane phytoremediator varieties.
Guo, Jinlong; Xu, Liping; Su, Yachun; Wang, Hengbo; Gao, Shiwu; Xu, Jingsheng; Que, Youxiong
Structural and bioinformatic analysis of the Roman snail Cd-Metallothionein gene uncovers molecular adaptation towards plasticity in coping with multifarious environmental stress.
Metallothioneins (MTs) are a family of multifunctional proteins involved, among others, in stress response. The Cadmium (Cd)-MT gene of the Roman snail (Helix pomatia), for example, encodes for a protein induced upon cadmium exposure. While our previous studies have demonstrated that the expressed Cd-MT isoform of Roman snails assists detoxification of cadmium, the present work focuses on the potential plasticity of this gene in response to a variety of environmental stressors playing a crucial role in the specific ecological niche of H. pomatia. Our hypothesis is based on a bioinformatic approach involving gene sequencing, structural and in silico analysis of transcription factor binding sites (TFBs), and a comparison of these features with other MT genes. Our results show that the Roman snail's Cd-MT gene not only is the largest known MT gene, but also contains--apart from the regulatory promoter region--several intronic repeat cassettes of putative TFBs suggested to be involved in environmental stress response, immune competence, and regulation of gene expression. Moreover, intronic scaffold/matrix attachment regions (S/MARs) and stress-induced duplex destabilization sites confer a high potential for epigenetic gene regulation. This suggested regulatory plasticity is also supported by physiological data showing that Cd-MT in Roman snails can be induced differentially not only after cadmium exposure, but also in response to nonmetallic environmental stressors. It is concluded that structural analysis combined with bioinformatic screening may constitute valuable tools for predicting the potential for plasticity and niche-specific adaptation of stress-responsive genes in populations living under rapidly changing environmental conditions. PMID:19457198
Egg, Margit; H�ckner, Martina; Brandst�tter, Anita; Schuler, Dietmar; Dallinger, Reinhard
Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium.
Due to its exemplary resistance to ionising radiation, oxidative stress, desiccation and several DNA damaging agents, Deinococcus radiodurans R1 (DR1) is considered as one of the most appropriate candidates for the bioremediation of the nuclear waste sites. However, the high sensitivity of this bacterium to heavy metals, which are usually preponderant at nuclear waste dump sites, precludes its application for bioremediation. This study deals with the expression two metal binding peptides in DR1 as an attractive strategy for developing metal tolerance in this bacterium. A synthetic gene (EC20) encoding a phytochelatin analogue with twenty repeating units of glutamate and cysteine was constructed by overlap extension and expressed in DR1. The cyanobacterial metallothionein (MT) gene, smtA was cloned for intracellular expression in DR1. Both the genes were expressed under the native groESL promoter. DR1 strain carrying the recombinant EC20 demonstrated 2.5-fold higher tolerance to Cd(2+) and accumulated 1.21-fold greater Cd(2+) as opposed to the control while the heterologous expression of MT SmtA in DR1 imparted the transformant superior tolerance to Cd(2+) amassing 2.5-fold greater Cd(2+) than DR1 expressing EC20. PMID:24578153
Chaturvedi, Ruchi; Archana, G
Metallothionein and the Biology of Aging
Metallothionein (MT) is a low molecular weight protein with anti-apoptotic properties that has been demonstrated to scavenge free radicals in vitro. MT has not been extensively investigated within the context of aging biology. The purpose of this review, therefore, is to discuss findings on MT that are relevant to basic aging mechanisms and to draw attention to the possible role of MT in pro-longevity interventions. MT is one of just a handful of proteins that, when overexpressed, has been demonstrated to increase mouse lifespan. MT also protects against development of obesity in mice provided a high fat diet as well as diet-induced oxidative stress damage. Abundance of MT is responsive to caloric restriction (CR) and inhibition of the insulin / insulin-like signaling (IIS) pathway, and elevated MT gene expression has been observed in tissues from fasted and CR-fed mice, long-lived dwarf mice, worms maintained under CR conditions, and long-lived daf-2 mutant worms. The dysregulation of MT in these systems is likely to have tissue-specific effects on aging outcomes. Further investigation will therefore be needed to understand how MT contributes to the response of invertebrates and mice to CR and the endocrine mutations studied by aging researchers.
Swindell, William R.
Determination of metallothionein in biological fluids using enzyme-linked immunoassay with commercial antibody.
Metallothionein (MT) is a low molecular weight cysteine-rich protein with a number of roles in the pro/antioxidant balance and homeostasis of essential metals, such as zinc and copper, and in the detoxification of heavy metals, such as cadmium and mercury. Until now, detection of metallothionein in biological fluids remained difficult because of a lack of a broadly reactive commercial test. Meaningful comparison of the values of metallothionein concentrations reported by different authors using their specific isolation procedures and different conditions of enzyme-linked immunoassay is difficult due to the absence of a reference material for metallothionein. Therefore in the present study, we describe a quantitative assay for metallothionein in biological fluids such as plasma and urine performed by a direct enzyme-linked immunoassay using a commercially available monoclonal mouse anti-metallothionein clone E9 antibody and commercial standards of metallothionein from rabbit liver and a custom preparation of metallothionein from human liver. The sensitivity of the assay for the standard containing two isoforms MT-I and MT-II from human liver was 140 pg/well. The reactivity of the commercial standards and standards containing two isoforms MT-I and MT-II isolated from human liver in our laboratory with a commercial monoclonal mouse anti-metallothionein clone E9 antibody were similar. This suggests that the described ELISA test can be useful for determination of metallothionein concentration in biological fluids. The concentrations of metallothionein in human plasma, erythrocyte lysate and in urine of smoking and non-smoking healthy volunteers are reported. Tobacco smoking increases the extracellular metallothionein concentration (plasma and urine) but does not affect the intracellular concentration (erythrocyte lysate). PMID:20349027
Milnerowicz, Halina; Bizo?, Anna
Mapping of the mouse homologue of the Wilson disease gene to mouse chromosome 8
ATP7B, the gene altered in Wilson disease (WD) patients, lies in a block of homology shared between human chromosome 13q14 and the central region of mouse chromosome 14. However, we have mapped the murine homologue of ATP7B (Atp7b) to mouse chromosome 8 by somatic cell hybrid analysis. Analysis of 80 interspecific backcross offspring was used to position Atp7b close to D8Mit3 and another ATPase locus, Atp4b, on mouse chromosome 8. ATP4B lies in 13q34 and is separated from ATP7B by several loci whose mouse homologues map to mouse chromosome 14. The assignment of Atp7b to mouse chromosome 8 identifies a previously unrecognized region of homology between this chromosome and human chromosome 13. This assignment suggests a possible location for the toxic milk mutation in the mouse, which has been proposed as a homologue of WD. 17 refs., 2 figs.
Reed, V.; Williamson, P.; Boyd, Y. [MRC Radiobiology Unit, Toronto (Canada)] [and others] [MRC Radiobiology Unit, Toronto (Canada); and others
Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli.
Salicornia brachiata is an extreme halophyte growing luxuriantly in the coastal marshes and frequently exposed to various abiotic stresses including heavy metals. A full length type 2 metallothionein (SbMT-2) gene was isolated using RACE and its copy number was confirmed by southern blot analysis. Transcript expression of SbMT-2 gene was analyzed by semi-quantitative Rt-PCR and real time quantitative (qRT) PCR. Expression of SbMT-2 gene was up-regulated concurrently with zinc, copper, salt, heat and drought stress, down regulated by cold stress while unaffected under cadmium stress. Heterologous expression of SbMT-2 gene enhances metal accumulation and tolerance in E. coli. Metal-binding characteristics of SbMT-2 protein show its possible role in homeostasis and/or detoxification of heavy metals. Significant tolerance was observed by E. coli cells expressing recombinant SbMT-2 for Zn(++), Cu(++) and Cd(++) compared to cells expressing GST only. Sequestration of zinc was 4-fold higher compared to copper and in contrast SbMT-2 inhibits the relative accumulation of cadmium by 1.23-fold compared to GST protein. Fusion protein SbMT-2 showed utmost affinity to zinc (approx. 2.5 fold to Cu(++) and Cd(++)) followed by copper and cadmium ions with same affinity. Halophyte S. brachiata has inherent resilience of varying abiotic tolerance therefore SbMT-2 gene could be a potential candidate to be used for enhanced metal tolerance and heavy metal phytoremediation. PMID:22441126
Chaturvedi, Amit Kumar; Mishra, Avinash; Tiwari, Vivekanand; Jha, Bhavanath
Light damage induced changes in mouse retinal gene expression.
Oxidative stress plays a role in the light damage model of retinal degeneration as well as in age-related macular degeneration. The purpose of this study is to identify retinal genes induced by acute photo-oxidative stress, which may function as mediators of apoptosis or as survival factors. To accomplish this, Balb/c mice were exposed to bright cool white fluorescent light for 7 hr. Retinas were then isolated for total RNA preparation followed by Affymetrix DNA microarray analysis to compare gene expression in light damaged mice to unexposed controls. Three independent light damage experiments were carried out and statistical filters were applied to detect genes with expression changes averaging at least two-fold. Quantitative PCR was carried out to confirm altered gene expression. Seventy genes were upregulated at least two-fold immediately following light damage. QPCR confirmed upregulation of all 10 genes tested. The upregulated genes fall into several categories including antioxidants: ceruloplasmin, metallothionein, and heme oxygenase; antiapoptotic gene: bag3, chloride channels: clic1 and clic4; transcription factors: c-fos, fra1, junB, stat1, krox-24 and c/ebp; secreted signaling molecules: chitinase 3-like protein 1 and osteopontin; inflammation related genes: MCP-1 and ICAM1 and others. Upregulation of five interferon-gamma responsive genes suggests elevated interferon levels after light damage. Upregulation of three components of the AP-1 transcription factor is consistent with previous evidence implicating AP-1 in light damage pathogenesis. Four copper or iron binding proteins were upregulated, suggesting that photo-oxidative stress may affect metal homeostasis. The genes found upregulated by light damage may affect the survival of photoreceptors subjected to photo-oxidative stress. PMID:15325571
Chen, Lin; Wu, Wayne; Dentchev, Tzvete; Zeng, Yong; Wang, Jianhua; Tsui, Irena; Tobias, John W; Bennett, Jean; Baldwin, Donald; Dunaief, Joshua L
Characterization and expression of a metallothionein gene in the aquatic fern Azolla filiculoides under heavy metal stress.
A cDNA encoding a type 2 metallothionein (MT) was isolated from Azolla filiculoides, termed AzMT2, accession no. AF482470. The AzMT2 transcript was expressed in sterile A. filiculoides that were free of the cyanobiont Anabaena azollae after erythromycin treatment, proving that AzMT2 is encoded by the fern genome. AzMT2 RNA expression was enhanced by the addition of Cd(+2), Cu(+2), Zn(+2) and Ni(+2) to the growth medium. The transcript level of AzMT2 correlated with the metal content in the plants. Temporal analysis of AzMT2 expression demonstrated that Cd(2+) and Ni(2+) induction of AzMT2 RNA expression occurred within 48 h. AzMT2-enhanced expression responded more intensely to the toxic Cd and Ni ions in A. filiculoides suggesting that AzMT2 may participate in detoxification mechanism. The more moderate response of AzMT2 to Zn and Cu ions, which are essential micronutrients, suggest a role for AzMT2 in metal homeostasis. PMID:16133213
Schor-Fumbarov, Tamar; Goldsbrough, Peter B; Adam, Zach; Tel-Or, Elisha
Algal metallothioneins: secondary metabolites and proteins
Metallothioneins, MT's, are low-molecular-weight, cysteine-rich, polypeptides that complex �soft� metal ions in thiol clusters.\\u000a They are structurally diverse. Some MT's are gene products, while others are secondary metabolites. Two of the three classes\\u000a of MT have been identified in algae. Eukaryotic algae possess the secondary metabolites referred to as class III MT. There\\u000a is no unequivocal evidence that MT genes
Nigel J. Robinson
Tryphostin AG879, a tyrosine kinase inhibitor: prevention of transcriptional activation of the electrophile and the aromatic hydrocarbon response elements 1 1 Abbreviations: EPRE, electrophile response element; AHRE, aromatic hydrocarbon response element; MRE, metal response element; Nqo1 and NQO1, mouse NAD(P)H:quinone oxidoreductase [also called NMO1, quinone reductase, DT-diaphorase] gene and mRNA; Cyp1a1 and CYP1A1, mouse cytochrome P450 1A1 gene and mRNA; Mt1 and MT1, mouse metallothionein-1 gene and mRNA; Sod, mouse Cu,Zn-superoxide dismutase gene; SOD, rat Cu,Zn-superoxide dismutase gene; SOD, mouse and rat Cu,Zn-superoxide dismutase mRNA and protein; Luc and LUC, firefly luciferase gene and enzyme; BGAL, ?-galactosidase enzyme activity; tBHQ, tert-butylhydroquinone; dioxin (also TCDD), 2,3,7,8-tetrahydro chlorodibenzo- p-dioxin; SSC, standard sodium citrate; and SET, sodium ethylenediamine tetraacetic acid (EDTA) Tris buffer
To investigate a possible role of phosphorylation in the signal transduction pathways responsible for transcriptional regulation of drug-metabolizing enzymes, we tested seven specific tyrosine kinase inhibitors (tyrphostins) for their effects on NAD(P)H:quinone oxidoreductase-1 (NQO1) mRNA levels in mouse hepatoma Hepa-1c1c7 (Hepa-1) cells and chose to study AG879 further. The potent electrophile tert-butylhydroquinone (tBHQ) is known to activate NQO1 gene transcription
Matthew Z Dieter; Sarah L Freshwater; Willy A Solis; Daniel W Nebert; Timothy P Dalton
Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase
Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. There the authors report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 {mu}g mL{sup {minus}1} aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.
D'Anna, J.A.; Tobey, R.A. (Los Alamos National Lab., NM (USA))
Improved human disease candidate gene prioritization using mouse phenotype
Background The majority of common diseases are multi-factorial and modified by genetically and mechanistically complex polygenic interactions and environmental factors. High-throughput genome-wide studies like linkage analysis and gene expression profiling, tend to be most useful for classification and characterization but do not provide sufficient information to identify or prioritize specific disease causal genes. Results Extending on an earlier hypothesis that the majority of genes that impact or cause disease share membership in any of several functional relationships we, for the first time, show the utility of mouse phenotype data in human disease gene prioritization. We study the effect of different data integration methods, and based on the validation studies, we show that our approach, ToppGene , outperforms two of the existing candidate gene prioritization methods, SUSPECTS and ENDEAVOUR. Conclusion The incorporation of phenotype information for mouse orthologs of human genes greatly improves the human disease candidate gene analysis and prioritization.
Chen, Jing; Xu, Huan; Aronow, Bruce J; Jegga, Anil G
Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR
Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv{sub BAS}) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv{sub BAS} increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.
Zorita, I. [Lab. Cell Biology and Histology, Dept. Zoology and Animal Cell Biology, University of the Basque Country, PO Box 644, E-48080 Bilbao, Basque Country (Spain); Bilbao, E. [Lab. Cell Biology and Histology, Dept. Zoology and Animal Cell Biology, University of the Basque Country, PO Box 644, E-48080 Bilbao, Basque Country (Spain); Schad, A. [Institute of Pathology, Johannes Gutenberg University, 55101, Mainz (Germany); Cancio, I. [Lab. Cell Biology and Histology, Dept. Zoology and Animal Cell Biology, University of the Basque Country, PO Box 644, E-48080 Bilbao, Basque Country (Spain); Soto, M. [Lab. Cell Biology and Histology, Dept. Zoology and Animal Cell Biology, University of the Basque Country, PO Box 644, E-48080 Bilbao, Basque Country (Spain); Cajaraville, M.P. [Lab. Cell Biology and Histology, Dept. Zoology and Animal Cell Biology, University of the Basque Country, PO Box 644, E-48080 Bilbao, Basque Country (Spain)]. E-mail: email@example.com
Cloning, characterization and targeting of the mouse HEXA gene
The HEXA gene, encoding the {alpha} subunit of {beta}-hexosaminidase A, is essential for the metabolism of ganglioside G{sub M2}, and defects in this gene cause Tay-Sachs disease in humans. To elucidate the role of the gene in the nervous system of the mouse and to establish a mouse model of Tay-Sachs disease, we have cloned and characterized the HEXA gene and targeted a disruption of the gene in mouse ES cells. The mouse HEXA gene spans {approximately}26 kb and consists of 14 exons, similar to the human gene. A heterogeneous transcription initiation site was identified 21-42 bp 5{prime} of the initiator ATG, with two of the sites fitting the consensus CTCA (A = start) as seen for some weak initiator systems. Promoter analysis showed that the first 150 bp 5{prime} of the ATG contained 85% of promoter activity observed in constructs containing up to 1050 bp of 5{prime} sequence. The active region contained a sequence matching that of the adenovirus major late promoter upstream element factor. A survey of mouse tissues showed that the highest mRNA levels were in (max to min): testis (5.5 x brain cortex), adrenal, epididymis, heart, brain, lung, kidney, and liver (0.3 x brain cortex). A 12 kb BstI/SalI fragment containing nine exons was disrupted with the insertion of the bacterial neo{sup r} gene in exon 11 and was targeted into 129/Sv ES cells by homologous recombination. Nine of 153 G418 resistant clones were correctly targeted as confirmed by Southern blotting. The heterozygous ES cells were microinjected into mouse blastocysts and implanted into pseudo-pregnant mice. Nine male chimeric mice, showing that 40-95% chimerism for the 129/Sv agouti coat color marker, are being bred in an effort to generate germline transmission of the disrupted HEXA gene.
Wakamatsu, N.; Trasler, J.M.; Gravel, R.A. [McGill Univ., Quebec (Canada)] [and others
The mammalian gene function resource: the International Knockout Mouse Consortium.
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research. PMID:22968824
Bradley, Allan; Anastassiadis, Konstantinos; Ayadi, Abdelkader; Battey, James F; Bell, Cindy; Birling, Marie-Christine; Bottomley, Joanna; Brown, Steve D; B�rger, Antje; Bult, Carol J; Bushell, Wendy; Collins, Francis S; Desaintes, Christian; Doe, Brendan; Economides, Aris; Eppig, Janan T; Finnell, Richard H; Fletcher, Colin; Fray, Martin; Frendewey, David; Friedel, Roland H; Grosveld, Frank G; Hansen, Jens; H�rault, Yann; Hicks, Geoffrey; H�rlein, Andreas; Houghton, Richard; Hrab� de Angelis, Martin; Huylebroeck, Danny; Iyer, Vivek; de Jong, Pieter J; Kadin, James A; Kaloff, Cornelia; Kennedy, Karen; Koutsourakis, Manousos; Lloyd, K C Kent; Marschall, Susan; Mason, Jeremy; McKerlie, Colin; McLeod, Michael P; von Melchner, Harald; Moore, Mark; Mujica, Alejandro O; Nagy, Andras; Nefedov, Mikhail; Nutter, Lauryl M; Pavlovic, Guillaume; Peterson, Jane L; Pollock, Jonathan; Ramirez-Solis, Ramiro; Rancourt, Derrick E; Raspa, Marcello; Remacle, Jacques E; Ringwald, Martin; Rosen, Barry; Rosenthal, Nadia; Rossant, Janet; Ruiz Noppinger, Patricia; Ryder, Ed; Schick, Joel Zupicich; Schn�tgen, Frank; Schofield, Paul; Seisenberger, Claudia; Selloum, Mohammed; Simpson, Elizabeth M; Skarnes, William C; Smedley, Damian; Stanford, William L; Stewart, A Francis; Stone, Kevin; Swan, Kate; Tadepally, Hamsa; Teboul, Lydia; Tocchini-Valentini, Glauco P; Valenzuela, David; West, Anthony P; Yamamura, Ken-ichi; Yoshinaga, Yuko; Wurst, Wolfgang
Metallothionein and liver cell regeneration.
Hepatocytes in adults are in a nonproliferative state but they have high capacity to regenerate within few hours after an injury. After partial hepatectomy or chemical injury, hepatocytes undergo a synchronized multistep process consisting of priming/initiation, proliferation, and termination. These distinct steps are essential for restoring the structure and functions of liver. The mechanisms involved in each of these steps of regeneration are well documented from various laboratories and are described in several reviews. We briefly describe these steps and the involvement of various cytokines and growth factors for cell regeneration in this short review. Liver cell regeneration may also involve stem cell proliferation. The regenerating cells require large amounts of zinc within a short time, and this requirement is met by induction of a zinc and copper binding protein, metallothionein (MT), during the priming step, soon after an injury. There are several reports on the transfer of zinc from MT to various metalloenzymes and transcription factors. Genetically modified mouse models have been used to study the involvement of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in cell regeneration. The use of an MT-knockout mouse has enabled us to investigate the specific role of MT in liver regeneration after partial hepatectomy, chemical injury, and fibrosis. Several studies have suggested a defective liver regeneration after an injury in MT-knockout mice. There is cumulative evidence that indicates an essential role for MT in liver cell regeneration. PMID:16446489
Cherian, M George; Kang, Y James
Paracetamol hepatotoxicity in metallothionein-null mice
The role of metallothionein (MT) in protecting the liver against paracetamol (PCT) toxicity was investigated in vivo and in vitro in mice lacking expression of MT?1 and MT?2 genes (MT?\\/?). In the fed, glycogen replete state, hepatotoxicity (PCT 300 mg\\/kg i.p.) at 6 h was significantly greater in MT?\\/? than MT+\\/+ mice. Plasma lactate dehydrogenase (LD) and alanine aminotransferase (ALT)
A. M Rofe; E. F Barry; T. L Shelton; J. C Philcox; P Coyle
Project normal: Defining normal variance in mouse gene expression
The mouse has become an indispensable and versatile model organism for the study of development, genetics, behavior, and disease. The application of comprehensive gene expression profiling technologies to compare normal and diseased tissues or to assess molecular alterations resulting from various experimental interventions has the potential to provide highly detailed qualitative and quantitative descriptions of these processes. Ideally, to interpret experimental data, the magnitude and diversity of gene expression for the system under study should be well characterized, yet little is known about the normal variation of mouse gene expression in vivo. To assess natural differences in murine gene expression, we used a 5406-clone spotted cDNA microarray to quantitate transcript levels in the kidney, liver, and testis from each of 6 normal male C57BL6 mice. We used ANOVA to compare the variance across the six mice to the variance among four replicate experiments performed for each mouse tissue. For the 6 kidney samples, 102 of 3,088 genes (3.3%) exhibited a statistically significant mouse variance at a level of 0.05. In the testis, 62 of 3,252 genes (1.9%) showed statistically significant variance, and in the liver, there were 21 of 2,514 (0.8%) genes with significantly variable expression. Immune-modulated, stress-induced, and hormonally regulated genes were highly represented among the transcripts that were most variable. The expression levels of several genes varied significantly in more than one tissue. These studies help to define the baseline level of variability in mouse gene expression and emphasize the importance of replicate microarray experiments.
Pritchard, Colin C.; Hsu, Li; Delrow, Jeffrey; Nelson, Peter S.
Structure and function of the mouse surfactant protein B gene.
The mouse surfactant protein B (SP-B) gene was isolated from a DBA/2J EMBL3 genomic library, and the nucleotide sequence and transcriptional start site was determined. The SP-B gene was comprised of 11 exons and 10 introns and spanned 9,882 bases. Repetitive elements of the B1, B2, and R families and several other motifs were located within nontranslated regions of the gene. The 5' flanking region of the gene was utilized to express a bacterial chloramphenicol acetyl transferase reporter gene in mouse and human pulmonary adenocarcinoma cell lines. Transcriptionally active regions of the promoter were identified that determined lung epithelial cell-specific gene expression. Regions of the gene promoter shared between the mouse and human SP-B gene were used to locate putative transcription factor binding sites for thyroid transcription factor-1 and hepatocyte nuclear factor-3. The 5' flanking region of the murine SP-B contains cell-specific cis-active elements that regulate SP-B expression in the respiratory epithelium. PMID:7900819
Bruno, M A; Bohinski, R J; Carter, J E; Foss, K A; Whitsett, J A
Model for Mammalian Metallothionein Structure
The results of physicochemical studies of mammalian metallothioneins are summarized and used to propose a model of the protein. The primary structures of all mammalian metallothioneins are very homologous; there are 38 invariant residues and 20 of them are cysteines. The results of UV and CD optical studies indicated that all 20 cysteines are involved in the ligation of 7
Yvan Boulanger; Christopher M. Goodman; Carla P. Forte; Stephen W. Fesik; Ian M. Armitage
Mouse zona pellucida genes and glycoproteins
The zona pellucida (ZP) is a thick extracellular coat that surrounds all mammalian eggs. The ZP plays important roles during oogenesis, fertilization, and preimplantation development. The mouse ZP consists of only three glycoproteins, called ZP1, ZP2, and ZP3. All three glycoproteins are essential structural components of the ZP. Additionally, ZP3 serves as a primary sperm receptor and acrosome reaction-inducer, and
P. M. Wassarman; L. Jovine; E. S. Litscher
Activation of genes involved in xenobiotic metabolism is a shared signature of mouse models with extended lifespan.
Xenobiotic metabolism has been proposed to play a role in modulating the rate of aging. Xenobiotic metabolizing enzymes (XME) are expressed at higher levels in calorically restricted mice (CR) and in GH/IGF-I-deficient, long-lived mutant mice. In this study, we show that many phase I XME genes are similarly upregulated in additional long-lived mouse models, including "crowded litter" (CL) mice, whose lifespan has been increased by food restriction limited to the first 3 wk of life, and in mice treated with rapamycin. Induction in the CL mice lasts at least through 22 mo of age, but induction by rapamycin is transient for many of the mRNAs. Cytochrome P-450s, flavin monooxygenases, hydroxyacid oxidase, and metallothioneins were found to be significantly elevated in similar proportions in each of the models of delayed aging tested, whether these were based on mutation, diet, drug treatment, or transient early intervention. The same pattern of mRNA elevation could be induced by 2 wk of treatment with tert-butylhydroquinone, an oxidative toxin known to activate Nrf2-dependent target genes. These results suggest that elevation of phase I XMEs is a hallmark of long-lived mice and may facilitate screens for agents worth testing in intervention-based lifespan studies. PMID:22693205
Steinbaugh, Michael J; Sun, Liou Y; Bartke, Andrzej; Miller, Richard A
Metallothionein III (MT3) is a putative tumor suppressor gene that is frequently inactivated in pediatric acute myeloid leukemia by promoter hypermethylation
Background Acute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear. Methods Eleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis. Results The MT3 promoter was hypermethylated in leukemia cell lines. More CpG�s methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P?0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P?=?0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P?0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1. Conclusion MT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.
Inducible and reversible regulation of endogenous gene in mouse
Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene�s promoter region turns the gene on and off in a tetracycline-inducible manner. NmycTRE mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. NmycTRE mice were crossed to tTS transgenic mice to produce NmycTRE/TRE: tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS�dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed.
Sun, Ruilin; Zhao, Kai; Shen, Ruling; Cai, Lei; Yang, Xingyu; Kuang, Ying; Mao, Jifang; Huang, Fang; Wang, Zhugang; Fei, Jian
The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO4 by addit...
Recent segmental and gene duplications in the mouse genome
Background The high quality of the mouse genome draft sequence and its associated annotations are an invaluable biological resource. Identifying recent duplications in the mouse genome, especially in regions containing genes, may highlight important events in recent murine evolution. In addition, detecting recent sequence duplications can reveal potentially problematic regions of the genome assembly. We use BLAST-based computational heuristics to identify large (? 5 kb) and recent (? 90% sequence identity) segmental duplications in the mouse genome sequence. Here we present a database of recently duplicated regions of the mouse genome found in the mouse genome sequencing consortium (MGSC) February 2002 and February 2003 assemblies. Results We determined that 33.6 Mb of 2,695 Mb (1.2%) of sequence from the February 2003 mouse genome sequence assembly is involved in recent segmental duplications, which is less than that observed in the human genome (around 3.5-5%). From this dataset, 8.9 Mb (26%) of the duplication content consisted of 'unmapped' chromosome sequence. Moreover, we suspect that an additional 18.5 Mb of sequence is involved in duplication artifacts arising from sequence misassignment errors in this genome assembly. By searching for genes that are located within these regions, we identified 675 genes that mapped to duplicated regions of the mouse genome. Sixteen of these genes appear to have been duplicated independently in the human genome. From our dataset we further characterized a 42 kb recent segmental duplication of Mater, a maternal-effect gene essential for embryogenesis in mice. Conclusion Our results provide an initial analysis of the recently duplicated sequence and gene content of the mouse genome. Many of these duplicated loci, as well as regions identified to be involved in potential sequence misassignment errors, will require further mapping and sequencing to achieve accuracy. A Genome Browser database was set up to display the identified duplication content presented in this work. This data will also be relevant to the growing number of investigators who use the draft genome sequence for experimental design and analysis.
Cheung, Joseph; Wilson, Michael D; Zhang, Junjun; Khaja, Razi; MacDonald, Jeffrey R; Heng, Henry HQ; Koop, Ben F; Scherer, Stephen W
Differential gene expression in the developing mouse ureter.
In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations. PMID:16459152
Mitchell, Eleanor K L; Taylor, Darrin F; Woods, Kyra; Davis, Melissa J; Nelson, Amy L; Teasdale, Rohan D; Grimmond, Sean M; Little, Melissa H; Bertram, John F; Caruana, Georgina
Identification and characterization of a second mouse Nramp gene
The Nramp gene was isolated as a candidate for the host resistance locus Bcg\\/Ity\\/Lsh, which controls natural resistance of mice to several types of infections. We have isolated by cross-hybridization cDNA clones corresponding to a second mouse Nramp gene, which we designate Nramp2. Nucleotide and predicted amino acid sequence analyses of full-length cDNA clones for Nramp2 indicate that this novel
Samantha Gruenheid; Mathieu Cellier; Silvia Vidal; Philippe Gros
Human monocyte�mouse melanoma fusion hybrids express human gene
Artificial fusion of human monocyte with Cloudman S91 mouse melanoma cells resulted in hybrids that showed increased motility in vitro, enhanced metastatic potential in vivo, and also tended to be super melanotic (Rachkovsky et al., Clin. Exp. Metastasis 16 (1998) 299). However, no gene derived from monocytes has been shown to be expressed in these hybrids until now. Similar observations
Ashok K. Chakraborty; Josany de Freitas Sousa; Enilza M. Espreafico; John M. Pawelek
Direct Gene Transfer into Mouse Muscle in Vivo
RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in
Jon A. Wolff; Robert W. Malone; Phillip Williams; Wang Chong; Gyula Acsadi; Agnes Jani; Philip L. Felgner
Genes and gene expression modules associated with caloric restriction and aging in the laboratory mouse
BACKGROUND: Caloric restriction (CR) counters deleterious effects of aging and, for most mouse genotypes, increases mean and maximum lifespan. Previous analyses of microarray data have identified gene expression responses to CR that are shared among multiple mouse tissues, including the activation of anti-oxidant, tumor suppressor and anti-inflammatory pathways. These analyses have provided useful research directions, but have been restricted to
William R Swindell
Ecological Risk Assessment of Alfalfa Medicago Varia L.) Genetically Engineered to Express a Human Metallothionein ( hMT ) Gene
The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO4 by addition of CuSO4 solutions to soil and (2) to identify potential unintended effects of the genetic engineering
Lidia S. Watrud; Santosh Misra; Leshitew Gedamu; Tamotsu Shiroyama; Sharon Maggard; George Di Giovanni
Inducible and reversible regulation of endogenous gene in mouse.
Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene's promoter region turns the gene on and off in a tetracycline-inducible manner. Nmyc(TRE) mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. Nmyc(TRE) mice were crossed to tTS transgenic mice to produce Nmyc(TRE/TRE): tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS-dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed. PMID:22879379
Sun, Ruilin; Zhao, Kai; Shen, Ruling; Cai, Lei; Yang, Xingyu; Kuang, Ying; Mao, Jifang; Huang, Fang; Wang, Zhugang; Fei, Jian
The mouse (Mus musculus) T cell receptor delta variable (TRDV), diversity (TRDD) and joining (TRDJ) genes.
'The Mouse (Mus musculus) T Cell Receptor Delta Variable (TRDV), Diversity (TRDD) and Joining (TRDJ) Genes', the 15th report of the 'IMGT Locus in Focus' section, comprises 7 tables entitled: (1) 'Number of mouse (Mus musculus) germline TRDV genes at 14D1-D2 and potential repertoire'; (2) 'Mouse (Mus musculus) germline TRDV genes at 14D1-D2'; (3) 'Mouse (Mus musculus) TRDV allele table'; (4) 'Mouse (Mus musculus) germline TRDD genes and alleles'; (5) 'Mouse (Mus musculus) germline TRDJ genes'; (6) 'Mouse (Mus musculus) TRDJ allele table', and (7) 'Correspondence between the different mouse (Mus musculus) TRDV gene nomenclatures'. These tables are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cines. fr:8104) created by Marie-Paule Lefranc, Universit� Montpellier II, CNRS, Montpellier, France. PMID:11150853
Bosc, N; Contet, V; Lefranc, M P
Cellular Functions of Genetically Imprinted Genes in Human and Mouse as Annotated in the Gene Ontology
By analyzing the cellular functions of genetically imprinted genes as annotated in the Gene Ontology for human and mouse, we found that imprinted genes are often involved in developmental, transport and regulatory processes. In the human, paternally expressed genes are enriched in GO terms related to the development of organs and of anatomical structures. In the mouse, maternally expressed genes regulate cation transport as well as G-protein signaling processes. Furthermore, we investigated if imprinted genes are regulated by common transcription factors. We identified 25 TF families that showed an enrichment of binding sites in the set of imprinted genes in human and 40 TF families in mouse. In general, maternally and paternally expressed genes are not regulated by different transcription factors. The genes Nnat, Klf14, Blcap, Gnas and Ube3a contribute most to the enrichment of TF families. In the mouse, genes that are maternally expressed in placenta are enriched for AP1 binding sites. In the human, we found that these genes possessed binding sites for both, AP1 and SP1.
Hamed, Mohamed; Ismael, Siba; Paulsen, Martina; Helms, Volkhard
Mouse chromosome 17 candidate modifier genes for thrombosis
Two overlapping quantitative trait loci (QTLs) for clot stability, Hmtb8 and Hmtb9, were identified on mouse chromosome 17 in an F2 intercross derived from C57BL/6J (B6) and B6-Chr17A/J (B6-Chr17) mouse strains. The intervals were in synteny with a QTL for thrombotic susceptibility on chromosome 18 in a human study, and there were 23 homologs between mouse and human. The objective of this study was to determine whether any of these genes in the syntenic region are likely candidates as modifiers for clot stability. Seven genes, Twsg1, Zfp161, Dlgap1, Ralbp1, Myom1, Rab31, and Emilin2, of the 23 genes with single nucleotide polymorphisms (SNPs) in the mRNA-UTR had differential expression in B6 and A/J mice. Dlgap1, Ralbp1, Myom1, and Emilin2 also had nonsynonymous SNPs. In addition, two other genes had nonsynonymous SNPs, Lama1 and Ndc80. Of these nine candidate genes, Emilin2 was selected for further analysis since other EMILIN (Elastin Microfibril Interface Located Protein) proteins have known functions in vascular structure and coagulation. Differences were found between B6 and A/J mice in vessel wall architecture and EMILIN2 protein in plasma, carotid vessel wall, and thrombi formed after ferric chloride injury. In B6-Chr17A/J mice both clot stability and Emilin2 mRNA expression were higher compared to those in B6 and A/J mice, suggesting the exposure of epistatic interactions. Although other homologous genes in the QTL region cannot be ruled out as causative genes, further investigation of Emilin2 as a candidate gene for thrombosis susceptibility is warranted. Electronic supplementary material The online version of this article (doi:10.1007/s00335-010-9274-6) contains supplementary material, which is available to authorized users.
Sa, Qila; Hart, Erika; Nadeau, Joseph H.
Identification and characterization of a second mouse Nramp gene
Nramp gene was isolated as a candidate for the host resistance locus Bcg/Ity/Lsh, which controls natural resistance of mice to several types of infections. We have isolated by cross-hybridization cDNA clones corresponding to a second mouse Nramp gene, which we designate Nramp2. Nucleotide and predicted amino acid sequence analyses of full-length cDNA clones for Nramp2 indicate that this novel Nramp protein is closely homologous to the previously described Nramp and that the two genes form part of a small gene family. The two Nramp proteins encode integral membrane proteins that share 63% identical residues and an overall homology of 78%. They share very similar secondary structure, including identical hydropathy profiles and predicted membrane organization, with a minimum of 10 and most probably 12 transmembrane domains, a cluster of predicted N-linked glycosylation sites, and a consensus transport motif. Analysis of the distribution of Nramp2 mRNA transcripts in normal mouse tissues by Northern blotting revealed that the Nramp2 gene produces several mRNAs, including prominent 3.3- and 2.3-kb species generated by the use of alternative polyadenylation signals. In contrast to the previously described macrophage-specific Nramp gene, Nramp2 mRNAs were found to be expressed at low levels in all tissues tested. Using a polymorphic (GT)26 dinucleotide repeat identified in the 3{prime} untranslated region of the mRNA, we have mapped the Nramp2 gene to the distal part of mouse chromosome 15 between markers D15Mit41 and D15Mit15, with the gene order and intergene distance (in cM): centromere-56.1-D15Mit41-(1{+-}1)-Nramp2-(5{+-}2)-D15Mit15. 59 refs., 5 figs., 1 tab.
Gruenheid, S.; Cellier, M.; Vidal, S. [McGill Univ., Montreal, Quebec (Canada)] [and others] [McGill Univ., Montreal, Quebec (Canada); and others
Sexual dimorphisms in zonal gene expression in mouse liver.
Many of the metabolic functions of the liver are localized either in the pericentral region (zone 3) or in the periportal region (zone 1). However, a systematic analysis of the heterogeneity and sexual dimorphism of gene expression in the liver is lacking. Our objective was to obtain sections of intact tissue from zone 1 and zone 3 from both male and female mouse liver, and to measure the patterns of gene expression in these sections. Zone 1 and zone 3 areas were isolated by laser capture microdissection of liver sections, total RNA was isolated and microarray analysis was conducted using Agilent Whole Mouse Genome oligo arrays. To investigate functional characteristics as well as upstream regulators of specific gene lists, we used Ingenuity Pathway Analysis. We identified more than 925 genes in zone 1 and more than 450 genes in zone 3 of both male and female mice. Sexual dimorphism in metabolic functions was present in zone 1 but not zone 3. In zone 1, canonical pathways related to gluconeogenesis were male predominant, while canonical pathways related to hepatic progenitor cells were female predominant. In addition, we also analyzed the upstream regulators of zone-specific genes. SREBF1 was male-specific in zone 1, while TRIM24 was female-specific in zone 3. These results demonstrate the heterogeneity and sexually dimorphic differences in gene expression in the liver. PMID:23791742
Saito, Kosuke; Negishi, Masahiko; James Squires, E
Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins
Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least\\u000a five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which\\u000a has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for
Roger S. Holmes; Matthew W. Wright; Stanley J. F. Laulederkind; Laura A. Cox; Masakiyo Hosokawa; Teruko Imai; Shun Ishibashi; Richard Lehner; Masao Miyazaki; Everett J. Perkins; Phillip M. Potter; Matthew R. Redinbo; Jacques Robert; Tetsuo Satoh; Tetsuro Yamashita; Bingfan Yan; Tsuyoshi Yokoi; Rudolf Zechner; Lois J. Maltais
EMAGE mouse embryo spatial gene expression database: 2014 update.
EMAGE (http://www.emouseatlas.org/emage/) is a freely available database of in situ gene expression patterns that allows users to perform online queries of mouse developmental gene expression. EMAGE is unique in providing both text-based descriptions of gene expression plus spatial maps of gene expression patterns. This mapping allows spatial queries to be accomplished alongside more traditional text-based queries. Here, we describe our recent progress in spatial mapping and data integration. EMAGE has developed a method of spatially mapping 3D embryo images captured using optical projection tomography, and through the use of an IIP3D viewer allows users to view arbitrary sections of raw and mapped 3D image data in the context of a web browser. EMAGE now includes enhancer data, and we have spatially mapped images from a comprehensive screen of transgenic reporter mice that detail the expression of mouse non-coding genomic DNA fragments with enhancer activity. We have integrated the eMouseAtlas anatomical atlas and the EMAGE database so that a user of the atlas can query the EMAGE database easily. In addition, we have extended the atlas framework to enable EMAGE to spatially cross-index EMBRYS whole mount in situ hybridization data. We additionally report on recent developments to the EMAGE web interface, including new query and analysis capabilities. PMID:24265223
Richardson, Lorna; Venkataraman, Shanmugasundaram; Stevenson, Peter; Yang, Yiya; Moss, Julie; Graham, Liz; Burton, Nicholas; Hill, Bill; Rao, Jianguo; Baldock, Richard A; Armit, Chris
Mapping of the mouse Tdgf1 gene and Tdgf pseudogenes.
Teratocarcinoma-derived growth factor-1 (Tdgf1), a member of the "EGF family" of growth factors, is expressed during mouse gastrulation in the forming mesoderm and later in the truncus arteriosus of the developing heart. In humans, TDGF1 is highly expressed in germ cell tumors and in colon and mammary carcinomas. In mouse, one gene (Tdgf1) and two pseudogenes (Tdgf1-ps1 and Tdgf1-ps2) have been isolated and characterized. Tdgf1 corresponds to the gene expressed in F9 teratocarcinoma cells. Tdgf1-ps1 and Tdgf1-ps2 are two intronless sequences with all the characteristics of retroposons. In the present paper, we assign the chromosomal location for Tdgf1, Tdgf1-ps1, and Tdgf1-ps2 sequences to Chromosomes (Chrs) 9, 16, and 17, respectively. Two previously described mouse mutants, scant hair (sch) and fur deficient (fd), map near the Tdgf1 gene. Analysis of their DNA coding region provided no evidence that Tdgf1 could be the responsible gene for these phenotypes. Finally, analysis of the DNA from several Mus musculus strains and from Mus spretus mice revealed a highly variable restriction pattern and the absence of the Tdgf1-ps1 genomic sequence from the Mus spretus genome. PMID:9195995
Liguori, G; De Gregorio, L; Tucci, M; Lago, C T; Barra, A; Dragani, T A; Persico, M
A new spontaneous mouse mutation in the Kcne1 gene.
A new mouse mutant, punk rocker (allele symbol Kcne1(pkr)), arose spontaneously on a C57BL/10J inbred strain background and is characterized by a distinctive head-tossing, circling, and ataxic phenotype. It is also profoundly and bilaterally deaf. The mutation resides in the Kcne1 gene on Chromosome (Chr) 16 and has been identified as a single base change within the coding region of the third exon. The C to T nucleotide substitution causes an arginine to be altered to a termination codon at amino acid position 67, and predictably this will result in a significantly truncated protein product. The Kcne1(pkr) mutant represents the first spontaneous mouse model for the human disorder, Jervell and Lange-Nielsen syndrome, associated with mutations in the homologous KCNE1 gene on human Chr 21. PMID:11003695
Letts, V A; Valenzuela, A; Dunbar, C; Zheng, Q Y; Johnson, K R; Frankel, W N
A new spontaneous mouse mutation in the Kcne1 gene
A new mouse mutant, punk rocker (allele symbol Kcne1pkr), arose spontaneously on a C57BL/10J inbred strain background and is characterized by a distinctive head-tossing, circling, and ataxic phenotype. It is also profoundly and bilaterally deaf. The mutation resides in the Kcne1 gene on Chromosome (Chr) 16 and has been identified as a single base change within the coding region of the third exon. The C to T nucleotide substitution causes an arginine to be altered to a termination codon at amino acid position 67, and predictably this will result in a significantly truncated protein product. The Kcne1pkr mutant represents the first spontaneous mouse model for the human disorder, Jervell and Lange-Nielsen syndrome, associated with mutations in the homologous KCNE1 gene on human Chr 21.
Letts, V.A.; Valenzuela, A.; Dunbar, C.; Zheng, Q.Y.; Johnson, K.R.; Frankel, W.N.
Cloning and chromosome localization of the mouse Ews gene
The human EWS gene encodes a putative RNA binding protein. As a result of acquired chromosome rearrangement, the N-terminal portion of the EWS protein is fused to the DNA binding domain of either FLI1-or ERG in the Ewing family of tumors and to the DNA binding domain of ATF1 in malignant melanoma of soft parts. We have determined the cDNA sequence of the mouse Ews gene. Its nucleotide sequence and its translation product demonstrate 93 and 98% homology with the human EWS cDNA and protein, respectively. The murine Ews locus lies within a conserved synteny segment between human chromosome 22q12 and mouse chromosome 11A1-A3.
Plougastel, B.; Thomas, G.; Delattre, O. [Laboratoire de Genetique des Tumeurs, Paris (France)] [Laboratoire de Genetique des Tumeurs, Paris (France); Mattei, M.G. [Faculte de Medicine de la Timone, Marseille (France)] [Faculte de Medicine de la Timone, Marseille (France)
Metallothioneins in human tumors and potential roles in carcinogenesis
Metallothioneins (MT) are a group of low-molecular weight, cysteine rich intracellular proteins, which are encoded by a family of genes containing at least 10 functional isoforms in human. The expression and induction of these proteins have been associated with protection against DNA damage, oxidative stress and apoptosis. Moreover, MT may potentially activate certain transcriptional factors by donating zinc. Although MT
M. George Cherian; A. Jayasurya; Boon-Huat Bay
Metallothionein expression in human neoplasia.
The metallothionein family is a class of low-molecular-weight, cysteine-rich proteins with high affinity for metal ions. Four major isoforms (metallothionein-1, -2, -3, and -4) have been identified in mammals, involved in many pathophysiological processes, including metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, drug and radiotherapy resistance and several aspects of the carcinogenic process. In the present review we examine the expression of metallothionein in different human tumours and its correlation with histopathological variables, tumour cell proliferation or apoptosis, resistance to radiation or chemotherapy, patient survival and prognosis. A variable profile of metallothionein and its isoforms' expression has been observed in different cancer types. Although metallothionein expression has been implicated in carcinogenic evolution, its use as a marker of tumour differentiation, cell proliferation and prognosis predictor remains unclear. Detailed studies focused on the expression of metallothionein isoforms and isotypes in different tumour types could elucidate the role of this group of proteins in the carcinogenic process, delineating its possible clinical significance for the management of patients. PMID:15279628
Theocharis, S E; Margeli, A P; Klijanienko, J T; Kouraklis, G P
Expression of copper trafficking genes in the mouse brain.
Copper homeostasis in the brain must be strictly maintained, since copper is an essential trace element and is potentially toxic. To understand the mechanism of copper homeostasis in the brain, we cloned several mouse homologues of copper trafficking genes and performed in situ hybridization histochemistry. mCTR1, mATX1, and mATP7a were highly expressed in the choroid plexus, indicating that the choroid plexus uses the trafficking pathway from uptake to efflux to transport copper to the cerebrospinal fluids. We suggest that these genes may regulate copper concentration in the brain through the choroid plexus. PMID:9831461
Nishihara, E; Furuyama, T; Yamashita, S; Mori, N
Update of the human and mouse SERPIN gene superfamily.
The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease. PMID:24172014
Heit, Claire; Jackson, Brian C; McAndrews, Monica; Wright, Mathew W; Thompson, David C; Silverman, Gary A; Nebert, Daniel W; Vasiliou, Vasilis
Update of the human and mouse SERPIN gene superfamily
The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease.
Mouse lysozyme M gene: isolation, characterization, and expression studies.
We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels. Images
Cross, M; Mangelsdorf, I; Wedel, A; Renkawitz, R
Genetic mapping of tumor susceptibility genes involved in mouse plasmacytomagenesis
Plasmacytomas (PCTs) were induced in 47% of BALB/cAnPt mice by the intraperitoneal injection of pristane, in 2% of (BALB/c [times] DBA/2N)F[sub 1], and in 11% of 773 BALB/cAnPt [times] (BALB/cAnPt [times] DBA/2N)F[sub 1]N[sub 2] backcross mice. This result indicates a multigenic mode of inheritance for PCT susceptibility. To locate genes controlling this complex genetic trait, tumor susceptibility in backcross progeny generated from BALB/c and DBA/2N (resistant) mice was correlated with alleles of 83 marker loci. The genotypes of the PCT-susceptible progeny displayed an excess homozygosity for BALB/c alleles with a 32-centimorgan stretch of mouse chromosome 4 (>95% probability of linkage) with minimal recombination (12%) near Gt10. Another susceptibility gene on mouse chromosome 1 may be linked to Fcgr2 (90% probability of linkage); there were excess heterozygotes for Fcgr2 among the susceptible progeny and excess homozygotes among the resistant progeny. Regions of mouse chromosomes 4 and 1 that are correlated with PCT susceptibility share extensive linkage homology with regions of human chromosome 1 that have been associated with cytogenetic abnormalities in multiple myeloma and lymphoid, breast, and endocrine tumors. 68 refs., 2 figs., 1 tab.
Mock, B.A.; Krall, M.M.; Dosik, J.K. (National Institutes of Health, Bethesda, MD (United States))
Epigenetic interplay between mouse endogenous retroviruses and host genes
Background Transposable elements are often the targets of repressive epigenetic modifications such as DNA methylation that, in theory, have the potential to spread toward nearby genes and induce epigenetic silencing. To better understand the role of DNA methylation in the relationship between transposable elements and genes, we assessed the methylation state of mouse endogenous retroviruses (ERVs) located near genes. Results We found that ERVs of the ETn/MusD family show decreased DNA methylation when near transcription start sites in tissues where the nearby gene is expressed. ERVs belonging to the IAP family, however, are generally heavily methylated, regardless of the genomic environment and the tissue studied. Furthermore, we found full-length ETn and IAP copies that display differential DNA methylation between their two long terminal repeats (LTRs), suggesting that the environment surrounding gene promoters can prevent methylation of the nearby LTR. Spreading from methylated ERV copies to nearby genes was rarely observed, with the regions between the ERVs and genes apparently acting as a boundary, enriched in H3K4me3 and CTCF, which possibly protects the unmethylated gene promoter. Furthermore, the flanking regions of unmethylated ERV copies harbor H3K4me3, consistent with spreading of euchromatin from the host gene toward ERV insertions. Conclusions We have shown that spreading of DNA methylation from ERV copies toward active gene promoters is rare. We provide evidence that genes can be protected from ERV-induced heterochromatin spreading by either blocking the invasion of repressive marks or by spreading euchromatin toward the ERV copy.
Mammalian MT1 and MT2 metallothioneins differ in their metal binding abilities.
Metallothioneins (MTs) constitute a universal family of polymorphic, ubiquitous small Cys-rich metal-binding polypeptides that in mammals are represented by four highly similar isoforms (MT1 to MT4). MT1 and MT2 have generally been considered as equivalent proteins, so that they are commonly referred to as MT1/MT2. However, transcription data have suggested a differential behavior for both gene products. In the present study, the metal binding abilities of mouse MT2 (mMT2) with divalent (Zn(II) and Cd(II)) and monovalent (Cu(I)) ions were analyzed and compared to those of the mouse MT1 (mMT1) isoform, previously determined using the same methodological approach. The comprehensive consideration of all the results obtained in this work experimentally demonstrates that the mMT2 isoform exhibits metal ion binding abilities distinct from those of mMT1, with a clear preference for Zn(II) coordination, if compared to Cu(I) or even to Cd(II). This is in full agreement with the gene expression regulation pattern for the MT1 and MT2 genes, as well as with the hypothesized preferential role of mMT2 in Zn(II) homeostasis mechanisms, while MT1, possibly differentiated from a most recent duplication event in the mammalian MT gene cluster, would have evolved to detoxify Cd(II), and probably other divalent metal ions. PMID:23925449
Artells, Ester; Palacios, �scar; Capdevila, Merc�; Atrian, S�lvia
Maternal effect genes: Findings and effects on mouse embryo development
Stored maternal factors in oocytes regulate oocyte differentiation into embryos during early embryonic development. Before zygotic gene activation (ZGA), these early embryos are mainly dependent on maternal factors for survival, such as macromolecules and subcellular organelles in oocytes. The genes encoding these essential maternal products are referred to as maternal effect genes (MEGs). MEGs accumulate maternal factors during oogenesis and enable ZGA, progression of early embryo development, and the initial establishment of embryonic cell lineages. Disruption of MEGs results in defective embryogenesis. Despite their important functions, only a few mammalian MEGs have been identified. In this review we summarize the roles of known MEGs in mouse fertility, with a particular emphasis on oocytes and early embryonic development. An increased knowledge of the working mechanism of MEGs could ultimately provide a means to regulate oocyte maturation and subsequent early embryonic development.
Kim, Kyeoung-Hwa
Fibrillin genes map to regions of conserved mouse/human synteny on mouse chromosomes 2 and 18.
Fibrillin proteins are major structural components of the 10-nm microfibrils found in elastic and nonelastic connective tissues. Previous studies have mapped the human genes for two fibrillins to chromosome bands 15q21 (FBN1) and 5q23-q31 (FBN2) and have demonstrated that FBN1 mutations are associated with Marfan syndrome, while FBN2 is linked to the gene for congenital contractural arachnodactyly. Here, we report the isolation of genomic clones of the corresponding mouse fibrillin genes (Fbn-1 and Fbn-2). By analyzing a mapping panel of mouse x rodent somatic hybrid cell lines, we have assigned the Fbn-1 gene to mouse chromosome 2 and the Fbn-2 gene to mouse chromosome 18. We then sublocalized the fibrillin genes to bands 2F (Fbn-1) and 18D-E1 (Fbn-2) by fluorescence in situ hybridization. These regions are known to exhibit conserved synteny with the regions on human chromosomes 15 and 5 that carry the homologous human fibrillin genes. In addition, the Fbn-1 gene maps in the vicinity of the gene for a connective tissue disorder on mouse chromosome 2 called Tight-skin (Tsk). PMID:8307578
Li, X; Pereira, L; Zhang, H; Sanguineti, C; Ramirez, F; Bonadio, J; Francke, U
The mouse angiogenin gene family: Structures of an angiogenin-related protein gene and two pseudogenes
Angiogenin, a homologue of pancreatic ribonuclease, is a potent inducer of blood vessel formation. As an initial step toward investigating the in vivo functional role of this protein via gene disruption, we undertook the isolation of the angiogenin gene (Ang) from the 129 strain mouse, which will be used for generating targeting constructs. Unexpectedly, screening of a genomic library with an Ang gene probe obtained previously from the BALB/c strain yielded two new genes closely similar to Ang rather than Ang itself. One of these encodes a protein with 78% sequence identity to angiogenin and is designated {open_quotes}Angrp{close_quotes} for {open_quotes}angiogenin-related protein.{close_quotes} The ribonucleolytic active site of angiogenin, which is critical for angiogenic activity, is completely conserved in Angrp, whereas a second essential site, thought to bind cellular receptors, is considerably different. Thus, the Angrp product may have a function distinct from that of angiogenin. The second gene obtained by library screening is a pseudogene, designated {open_quotes}Ang-ps1,{close_quotes} that contains a frame shift mutation in the early part of the coding region. Although the Ang gene was not isolated from this library, it was possible to amplify this gene from 129 mouse genomic DNA by the polymerase chain reaction (PCR). Sequence analysis showed that the 129 strain Ang gene is identical to the BALB/c gene throughout the coding region. PCR cloning also yielded a second Ang-like pseudogene, designated {open_quotes}Ang-ps2.{close_quotes} Southern blotting of genomic DNA confirmed the presence of Ang, Angrp, and at least one of the pseudogenes in an individual mouse and suggested that the mouse Ang gene family may contain more than the four members identified here. 31 refs., 4 figs., 1 tab.
Brown, W.E.; Nobile, V.; Shapiro, R. [Harvard Medical School, Boston, MA (United States)] [and others] [Harvard Medical School, Boston, MA (United States); and others
tRNA genes protect a reporter gene from epigenetic silencing in mouse cells
It is a well-established fact that the tRNA genes in yeast can function as chromatin barrier elements. However, so far there is no experimental evidence that tRNA and other Pol III-transcribed genes exhibit barrier activity in mammals. This study utilizes a recently developed reporter gene assay to test a set of Pol III-transcribed genes and gene clusters with variable promoter and intergenic regions for their ability to prevent heterochromatin-mediated reporter gene silencing in mouse cells. The results show that functional copies of mouse tRNA genes are effective barrier elements. The number of tRNA genes as well as their orientation influence barrier function. Furthermore, the DNA sequence composition of intervening and flanking regions affects barrier activity of tRNA genes. Barrier activity was maintained for much longer time when the intervening and flanking regions of tRNA genes were replaced by AT-rich sequences, suggesting a negative role of DNA methylation in the establishment of a functional barrier. Thus, our results suggest that tRNA genes are essential elements in establishment and maintenance of chromatin domain architecture in mammalian cells.
Ebersole, Thomas; Kim, Jung-Hyun; Samoshkin, Alexander; Kouprina, Natalay; Pavlicek, Adam; White, Robert J
A new Pax gene, Pax9 , maps to mouse Chromosome 12
Members of the Pax gene family have recently been shown to play important roles in mouse embryogenesis. Of eight so far characterized Pax genes, three have been associated with mouse developmental mutants. Here we report the cloning of a new Pax gene, Pax-9. Most of the DNA sequence encoding the highly conserved paired domain has been determined and compared with
Johan Wallin; Yoko Mizutani; Kenji Imai; Nobumotu Miyashita; Kazuo Moriwaki; Masaru Taniguchi; Haruhiko Koseki; Rudi Balling
In vitro Methylation of the Hamster Adenine Phosphoribosyltransferase Gene Inhibits Its Expression in Mouse L Cells
The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector.
R. Stein; A. Razin; H. Cedar
Human Jk recombination signal binding protein gene (IGKJRB): Comparison with its mouse homologue
The mouse Igkjrb protein specifically binds to the immunoglobulin Jk recombination signal sequence. The IGKJRB gene is highly conserved among many species such as human, Xenopus, and Drosophila. Using cDNA fragments of the mouse Igkjrb gene, the authors isolated its human counterpart, IGKJRB. The human genome contains one functional IGKJRB gene and two types of processed pseudogenes. In situ chromosome
Ryuichi Amakawa; Wu Jing; Norisada Matsunami; Yasushi Hamaguchi; Fumihiko Matsuda; Masashi Kawaichi; Tasuku Honjo; Kazuo Ozawa
Tissue specific expression of mouse alpha-amylase genes.
Two distinct alpha-amylase genes, Amy-1a and Amy-2a, are expressed in the mouse strain A/J. Amy-1a and Amy-2a are interrupted by 10 and 9 introns, respectively. With the exception of the first Amy-1a intron, which has no counterpart in Amy-2a, introns are located at analogous positions within the two genes. Comparable exons of Amy-1a and Amy-2a are more highly conserved in sequence than analogous introns. Amy-2a specifies pancreatic alpha-amylase mRNA. Two apparently identical copies of this gene exist in the haploid mouse genome. The single copy of Amy-1a is expressed in a tissue specific fashion in the salivary gland and the liver. It specifies alpha-amylase mRNA with identical translated and 3' nontranslated sequences but different 5' nontranslated sequences in the two tissues. These different mRNAs are generated by tissue specific splicing events. S1 nuclease mapping of nuclear transcripts from salivary gland and liver suggests the presence of at least two promotors of different strength in Amy-1a. A strong promoter appears to be active in the salivary gland exclusively, while a weak promoter is apparently used in both the salivary gland and the liver. The data suggest that regulation of Amy-1a expression occurs primarily at the transcriptional level. PMID:6186131
Schibler, U; Hagenb�chle, O; Young, R A; Tosi, M; Wellauer, P K
ATM localization and gene expression in the adult mouse eye
Purpose High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Methods Atm gene expression was analyzed by RT�PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. Results Using RT�PCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. Conclusions We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity.
Leemput, Julia; Masson, Christel; Bigot, Karine; Errachid, Abdelmounaim; Dansault, Anouk; Provost, Alexandra; Gadin, Stephanie; Aoufouchi, Said; Menasche, Maurice
Genomic Cloning of Mouse MIF (Macrophage Inhibitory Factor) and Genetic Mapping of the Human and Mouse Expressed Gene and Nine Mouse Pseudogenes
The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon\\/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus musculus or Mus spretus. Nine additional loci containing related sequences,
Christine A. Kozak; M. Charlene Adamson; Charles E. Buckler; Lorenzo Segovia; Vishwas Paralkar; Graeme Wistow
Mucin gene expression and mouse middle ear epithelium
Objectives To investigate the expression of recently identified human mucin genes in an in vitro model of cultured mouse middle ear epithelial cells (MMEEC). Methods MMEEC were established, RNA was extracted and primers were designed for RT-PCR to assess for expression of mucin genes Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc6, Muc7, Muc8, Muc9, Muc10, Muc11/12, Muc13, Muc15, Muc16, Muc17, Muc18, Muc19 and Muc20 expression. Results Mucin genes Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc9, Muc10, Muc13, Muc15, Muc16, Muc18, Muc19 and Muc20 were identified and expressed in MMEEC. The genes Muc6, Muc7, Muc8, Muc11/12 and Muc17 were not identified. Conclusion Many of the mucin genes that have been recently identified in human MEE and chinchilla MEE are also expressed in MMEEC. There are differences in expression, however, which may have implications in utilizing various animal models for study of middle ear physiology and pathogenesis; specifically as it relates to mucin gene expression.
Kerschner, Joseph E.; Lin, Jizhen; Tsushiya, Katsuyuki; Khampang, P.
PHYTOCHELATINS AND METALLOTHIONEINS: Roles in Heavy Metal Detoxification and Homeostasis
? Abstract Among,the heavy metal-binding ligands in plant cells the phytochelatins (PCs) and metallothioneins,(MTs) are the best characterized. PCs and MTs are different classes of cysteine-rich, heavy metal-binding protein molecules. PCs are enzymatically synthesized peptides, whereas MTs are gene-encoded polypeptides. Recently, genes encoding,the enzyme,PC synthase have been identified in plants and other species while the completion,of the Arabidopsis genome,sequence,has allowed
Christopher Cobbett; Peter Goldsbrough
Identification of a Novel SCD Gene and Expression of the SCD Gene Family in Mouse Skin
We have refined the position of asebia locus by genotyping DNA from more than 600 backcross mice derived from asebia mouse and a genetically unrelated strain. One of the candidate genes in the locus is stearoyl-CoA desaturase (SCD). Previously two members of this gene family, namely SCD1 and SCD2, have been described. We have found, for the first time, that
Satish Parimoo; Ying Zheng; Ken Eilertsen; Lan Ge; Stephen Prouty; John Sundberg; Kurt Stenn; Sathish Parimmo
Genetic Mapping of Four Mouse bHLH Genes Related to DrosophilaProneural Gene atonal
It has been shown that mammalian neurogenesis is partly controlled by multiple basic helix�loop�helix (bHLH) genes, as inDrosophila.Recently, mouse homologs ofDrosophila atonal,a proneural gene encoding a bHLH protein required for chordotonal organ and photoreceptor development, have been characterized to obtain further insights into the molecular nature of mammalian neurogenesis. Here, to assess their potential involvement in genetic neural disorders, we
Fumiaki Isaka; Chikara Shimizu; Shigetada Nakanishi; Ryoichiro Kageyama
Screening Helicobacter pylori genes induced during infection of mouse stomachs
AIM: To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H. pylori) as it relates to its survival in the host. METHODS: In vivo expression technology (IVET) systems are used to identify microbial virulence genes. We modified the IVET-transcriptional fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors, pIVET11 and pIVET12. Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H. pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase. Additionally, each vector contains a kanamycin resistance gene. We used a mouse macrophage cell line, RAW 264.7 and mice, as selective media to identify specific genes that H. pylori expresses in vivo. Gene expression studies were conducted by infecting RAW 264.7 cells with H. pylori. This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes. RESULTS: In this study, we have identified 31 in vivo induced (ivi) genes in the initial screens. These 31 genes belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Their detection validates the efficacy of these screening systems. Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H. pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264.7 coculture with that of H. pylori only. Some genes such as cagA, vacA, lpxC, murI, tlpC, trxB, sodB, tnpB, pgi, rbfA and infB showed a 2-20 fold upregulation. Statistically significant upregulation was obtained for all the above mentioned genes (P < 0.05). tlpC, cagA, vacA, sodB, rbfA, infB, tnpB, lpxC and murI were also significantly upregulated (P < 0.01). These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal. CONCLUSION: The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H. pylori gene expression in the host environment.
Singh, Aparna; Hodgson, Nathaniel; Yan, Ming; Joo, Jungsoo; Gu, Lei; Sang, Hong; Gregory-Bryson, Emmalena; Wood, William G; Ni, Yisheng; Smith, Kimberly; Jackson, Sharon H; Coleman, William G
Expression patterns of metallothionein, cytochrome P450 1A and vitellogenin genes in western mosquitofish (Gambusia affinis) in response to heavy metals.
The aim of this study was to evaluate the effects of three metals (Zn, Cd and Pb) on hepatic metallothionein (MT), cytochrome P450 1A (CYP1A) and vitellogenin (Vtg) mRNA expression in the liver of adult female mosquitofish (Gambusia affinis) after 1, 3 or 8d. Both concentration-response and time-course effects of hepatic MT, CYP1A and Vtg at the transcription level were determined by quantitative real-time PCR. The results from this study showed that Zn, Cd and Pb could significantly induced MT, CYP1A and Vtg mRNA expression levels in mosquitofish. In general, this study demonstrated that heavy metals modulate MT, CYP1A and Vtg mRNA expression levels in a metal-, concentration- or time-dependent manner. PMID:24793519
Huang, Guo-Yong; Ying, Guang-Guo; Liang, Yan-Qiu; Liu, Shuang-Shuang; Liu, You-Sheng
A combination of derepression of the lac operator-repressor system with positive induction by glucocorticoid and metal ions provides a high-level-inducible gene expression system based on the human metallothionein-IIA promoter.
We and others have introduced the use of the lac operator-repressor system as a method for providing inducible gene expression for gene transfer experiments in animal cells (M. C.-T. Hu, and N. Davidson, Cell 48:555-566, 1987; J. Figge, C. Wright, C. J. Collins, T. M. Roberts, and D. M. Livingston, Cell 52:713-722, 1988). To improve the dynamic range of such an inducible system, we have investigated the effects of combining the relief by isopropyl-beta-D-thiogalactoside (IPTG) of negative control by the lac system with positive induction by the natural inducers glucocorticoids and cadmium ion for a system based on the human metallothionein-IIA gene promoter. We used the chloramphenicol acetyltransferase gene as a reporter gene and inserted a lacO sequence into the promoter between the GC box and metal-responsive element 1, between metal-responsive element 1 and the TATA box, or between the TATA box and the transcription start site. Surprisingly, all of these insertions had a significant inhibitory effect on promoter activity even in the absence of repressor. However, with these lacO-containing constructs, the levels of gene expression after induction by glucocorticoid, Cd2+, or both were considerably reduced in cells engineered to express the lac repressor. Derepression by IPTG, coupled with induction by both dexamethasone and Cd2+ ion, then provided a high level of induced expression, i.e., by a factor of approximately 100 over the basal level of expression. However, inserting the lacO sequence well upstream just before the glucocorticoid-responsive element had much smaller effects on expression levels in both repressor-negative and repressor-positive cells. This study describes a new, high-level-inducible promoter system for gene transfer experiments. The observed effects are discussed in terms of current models of the mechanisms by which transcription factors control gene expression. Images
Hu, M C; Davidson, N
Viral gene transfer to developing mouse salivary glands.
Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis. PMID:22095070
Hsu, J C; Di Pasquale, G; Harunaga, J S; Onodera, T; Hoffman, M P; Chiorini, J A; Yamada, K M
Viral Gene Transfer to Developing Mouse Salivary Glands
Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.
Hsu, J.C.; Di Pasquale, G.; Harunaga, J.S.; Onodera, T.; Hoffman, M.P.; Chiorini, J.A.; Yamada, K.M.
Transcriptional termination sequences in the mouse serum albumin gene
Poly(A) signals are required for efficient 3? end formation and transcriptional termination of most protein-encoding genes transcribed by RNA polymerase II. However, transcription can extend far beyond the poly(A) site before termination occurs. This implies the existence of further downstream termination signals. In mammals, a variety of sequence elements, in addition to the poly(A) site, have been implicated in the termination process. For example, termination of the human ?- and ?-globin genes is mediated by a sequence downstream of the poly(A) site that promotes an RNA cotranscriptional cleavage (CoTC). Here we report the identification of multiple termination sequences in the mouse serum albumin (MSA) 3? flanking region. Many transcripts from this region are cleaved cotranscriptionally, implying that such cleavage of pre-mRNA may be a more general feature of transcriptional termination.
Multiple polyadenylation sites in a mouse alpha-amylase gene.
Two alpha-amylase mRNAs which differ in the length of their 3' non-translated region accumulate in the cytoplasm in both mouse liver and salivary gland tissues. The two species in each tissue are transcribed from the same gene (Amy-1A). The minor species is approximately 20-nucleotides preceding the poly(A) tract. Sequence analysis of genomic DNA shows that these extra 237 nucleotides are specified by sequences contiguous to those shared by the two mRNAs. These data demonstrate that transcription can proceed through the major polyadenylation site and that alternative polyadenylation sites are used in the Amy-1A gene. Sequences which trail the two polyadenylation sites exhibit extensive homology and might therefore be involved in polyadenylation or transcription termination. PMID:6166922
Tosi, M; Young, R A; Hagenb�chle, O; Schibler, U
Resveratrol induces insulin gene expression in mouse pancreatic ?-cells
Background Type 1 and type 2 diabetes are characterized by loss of ?-cells; therefore, ?-cell regeneration has become one of the primary approaches to diabetes therapy. Resveratrol, a naturally occurring polyphenolic compound, has been shown to improve glycaemic control in diabetic patients, but its action on pancreatic ?-cells is not well understood. Findings Using mouse ?-cells (?TC9), we showed that resveratrol induces expression of pancreatic ?-cell genes such as Pdx1 and Ins2 in a SirT1-dependent manner. The mRNA and protein levels of insulin were further increased by histone deacetylase (HDAC) inhibition. Conclusion In summary, we provide new mechanistic insight into the anti-diabetic action of resveratrol through its ability to express ?-cell genes in ?-cells.
Mouse Chromosomal location of four Na/H exchanger isoform genes
The Na/H exchanger genes Slc9a1, Slc9a2, Slc9a3, and Slc9a4 have been mapped in the mouse using an interspecific backcross panel. These loci map to previously defined homologous regions between human and mouse chromosomes and provide additional information regarding human/mouse comparative mapping. 13 refs., 1 fig., 1 tab.
Pathak, B.G.; Jenkins, N.A.; Copeland, N.G. [NCI-Frederick Cancer Research and Development Center, MD (United States)] [and others] [NCI-Frederick Cancer Research and Development Center, MD (United States); and others
Mouse chromosomal location of three epithelial sodium channel subunit genes and an apical sodium chloride cotransporter gene
The amiloride-sensitive epithelial sodium channel {alpha}, {beta}, and {gamma} subunit genes, Scnn1a, Scnn1b, and Scnn1g, and the thiazide-sensitive sodium chloride cotransporter gene, Slc12a1, have been mapped in the mouse using an interspecific backcross panel. These loci map to previously defined homologous regions between human and mouse chromosomes and provide additional information regarding human/mouse comparative mapping. 19 refs., 1 fig., 1 tab.
Pathak, B.G.; Shaughnessy, J.D. Jr.; Jenkins, N.A. [NCI-Frederick Cancer Research and Development Center, MD (United States)] [NCI-Frederick Cancer Research and Development Center, MD (United States)
Trmt112 Gene Expression in Mouse Embryonic Development
Mouse Trmt112, the homologous gene of yeast Trm112 (tRNA methyltransferase 11-2), was initially cloned from RIKEN with uncertain function. The yeast TRM112 is now known to play important roles in RNA methylation. Here, we studied the expression of Trmt112 by in situ hybridization and quantitative real-time RT-PCR (QRT-PCR). A higher expression level of Trmt112 was observed in the brain and nervous system by whole mount in situ hybridization from embryonic day 10.5 (E10.5) to E11.5. At later developmental stages E13.5 and E16.5, abundant expression was prominently found in various organs and tissues including developing brain, nervous system, thymus, lung, liver, intestine, kidney, and cartilage. Furthermore, Trmt112 was persistently expressed from E9.5 to E18.5 on whole embryos and highly expressed in multiple organs at E12.5, E15.5 and E18.5 by QRT-PCR. These results showed that Trmt112 gene was highly and ubiquitously expressed during mouse embryonic development, implying that it might be involved in the morphogenesis of diverse organs and tissues and numerous physiological functions.
Gu, Tiantian; He, Hongjuan; Zhang, Yan; Han, Zhengbin; Hou, Guangyuan; Zeng, Tiebo; Liu, Qi; Wu, Qiong
Increased metallothionein expression reflects steroid resistance in renal allograft recipients.
Steroid-refractory acute rejection is a risk factor for inferior renal allograft outcome. We aimed to gain insight into the mechanisms underlying steroid resistance by identifying novel molecular markers of steroid-refractory acute rejection. Eighty-three kidney transplant recipients (1995-2005), who were treated with methylprednisolone during a first acute rejection episode, were included in this study. Gene expression patterns were investigated in a discovery cohort of 36 acute rejection biopsies, and verified in a validation cohort of 47 acute rejection biopsies. In the discovery set, expression of metallothioneins (MT) was significantly (p < 0.000001) associated with decreased response to steroid treatment. Multivariate analysis resulted in a predictive model containing MT-1 as an independent covariate (AUC = 0.88, p < 0.0000001). In the validation set, MT-1 expression was also significantly associated with steroid resistance (p = 0.029). Metallothionein expression was detected in macrophages and tubular epithelial cells. Parallel to the findings in patients, in vitro experiments of peripheral blood mononuclear cells from 11 donors showed that nonresponse to methylprednisolone treatment is related to highly elevated MT levels. High expression of metallothioneins in renal allografts is associated with resistance to steroid treatment. Metallothioneins regulate intracellular concentrations of zinc, through which they may diminish the zinc-requiring anti-inflammatory effect of the glucocorticoid receptor. PMID:23763497
Rekers, N V; Bajema, I M; Mallat, M J K; Anholts, J D H; de Vaal, Y J H; Zandbergen, M; Haasnoot, G W; van Zwet, E W; de Fijter, J W; Claas, F H J; Eikmans, M
Attomole voltammetric determination of metallothionein
Number of authors have concerned with electrochemical analysis of metallothionein. Recently new electroanalytical techniques enabling determination of MT at picomole level has been suggested. The aim of the presented work was to show advantages and disadvantages of the different electrochemical procedures, which are commonly used for the detection of MT�(i) cyclic voltammetry, (ii) differential pulse voltammetry, and (iii) Brdicka reaction.
Jitka Petrlova; David Potesil; Radka Mikelova; Ondrej Blastik; Vojtech Adam; Libuse Trnkova; Frantisek Jelen; Richard Prusa; Jiri Kukacka; Rene Kizek
Gene Targeting of Mouse Tardbp Negatively Affects Masp2 Expression
Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function. To investigate this hypothesis, we attempted to generate a mouse conditional knockout of the Tardbp gene using the classical Cre-loxP technology. Even though heterozygote mice for the targeted allele were successfully generated, we were unable to obtain homozygotes. Here we show that although the targeting vector was specifically designed to not overlap with Tardbp adjacent genes, the homologous recombination event affected the expression of a downstream gene, Masp2. This may explain the inability to obtain homozygote mice with targeted Tardbp.
Dib, Samar; Xiao, Shangxi; Miletic, Denise; Robertson, Janice
Coping with cadmium exposure in various ways: the two helicid snails Helix pomatia and Cantareus aspersus share the metal transcription factor-2, but differ in promoter organization and transcription of their Cd-metallothionein genes.
Gastropods are able to withstand fluctuating availabilities of nonessential trace elements such as cadmium by induction of Cd-specific metallothionein isoform (Cd-MT) expression. As in other species, the induction mechanism involves the binding of metal-regulatory transcription factors (MTF-1 or MTF-2) to metal responsive elements (MREs) in the MT promoter regions. Cd-dependent transcription of Cd-MT genes was assessed by quantitative real time PCR in two helicid gastropods, Helix pomatia and Cantareus aspersus, over a period of eight days. The promoter regions of the Cd-MT genes of the two species were sequenced and compared regarding the position of MREs and other relevant potential transcription factor binding sites (TFBs). Cd-MT gene transcription is induced after Cd exposure in Helix pomatia and Cantareus aspersus, showing a transient peak in Helix pomatia, contrasting with a persistent induction rate in Cantareus aspersus. Since the existence of MTF-2 was verified in both species, differing transcription patterns of Cd-MT genes must be due to functional differences in their metal-responsive promoter regions. Both promoters contain a proximal cluster of three MREs overlapping with TFBs for the transcriptional regulator Sp1. In contrast to Cantareus aspersus, however, the Cd-MT gene of Helix pomatia hosts an additional distal MRE overlapping with a Sp1 binding site and a CACCC box. Inhibitory effects of MRE overlapping Sp1 binding sites were observed in other MT genes. We therefore suggest that transient Cd-MT transcription upon Cd(2+) exposure in Helix pomatia may be the result of an inhibitory action of the distal MRE cluster. PMID:19691054
H�ckner, M; Stefanon, K; Schuler, D; Fantur, R; de Vaufleury, A; Dallinger, R
Clustered metallothionein genes are co-regulated in rice and ectopic expression of OsMT1e-P confers multiple abiotic stress tolerance in tobacco via ROS scavenging
Background Metallothioneins (MT) are low molecular weight, cysteine rich metal binding proteins, found across genera and species, but their function(s) in abiotic stress tolerance are not well documented. Results We have characterized a rice MT gene, OsMT1e-P, isolated from a subtractive library generated from a stressed salinity tolerant rice genotype, Pokkali. Bioinformatics analysis of the rice genome sequence revealed that this gene belongs to a multigenic family, which consists of 13 genes with 15 protein products. OsMT1e-P is located on chromosome XI, away from the majority of other type I genes that are clustered on chromosome XII. Various members of this MT gene cluster showed a tight co-regulation pattern under several abiotic stresses. Sequence analysis revealed the presence of conserved cysteine residues in OsMT1e-P protein. Salinity stress was found to regulate the transcript abundance of OsMT1e-P in a developmental and organ specific manner. Using transgenic approach, we found a positive correlation between ectopic expression of OsMT1e-P and stress tolerance. Our experiments further suggest ROS scavenging to be the possible mechanism for multiple stress tolerance conferred by OsMT1e-P. Conclusion We present an overview of MTs, describing their gene structure, genome localization and expression patterns under salinity and development in rice. We have found that ectopic expression of OsMT1e-P enhances tolerance towards multiple abiotic stresses in transgenic tobacco and the resultant plants could survive and set viable seeds under saline conditions. Taken together, the experiments presented here have indicated that ectopic expression of OsMT1e-P protects against oxidative stress primarily through efficient scavenging of reactive oxygen species.
Gene Expression and Functional Annotation of the Human and Mouse Choroid Plexus Epithelium
Background The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and man differ with respect to transport and metabolic functions.
Janssen, Sarah F.; van der Spek, Sophie J. F.; ten Brink, Jacoline B.; Essing, Anke H. W.; Gorgels, Theo G. M. F.; van der Spek, Peter J.; Jansonius, Nomdo M.; Bergen, Arthur A. B.
Mouse histamine N-methyltransferase: cDNA cloning, expression, gene cloning and chromosomal localization
Objective: Histamine N-methyltransferase (HNMT) catalyzes the N t -methylation of histamine. We set out to clone a mouse liver HNMT cDNA and the mouse HNMT gene as steps toward characterizing molecular genetic mechanisms involved in the regulation of this important histamine-metabolizing enzyme. Design: A PCR-based strategy was used to clone both the mouse HNMT cDNA and the gene encoding that
L. Wang; L. Yan; C. McGuire; C. A. Kozak; M. Wang; U.-J. Kim; M. Siciliano; R. M. Weinshilboum
Cloning of mouse sepiapterin reductase gene and characterization of its promoter region
We have isolated and characterized ?5 kb mouse sepiapterin reductase gene (Spr) and a highly homologous pseudogene (Sprp). The authentic Spr gene is present as a single copy in the mouse genome and is composed of three exons containing the entire coding region. The primer extension experiment located the transcription initiation site in a putative pyrimidine-rich Inr element. The promoter
Soo Woong Lee; In Young Park; Yoonsoo Hahn; Ji Eun Lee; Chang Soo Seong; Jae Hoon Chung; Young Shik Park
Bacterium-based heavy metal biosorbents: enhanced uptake of cadmium by E. coli expressing a metallothionein fused to beta-galactosidase.
We investigated the potential utility of a recombinant E. coli that expresses the human metallothionein II gene as a fusion protein with beta-galactosidase as a heavy metal biosorbent. E. coli cells expressing the metallothionein fusion demonstrated enhanced binding of Cd2+ compared to cells that lack the metallothionein. It was shown that the metallothionein fusion was capable of efficiently removing Cd2+ from solutions. Approximately 40% of the Cd2+ accumulated by the recombinant cells free in suspension was associated with the outer cell membrane, and 60% of that was present in the cytoplasm. PMID:11911659
Yoshida, N; Kato, T; Yoshida, T; Ogawa, K; Yamashita, M; Murooka, Y
Genomic organization of the mouse dystrobrevin gene: Comparative analysis with the dystrophin gene
Dystrobrevin, the mammalian orthologue of the Torpedo 87-kDa postsynaptic protein, is a member of the dystrophin gene family with homology to the cysteine-rich carboxy-terminal domain of dystrophin. Torpedo dystrobrevin copurifies with the acetylcholine receptors and is thought to form a complex with dystrophin and syntrophin. This complex is also found at the sarcolemma in vertebrates and defines the cytoplasmic component of the dystrophin-associated protein complex. Previously we have cloned several dystrobrevin isoforms from mouse brain and muscle. Here we show that these transcripts are the products of a single gene located on proximal mouse chromosome 18. To investigate the diversity of dystrobrevin transcripts we have determined that the mouse dystrobrevin gene is organized into 24 coding exons that span between 130 and 170 kb at the genomic level. The gene encodes at least three distinct protein isoforms that are expressed in a tissue-specific manner. Interestingly, although there is only 27% amino acid identity between the homologous regions of dystrobrevin and dystrophin, the positions of 8 of the 15 exon-intron junctions are identical. 47 refs., 4 figs., 2 tabs.
Ambrose, H.J.; Blake, D.J.; Nawrotzki, R.A.; Davies, K.E. [Univ. of Oxford (United Kingdom)] [Univ. of Oxford (United Kingdom)
Endothelial metallothionein expression and intracellular free zinc levels are regulated by shear stress
We examined the effects of fluid shear stress on metallothionein (MT) gene and protein expression and intracellular free zinc in mouse aorta and in human umbilical vein endothelial cells (HUVECs). Immunostaining of the endothelial surface of mouse aorta revealed increased expression of MT protein in the lesser curvature of the aorta relative to the descending thoracic aorta. HUVECs were exposed to high steady shear stress (15 dyn/cm2), low steady shear stress (1 dyn/cm2), or reversing shear stress (mean of 1 dyn/cm2, 1 Hz) for 24 h. Gene expression of three MT-1 isoforms, MT-2A, and zinc transporter-1 was upregulated by low steady shear stress and reversing shear stress. HUVECs exposed to 15 dyn/cm2 had increased levels of free zinc compared with cells under other shear stress regimes and static conditions. The increase in free zinc was partially blocked with an inhibitor of nitric oxide synthesis, suggesting a role for shear stress-induced endothelial nitric oxide synthase activity. Cells subjected to reversing shear stress in zinc-supplemented media (50 ?M ZnSO4) had increased intracellular free zinc, reduced surface intercellular adhesion molecule-1 expression, and reduced monocyte adhesion compared with cells exposed to reversing shear stress in normal media. The sensitivity of intracellular free zinc to differences in shear stress suggests that intracellular zinc levels are important in the regulation of the endothelium and in the progression of vascular disease.
Conway, Daniel E.; Lee, Sungmun; Eskin, Suzanne G.; Shah, Ankit K.; Jo, Hanjoong
Gene silencing by RNAi in mouse Sertoli cells
Background RNA interference (RNAi) is a valuable tool in the investigation of gene function. The purpose of this study was to examine the availability, target cell types and efficiency of RNAi in the mouse seminiferous epithelium. Methods The experimental model was based on transgenic mice expressing EGFP (enhanced green fluorescent protein). RNAi was induced by in vivo transfection of plasmid vectors encoding for short hairpin RNAs (shRNAs) targeting EGFP. shRNAs were transfected in vivo by microinjection into the seminiferous tubules via the rete testis followed by square wave electroporation. As a transfection reporter, expression of red fluorescent protein (HcRed 1) was used. Cell types, the efficiency of both transfections and RNAi were all evaluated. Results Sertoli cells were the main transfected cells. A reduction of about 40% in the level of EGFP protein was detected in cells successfully transfected both in vivo and in vitro. However, the efficiency of in vivo transfection was low. Conclusion In adult seminiferous epithelial cells, in vivo post-transcriptional gene silencing mediated by RNAi via shRNA is efficient in Sertoli cells. Similar levels of RNAi were detected both in vivo and in vitro. This also indicates that Sertoli cells have the necessary silencing machinery to repress the expression of endogenous genes via RNAi.
Gonzalez-Gonzalez, Emilio; Lopez-Casas, Pedro P; del Mazo, Jesus
Identification of a set of genes showing regionally enriched expression in the mouse brain
Background The Pleiades Promoter Project aims to improve gene therapy by designing human mini-promoters (< 4 kb) that drive gene expression in specific brain regions or cell-types of therapeutic interest. Our goal was to first identify genes displaying regionally enriched expression in the mouse brain so that promoters designed from orthologous human genes can then be tested to drive reporter expression in a similar pattern in the mouse brain. Results We have utilized LongSAGE to identify regionally enriched transcripts in the adult mouse brain. As supplemental strategies, we also performed a meta-analysis of published literature and inspected the Allen Brain Atlas in situ hybridization data. From a set of approximately 30,000 mouse genes, 237 were identified as showing specific or enriched expression in 30 target regions of the mouse brain. GO term over-representation among these genes revealed co-involvement in various aspects of central nervous system development and physiology. Conclusion Using a multi-faceted expression validation approach, we have identified mouse genes whose human orthologs are good candidates for design of mini-promoters. These mouse genes represent molecular markers in several discrete brain regions/cell-types, which could potentially provide a mechanistic explanation of unique functions performed by each region. This set of markers may also serve as a resource for further studies of gene regulatory elements influencing brain expression.
D'Souza, Cletus A; Chopra, Vikramjit; Varhol, Richard; Xie, Yuan-Yun; Bohacec, Slavita; Zhao, Yongjun; Lee, Lisa LC; Bilenky, Mikhail; Portales-Casamar, Elodie; He, An; Wasserman, Wyeth W; Goldowitz, Daniel; Marra, Marco A; Holt, Robert A; Simpson, Elizabeth M; Jones, Steven JM
The four members of the Drosophila metallothionein family exhibit distinct yet overlapping roles in heavy metal homeostasis and detoxification
Four metallothionein genes are present in the Drosophila melanogaster genome, designated MtnA , MtnB , MtnC , MtnD , all of which are transcriptionally induced by heavy metals through the same metal-responsive transcription factor, MTF-1. Here we show, by targeted mutagenesis, that the four metallothionein genes exhibit distinct, yet overlapping, roles in heavy metal homeostasis and toxicity prevention. Among the
Dieter Egli; Jordi Dom�nech; Anand Selvaraj; Kuppusamy Balamurugan; Haiqing Hua; Merc� Capdevila; Oleg Georgiev; Walter Schaffner; S�lvia Atrian
Genomic cloning of mouse MIF (macrophage inhibitory factor) and genetic mapping of the human and mouse expressed gene and nine mouse pseudogenes
The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus or Mus spretus. Nine additional loci containing related sequences, apparently all processed pseudogenes, were also mapped to chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19. While most of these pseudogenes were also found in inbred mice and M. spretus, some are species specific. This suggests that there have been active phases of pseudogene formation in Mus both before and after the separation of musculus and spretus. The human gene contains no pseudogene; we assigned the human gene to chromosome 19, consistent with the location of mouse and human functional genes for MIF in a region of conserved linkage. 43 refs., 4 figs., 1 tab.
Kozak, C.A.; Adamson, M.C.; Buckler, C.E. [National Institute of Allergy and Infectious Diseases, Bethesda, MD (United States)] [and others] [National Institute of Allergy and Infectious Diseases, Bethesda, MD (United States); and others
The mouse homologue of the polycystic kidney disease gene (Pkd1) is a single-copy gene
The mouse homologue of the polycystic kidney disease 1 gene (PKD1) was mapped to chromosome 17 using somatic cell hybrid, BXD recombinant inbred strains, and FISH. The gene is located within a previously defined conserved synteny group that includes the mouse homologue of tuberous sclerosis 2 (TSC2) and is linked to the {alpha} globin pseudogene Hba-ps4. Although the human genome contains multiple copies of genes related to PKD1, there is no evidence for more than one copy in the mouse genome. Like their human counterparts, the mouse Tsc2 and Pkd1 genes are arranged in a tail-to-tail orientation with a distance of only 63 bp between the polyadenylation signals of the two genes. 17 refs., 3 figs.
Olsson, P.G.; Loehning, C.; Frischauf, A.M. [Imperial Cancer Research Fund, London (United Kingdom)] [and others] [Imperial Cancer Research Fund, London (United Kingdom); and others
Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression
Background and Aims It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant�fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil. Methods In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal- (Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription�PCR. Free and conjugated foliar PA concentrations were determined in parallel. Results On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil. Conclusions Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs.
Cicatelli, Angela; Lingua, Guido; Todeschini, Valeria; Biondi, Stefania; Torrigiani, Patrizia; Castiglione, Stefano
Structure, expression, and conserved physical linkage of mouse testicular cell adhesion molecule-1 (TCAM-1) gene.
Isolation and characterization were performed for cDNA encoding mouse testicular cell adhesion molecule-1 (TCAM-1) using 2908 bases coding for a protein having 548 amino acids (60 kDa). Mouse TCAM-1 protein was found to consist of seven domains for signal sequence, five immunoglobulin (Ig) domains, and the transmembrane plus cytoplasmic domain. TCAM-1 gene and the region linking it to growth hormone (GH) gene located downstream from the TCAM-1 gene were then analyzed. The mouse TCAM-1 gene was 11.6 kb in length with 8 exons; the same as for the 12.0 kb rat gene. The distance from the TCAM-1 to GH gene was 12.5 kb in the mouse genome, and 7.6 kb in the rat. By Northern hybridization, 3.1-kb TCAM-1 mRNA was detected in 17-day testis and would appear present in pachytene spermatocytes and round spermatids. PMID:11195349
Sakatani, S; Takahashi, R; Okuda, Y; Aizawa, A; Otsuka, A; Komatsu, A; Ono, M
Comparative gene mapping of the human and mouse TEP1 genes, which encode one protein component of telomerases.
The chromosomal locations of the human TEP1 (telomerase protein component 1) and mouse Tep1 genes, which were originally named TLP1 (telomerase protein 1) or TP1 (telomerase-associated protein 1), were determined by direct R-banding FISH and a molecular linkage analysis with interspecific backcross mice. The human TEP1 and mouse Tep1 genes were mapped by FISH to human chromosome 14q11.2 and to the C2D1 band of mouse chromosome 14, respectively. By means of genetic linkage mapping, the mouse gene was further localized as being 2.7 cM distal to D14Mit18 and D14Mit134 and 2.0 cM proximal to D14Mit5 on mouse chromosome 14, where conserved linkage homology with human chromosome 14q11-q12 has been identified. PMID:9403057
Saito, T; Matsuda, Y; Suzuki, T; Hayashi, A; Yuan, X; Saito, M; Nakayama, J; Hori, T; Ishikawa, F
Characterization of the cDNA coding for mouse plasminogen and localization of the gene to mouse chromosome 17.
A full-length cDNA coding for mouse plasminogen has been isolated and characterized. The cDNA is 2720 bp in length (excluding the poly(A) tail) and contains a 24-bp 5' noncoding region, an open reading frame of 2436 bp, and a 3' noncoding region of 257 bp. The open reading frame codes for 812 amino acids and includes a signal peptide that is likely 19 amino acids in length and the mature protein of 793 amino acids. The calculated Mr of mouse plasminogen is 88,706 excluding carbohydrate. There are two potential N-linked carbohydrate addition sites; one of which is glycosylated in human, bovine, and porcine plasminogens. Mouse plasminogen was found to contain two additional amino acids compared to the human protein. In addition, mouse and human plasminogens were found to be 79 and 76% identical at the protein and DNA levels, respectively. Analysis of the segregation of two allelic forms, Plgb and Plgd, of plasminogen DNA in three sets of recombinant inbred strains has allowed the localization of the mouse plasminogen gene to the proximal end of mouse chromosome 17 within the t complex and close to the locus D17Rp17. The Plg gene is deleted in the semidominant deletion mutant, hair-pintail (Thp). PMID:2081600
Degen, S J; Bell, S M; Schaefer, L A; Elliott, R W
Zeptomole Electrochemical Detection of Metallothioneins
BackgroundThiol-rich peptides and proteins possess a large number of biological activities and may serve as markers for numerous health problems including cancer. Metallothionein (MT), a small molecular mass protein rich in cysteine, may be considered as one of the promising tumour markers. The aim of this paper was to employ chronopotentiometric stripping analysis (CPSA) for highly sensitive detection of MT.Methodology\\/Principal
Vojtech Adam; Jitka Petrlova; Joseph Wang; Tomas Eckschlager; Libuse Trnkova; Rene Kizek; Yann Astier
Global gene expression analysis of lenses from different mouse strains and in the ?3Cx46 knockout mouse
Purpose Disruption of the mouse gene encoding the gap junction subunit ?3 connexin 46 (?3Cx46) results in the formation of lens cataracts that have a severity affected by the genetic background of the mouse strain. To identify the genes that influence the severity of the nuclear opacity, global gene expression was analyzed in lenses from the 129SvJae strain and compared to the C57BL/6J strain. Methods Lens transcripts were subjected to cDNA microarray analysis. Results on selected genes were confirmed by real-time PCR. Results Genes that were determined to be altered in expression levels as a result of strain differences could be clustered into three groups: energy metabolism, stress response, and cell growth. Conclusions There were no observed changes in gene expression as a result of the lack of ?3Cx46 in the different mouse strains, suggesting that the pathways mediated by this connexin do not influence gene transcription in the lens. Analysis of the transcript changes due to strain differences provides new insights into potential genetic modifiers of cataractogenesis. More detailed experimentation will be needed to determine if these observed changes do indeed affect cataractogenesis.
Crowley, Thomas E.; Kumar, Nalin M.
ENU-based gene-driven mutagenesis in the mouse: a next-generation gene-targeting system.
As a new mouse mutant resource, the RIKEN ENU-based gene-driven mutagenesis system in the mouse has been available to the research community since 2002. By using random base-substitution mutagenesis with ENU, a new reverse genetics infrastructure has been developed as a next-generation gene-targeting system. The construction of a large-scale mutant mouse library and high-throughput mutation discovery systems were the keys making it practically feasible. The RIKEN mutant mouse library consists of ~ 10,000 G1 mice, within which 100-150 mutant strains have been established based on users' requests every year. Use of the system is very simple: users 1) download an application form from our web site and send to us, and 2) design the PCR primers for the target gene. Then, we screen the RIKEN mutant mouse library and report all the detected mutations to the user. From among the allelic series of discovered mutations, users decide which mutant strain(s) to analyze and request the live mutant strain for functional studies of the target gene. Users have been reporting various functional mutations in the RIKEN mutant mouse library: e.g., missense, knockout-type and even functional non-coding mutations. In the near future, next-generation re-sequencing systems should drastically enhance the utility of the ENU-based gene-driven mutagenesis not only for the mouse but also for other species. PMID:21030782
Gondo, Yoichi; Fukumura, Ryutaro; Murata, Takuya; Makino, Shigeru
Mapping of the mouse macrophage inflammatory protein-1{alpha} receptor gene Scya3r and two related mouse {beta} chemokine receptor-like genes to chromosome 9
Macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) and RANTES are members of the {beta} chemokine family of leukocyte chemoattractants. We have previously cloned three mouse genes by cross-hybridization with the human MIP-1{alpha}/RANTES receptor gene CMKBR1. One of the mouse genes, Scya3r, encodes a functional MIP-1{alpha} receptor. The functions of the other two, Scya3r-rs1 and Scya3r-rs2, are not known. We have now mapped Scya3r, Scya3r-rs1, and Scya3r-rs2 to chromosome 9, in a region of conserved synteny with the location of CMKBR1. Thus, like chemokine genes and {alpha} chemokine receptor genes, this group of {Beta} chemokine receptor genes arose by tandem duplication. 17 refs., 2 figs.
Kozak, C.A.; Gao, Ji-Liang; Murphy, P.M. [National Institutes of Health, Bethesda, MD (United States)] [National Institutes of Health, Bethesda, MD (United States)
The top skin-associated genes: a comparative analysis of human and mouse skin transcriptomes.
The mouse represents a key model system for the study of the physiology and biochemistry of skin. Comparison of skin between mouse and human is critical for interpretation and application of data from mouse experiments to human disease. Here, we review the current knowledge on structure and immunology of mouse and human skin. Moreover, we present a systematic comparison of human and mouse skin transcriptomes. To this end, we have recently used a genome-wide database of human gene expression to identify genes highly expressed in skin, with no, or limited expression elsewhere - human skin-associated genes (hSAGs). Analysis of our set of hSAGs allowed us to generate a comprehensive molecular characterization of healthy human skin. Here, we used a similar database to generate a list of mouse skin-associated genes (mSAGs). A comparative analysis between the top human (n=666) and mouse (n=873) skin-associated genes (SAGs) revealed a total of only 30.2% identity between the two lists. The majority of shared genes encode proteins that participate in structural and barrier functions. Analysis of the top functional annotation terms revealed an overlap for morphogenesis, cell adhesion, structure, and signal transduction. The results of this analysis, discussed in the context of published data, illustrate the diversity between the molecular make up of skin of both species and grants a probable explanation, why results generated in murine in vivo models often fail to translate into the human. PMID:24497224
Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Homey, Bernhard; Zlotnik, Albert; Hevezi, Peter
The Begain gene marks the centromeric boundary of the imprinted region on mouse chromosome 12.
Although the central portion of the imprinted region on mouse chromosome 12 has been intensively analysed in the past, little is known about the neighbouring centromeric genes. A DNA sequence comparison shows that the region upstream of Dlk1 and Gtl2 is dominated by an expanded cluster of repetitive elements in the mouse. These elements separate the paternally expressed Dlk1 gene from the centromeric Begain gene. Despite the long physical distance to the IG-DMR imprinting centre, Begain is subjected to genomic imprinting. Similar to the ovine Begain gene, the homologous mouse gene encodes two different transcript variants, one of which shows a strong bias toward paternal transcription. Nevertheless, imprinting effects do not spread further centromeric to the Wdr25 gene, which is biallelically expressed as the previously studied neighbouring Wars and Yy1 genes. PMID:19641963
Tierling, Sascha; Gasparoni, Gilles; Youngson, Neil; Paulsen, Martina
Structural determinant of the species-specific transcription of the mouse rRNA gene promoter.
Mammalian ribosomal DNA (rDNA) transcription has a certain species specificity such that, both in vivo and in vitro, human rDNA cannot be transcribed by mouse machinery and vice versa. This is due to a species-dependent transcription factor, TFID (Y. Mishima, I. Financsek, R. Kominami, and M. Muramatsu, Nucleic Acids Res. 10:6659-6670, 1982). On the basis of the information obtained from 5' and 3' substitution mutants, we prepared a chimeric gene in which the mouse sequence from positions -32 to -14 was inserted into the corresponding location of the human rDNA promoter. The chimeric gene could be transcribed by mouse extracts nearly as efficiently as the wild-type mouse promoter. The chimeric gene could also sequester transcription factor TFID at an efficiency similar to that for the mouse promoter. Partially purified mouse TFID that could not protect the human rDNA promoter against DNase I produced a clear footprint on this chimeric gene that was similar to that on mouse rDNA promoter. The basic structure of the mouse rDNA core promoter is discussed in relation to the interaction with TFID. Images
Safrany, G; Tanaka, N; Kishimoto, T; Ishikawa, Y; Kato, H; Kominami, R; Muramatsu, M
Gene Transfer Mediated by Recombinant Baculovirus into Mouse Eye
Purpose To determine the efficiency of baculoviruses (BVs) to transfer recombinant genes in vivo into murine ocular tissues. Methods Recombinant (r)BVs carrying fluorescent protein (FP) cDNA under the control of cytomegalovirus (CMV) immediate early promoter were constructed. Initially, cultured HEK293 and ARPE19 cells were infected with these rBVs and analyzed for efficiency and stability of transgene expression. The rBV-CMV green (G)FP was also injected into the intravitreal and subretinal space of mouse eye. Mice were periodically analyzed to determine the efficiency and stability of expression by histologic examination under fluorescence microscopy. The effect of rBV-CMV-GFP on the physiology of the retina was analyzed by electroretinography. Results cDNAs encoding fluorescent proteins were efficiently transduced in HEK293 and ARPE19 cells in vitro. GFP expression in vivo was observed exclusively in retinal pigment epithelial (RPE) cells after subretinal injections. Intravitreal injections of rBV resulted in GFP expression in the corneal endothelium, lens, RPE, and retina. GFP expression was observed for up to 14 days after injection. The infiltration of macrophages, observed 2 days after injection in the area of GFP transduction, had dissipated by day 8 after injection. No alteration in ERG responses was observed 6 weeks after injection of rBV-CMV-GFP. Conclusions BV efficiently transduces cultured RPE cells and many cell types in vivo in the eye, including endothelial, epithelial, and neuronal cells. BV may be a useful vector for transferring genes in cultured cells and in vivo into ocular tissue.
Haeseleer, Francoise; Imanishi, Yoshikazu; Saperstein, David A.; Palczewski, Krzysztof
In vitro and in vivo promoter analyses of the mouse phospholamban gene
To determine the mechanisms responsible for regulation of the phospholamban (PLB) gene expression, a critical regulatory phosphoprotein in cardiac muscle, the mouse PLB gene was isolated and promoter analysis was performed in vitro and in vivo. The PLB gene consists of two exons separated by a single large intron. Deletion analysis revealed that a 7-kb 5? flanking fragment (including exon
Kobra Haghighi; Vivek J Kadambi; Kimberly L Koss; Wusheng Luo; Judy M Harrer; Sathivel Ponniah; Zuoping Zhou; Evangelia G Kranias
Global Gene Expression Analysis in a Mouse Model for Norrie Disease: Late Involvement of Photoreceptor Cells
PURPOSE. Mutations in the NDP gene give rise to a variety of eye diseases, including classic Norrie disease (ND), X-linked exu- dative vitreoretinopathy (EVRX), retinal telangiectasis (Coats disease), and advanced retinopathy of prematurity (ROP). The gene product is a cystine-knot- containing extracellular signal- ing molecule of unknown function. In the current study, gene expression was determined in a mouse model
Steffen Lenzner; Sandra Prietz; Silke Feil; Ulrike A. Nuber; Wolfgang Berger
Number of CpG Islands and Genes in Human and Mouse
Estimation of gene number in mammals is difficult due to the high proportion of noncoding DNA within the nucleus. In this study, we provide a direct measurement of the number of genes in human and mouse. We have taken advantage of the fact that many mammalian genes are associated with CpG islands whose distinctive properties allow their physical separation from
Francisco Antequera; Adrian Bird
Gene transfer techniques in whole embryo cultured post-implantation mouse embryos.
Gene transfer techniques such as electroporation and lipofection are powerful systems for investigating gene function. In this chapter we focus on the methods and applications of gene transfer into specific cells and tissues of post-implantation mouse embryos. PMID:24318824
Sakai, Daisuke; Trainor, Paul A
In vivo selection for metastasis promoting genes in the mouse
Here, we report the identification of a metastasis promoting factor by a forward genetic screen in mice. A retroviral cDNA library was introduced into the nonmetastatic cancer cell line 168FARN, which was then orthotopically transplanted into mouse mammary fat pads, followed by selection for cells that metastasize to the lung. The genes encoding the disulfide isomerase ERp5 and ?-catenin were found to promote breast cancer invasion and metastasis. Disulfide isomerases (thiol isomerases), which catalyze disulfide bond formation, reduction, and isomerization, have not previously been implicated in cancer cell signaling and tumor metastasis. Overexpression of ERp5 promotes both in vitro migration and invasion and in vivo metastasis of breast cancer cells. These effects were shown to involve activation of ErbB2 and phosphoinositide 3-kinase (PI3K) pathways through dimerization of ErbB2. Activation of ErbB2 and PI3K subsequently stimulates RhoA and ?-catenin, which mediate the migration and invasion of tumor cells. Inhibition of ErbB2 and PI3K reverses the phenotypes induced by ERp5. Finally, ERp5 was shown to be up-regulated in human surgical samples of invasive breast cancers. These data identify a link between disulfide isomerases and tumor development, and provide a mechanism that modulates ErbB2 and PI3K signaling in the promotion of cancer progression.
Gumireddy, Kiranmai; Sun, Fangxian; Klein-Szanto, Andres J.; Gibbins, Jonathan M.; Gimotty, Phyllis A.; Saunders, Aleister J.; Schultz, Peter G.; Huang, Qihong
Localization of the murine activating transcription factor 4 gene to mouse chromosome 15
Restriction fragment length variant analysis employing a mouse cDNA probe was used to localize the gene encoding murine activating transcription factor 4 (ATF-4) to mouse chromosome 15 in close proximity to Sis (the cellular homolog of the simian sarcoma virus oncoprotein). Previous studies suggest that conserved linkage relationships exist between this region of mouse chromosome 15 and human chromosome 22q. The chromosomal locations of genes encoding most members of the ATF and cyclic AMP response element binding protein (CREB) subfamilv of b-zip proteins have not been determined. This study demonstrates that the location of the gene for murine ATF-4 is not linked to the genes for JUN family members, CREB1 and CREB2. Further mapping of individual ATF/ CREB subfamily members in the mouse will provide insight into the evolution of this multigene family. 15 refs., 1 fig., 1 tab.
Mielnicki, L.M.; Elliott, R.W.; Pruitt, S.C. (Roswell Park Cancer Institute, Buffalo, NY (United States))
Augmented hepatic injury followed by impaired regeneration in metallothionein-I\\/II knockout mice after treatment with thioacetamide
A previous study (Oliver, J.R., Mara, T.W., Cherian, M.G. 2005. Impaired hepatic regeneration in metallothionein-I\\/II knockout mice after partial hepatectomy. Exp. Biol. Med. 230, 61�67) has shown an impairment of liver regeneration following partial hepatectomy (PH) in metallothionein (MT)-I and MT-II gene knockout (MT-null) mice, thus suggesting a requirement for MT in cellular growth. The present study was undertaken to
Jordan R. Oliver; Sean Jiang; M. George. Cherian
Genomic Organization, Expression, and Chromosomal Mapping of the Mouse Adrenomedullin Gene
We have isolated and characterized the mouse adrenomedullin (AM) gene (Adm) and determined its chromosomal location. The gene spans approximately 2.1 kb and is organized into four exons separated by three introns. The transcription start site was determined to be the adenine nucleotide at ?618. The mouse AM 5?-flanking region contains a TATA box-like sequence and severalcis-acting regulatory elements. Analysis
Taku Okazaki; Yoshihiro Ogawa; Naohisa Tamura; Kiyoshi Mori; Naohi Isse; Tomohiro Aoki; Julie M. Rochelle; Makoto M. Taketo; Michael F. Seldin; Kazuwa Nakao
Characterization of the mouse neutrophil elastase gene and localization to Chromosome 10
Neutrophil elastase (NE), a serine proteinase, is considered to play a role in normal tissue turnover and host defense. NE\\u000a may also cause tissue damage in acute and chronic inflammatory diseases. We have isolated and characterized the gene for mouse\\u000a NE and determined its chromosomal location. The mouse NE gene has been localized by interspecific backcross analysis to Chromosome (Chr)
A. Belaaouaj; B. C. Walsh; N. A. Jenkins; N. G. Copeland; S. D. Shapiro
Gene expression profiles of the collecting duct in the mouse renal inner medulla
Gene expression profiles of the collecting duct in the mouse renal inner medulla.BackgroundGene expression profiles, constructed from 1000 to 2000 cloned cDNA sequences, depict their relative abundance of expression in a tissue. Establishing such a profile for mouse inner renal medullary collecting ducts (IMCDs), we compared expression patterns with those in other tissues including proximal tubule.MethodsA nonbiased 3?-end cDNA library
Masaru Takenaka; Enyu Imai; Yasuyuki Nagasawa; Yasuko Matsuoka; Toshiki Moriyama; Tetsuya Kaneko; Masatsugu Hori; Shoko Kawamoto; Kousaku Okubo
Assignment of the mouse tartrate-resistant acid phosphatase gene (Acp5) to chromosome 9
Tartrate-resistant acid phosphatase is a marker enzyme for osteoclasts, the multinucleated cell responsible for bone resorption. Interspecific somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map the gene encoding tartrate-resistant acid phosphatse (acp5) to mouse Chromosome 9. Acp5 is therefore a member of a syntenic family of genes that map to human chromosome 19p13.1-p13.3 and mouse Chromosome 9. 8 refs., 1 fig., 1 tab.
Grimes, R.; Reddy, S.V.; Leach, R.J.; Scarcez, T.; Sakaguchi, A.Y. (Univ. of Texas Health Science Center, San Antonio (United States)); Roodman, G.D. (Univ. of Texas Health Science Center, San Antonio (United States) Audie Murphy Veterans Administration Hospital, San Antonio, TX (United States)); Lalley, P.A. (Wayne State Univ. of School of Medicine, Detroit, MI (United States)); Windle, J.J. (Cancer Therapy and Research Center, San Antonio, TX (United States))
Persistent gene expression in mouse nasal epithelia following feline immunodeficiency virus-based vector gene transfer.
Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 10(6) transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 10(7) to 10(9) TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (approximately 10(9) TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for approximately 1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity. PMID:16188984
Sinn, Patrick L; Burnight, Erin R; Hickey, Melissa A; Blissard, Gary W; McCray, Paul B
Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes
Background Thioredoxin reductase (TR) is a redox active protein involved in many cellular processes as part of the thioredoxin system. Presently there are three recognised forms of mammalian thioredoxin reductase designated as TR1, TR3 and TGR, that represent the cytosolic, mitochondrial and novel forms respectively. In this study we elucidated the genomic organisation of the mouse (Txnrd1) and human thioredoxin reductase 1 genes (TXNRD1) through library screening, restriction mapping and database mining. Results The human TXNRD1 gene spans 100 kb of genomic DNA organised into 16 exons and the mouse Txnrd1 gene has a similar exon/intron arrangement. We also analysed the alternative splicing patterns displayed by the mouse and human thioredoxin reductase 1 genes and mapped the different mRNA isoforms with respect to genomic organisation. These isoforms differ at the 5' end and encode putative proteins of different molecular mass. Genomic DNA sequences upstream of mouse exon 1 were compared to the human promoter to identify conserved elements. Conclusions The human and mouse thioredoxin reductase 1 gene organisation is highly conserved and both genes exhibit alternative splicing at the 5' end. The mouse and human promoters share some conserved sequences.
Osborne, Simone A; Tonissen, Kathryn F
Human Jk recombination signal binding protein gene (IGKJRB): Comparison with its mouse homologue
The mouse Igkjrb protein specifically binds to the immunoglobulin Jk recombination signal sequence. The IGKJRB gene is highly conserved among many species such as human, Xenopus, and Drosophila. Using cDNA fragments of the mouse Igkjrb gene, the authors isolated its human counterpart, IGKJRB. The human genome contains one functional IGKJRB gene and two types of processed pseudogenes. In situ chromosome hybridization analysis demonstrated that the functional gene is localized at chromosome 3q25, and the pseudogenes (IGKJRBP1 and IGKJRBP2, respectively) are located at chromosomes 9p13 and 9q13. The functional gene is composed of 13 exons spanning at least 67 kb. Three types of cDNA with different 5[prime] sequences were isolated by rapid amplification of cDNA ends, suggesting the presence of three proteins. The aPCR-1 protein, which possessed the exon 1 sequence, was the counterpart of the mouse RBP-2 type protein. The aPCR-2 and 3 proteins may be specific to human cells because the mouse counterparts were not detected. The amino acid sequences of the human and mouse IGKJRB genes were 98% homologous in exons 2-11, whereas the homology of the human and mouse exon 1 sequences was 75%. 40 refs., 7 figs.
Amakawa, Ryuichi; Jing, Wu; Matsunami, Norisada; Hamaguchi, Yasushi; Matsuda, Fumihiko; Kawaichi, Masashi; Honjo, Tasuku (Kyoto Univ., Sakyo-ku, Kyoto (Japan)); Ozawa, Kazuo (Tsukuba Life Science Center, Tsukuba, Ibraraki (Japan))
Mouse Palmitoyl Protein Thioesterase: Gene Structure and Expression of cDNA
Palmitoyl protein thioesterase (PPT) is the defective enzyme in infantile neuronal ceroid lipofuscinosis (INCL), which is a recessively inherited, progressive neurodegenerative disorder. We present here the cloning, chromosomal mapping, genomic structure, and the expression of the cDNA of mouse PPT. The mouse PPT gene spans >21 kb of genomic DNA and contains nine exons with a coding sequence of 918 bp. Fluorescence in situ hybridization to metaphase chromosomes localized the mouse PPT gene to the chromosome 4 conserved syntenic region with human chromosome 1p32 where the human PPT is located. PPT is expressed widely in a variety of mouse tissues. The mouse PPT cDNA is conserved highly with the human and rat PPT both at the nucleotide and amino acid sequence level. Transient expression of mouse PPT in COS-1 cells yielded a 38/36-kD differentially glycosylated polypeptide that was also secreted into culture media. Immunofluorescence analysis of transiently transfected HeLa cells indicated lysosomal localization of mouse PPT. Based on the high conservation of the gene and polypeptide structure as well as similar processing and intracellular localization, the function of PPT in mouse and human are likely to be very similar. [The sequence data described in this paper have been submitted to GenBank under accession no. AF071O25.
Salonen, Tarja; Hellsten, Elina; Horelli-Kuitunen, Nina; Peltonen, Leena; Jalanko, Anu
Three new immunoglobulin kappa variable (Igk-V) gene segments in the mouse
In the mouse, the immunoglobulin (Ig) kappa variable (Igk-V) gene segments make a large contribution to the antibody repertoire, because the light chain in over 90% of serum Ig molecules is of the kappa type. The number of Igk-V gene segments has been the subject of some controversy. Here we report three new Igk-V gene segments found among rearranged kappa genes from day 14 - 16 fetal liver of the C57BL/6J mouse. As the segments are potentially productive, the new Igk-V segments are presumable used. 8 refs., 1 fig.
Ramsden, D.A.; Wu, G.E. [Univ. of Toronto, Ontario (Canada)
Structural organization and chromosome location of the mouse elongin A gene (Tceb3).
Elongin A is the transcriptionally active subunit of the Elongin complex, which strongly increases the rate of elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites within transcription units. In the present study, we obtained the cDNA sequence of the mouse Elongin A gene (Tceb3) and characterized its genomic structure. The deduced 773-amino acid sequence of mouse Elongin A shows 91% and 81% identity with rat and human Elongin A, respectively. The Elongin A gene was mapped to mouse chromosome 4D3 by fluorescence in situ hybridization. PMID:10575222
Aso, T; Amimoto, K; Takebayashi, S; Okumura, K; Hatakeyama, M
Chromosomal localization of the gene encoding the human DNA helicase RECQL and its mouse homologue
We have determined the chromosomal location of the human and mouse genes encoding the RECQL protein, a putative DNA helicase homologous to the bacterial DNA helicase, RecQ. RECQL was localized to human chromosome 12 by analysis of human-rodent somatic cell hybrid DNA, fine mapping of RECQL by fluorescence in situ hybridization revealed its chromosomal location to be 12p11-p12. The corresponding mouse gene, Recql, was mapped to the telomeric end of mouse chromosome 6 by analysis of DNA from an interspecific cross. 19 refs., 2 figs.
Puranam, K.L.; Kennington, E.; Blackshear, P.J. [Duke Univ., Durham, NC (United States)] [and others] [Duke Univ., Durham, NC (United States); and others
The PPCD1 Mouse: Characterization of a Mouse Model for Posterior Polymorphous Corneal Dystrophy and Identification of a Candidate Gene
The PPCD1 mouse, a spontaneous mutant that arose in our mouse colony, is characterized by an enlarged anterior chamber resulting from metaplasia of the corneal endothelium and blockage of the iridocorneal angle by epithelialized corneal endothelial cells. The presence of stratified multilayered corneal endothelial cells with abnormal patterns of cytokeratin expression are remarkably similar to those observed in human posterior polymorphous corneal dystrophy (PPCD) and the sporadic condition, iridocorneal endothelial syndrome. Affected eyes exhibit epithelialized corneal endothelial cells, with inappropriate cytokeratin expression and proliferation over the iridocorneal angle and posterior cornea. We have termed this the �mouse PPCD1� phenotype and mapped the mouse locus for this phenotype, designated �Ppcd1�, to a 6.1 Mbp interval on Chromosome 2, which is syntenic to the human Chromosome 20 PPCD1 interval. Inheritance of the mouse PPCD1 phenotype is autosomal dominant, with complete penetrance on the sensitive DBA/2J background and decreased penetrance on the C57BL/6J background. Comparative genome hybridization has identified a hemizygous 78 Kbp duplication in the mapped interval. The endpoints of the duplication are located in positions that disrupt the genes Csrp2bp and 6330439K17Rik and lead to duplication of the pseudogene LOC100043552. Quantitative reverse transcriptase-PCR indicates that expression levels of Csrp2bp and 6330439K17Rik are decreased in eyes of PPCD1 mice. Based on the observations of decreased gene expression levels, association with ZEB1-related pathways, and the report of corneal opacities in Csrp2bptm1a(KOMP)Wtsi heterozygotes and embryonic lethality in nulls, we postulate that duplication of the 78 Kbp segment leading to haploinsufficiency of Csrp2bp is responsible for the mouse PPCD1 phenotype. Similarly, CSRP2BP haploinsufficiency may lead to human PPCD.
Shen, Anna L.; O'Leary, Kathleen A.; Dubielzig, Richard R.; Drinkwater, Norman; Murphy, Christopher J.; Kasper, Charles B.; Bradfield, Christopher A.
Meta-Analysis of Differentiating Mouse Embryonic Stem Cell Gene Expression Kinetics Reveals Early Change of a Small Gene Set
Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time
Clive H. Glover; Michael Marin; Connie J. Eaves; Cheryl D. Helgason; James M. Piret; Jennifer Bryan
Identification and characterization of promoter and regulatory regions for mouse Adam2 gene expression.
ADAM2, a member of the 'a disintegrin and metalloprotease' (ADAM) family, is a key protein in mammalian fertilization that is specifically expressed in testicular germ cells. Here, we investigated the transcriptional regulation of the mouse Adam2 gene. An in silico analysis identified two conserved non-coding sequences located upstream of the mouse and human ADAM2 genes. The upstream region of the mouse Adam2 gene was found to lack typical TATA and CAAT boxes, and to have a high GC content. Our in vitro transient transfection-reporter analysis identified a promoter in this region of the mouse Adam2 gene, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Site-directed mutagenesis revealed that the caudal-type homeobox 1 and CCTC-binding factor motifs are responsible for the inhibitory activities of the repressor regions. Finally, electrophoretic mobility shift assays showed putative transcription factor-promoter DNA complexes, and DNA-affinity chromatography and proteomic analyses identified myelin gene regulatory factor as a binding partner of the Adam2 promoter. This provides the first identification and characterization of promoter and repressor regions that regulate the transcription of the mouse Adam2 gene, and offers insights into the regulation of this germ-cell-specific gene. PMID:23065232
Choi, Heejin; Lee, Boyeon; Jin, Sora; Kwon, Jun Tae; Kim, Jihye; Jeong, Juri; Oh, Seungmin; Cho, Byung-Nam; Park, Zee Yong; Cho, Chunghee
Functional Conservation of Gsdma Cluster Genes Specifically Duplicated in the Mouse Genome
Mouse Gasdermin A3 (Gsdma3) is the causative gene for dominant skin mutations exhibiting alopecia. Mouse has two other Gsdma3-related genes, Gsdma and Gsdma2, whereas human and rat have only one related gene. To date, no skin mutation has been reported for human GSDMA and rat Gsdma as well as mouse Gsdma and Gsdma2. Therefore, it is possible that only Gsdma3 has gain-of-function type mutations to cause dominant skin phenotype. To elucidate functional divergence among the Gsdma-related genes in mice, and to infer the function of the human and rat orthologs, we examined in vivo function of mouse Gsdma by generating Gsdma knockout mice and transgenic mice that overexpress wild-type Gsdma or Gsdma harboring a point mutation (Alanine339Threonine). The Gsdma knockout mice shows no visible phenotype, indicating that Gsdma is not essential for differentiation of epidermal cells and maintenance of the hair cycle, and that Gsdma is expressed specifically both in the inner root sheath of hair follicles and in suprabasal cell layers, whereas Gsdma3 is expressed only in suprabasal layers. By contrast, both types of the transgenic mice exhibited epidermal hyperplasia resembling the Gsdma3 mutations, although the phenotype depended on the genetic background. These results indicate that the mouse Gsdma and Gsdma3 genes share common function to regulate epithelial maintenance and/or homeostasis, and suggest that the function of human GSDMA and rat Gsdma, which are orthologs of mouse Gsdma, is conserved as well.
Tanaka, Shigekazu; Mizushina, Youichi; Kato, Yoriko; Tamura, Masaru; Shiroishi, Toshihiko
Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes
Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome�based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and ? light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.
Macdonald, Lynn E.; Karow, Margaret; Stevens, Sean; Auerbach, Wojtek; Poueymirou, William T.; Yasenchak, Jason; Frendewey, David; Valenzuela, David M.; Giallourakis, Cosmas C.; Alt, Frederick W.; Yancopoulos, George D.; Murphy, Andrew J.
A Survey for Novel Imprinted Genes in the Mouse Placenta by mRNA-seq
Many questions about the regulation, functional specialization, computational prediction, and evolution of genomic imprinting would be better addressed by having an exhaustive genome-wide catalog of genes that display parent-of-origin differential expression. As a first-pass scan for novel imprinted genes, we performed mRNA-seq experiments on embryonic day 17.5 (E17.5) mouse placenta cDNA samples from reciprocal cross F1 progeny of AKR and PWD mouse strains and quantified the allele-specific expression and the degree of parent-of-origin allelic imbalance. We confirmed the imprinting status of 23 known imprinted genes in the placenta and found that 12 genes reported previously to be imprinted in other tissues are also imprinted in mouse placenta. Through a well-replicated design using an orthogonal allelic-expression technology, we verified 5 novel imprinted genes that were not previously known to be imprinted in mouse (Pde10, Phf17, Phactr2, Zfp64, and Htra3). Our data suggest that most of the strongly imprinted genes have already been identified, at least in the placenta, and that evidence supports perhaps 100 additional weakly imprinted genes. Despite previous appearance that the placenta tends to display an excess of maternally expressed imprinted genes, with the addition of our validated set of placenta-imprinted genes, this maternal bias has disappeared.
Wang, Xu; Soloway, Paul D.; Clark, Andrew G.
Yeast and Mammalian Metallothioneins Functionally Substitute for Yeast Copper-Zinc Superoxide Dismutase
NASA Astrophysics Data System (ADS)
Copper-zinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copper-zinc superoxide dismutase, which is encoded by the SOD1 gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxide-generating drugs such as paraquat. We have found that in addition to these known phenotypes, SOD1-deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence of copper suppresses the lactate growth defect and some other phenotypes associated with SOD1-deletion strains, indicating that copper metallothioneins substitute for copper-zinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vitro assays demonstrate that yeast metallothionein, purified or from whole-cell extracts, exhibits copper-dependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants.
Tamai, Katherine T.; Gralla, Edith B.; Ellerby, Lisa M.; Valentine, Joan S.; Thiele, Dennis J.
Gene Expression From the Aneuploid Chromosome in a Trisomy Mouse Model of Down Syndrome
Trisomy 21 is the prototype of human aneuploidies. Since its discovery in 1959, the hypothesis has been that overexpression of the ?230 human chromosome 21 (Hsa21) genes result in the complex phenotype. However, the level of overexpression of Hsa21 genes in trisomic individuals is presently unknown. We have used Taqman real-time quantitative PCR to accurately measure expression of the mouse orthologs of Hsa21 in the partial trisomy mouse model Ts65Dn. The transcript levels of 78 protein-coding genes present in three copies in Ts65Dn and 21 control genes were compared between Ts65Dn and normal mouse littermates. The mean overexpression of the aneuploid genes is very close to the expected 1.5-fold in all six tissues studied. However, only approximately a third of the genes (37%) are expressed at the theoretical value of 1.5-fold. On average, 45% of the genes are expressed at significantly lower than 1.5-fold, and 9% are not significantly different from 1.0. Interestingly, 18% of the aneuploid genes were expressed at levels significantly greater than 1.5-fold. These data provide candidate genes that might be involved in the phenotypes of Down syndrome, and reveal a complex regulation of gene expression that is not only related to gene copy number.
Lyle, Robert; Gehrig, Corinne; Neergaard-Henrichsen, Charlotte; Deutsch, Samuel; Antonarakis, Stylianos E.
Plasmid-based gene transfer ameliorates visceral storage in a mouse model of Sandhoff disease
Sandhoff disease is a severe neurodegenerative disorder with visceral involvement caused by mutations in the HEXB gene coding for the g subunit of the lysosomal hexosaminidases A and B. HEXB mutations result in the accumulation of undegraded substrates such as GM2 and GA2 in lysosomes. We evaluated the efficacy of cationic liposome-mediated plasmid gene therapy using the Sandhoff disease mouse,
Akira Yamaguchi; Kayoko Katsuyama; Kyoko Suzuki; Kenji Kosaka; Ichiro Aoki; Shoji Yamanaka
Mapping of the Tuple1 gene to mouse chromosome 16A-B1
The human TUPLE1 gene encodes a putative transcriptional regulator and maps to chromosome 22, and therefore may play a role in Di-George syndrome (DGS), relo-cardio-facial syndrome (VCFS), or a related pathology. The murine TUPLE1 gene has also been cloned and shows strong sequence similarity to TUPLE1. Comparative mapping is useful in the study of chromosome evolution and is sometimes able to indicate possible mouse mutations that are potential models of human genetic disorders. As TIPLE1 is a candidate gene for the haploinsufficient phenotype in DGS, we mapped TUPLE1 to mouse chromosome 16A-B1. 6 refs., 1 fig.
Mattei, M.G. [Faculte de Medicine, Marseille (France)] [Faculte de Medicine, Marseille (France); Halford, S.; Scambler, P.J. [Institute of Child Health, London (United Kingdom)] [Institute of Child Health, London (United Kingdom)
A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes
A gene mapping to the sex-determining region of the mouse Y chromosome is deleted in a line of XY female mice mutant for Tdy, and is expressed at a stage during male gonadal development consistent with its having a role in testis determination. This gene is a member of a new family of at least five mouse genes, related by
John Gubbay; J�r�me Collignon; Peter Koopman; Blanche Capel; Androulla Economou; Andrea M�nsterberg; Nigel Vivian; Peter Goodfellow; Robin Lovell-Badge
Aberrant gene expression profile in a mouse model of endometriosis mirrors that observed in women
Objective To define the altered gene expression profile of endometriotic lesions in a mouse model of surgically-induced endometriosis Design Autologous experimental mouse model. Setting Medical school department. Animals Adult C57Bl6 mice. Intervention(s) Endometriosis was surgically-induced by auto-transplantation of uterine tissue to the intestinal mesentery. Endometriotic lesions and eutopic uteri were recovered at 3 or 29 days post-induction. Main Outcome Measure(s) Altered gene expression was measured in the endometriotic lesion relative to the eutopic uterus by genome wide cDNA microarray analysis and was confirmed by real time RT-PCR for six genes. Relevant categories of altered genes were identified using gene ontology analysis to determine groups of genes enriched for altered expression. Result(s) The expression of 479 and 114 genes was altered in the endometriotic lesion compared to the eutopic uterus at 3 or 29 days post-induction, respectively. Gene ontology enrichment analysis revealed that genes associated with the extracellular matrix, cell adhesions, immune function, cell growth, and angiogenesis were altered in the endometriotic lesion compared to the eutopic uterus. Conclusion(s) Based on gene expression analysis, the mouse model of surgically-induced endometriosis appears to be a good model for studying the pathophysiology and treatment of endometriosis.
Pelch, Katherine E.; Schroder, Amy L.; Kimball, Paul A.; Sharpe-Timms, Kathy L.; Davis, J. W.; Nagel, Susan C.
Gene Expression Analyses of Mouse Aortic Endothelium in Response to Atherogenic Stimuli
Objective Endothelial cells are central to the initiation of atherosclerosis, yet there has been limited success in studying their gene expression in the mouse aorta. To address this, we developed a method for determining the global transcriptional changes that occur in the mouse endothelium in response to atherogenic conditions and applied it to investigate inflammatory stimuli. Approach and Results We characterized a method for the isolation of endothelial cell RNA with high purity directly from mouse aortas and adapted this method to allow for the treatment of aortas ex vivo before RNA collection. Expression array analysis was performed on endothelial cell RNA isolated from control and hyperlipidemic prelesion mouse aortas, and 797 differentially expressed genes were identified. We also examined the effect of additional atherogenic conditions on endothelial gene expression, including ex vivo treatment with inflammatory stimuli, acute hyperlipidemia, and age. Of the 14 most highly differentially expressed genes in endothelium from prelesion aortas, 8 were also perturbed significantly by ?1 atherogenic conditions: 2610019E17Rik, Abca1, H2-Ab1, H2-D1, Pf4, Ppbp, Pvrl2, and Tnnt2. Conclusions We demonstrated that RNA can be isolated from mouse aortic endothelial cells after in vivo and ex vivo treatments of the murine vessel wall. We applied these methods to identify a group of genes, many of which have not been described previously as having a direct role in atherosclerosis, that were highly regulated by atherogenic stimuli and may play a role in early atherogenesis.
Erbilgin, Ayca; Siemers, Nathan; Kayne, Paul; Yang, Wen-pin; Berliner, Judith; Lusis, Aldons J.
Identifying essential genes in mouse development via an ENU-based forward genetic approach.
The completion of the human and mouse genome projects at the beginning of the past decade represented a very important step forward in our pursuit of a comprehensive understanding of the genetic control of mammalian development. Nevertheless, genetic analyses of mutant phenotypes are still needed to understand the function of individual genes. The genotype-based approaches, including gene-trapping and gene-targeting, promise a mutant embryonic stem (ES) cell resource for all the genes in mouse genome; however, the phenotypic consequences of these mutations will not be addressed until mutant mice are derived from these ES cells, which is not trivial. An efficient and non-biased, N-ethyl-N-nitrosourea (ENU)-based forward genetic approach in mouse provides a unique tool for the identification of genes essential for development and adult physiology. We have had great success in identifying genes essential for morphogenesis and early patterning of mouse via this approach. Combined with complete genome information and numerous genetic resources available, ENU-based mutagenesis has become a powerful tool in deciphering gene functions. PMID:24318816
Liu, Aimin; Eggenschwiler, Jonathan
Six members of the mouse forkhead gene family are developmentally regulated.
The 110-aa forkhead domain defines a class of transcription factors that have been shown to be developmentally regulated in Drosophila melanogaster and Xenopus laevis. The forkhead domain is necessary and sufficient for target DNA binding as shown for the rat hepatic nuclear factor 3 (HNF3) gene family. We have cloned six forkhead gene family members from a mouse genomic library in addition to the mouse equivalents of the genes for HNF3 alpha, -beta, and -gamma. The six genes, termed fkh-1 to fkh-6, share a high degree of similarity with the Drosophila forkhead gene, having 57-67% amino acid identity within the forkhead domain. fkh-1 seems to be the mammalian homologue of the Drosophila FD1 gene, as the sequences are 86% identical. fkh-1 to fkh-6 show distinct spatial patterns of expression in adult tissues and are expressed during embryogenesis. Images Fig. 2 Fig. 3
Kaestner, K H; Lee, K H; Schlondorff, J; Hiemisch, H; Monaghan, A P; Schutz, G
Gene structure and expression of the mouse dyskeratosis congenita gene, dkc1.
Mutations in the DKC1 gene are responsible for causing X-linked recessive dyskeratosis congenita (DKC) and a more severe allelic variant of the disease, Hoyeraal-Hreidarsson syndrome. Both diseases are characterized by progressive and fatal bone marrow failure. The nucleolar protein dyskerin is the pseudouridine synthase component of the box H+ACA snoRNAs and also interacts with the RNA component (human telomerase, hTR) of the telomerase complex. Dyskerin is therefore thought to function in the processing of pre-rRNA and of the hTR, strengthening the notion that the underlying mechanism of DKC is a premature senescence of cells, especially of the rapidly dividing epithelial and hemopoietic cells. To examine the functions of dyskerin in vivo, it will be necessary to generate mouse models. As a first step, we here provide the genomic structure of the mouse Dkc1 gene and expression analysis of the transcript. Northern hybridizations revealed the tissue-specific expression of an alternative 4.5-kb transcript, in addition to the ubiquitous 2.6-kb transcript. RNA in situ hybridizations on day 10.5-18.5 postconception embryos showed a ubiquitous expression of Dkc1 with a notably higher level of expression confined to the epithelial tissues. In addition, higher level Dkc1 expression was confined to embryonic neural tissues as well as to specific neurons in the cerebellum (Purkinje cells) and the olfactory bulb (mitral cells) of the adult brain. In adult testis, elevated expression was limited to the Leydig cells. The results indicate that some of the pertinent functions of dyskerin may be more tissue-specific than previously thought and are not limited to rapidly dividing cells. PMID:10903840
Heiss, N S; B�chner, D; Salowsky, R; Kolb, A; Kioschis, P; Poustka, A
The chromosomal mapping of four genes encoding winged helix proteins expressed early in mouse development.
Members of the winged helix family of transcription factors are required for the normal embryonic development of the mouse. Using the interspecific backcross panel from The Jackson Laboratory, we have determined the chromosomal locations of four genes that encode winged helix containing proteins. Mf1 was assigned to mouse Chromosome 8, Mf2 to Chromosome 4, Mf3 to Chromosome 9, and Mf4 to Chromosome 13. Since Mf3 is located in a region of Chromosome 9 containing many well-characterized mouse mutations such as short ear (se), ashen (ash), and dilute (d), we have analyzed deletion mutants to determine the location of Mf3 more precisely. PMID:8661058
Labosky, P A; Winnier, G E; Sasaki, H; Blessing, M; Hogan, B L
Orthology-Based Multilevel Modeling of Differentially Expressed Mouse and Human Gene Pairs
There is great interest in finding human genes expressed through pharmaceutical intervention, thus opening a genomic window into benefit and side-effect profiles of a drug. Human insight gained from FDA-required animal experiments has historically been limited, but in the case of gene expression measurements, proposed biological orthologies between mouse and human genes provide a foothold for animal-to-human extrapolation. We have
Benjamin A. Ogorek; Leonard A. Stefanski
Assessment of orthologous splicing isoforms in human and mouse orthologous genes
Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still maintaining the conventional definition of gene orthology, a new concept of "splicing orthology" can be defined at transcript level.
RNA transcript profiling during zygotic gene activation in the preimplantation mouse embryo
Zygotic gene activation is essential for development beyond the 2-cell stage in the preimplantation mouse embryo. Based on ?-amanitin-sensitive BrUTP incorporation, transcription initiates in the 1-cell embryo and a major reprogramming of gene expression driven by newly expressed genes is prominently observed during the 2-cell stage. Superimposed on genome activation is the development of a transcriptionally repressive state that is
Fanyi Zeng; Richard M. Schultz
Significance of metallothioneins in aging brain.
Aging is an inevitable biological process, associated with gradual and spontaneous biochemical and physiological changes, and increased susceptibility to diseases. Chronic inflammation and oxidative stress are hallmarks of aging. Metallothioneins (MTs) are low molecular weight, zinc-binding, anti-inflammatory, and antioxidant proteins that provide neuroprotection in the aging brain through zinc-mediated transcriptional regulation of genes involved in cell growth, proliferation, and differentiation. In addition to Zn(2+) homeostasis, antioxidant role of MTs is routed through -SH moieties on cysteine residues. MTs are induced in aging brain as a defensive mechanism to attenuate oxidative and nitrative stress implicated in broadly classified neurodegenerative ?-synucleinopathies. In addition, MTs as free radical scavengers inhibit Charnoly body (CB) formation to provide mitochondrial neuroprotection in the aging brain. In general, MT-1 and MT-2 induce cell growth and differentiation, whereas MT-3 is a growth inhibitory factor, which is reduced in Alzheimer's disease. MTs are down-regulated in homozygous weaver (wv/wv) mice exhibiting progressive neurodegeneration, early aging, morbidity, and mortality. These neurodegenerative changes are attenuated in MTs over-expressing wv/wv mice, suggesting the neuroprotective role of MTs in aging. This report provides recent knowledge regarding the therapeutic potential of MTs in neurodegenerative disorders of aging such as Parkinson's disease and Alzheimer's disease. PMID:24389356
Sharma, Sushil; Ebadi, Manuchair
Hypoxia acts through multiple signaling pathways to induce metallothionein transactivation by the metal-responsive transcription factor-1 (MTF-1)
Metal-responsive transcription factor-1 (MTF-1) is essential for the induction of genes encoding metallothionein by metals and hypoxia. Here, we studied the mechanism controlling the activation of MTF-1 by hypoxia. Hypoxia activation of Mt gene transcription is dependent on the presence of metal regulatory elements (MREs) in the promoter of Mt genes. We showed that MREa and MREd are the main elements controlling mouse Mt-1 gene induction by hypoxia. Transfection experiments in Mtf-1-null cells showed that MTF-1 is essential for induction by hypoxia. Chromatin immunoprecipitation analysis showed that MTF-1 DNA-binding activity was strongly enhanced in the presence of zinc but not by hypoxia. Notably, hypoxia inducible factor- (HIF) 1? was recruited to the Mt-1 promoter in response to hypoxia but not to zinc. MTF-1 activation was inhibited by PKC, JNK, and PI3K inhibitors and by the electron transport chain inhibitors rotenone and myxothiazol, but not by the antioxidant N-acetylcysteine. We showed that prolyl-hydroxylase inhibitors can activate MTF-1, but this activation requires the presence of HIF-1?. Finally, HIF-dependent transcription is enhanced in the presence of MTF-1 and induction of an MRE promoter is stimulated by HIF-1?, thus indicating cooperation between these 2 factors. However, coimmunoprecipitation experiments did not suggest direct interaction between MTF-1 and HIF-1?.
Dube, Annie; Harrisson, Jean-Francois; Saint-Gelais, Genevieve; Seguin, Carl
Rational design of murine secreted alkaline phosphatase for enhanced performance as a reporter gene in mouse gene therapy preclinical studies.
Many preclinical gene therapy studies use a reporter gene to evaluate vector design and performance in mouse models of human disease. Unfortunately, most commonly used reporter genes are immunogenic in mice, which confounds accurate evaluation of vector function. In previous studies, we showed that the murine secreted alkaline phosphatase (mSEAP) gene functions well as a simple and sensitive reporter gene in mice. In this study, we have used rational design to enhance mSEAP performance. The majority of native mSEAP remains attached to the outer surface of the cell through glycan phosphatidylinositol linkage; removal of the carboxy-terminal tail of mSEAP resulted in a dramatic enhancement of release of the protein into cell culture medium and into mouse plasma in whole animal experiments. We increased the heat stability of mSEAP through mutation of a key residue in the crown domain of the protein (H451E), thus allowing us to reduce endogenous, background AP activity through heat inactivation for enhanced sensitivity. We show that these alterations in mSEAP result in enhanced performance in tissue culture and mouse studies. Taken together, these data illustrate that mSEAP is a sensitive, nonimmunogenic reporter for preclinical mouse studies. PMID:21083426
Christou, Carin; Parks, Robin J
NSDL National Science Digital Library
Based in New York City, the MOUSE organization works to empower "underserved students to provide technology support and leadership in their schools, supporting their academic and career success." On their homepage, visitors can learn about their programs, learn about supporting the MOUSE organization, and read up on their resources. In the "Resources" area, visitors can learn about their outreach activities in New York City, Chicago, and California. Visitors working in educational outreach will appreciate the information offered here, including materials on how different groups can receive assistance from the MOUSE organization. Also, visitors can look over the "News" updates to learn about their new programs, their educational seminars, and their outreach activities.
Dual-mode enhancement of metallothionein protein with cell transduction and retention peptide fusion.
Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), have been developed as effective systems for delivering bio-active cargos such as proteins, genes and particles. Further improvements on cell-specific targeting, intracellular organelle targeting and intracellular retention are still necessary to enhance the therapeutic effect of PTD fusion proteins. In order to enhance the cell transduction and retention of anti-oxidative metallothionein protein (MT), MT was recombinantly fused with transcriptional activator (Tat) with or without a short peptide (sMTS) derived from mitochondria malate dehydrogenase (mMDH). Cellular uptake and retention time of fusion protein were significantly increased in the H9c2 cell by sMTS. The Tat-sMTS-MT (TMM) fusion protein protected H9c2 cells more effectively against hypoxia, hyperglycemia and combination compared with Tat-MT (TM) by reducing intracellular ROS level. It maintained the normal blood glucose level over an extended period of time in a streptozotocin-induced diabetic mouse model. PTD-sMTS-MT fusion protein has a potential to be used as a therapeutic protein for the treatment or prevention of diabetes and diabetic complications. PMID:23871961
Lim, Kwang Suk; Lim, Myoung-Hwa; Won, Young-Wook; Kim, Jang Kyoung; Kang, Young Cheol; Park, Eun Jeong; Chae, Ji-Won; Kim, So-Mi; Ryu, Seong-Eon; Pak, Youngmi Kim; Kim, Yong-Hee
Relationship of Metallothionein Induction to the Heat Shock Response.
National Technical Information Service (NTIS)
Heat shock proteins (hsps) and Metallothioneins (MTs) are sets of proteins specifically synthesized by cells in response to sublethal injury such as heat, heavy metals or radiations. This dissertation investigated the relationship of metallothionein induc...
J. M. Yang P. D. Bowman M. A. Deaton S. T. Schuschereba B. E. Stuck
[Metallothioneins: the structure and mechanisms of action].
Metallothioneins (MT)--4 groups (MT-I, II, III and IV) of low molecular mass (approximately 6.5 kDa, 61-62 amino acid residues) cytosol proteins. They are rich in sulphur--20 residues for cysteine, are found in cytosol and nuclei of eucaryotic cells. MT-I and MT-II are found in all animal tissues, MT-III and MT-IV--in the brain. The functions of MT are regulation and control of redox-homeostasis, thioldisulphide equilibrium in the cell in synergism with GSH. MT molecule involve two domains, alpha and beta. MT gene promoter have response elements to metals (MRE), to glucocorticoids (GRE) and to oxidative agents, electrophilic compounds and xenobiotics (ARE). Expression and synthesis of MT are induced for heavy metals (Zn, Cu, Cd and so Hg, Pb, As, Ni, Ag a.o.); glucocorticoids and other stress-hormones and cytokines; free radicals, peroxides, cancerogens and antitumor drugs, UV and ionizing radiation. Zn and partially Cu are physiological inductors of MT. Other inductors act more or less actively as stress-agents. Zinc stabilizes MT molecule, enhances some of their functional activities as a scavenger of metal ions, of free radicals, toxins and xenobiotics. MT are exceptional protection agents for embryo and adult from Cd and other heavy metals, from ionizing radiation, cancerogens, alkylating and DNA-linked agents, from oxidative stress. MT realizes negative control of immune system functions, of transcription factor NF-kB activity. The use of genetic engineering achievements (transgenic mice with defective MTF-1-genes and MT-overexpressing genes) enlarge the possibilities of MT study and application. PMID:14681972
Barabo?, V A; Petrina, L G
Effect of ICSI on gene expression and development of mouse preimplantation embryos
BACKGROUND In vitro culture (IVC) and IVF of preimplantation mouse embryos are associated with changes in gene expression. It is however not known whether ICSI has additional effects on the transcriptome of mouse blastocysts. METHODS We compared gene expression and development of mouse blastocysts produced by ICSI and cultured in Whitten's medium (ICSIWM) or KSOM medium with amino acids (ICSIKSOMaa) with control blastocysts flushed out of the uterus on post coital Day 3.5 (in vivo). In addition, we compared gene expression in embryos generated by IVF or ICSI using WM. Global pattern of gene expression was assessed using the Affymetrix 430 2.0 chip. RESULTS Blastocysts from ICSI fertilization have a reduction in the number of trophoblastic and inner cell mass cells compared with embryos generated in vivo. Approximately 1000 genes are differentially expressed between ICSI blastocyst and in vivo blastocysts; proliferation, apoptosis and morphogenetic pathways are the most common pathways altered after IVC. Unexpectedly, expression of only 41 genes was significantly different between embryo cultured in suboptimal conditions (WM) or optimal conditions (KSOMaa). CONCLUSIONS Our results suggest that fertilization by ICSI may play a more important role in shaping the transcriptome of the developing mouse embryo than the culture media used.
Giritharan, G.; Li, M.W.; De Sebastiano, F.; Esteban, F.J.; Horcajadas, J.A.; Lloyd, K.C.K.; Donjacour, A.; Maltepe, E.; Rinaudo, P.F.
The genomic organization of the mouse CD94 C-type lectin gene.
The mouse natural killer (NK) gene complex is located on chromosome 6 and contains a number of genes encoding C-type lectin receptors which have been found to regulate NK cell function. Among these are CD94 and four NKG2 genes. Like its human counterpart, the mouse CD94 protein associates with different NKG2 isoforms and recognizes the atypical MHC class I molecule Qa-1b. Here, the genomic organization of the mouse CD94 gene was determined by analysing a BAC clone containing the CD94 gene. The mouse CD94 gene contains six exons separated by five introns. Exons I and II encode the 5' untranslated region (UTR) and the transmembrane domain. Exon III encodes the stalk region and exons IV-VI encode the carbohydrate recognition domain (CRD). Furthermore, we cloned and sequenced the CD94 promoter region, and putative regulatory DNA elements were identified. Further studies on the CD94 promoter region may help to elucidate the restricted expression pattern of CD94 in NK cells and a subpopulation of T cells. PMID:10940084
Lohwasser, S; Wilhelm, B; Mager, D L; Takei, F
Mouse Ribosomal RNA Genes Contain Multiple Differentially Regulated Variants
Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning and characterization of seven mouse rDNA variants (v-rDNA). The identification of these v-rDNAs was based on restriction fragment length polymorphisms (RFLPs), which are conserved among individuals and mouse strains. The total copy number of the identified variants is less than 100 and the copy number of each individual variant ranges from 4 to 15. Sequence analysis of the cloned v-rDNA identified variant-specific single nucleotide polymorphisms (SNPs) in the transcribed region. These SNPs were used to develop a set of variant-specific PCR assays, which permitted analysis of the v-rDNAs' expression profiles in various tissues. These profiles show that three v-rDNAs are expressed in all tissues (constitutively active), two are expressed in some tissues (selectively active), and two are not expressed (silent). These expression profiles were observed in six individuals from three mouse strains, suggesting the pattern is not randomly determined. Thus, the mouse rDNA array likely consists of genetically distinct variants, and some are regulated tissue-specifically. Our results provide the first molecular evidence for cell-type-specific regulation of a subset of rDNA.
Tseng, Hung; Chou, Weichin; Wang, Junwen; Zhang, Xiaohong; Zhang, Shengliang; Schultz, Richard M.
Construction of a mouse model of factor VIII deficiency by gene targeting
To develop a small animal model of hemophilia A for gene therapy experiments, we set out to construct a mouse model for factor VIII deficiency by gene targeting. First, we screened a mouse liver cDNA library using a human FVIII cDNA probe. We cloned a 2.6 Kb partial mouse factor VIII cDNA which extends from 800 base pairs of the 3{prime} end of exon 14 to the 5{prime} end of exon 26. A mouse genomic library made from strain 129 was then screened to obtain genomic fragments covering the exons desired for homologous recombination. Two genomic clones were obtained, and one covering exon 15 through 22 was used for gene targeting. To make gene targeting constructs, a 5.8 Kb genomic DNA fragment covering exons 15 to 19 of the mouse FVIII gene was subcloned, and the neo expression cassette was inserted into exons 16 and 17 separately by different strategies. These two constructs were named MFVIIIC-16 and MFVIIIC-17. The constructs were linearized and transfected into strain 129 mouse ES cells by electroporation. Factor VIII gene-knockout ES cell lines were selected by G-418 and screened by genomic Southern blots. Eight exon 16 targeted cell lines and five exon 17 targeted cell lines were obtained. Three cell lines from each construct were injected into blastocysts and surgically transferred into foster mothers. Multiple chimeric mice with 70-90% hair color derived from the ES-cell genotype were seen with both constructs. Germ line transmission of the ES-cell genotype has been obtained for the MFVIIIC-16 construct, and multiple hemophilia A carrier females have been identified. Factor VIII-deficient males will be conceived soon.
Bi, L.; Lawler, A.; Gearhart, J. [Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States)] [and others
Systematic identification and integrative analysis of novel genes expressed specifically or predominantly in mouse epididymis
Background Maturation of spermatozoa, including development of motility and the ability to fertilize the oocyte, occurs during transit through the microenvironment of the epididymis. Comprehensive understanding of sperm maturation requires identification and characterization of unique genes expressed in the epididymis. Results We systematically identified 32 novel genes with epididymis-specific or -predominant expression in the mouse epididymis UniGene library, containing 1505 gene-oriented transcript clusters, by in silico and in vitro analyses. The Northern blot analysis revealed various characteristics of the genes at the transcript level, such as expression level, size and the presence of isoform. We found that expression of the half of the genes is regulated by androgens. Further expression analyses demonstrated that the novel genes are region-specific and developmentally regulated. Computational analysis showed that 15 of the genes lack human orthologues, suggesting their implication in male reproduction unique to the mouse. A number of the novel genes are putative epididymal protease inhibitors or ?-defensins. We also found that six of the genes have secretory activity, indicating that they may interact with sperm and have functional roles in sperm maturation. Conclusion We identified and characterized 32 novel epididymis-specific or -predominant genes by an integrative approach. Our study is unique in the aspect of systematic identification of novel epididymal genes and should be a firm basis for future investigation into molecular mechanisms underlying sperm maturation in the epididymis.
Oh, Jungsu; Lee, Jiae; Woo, Jong-Min; Choi, Eunyoung; Park, Inju; Han, Cecil; Baek, Namhoe; Lee, Hoyong; Kim, Do Han; Cho, Chunghee
Regulation of gene expression in the preimplantation mouse embryo
The maternal to zygotic transition, which entails activation of the embryonic genome, is arguably the first important event that must occur following fertilization for further successful development. In mammalian preimplantation embryos, zygotic gene activation occurs at different developmental stages. What regulates the timing of gene expression, however, is poorly understood and the spectrum of genes that are expressed is ill-defined.
R. M. Schultz; D. M. Worrad; W. Davis; P. A. De Sousa
Functional expression of yeast artificial chromosome-human multidrug resistance genes in mouse cells.
Multidrug resistance (MDR) genes, which are ATP-binding cassette family genes, encode the cell surface glycoprotein, P-glycoprotein, which functions as an energy-dependent drug efflux pump. Two relevant human genes, PGY1 and PGY3, are located on human chromosome 7, and three relevant mouse genes, mdr1a, mdr1b, and mdr2, are located on mouse chromosome 5. An LMD1 cell line was established after the transfer of a 580-kb yeast artificial chromosome (YAC) clone carrying the human MDR locus into mouse L cells; the cell line was shown to have stably integrated YAC DNA in an apparent intact form. Using LMD1 cells as the parental cell line, five vincristine-resistant sublines, designated LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000, were isolated by exposure to increasing concentrations of the drug. LMD1-V50, LMD1-V100, LMD1-V200, LMD1-V500, and LMD1-V1000 showed 3-, 7-, 13-, 45-, and 110-fold higher resistance to the cytotoxic effects of vincristine, respectively, than their parental counterpart, LMD1. Immunofluorescence, Western blot, and Northern blot analyses revealed that the human PGY1 gene or its product was overexpressed, accompanied by gene amplification. The human PGY3 gene was also overexpressed in the LMD1-V20, LMD1-V100, and LMD1-V1000 cell lines. Southern blot and fluorescence in situ hybridization (FISH) analyses demonstrated that although essentially the entire YAC DNA was integrated in mouse genome and amplified, the endogenous mouse mdr genes were not amplified in these drug-resistant cell lines. Similar results were obtained by the analyses of vincristine-resistant cell lines isolated from four independent subclones of LMD1 cells. Thus, in contrast to their mouse counterparts, the integrated human MDR genes retained susceptibility to both gene activation and amplification, during the selection of drug-resistant mouse cell lines. The possibility that transferred YACs may retain regulatory properties observed in the cells of origin, and may have a chromatin structure that favors augmented expression, is discussed. PMID:8593612
Kusaba, H; Kohno, K; Asakuno, K; Kuwano, M; Okumura, K; Green, E D; Schlessinger, D; Wada, M
Physical mapping of the retinoid X receptor B gene in mouse and human
Retinoid X receptors (RXRs) are zinc finger-containing nuclear transcription factors. They belong to the nuclear receptor superfamily that contains retinoid receptors, vitamin D receptors, thyroid hormone receptors, and steroid hormone receptors as well as the so-called orphan receptors. We previously mapped all three RXR genes on mouse chromosomes, using a panel of Mus spretus-Mus musculus interspecific backcross mice: namely, the RXRA-gene (Rxra) on Chr 2 near the centromere, the RXRB gene (Rxrb) on Chr 17 in the H2 region, and the RXRG gene (Rxrg) on distal Chr 1. Using cosmid clones that cover the major histocompatibility complex (MHC) region, we determined the precise physical map positions of the gene encoding mouse and human RXRB, respectively. The mouse gene (Rxrb) maps between H2-Ke4 and H2-Ke5: namely, immediately telomeric to H2-Ke4 which encodes a histidine-rich transmembrane protein, and 12 kilobases centromeric to H2-Ke5 which is expressed in lymphoid tissues, Rxrb and H2-Ke4 are transcribed into opposite directions from a CpG-rich promoter of about 250 base pairs. This gene organization is well conserved also in the human genome at the HLA-DP subregion of Chr 6p, underscoring the strong conservation of the gene organization in the MHC region between the two mammals. 54 refs., 4 figs.
Nagata, T.; Kitagawa, K.; Taketo, M. [Banyu Tsukuba Research Institute, Tsukuba (Japan); Weiss, E.H. [Ludwig-Maximilians-Univ., Munich (Germany); Abe, K. [Kumamoto Univ. School of Medicine, Kumamoto (Japan); Ando, A.; Yara-Kikuti, Y.; Inoko, H. [Tokai Univ. School of Medicine, Isehara (Japan); Seldin, M.F. [Duke Univ. Medical Center, Durham, NC (United States); Ozato, K. [National Institutes of Health, Bethesda, MD (United States)
A mouse atlas of gene expression: Large-scale digital gene-expression profiles from precisely defined developing C57BL/6J mouse tissues and cells
We analyzed 8.55 million LongSAGE tags generated from 72 libraries. Each LongSAGE library was prepared from a different mouse tissue. Analysis of the data revealed extensive overlap with existing gene data sets and evidence for the existence of ?24,000 previously undescribed genomic loci. The visual cortex, pancreas, mammary gland, preimplantation embryo, and placenta contain the largest number of differentially expressed transcripts, 25% of which are previously undescribed loci.
Siddiqui, Asim S.; Khattra, Jaswinder; Delaney, Allen D.; Zhao, Yongjun; Astell, Caroline; Asano, Jennifer; Babakaiff, Ryan; Barber, Sarah; Beland, Jaclyn; Bohacec, Slavita; Brown-John, Mabel; Chand, Steve; Charest, David; Charters, Anita M.; Cullum, Rebecca; Dhalla, Noreen; Featherstone, Ruth; Gerhard, Daniela S.; Hoffman, Brad; Holt, Robert A.; Hou, Juan; Kuo, Byron Y.-L.; Lee, Lisa L. C.; Lee, Stephanie; Leung, Derek; Ma, Kevin; Matsuo, Corey; Mayo, Michael; McDonald, Helen; Prabhu, Anna-liisa; Pandoh, Pawan; Riggins, Gregory J.; de Algara, Teresa Ruiz; Rupert, James L.; Smailus, Duane; Stott, Jeff; Tsai, Miranda; Varhol, Richard; Vrljicak, Pavle; Wong, David; Wu, Mona K.; Xie, Yuan-Yun; Yang, George; Zhang, Ida; Hirst, Martin; Jones, Steven J. M.; Helgason, Cheryl D.; Simpson, Elizabeth M.; Hoodless, Pamela A.; Marra, Marco A.
1 kb of 5? flanking sequence from mouse MC4R gene is sufficient for tissue specific expression in a transgenic mouse
The melanocortin 4 receptor (MC4R) plays a critical role in the regulation of energy homeostasis, and the MC4R knockout mouse and humans with MC4R defective mutations in only one allele indicate that there is a gene dosage effect. Alterations in gene expression levels for MC4R could, therefore, have significant effects on energy homeostasis. To begin to develop a mouse model
Philip B. Daniel; Chathurini Fernando; C.-S. Jenny Wu; Rebecca Marnane; Ric Broadhurst; Kathleen G. Mountjoy
Effects of metallothionein on mutagenicity and oxidation of quercetin.
The effects of four types of Zn/Cd-metallothioneins on the mutagenicity of quercetin in Salmonella typhimurium TA98 were studied. The four types of Zn/Cd-metallothionein used in this experiment were metallothionein I, metallothionein I/II and metallothionein II from rabbit liver and metallothionein I/II from horse kidney. All four of the metallothioneins enhanced the mutagenicity of quercetin. Metallothionein II from rabbit liver, in which Zn content was the highest of the four metallothioneins, enhanced the mutagenicity of quercetin most effectively. Metallothioneins as well as Zn/Cu-SOD prevented quercetin oxidation under aerobic conditions. The action of these metallothioneins, which enhances the mutagenicity of quercetin, was fairly proportional to the activity which prevents quercetin from being oxidized. Reduced glutathione did not enhance the mutagenicity of quercetin although it did inhibit the quercetin oxidation. These results suggest that metallothioneins have superoxide (O2-) scavenging ability and act as "SOD-like" proteins in vivo. A simple and reliable system to detect biological substances which have antioxidant activity for the superoxide (O2-) was devised. PMID:1488255
Okamoto, A
The Rab protein family: Genetic mapping of six Rab genes in the mouse
Rab proteins constitute a family of GTP-binding proteins that are located in distinct intracellular compartments and play a role in the regulation of vesicular trafficking. Yeast mutations in Rab gene homologs cause defects in vesicular transport similar to those observed in beige (bg) mice. To investigate Rab genes as candidates for mouse mutations characterized by defects in vesicular trafficking, we utilized an intersubspecific backcross [C57BL/6J-bg{sup J} X (C57BL/6J-bg{sup J} X CAST/Ei)F{sub 1}] segregating for the bg locus. Restriction fragment length polymorphisms (RFLPs) were obtained through Southern hybridization of F{sub 1} and C57BL/6J chromosomal DNA with the coding sequences of Rab genes. These RFLPs and 12 polymorphic microsatellites were used to determine the segregation of the Rab genes in 93 backcross mice. Rab4a, Rab4b, Rab7, Rab10, Rab22, and Rab24 were localized on mouse chromosomes 8, 7, 9, 12, 2, and 13, respectively. Although the results exclude these loci as candidates for bg, they demonstrate a wide dispersion of Rab genes throughout the mouse genome and reveal that Rab4b and Rab24 are possible candidates for the mouse mutations reduced pigmentation (rp) and purkinje cell degeneration (pcd), respectively. 31 refs., 3 figs., 2 tabs.
Barbosa, M.D.F.S.; Gutierrez, M.J.; Kingsmore, S.F. [Univ. of Florida, Gainesville (United States)] [and others] [Univ. of Florida, Gainesville (United States); and others
Temporally and spatially controllable gene expression and knockout in mouse urothelium.
Urothelium that lines almost the entire urinary tract performs important functions and is prone to assaults by urinary microbials, metabolites, and carcinogens. To improve our understanding of urothelial physiology and disease pathogenesis, we sought to develop two novel transgenic systems, one that would allow inducible and urothelium-specific gene expression, and another that would allow inducible and urothelium-specific knockout. Toward this end, we combined the ability of the mouse uroplakin II promoter (mUPII) to drive urothelium-specific gene expression with a versatile tetracycline-mediated inducible system. We found that, when constructed under the control of mUPII, only a modified, reverse tetracycline trans-activator (rtTA-M2), but not its original version (rtTA), could efficiently trans-activate reporter gene expression in mouse urothelium on doxycycline (Dox) induction. The mUPII/rtTA-M2-inducible system retained its strict urothelial specificity, had no background activity in the absence of Dox, and responded rapidly to Dox administration. Using a reporter gene whose expression was secondarily controlled by histone remodeling, we were able to identify, colocalize with 5-bromo-2-deoxyuridine incorporation, and semiquantify newly divided urothelial cells. Finally, we established that, when combined with a Cre recombinase under the control of the tetracycline operon, the mUPII-driven rtTA-M2 could inducibly inactivate any gene of interest in mouse urothelium. The establishment of these two new transgenic mouse systems enables the manipulation of gene expression and/or inactivation in adult mouse urothelium at any given time, thus minimizing potential compensatory effects due to gene overexpression or loss and allowing more accurate modeling of urothelial diseases than previously reported constitutive systems. PMID:20427471
Zhou, Haiping; Liu, Yan; He, Feng; Mo, Lan; Sun, Tung-Tien; Wu, Xue-Ru
Manual Gene Ontology annotation workflow at the Mouse Genome Informatics Database
The Mouse Genome Database, the Gene Expression Database and the Mouse Tumor Biology database are integrated components of the Mouse Genome Informatics (MGI) resource (http://www.informatics.jax.org). The MGI system presents both a consensus view and an experimental view of the knowledge concerning the genetics and genomics of the laboratory mouse. From genotype to phenotype, this information resource integrates information about genes, sequences, maps, expression analyses, alleles, strains and mutant phenotypes. Comparative mammalian data are also presented particularly in regards to the use of the mouse as a model for the investigation of molecular and genetic components of human diseases. These data are collected from literature curation as well as downloads of large datasets (SwissProt, LocusLink, etc.). MGI is one of the founding members of the Gene Ontology (GO) and uses the GO for functional annotation of genes. Here, we discuss the workflow associated with manual GO annotation at MGI, from literature collection to display of the annotations. Peer-reviewed literature is collected mostly from a set of journals available electronically. Selected articles are entered into a master bibliography and indexed to one of eight areas of interest such as �GO� or �homology� or �phenotype�. Each article is then either indexed to a gene already contained in the database or funneled through a separate nomenclature database to add genes. The master bibliography and associated indexing provide information for various curator-reports such as �papers selected for GO that refer to genes with NO GO annotation�. Once indexed, curators who have expertise in appropriate disciplines enter pertinent information. MGI makes use of several controlled vocabularies that ensure uniform data encoding, enable robust analysis and support the construction of complex queries. These vocabularies range from pick-lists to structured vocabularies such as the GO. All data associations are supported with statements of evidence as well as access to source publications.
Drabkin, Harold J.; Blake, Judith A.
Mouse Ribosomal RNA Genes Contain Multiple Differentially Regulated Variants
Previous cytogenetic studies suggest that various rDNA chromosomal loci are not equally active in different cell types. Consistent with this variability, rDNA polymorphism is well documented in human and mouse. However, attempts to identify molecularly rDNA variant types, which are regulated individually (i.e., independent of other rDNA variants) and tissue-specifically, have not been successful. We report here the molecular cloning
Hung Tseng; Weichin Chou; Junwen Wang; Xiaohong Zhang; Shengliang Zhang; Richard M. Schultz; Peter Fraser
The Mouse Polyubiquitin Gene Ubb Is Essential for Meiotic Progression? �
Ubiquitin is encoded in mice by two polyubiquitin genes, Ubb and Ubc, that are considered to be stress inducible and two constitutively expressed monoubiquitin (Uba) genes. Here we report that targeted disruption of Ubb results in male and female infertility due to failure of germ cells to progress through meiosis I and hypogonadism. In the absence of Ubb, spermatocytes and oocytes arrest during meiotic prophase, before metaphase of the first meiotic division. Although cellular ubiquitin levels are believed to be maintained by a combination of functional redundancy among the four ubiquitin genes, stress inducibility of the two polyubiquitin genes, and ubiquitin recycling by proteasome-associated isopeptidases, our results indicate that ubiquitin is required for and consumed during meiotic progression. The striking similarity of the meiotic phenotype in Ubb?/? germ cells to the sporulation defect in fission yeast (Schizosaccharomyces pombe) lacking a polyubiquitin gene suggests that a meiotic role of the polyubiquitin gene has been conserved throughout eukaryotic evolution.
Ryu, Kwon-Yul; Sinnar, Shamim A.; Reinholdt, Laura G.; Vaccari, Sergio; Hall, Susan; Garcia, Manuel A.; Zaitseva, Tatiana S.; Bouley, Donna M.; Boekelheide, Kim; Handel, Mary Ann; Conti, Marco; Kopito, Ron R.
Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure.
The mouse is extensively used to model human folate metabolism and therapeutic outcomes with antifolates. However, the folylpoly-?-glutamate synthetase (fpgs) gene, whose product determines folate/antifolate intracellular retention and antifolate antitumor activity, displays a pronounced species difference. The human gene uses only a single promoter, whereas the mouse uses two: P2, akin to the human promoter, at low levels in most tissues; and P1, an upstream promoter used extensively in liver and kidney. We deleted the mouse P1 promoter through homologous recombination to study the dual-promoter mouse system and to create a mouse with a humanized fpgs gene structure. Despite the loss of the predominant fpgs mRNA species in liver and kidney (representing 95 and 75% of fpgs transcripts in these tissues, respectively), P1-knockout mice developed and reproduced normally. The survival of these mice was explained by increased P2 transcription due to relief of transcriptional interference, by a 3-fold more efficient translation of P2-derived than P1-derived transcripts, and by 2-fold higher stability of P2-derived FPGS. In combination, all 3 effects reinstated FPGS function, even in liver. By eliminating mouse P1, we created a mouse model that mimicked the human housekeeping pattern of fpgs gene expression. PMID:24532667
Yang, Chen; Xie, Lin-Ying; Windle, Jolene J; Taylor, Shirley M; Moran, Richard G
Comparative analysis of cortical gene expression in mouse models of Alzheimer's disease.
Three mouse models of Alzheimer's disease (AD) were used to assess changes in gene expression potentially critical to amyloid beta-peptide (Abeta)-induced neuronal dysfunction. One mouse model harbored homozygous familial AD (FAD) knock-in mutations in both, amyloid precursor protein (APP) and presenilin 1 (PS-1) genes (APP(NLh/NLh)/PS-1(P264L/P264L)), the other two models harbored APP over-expression of FAD mutations (Tg2576) with the PS-1 knock-in mutation at either one or two alleles. These mouse models of AD had varying levels of Abeta40 and Abeta42 and different latencies and rates of Abeta deposition in brain. To assess changes in gene expression associated with Abeta accumulation, the Affymetrix murine genome array U74A was used to survey gene expression in the cortex of these three models both prior to and following Abeta deposition. Altered genes were identified by comparing the AD models with age-matched control littermates. Thirty-four gene changes were identified in common among the three models in mice with Abeta deposition. Among the up-regulated genes, three major classes were identified that encoded for proteins involved in immune responses, carbohydrate metabolism, and proteolysis. Down-regulated genes of note included pituitary adenylate cyclase-activating peptide (PACAP), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor I receptor (IGF-IR). In young mice without detectable Abeta deposition, there were no regulated genes common among the three models, although 40 genes were similarly altered between the two Tg2576 models with the PS-1 FAD knock-in. Finally, changes in gene expression among the three mouse models of AD were compared with those reported in human AD samples. Sixty-nine up-regulated and 147 down-regulated genes were found in common with human AD brain. These comparisons across different genetic mouse models of AD and human AD brain provide greater support for the involvement of identified gene expression changes in the neuronal dysfunction and cognitive deficits accompanying amyloid deposition in mammalian brain. PMID:15927307
Wu, Zhi-Liang; Ciallella, John R; Flood, Dorothy G; O'Kane, Teresa M; Bozyczko-Coyne, Donna; Savage, Mary J
Extensive compensatory cis-trans regulation in the evolution of mouse gene expression
Gene expression levels are thought to diverge primarily via regulatory mutations in trans within species, and in cis between species. To test this hypothesis in mammals we used RNA-sequencing to measure gene expression divergence between C57BL/6J and CAST/EiJ mouse strains and allele-specific expression in their F1 progeny. We identified 535 genes with parent-of-origin specific expression patterns, although few of these showed full allelic silencing. This suggests that the number of imprinted genes in a typical mouse somatic tissue is relatively small. In the set of nonimprinted genes, 32% showed evidence of divergent expression between the two strains. Of these, 2% could be attributed purely to variants acting in trans, while 43% were attributable only to variants acting in cis. The genes with expression divergence driven by changes in trans showed significantly higher sequence constraint than genes where the divergence was explained by variants acting in cis. The remaining genes with divergent patterns of expression (55%) were regulated by a combination of variants acting in cis and variants acting in trans. Intriguingly, the changes in expression induced by the cis and trans variants were in opposite directions more frequently than expected by chance, implying that compensatory regulation to stabilize gene expression levels is widespread. We propose that expression levels of genes regulated by this mechanism are fine-tuned by cis variants that arise following regulatory changes in trans, suggesting that many cis variants are not the primary targets of natural selection.
Goncalves, Angela; Leigh-Brown, Sarah; Thybert, David; Stefflova, Klara; Turro, Ernest; Flicek, Paul; Brazma, Alvis; Odom, Duncan T.; Marioni, John C.
The context of T-cell receptor gamma chain genes among wild mouse species.
We have examined the context of mouse T-cell receptor gamma (Tcr gamma) chain variable (V gamma) and constant (C gamma) genes among a panel of geographically isolated species of mice. Our Southern hybridization survey with C gamma reveals that essentially three C gamma genes are found among mouse species extending phylogenetically from inbred mice through the feral species Mus pahari. However, a V gamma DNA probe detects three to nine V gamma restriction fragment bands among the same group of mice. These results suggest that certain feral mice such as M. pahari, M. platythrix, and M. shortridgei have amplified numbers of V gamma genes. Studies of individual mice from these particular species indicate the highly amplified V gamma content is not the result of a catastrophic gene duplication or deletion event. We conclude that certain species of mice maintain increased content of V gamma presumably for increased diversity in a T-cell response. PMID:3781572
Huppi, K; D'Hoostelaere, L; Kiefer, M; Steinmetz, M; Jouvin-Marche, E
Mapping of a liver phosphorylase kinase [alpha]-subunit gene on the mouse x chromosome
Phosphorylase kinase (PHK) is a regulatory enzyme of the glycogenolytic pathway composed of a complex of four subunits. We recently mapped the muscle [alpha]-subunit gene (Phka) to the mouse X chromosome in a region syntenic with the proximal long arm of the human X chromosome and containing the human homologue of this gene, PHKA. We now report the mapping of the liver [alpha]-subunit gene to the telomeric end of the mouse X chromosome. This mapping position would suggest a location for the human liver [alpha]-subunit gene on the proximal short arm of the X chromosome, a region recently implicated in X-linked liver glycogenosis (XLG). 20 refs., 2 figs.
Geng, Yan; Derry, J.M.J.; Barnard, P.J. (MRC Molecular Neurobiology Unit, Cambridge (United Kingdom)); Hendrickx, J.; Coucke, P.; Willems, P.R. (Univ. of Antwerp (Belgium))
Identification of a Copper-Binding Metallothionein in Pathogenic Mycobacteria
A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially bound up to 6 Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the gene�s expression in Mtb up to a thousand-fold. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionien (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function.
Gold, Ben; Deng, Haiteng; Bryk, Ruslana; Vargas, Diana; Eliezer, David; Roberts, Julia; Jiang, Xiuju; Nathan, Carl
Genetics of gene expression surveyed in maize, mouse and man
Treating messenger RNA transcript abundances as quantitative traits and mapping gene expression quantitative trait loci for these traits has been pursued in gene-specific ways. Transcript abundances often serve as a surrogate for classical quantitative traits in that the levels of expression are significantly correlated with the classical traits across members of a segregating population. The correlation structure between transcript abundances
Eric E. Schadt; Stephanie A. Monks; Thomas A. Drake; Aldons J. Lusis; Nam Che; Veronica Colinayo; Thomas G. Ruff; Stephen B. Milligan; John R. Lamb; Guy Cavet; Peter S. Linsley; Mao Mao; Roland B. Stoughton; Stephen H. Friend
Site and Time-Specific Gene Targeting in the Mouse
The efficient introduction of somatic mutations in a given gene, at a given time, in a specific cell type, will facilitate studies of gene function and the generation of animal models for human diseases. We have established a conditional site-specific recombination system in mice using a new version of the Cre\\/lox system. The Cre recombinase has been fused to a
Daniel Metzger; Pierre Chambon
Gene Delivery to the Retina: From Mouse to Man
With the recent progress in identifying disease-causing genes in humans and in animal models, there are more and more opportunities for using retinal gene transfer to learn more about retinal physiology and also to develop therapies for blinding disorders. Success in preclinical studies for one form of inherited blindness have led to testing in human clinical trials. This paves the way to consider a number of other retinal diseases as ultimate gene therapy targets in human studies. The information presented here is designed to assist scientists and clinicians to use gene transfer to probe the biology of the retina and/or to move appropriate gene-based treatment studies from the bench to the clinic.
Bennett, Jean; Chung, Daniel C.; Maguire, Albert
Characterization of the genomic structure of the mouse APLP1 gene
This article reports on the organization of the mouse APLP1 gene, an evolutionarily conserved amyloid precursor-like protein. The amyloid beta protein, important in Alzheimer diseases, is derived from these precursor proteins. By investigating the expression and structure of this murine gene, it is hoped that more will be learned about the function and regulation of the human homologue. 27 refs., 2 figs.
Zhong, Sue; Wu, Kuo; Black, I.B.; Schaar, D.G. [State Univ. of New Jersey, Piscataway, NJ (United States)] [State Univ. of New Jersey, Piscataway, NJ (United States)
Mouse Studies Identify Gene that May Influence Metastasis Risk in Breast Cancer
Researchers have identified a pattern of gene activity in mice that may help to predict individual risk for breast cancer metastasis and survival in humans. A single gene called bromodomain 4 (Brd4) regulates the expression of this pattern, also called a signature. The researchers found that one result of this Brd4 regulation is the suppression of tumor growth and metastasis in a mouse model of cancer.
Structural Organization and Chromosomal Assignment of the Mouse Embryonic TEA Domain-Containing Factor (ETF) Gene
Embryonic TEA domain-containing factor (ETF) belongs to the family of proteins structurally related to transcriptional enhancer factor-1 (TEF-1) and is implicated in neural development. Isolation and characterization of the cosmid clones encoding the mouse ETF gene (Etdf) revealed thatEtdfspans approximately 17.9 kb and consists of 12 exons. The exon�intron structure ofEtdfclosely resembles that of theDrosophila scallopedgene, indicating that these genes
Kazuo Suzuki; Michio Yasunami; Yoichi Matsuda; Takako Maeda; Hironori Kobayashi; Hidenori Terasaki; Hiroaki Ohkubo
Characterization of the Pituitary Tumor Transforming Gene 1 Knockout Mouse Retina
Recent gene expression studies on mouse models for retinal degeneration identified deregulation of Pituitary tumor transforming\\u000a gene 1 (Pttg1) as a potential susceptibility factor involved in photoreceptor cell death. Pttg1 is a transcription regulatory protein involved\\u000a in sister chromatid segregation, and Pttg1\\u000a ?\\/? mice exhibit testicular and splenic hypoplasia, thymic hyperplasia, aberrant cell cycle progression, chromosome instability,\\u000a and impaired glucose
Rosanne M. Yetemian; Cheryl M. Craft
Genomic Structure, Promoter Sequence, and Induction of Expression of the Mouse Nramp1 Gene in Macrophages
A candidate gene for the mouse chromosome 1 host resistance locus Bcg\\/Ity\\/Lsh was recently cloned and designated Nramp (natural resistance-associated macrophage protein). Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. Primer extension and cDNA cloning were used to isolate the complete 5? end of the Nramp1 mRNA. Analysis of genomic cosmid and bacteriophage
Greg Govoni; Silvia Vidal; Mathieu Cellier; Pierre Lepage; Danielle Malo; Philippe Gros
Transcriptome-Wide Identification of Novel Imprinted Genes in Neonatal Mouse Brain
Imprinted genes display differential allelic expression in a manner that depends on the sex of the transmitting parent. The degree of imprinting is often tissue-specific and\\/or developmental stage-specific, and may be altered in some diseases including cancer. Here we applied Illumina\\/Solexa sequencing of the transcriptomes of reciprocal F1 mouse neonatal brains and identified 26 genes with parent-of-origin dependent differential allelic
Xu Wang; Qi Sun; Sean D. McGrath; Elaine R. Mardis; Paul D. Soloway; Andrew G. Clark; Anne C. Ferguson-Smith
Expression of Hox cofactor genes during mouse ovarian follicular development and oocyte maturation
Very little is known about the expression and function of the HOX and HOX-cofactors genes in mammalian oogenesis. The aim of the present study was to determine the expression of PBX and PREP-1 gene products in the mouse ovary and their localization to particular ovarian compartment, specifically the oocyte-containing ovarian follicle.Immunocytochemical analysis demonstrated that PREP-1 was present in both granulosa
J. Carlos Villaescusa; Arturo C. Verrotti; Elisabetta Ferretti; Riaz Farookhi; Francesco Blasi
Functional studies of homeobox gene {\\\\it Cart1\\\\\\/} during mouse development
Cart1 is a paired-class homeobox-containing gene that is expressed in head mesenchyme, branchial arches, limb buds, and various cartilages during embryogenesis. To understand the role of Cart1 during mammalian development, I generated Cart1-mutant mice by gene targeting in mouse embryonic stem cells. Cart1-homozygous mutants were born alive but all died soon after birth. Most had acrania (absence of the cranial
Qi Zhao
Convenient and reproducible in vivo gene transfer to mouse parotid glands
OBJECTIVES Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands. METHODS The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen�s ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (107 vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR. RESULTS The chosen volume for mouse parotid vector delivery was 20 �L. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva. CONCLUSION These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.
Zheng, C; Shinomiya, T; Goldsmith, CM; Di Pasquale, G; Baum, BJ
Integrated Analysis of Protein Composition, Tissue Diversity, and Gene Regulation in Mouse Mitochondria
Mitochondria are tailored to meet the metabolic and signaling needs of each cell. To explore its molecular composition, we performed a proteomic survey of mitochondria from mouse brain, heart, kidney, and liver and combined the results with existing gene annotations to produce a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle. The protein expression
Vamsi K. Mootha; Jakob Bunkenborg; Jesper V. Olsen; Majbrit Hjerrild; Jacek R. Wisniewski; Erich Stahl; Marjan S. Bolouri; Heta N. Ray; Smita Sihag; Michael Kamal; Nick Patterson; Eric S. Lander; Matthias Mann
Genomic organisation and tissue specific expression of ABLIM2 gene in human, mouse and rat.
The exon-intron structures of the human, rat and mouse ABLIM2 gene were determined in silico. The experimental verification resulted in the revealing of two mRNA isoforms of the ABLIM2 gene. The isoforms a and b contained 20 exons and 18 exons, respectively. The highest expression of both isoforms was observed in rat brain and eye and in mouse embryos. The 5'-UTR region of the ABLIM2 gene was 127 bp in rat and mouse, but in human, it was 65 bp. The site of polyadenylation was shown to be present at a distance of 682 bp from the stop-codon in human and rat and 684 bp in mouse. The in silico analysis of the gene 5'-region was performed. The high density of brain and CNS specific transcription factors' binding sites in the promoter region was shown for all three organisms. The comparison of the amino acid sequences of the human ABLIM2 and ABLIM1 proteins showed that the number and arrangement of domains (four LIM-domains in the N-end region and the C-end VHP-domain) were similar. The structure of the ABLIM2 proteins was similar in all three organisms. On the basis of our data, it was assumed that the ABLIM2 protein was necessary for the normal functioning of neurons. PMID:16005990
Klimov, Eugene; Rud'ko, Olga; Rakhmanaliev, Elian; Sulimova, Galina
Isolation, characterization, and chromosomal localization of mouse and human COUP-TF I and II genes
Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologues have been cloned in many species, from Drosophila to human. The protein sequences of COUP-TFs are highly homologous across species, suggesting functional conservation. Two COUP-TF genes have been cloned from human, and their genomic organizations have been characterized. To determine whether the genomic organization is conserved between human and mouse, we isolated two mouse COUP-TF genes (I and II) and characterized their genomic structures. Both genes have relatively simple structures that are similar to those of their human counterparts. In addition, we mapped mouse COUP-TF I to the distal region of chromosome 13 and COUP-TF II to the central region of chromosome 7. Furthermore, we mapped human COUP-TF I to 5q14 of chromosome 5 and COUP-TF II to 15q26 of chromosome 15. The results demonstrate that COUP-TF genes are located in chromosomal regions that are syntenic between mouse and human. 25 refs., 5 figs.
Qiu, Y.; Krishnan, V.; Zeng, Z. [Baylor College of Medicine, Houston, TX (United States)] [and others] [Baylor College of Medicine, Houston, TX (United States); and others
Evolutionary conservation and selection of human disease gene orthologs in the rat and mouse genomes
BACKGROUND: Model organisms have contributed substantially to our understanding of the etiology of human disease as well as having assisted with the development of new treatment modalities. The availability of the human, mouse and, most recently, the rat genome sequences now permit the comprehensive investigation of the rodent orthologs of genes associated with human disease. Here, we investigate whether human
Hui Huang; Eitan E Winter; Huajun Wang; Keith G Weinstock; Heming Xing; Leo Goodstadt; Peter D Stenson; David N Cooper; Douglas Smith; M Mar Alb�; Chris P Ponting; Kim Fechtel
Mouse Model for the Cloning of a Tumor Suppressor Gene Mutated in Sporadic Breast Cancer.
National Technical Information Service (NTIS)
This report serves as the annual report for the period beginning July 1, 1996 for the project entitled A Mouse Model for the Cloning of a Tumor Suppressor Gene Mutated in Sporadic Breast Cancer. This report states the experimental progress made and propos...
H. Skinner
BioGPS and GXD: mouse gene expression data - the benefits and challenges of data integration
Mouse gene expression data are complex and voluminous. To maximize the utility of these data, they must be made readily accessible through databases, and those resources need to place the expression data in the larger biological context. Here we describe two community resources that approach these problems in different but complementary ways: BioGPS and the Mouse Gene Expression Database (GXD). BioGPS connects its large and homogenous microarray gene expression reference data sets via plugins with a heterogeneous collection of external gene centric resources, thus casting a wide but loose net. GXD acquires different types of expression data from many sources and integrates these data tightly with other types of data in the Mouse Genome Informatics (MGI) resource, with a strong emphasis on consistency checks and manual curation. We describe and contrast the �loose� and �tight� data integration strategies employed by BioGPS and GXD, respectively, and discuss the challenges and benefits of data integration. BioGPS is freely available at http://biogps.org. GXD is freely available through the Mouse Genome Informatics (MGI) web site (www.informatics.jax.org), or directly at www.informatics.jax.org/expression.shtml.
Wu, Chunlei
Global analysis of differential luminal epithelial gene expression at mouse implantation sites
Although implantation types differ between species, the interaction between blastocyst trophectoderm and apical surface of the uterine epithelium is a common event during the implantation process. In this study, uterine luminal epithelium at implantation and inter-implantation sites was isolated by enzymatic digestion and used for microarray analysis. In a mouse microarray containing 12 345 unigenes, we found 136 genes upregulated
Ying Chen; Hua Ni; Xing-Hong Ma; Shi-Jun Hu; Li-Ming Luan; Gang Ren; Yue-Chao Zhao; Shi-Jie Li; Hong-Lu Diao; Xiu Xu; Zhen-Ao Zhao; Zeng-Ming Yang
Interferon-? gene therapy improves survival in an immunocompetent mouse model of carcinomatosis
BackgroundInterferon-? (IFN?) has multiple antitumor effects; however, its use has been limited by its short half-life in vivo. This limitation may be overcome by IFN? gene therapy. We evaluated adenovirus-IFN? therapy in an immunocompetent mouse model of carcinomatosis.
Samantha K Hendren; Indira Prabakaran; Donald G Buerk; Giorgos Karakousis; Michael Feldman; Francis Spitz; Chandrakala Menon; Douglas L Fraker
Metallothionein protein evolution: a miniassay.
Metallothionein (MT) evolution is one of the most obscure yet fascinating aspects of the study of these atypical metal-binding peptides. The different members of the extremely heterogeneous MT protein superfamily probably evolved through a web of duplication, functional differentiation, and/or convergence events leading to the current scenario, which is particularly hard to interpret in terms of molecular evolution. Difficulties in drawing straight evolutionary relationships are reflected in the lack of definite MT classification criteria. Presently, MTs are categorized either according to a pure taxonomic clustering or depending on their metal binding preferences and specificities. Extremely well documented MT revisions were recently published. But beyond classic approaches, this review of MT protein evolution will bring together new aspects that have seldom been discussed before. Hence, the emergence of life on our planet, since metal ion utilization is accepted to be at the root of the emergence of living organisms, and global trends that underlie structural and functional MT diversification, will be presented. Major efforts are currently being devoted to identifying rules for function-constrained MT evolution that may be applied to different groups of organisms. PMID:21633816
Capdevila, Merc�; Atrian, S�lvia
Gene expression profiling in a mouse model for African trypanosomiasis
This study aimed to provide the foundation for an integrative approach to the identification of the mechanisms underlying the response to infection with Trypanosoma congolense, and to identify pathways that have previously been overlooked. We undertook a large-scale gene expression analysis study comparing susceptible A/J and more tolerant C57BL/6 mice. In an initial time course experiment, we monitored the development of parasitaemia and anaemia in every individual. Based on the kinetics of disease progression, we extracted total RNA from liver at days 0, 4, 7, 10 and 17 post infection and performed a microarray analysis. We identified 64 genes that were differentially expressed in the two strains in non-infected animals, of which nine genes remained largely unaffected by the disease. Gene expression profiling at stages of low, peak, clearance and recurrence of parasitaemia suggest that susceptibility is associated with high expression of genes coding for chemokines (e.g. Ccl24, Ccl27 and Cxcl13), complement components (C1q and C3) and interferon receptor alpha (Ifnar1). Additionally, susceptible A/J mice expressed higher levels of some potassium channel genes. In contrast, messenger RNA levels of a few immune response, metabolism and protease genes (e.g. Prss7 and Mmp13) were higher in the tolerant C57BL/6 strain as compared to A/J.
Kierstein, S; Noyes, H; Naessens, J; Nakamura, Y; Pritchard, C; Gibson, J; Kemp, S; Brass, A
Expression of Notch receptors, ligands and target genes during development of the mouse mammary gland
Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. Using an immunohistochemical approach with antibodies specific for each Notch receptor, we show that Notch proteins are temporally regulated in mammary epithelial cells during normal mammary gland development in the FVB/N mouse. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. These data define expression of the Notch ligand/receptor system throughout development of the mouse mammary gland and help set the stage for genetic analysis of Notch in this context.
Raafat, Ahmed; Goldhar, Anita S.; Klauzinska, Malgorzata; Xu, Keli; Amirjazil, Idean; McCurdy, David; Lashin, Karim; Salomon, David; Vonderhaar, Barbara K.; Egan, Sean; Callahan, Robert
Structural organization, chromosomal localization, expression and phylogenetic evaluation of mouse glutathione peroxidase encoding genes.
We have reported earlier the cloning and the chromosomal localization of 2 GPX-encoding sequences expressed differentially within the mouse epididymis, gpx5 and gpx3. Here, we have mapped on the mouse chromosomes the third known murine GPX-encoding gene, the cytosolic GPX or gpx1. We have compared the degree of identity of the 3 GPX proteins, the respective organization of the 3 corresponding single copy genes and, using degenerated oligonucleotides designed in highly conserved domains of the proteins, we have analyzed the expression of GPX-encoding genes in the mouse epididymis as well as in control tissues known to express GPX proteins (the liver for GPX1 and the kidney for GPX3). The 3 genes characterized to date were found expressed in each of the tissues tested but in a highly tissue-restricted manner. Nucleotidic sequences comparisons were carried out on GPX-encoding sequences from various species and were used to draw a dendrogram. Phylogenetic evaluation of the sequence information, as well as the chromosomal localizations, suggest that the GPX genes have evolved by duplication events followed by random insertions from a single ancestral gene. PMID:9011320
Dufaure, J P; Lareyre, J J; Schwaab, V; Mattei, M G; Drevet, J R
Locations of human and mouse genes encoding the RFX1 and RFX2 transcription factor proteins
RFX transcription factors constitute a highly conserved family of site-specific DNA binding proteins involved in the expression of a variety of cellular and viral genes, including major histocompatibility complex class II genes and genes in human hepatitis B virus. Five members of the RFX gene family have been isolated from human and mouse, and all share a highly characteristic DNA binding domain that is distinct from other known DNA binding motifs. The human RFX1 and RFX2 genes have been assigned by in situ hybridization to chromosome 19p13.1 and 19p13.3, respectively. In this paper, we present data that localize RFX1 and RFX2 precisely within the detailed physical map of human chromosome 19 and genetic data that assign Rfx1 and Rfx2 to homologous regions of mouse chromosomes 8 and 17, respectively. These data define the established relationships between these homologous mouse and human regions in further detail and provide new tools for linking cloned genes to phenotypes in both species. 26 ref., 2 figs.
Doyle, J.; Stubbs, L. [Oak Ridge National Lab., TN (United States)] [Oak Ridge National Lab., TN (United States); Hoffman, S. [Lawrence Livermore National Lab., CA (United States)] [and others] [Lawrence Livermore National Lab., CA (United States); and others
Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse
Cyclic nucleotides are important second messengers that mediate a number of cellular responses to external signals. Cyclic nucleotide phosphodiesterases play a role in signal transduction by regulating the cellular concentrations of these messengers. Here, the authors have applied Southern analyses of somatic cell hybrid lines and of recombinant inbred (RI) mouse strains as well as fluorescence chromosomal in situ hybridization (FISH) to chromosomally localize five cAMP-specific nucleotide phosphodiesterase genes in human and mouse. Genes DPDE1, DPDE2, DPDE3, and DPDE4 that share sequence homology with the Drosophila dunce gene were assigned to human chromosomes 19 (DPDE1 and DPDE2), ga12 (DPDE3), and 1p31 (DPDE4) and to mouse chromosomes 8, 9, 13, and 4, respectively. The high-affinity cAMP-specific phosphodiesterase gene (HCP1) was mapped to human chromosome 8q13-q22. Since these genes are potential candidates for involvement in psychiatric or behavioral disorders, knowledge of their chromosomal localizations will facilitate the discovery of their association with disease genes as they are being mapped by linkage studies.
Milatovich, A.; Francke, U. (Stanford Univ. Medical Center, CA (United States)); Bolger, G.; Michaeli, T. (Cold Spring Harbor Lab., NY (United States))
Manual annotation and analysis of the defensin gene cluster in the C57BL/6J mouse reference genome
Background Host defense peptides are a critical component of the innate immune system. Human alpha- and beta-defensin genes are subject to copy number variation (CNV) and historically the organization of mouse alpha-defensin genes has been poorly defined. Here we present the first full manual genomic annotation of the mouse defensin region on Chromosome 8 of the reference strain C57BL/6J, and the analysis of the orthologous regions of the human and rat genomes. Problems were identified with the reference assemblies of all three genomes. Defensins have been studied for over two decades and their naming has become a critical issue due to incorrect identification of defensin genes derived from different mouse strains and the duplicated nature of this region. Results The defensin gene cluster region on mouse Chromosome 8 A2 contains 98 gene loci: 53 are likely active defensin genes and 22 defensin pseudogenes. Several TATA box motifs were found for human and mouse defensin genes that likely impact gene expression. Three novel defensin genes belonging to the Cryptdin Related Sequences (CRS) family were identified. All additional mouse defensin loci on Chromosomes 1, 2 and 14 were annotated and unusual splice variants identified. Comparison of the mouse alpha-defensins in the three main mouse reference gene sets Ensembl, Mouse Genome Informatics (MGI), and NCBI RefSeq reveals significant inconsistencies in annotation and nomenclature. We are collaborating with the Mouse Genome Nomenclature Committee (MGNC) to establish a standardized naming scheme for alpha-defensins. Conclusions Prior to this analysis, there was no reliable reference gene set available for the mouse strain C57BL/6J defensin genes, demonstrating that manual intervention is still critical for the annotation of complex gene families and heavily duplicated regions. Accurate gene annotation is facilitated by the annotation of pseudogenes and regulatory elements. Manually curated gene models will be incorporated into the Ensembl and Consensus Coding Sequence (CCDS) reference sets. Elucidation of the genomic structure of this complex gene cluster on the mouse reference sequence, and adoption of a clear and unambiguous naming scheme, will provide a valuable tool to support studies on the evolution, regulatory mechanisms and biological functions of defensins in vivo.
Reference gene selection for real-time RT-PCR in regenerating mouse livers
The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.
Tatsumi, Kohei [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan); Ohashi, Kazuo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan)], E-mail: firstname.lastname@example.com; Taminishi, Sanae [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan); Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Yoshioka, Akira; Shima, Midori [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan)
Voltage-gated potassium channel genes are clustered in paralogous regions of the mouse genome
Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, the authors report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. They find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. They also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes. 78 refs., 5 figs., 1 tab.
Lock, L.F.; Gilbert, D.J.; Jenkins, N.A.; Copeland, N.G. (ABL-Basic Research Program, Frederick, MD (United States)); Street, V.A.; Migeon, M.B.; Tempel, B.L. (VA Medical Center, Seattle, WA (United States) Univ. of Washington School of Medicine, Seattle, WA (United States))
Apoptosis-related genes change their expression with age and hearing loss in the mouse cochlea
To understand possible causative roles of apoptosis gene regulation in age-related hearing loss (presbycusis), apoptotic gene expression patterns in the CBA mouse cochlea of four different age and hearing loss groups were compared, using GeneChip and real-time (qPCR) microarrays. GeneChip transcriptional expression patterns of 318 apoptosis-related genes were analyzed. Thirty eight probes (35 genes) showed significant differences in expression. The significant gene families include Caspases, B-cell leukemia/lymphoma2 family, P53, Cal-pains, Mitogen activated protein kinase family, Jun oncogene, Nuclear factor of kappa light chain gene enhancer in B-cells inhibitor-related and tumor necrosis factor-related genes. The GeneChip results of 31 genes were validated using the new TaqMan� Low Density Array (TLDA). Eight genes showed highly correlated results with the GeneChip data. These genes are: activating transcription factor3, B-cell leukemia/lymphoma2, Bcl2-like1, caspase4 apoptosis-related cysteine protease 4, Calpain2, dual specificity phosphatase9, tumor necrosis factor receptor superfamily member12a, and Tumor necrosis factor superfamily member13b, suggesting they may play critical roles in inner ear aging.
Tadros, Sherif F.; D'Souza, Mary; Zhu, Xiaoxia
Structural characterization and chromosomal location of the mouse macrophage migration inhibitory factor gene and pseudogenes
Macrophage migration inhibitory factor, MIF, is a cytokine released by T-lymphocytes, macrophages, and the pituitary gland that serves to integrate peripheral and central inflammatory responses. Ubiquitous expression and developmental regulation suggest that MIF may have additional roles outside of the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5{prime} untranslated region of the cDNA, a 3{prime} poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition. 46 refs., 5 figs., 1 tab.
Bozza, M.; Gerard, C.; Kolakowski, L.F. Jr. [Children`s Hospital, Boston, MA (United States)] [and others] [Children`s Hospital, Boston, MA (United States); and others
The Paralogous Hox Genes Hoxa10 and Hoxd10 Interact to Pattern the Mouse Hindlimb Peripheral Nervous System and Skeleton
The most 5? mouse Hoxa and Hoxd genes, which occupy positions 9�13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and
George M. Wahba; Sirkka Liisa Hostikka; Ellen M. Carpenter
Identification and characterization of the genes encoding human and mouse osteoactivin.
Osteoactivin (OA) is more highly expressed in the bones of osteopetrotic mutant rats (op/op) than in those of their normal littermates and is the homologue of human nmb, a cDNA more highly expressed in melanoma-derived cell lines of low metastatic potential, and of mouse DC-HIL, which has been implicated in endothelial cell adhesion. The human OA gene is found on chromosome 7p15.1 and consists of 11 exons spanning 28.3 kb. Murine OA is encoded by a highly similar gene of 11 exons spanning 20.2 kb on mouse chromosome 6. Human OA uses the same transcriptional initiation site in both bone and kidney as was reported for melanoma cells. OA is expressed in primary human and mouse osteoblast cultures at all stages of differentiation, with increased levels observed concurrently with the expression of osteoblast phenotype markers. OA is also expressed in a wide variety of human and mouse tissues as determined by RT-PCR analysis. Immunohistochemical investigation of OA expression in late mouse embryonic development showed very high, cell-specific expression in the nervous system, basal layer of the skin, germinal cells of hair follicles, and in the forming nephrons of the kidney. Continuing investigation of the cell-specific expression of OA in bone as well as in other tissues will lead to a better understanding of its function in the development of these cell types. PMID:14696968
Owen, T A; Smock, S L; Prakash, S; Pinder, L; Brees, D; Krull, D; Castleberry, T A; Clancy, Y C; Marks, S C; Safadi, F F; Popoff, S N
Patterned expression of ion channel genes in mouse dorsal raphe nucleus determined with the Allen Mouse Brain Atlas.
The dorsal raphe nucleus (DR) is the major source of serotonin (5-hydroxytryptamine, 5-HT) in the forebrain and dysfunction of this midbrain structure is implicated in affective disorders. The DR is composed of several types of 5-HT and non-5-HT neurons and their excitable-membrane properties are heterogeneous and overlapping. In order to understand how these properties may be generated, we examined the mRNA expression patterns of voltage- and ligand-gated ion channels in the DR using the Allen Mouse Brain Atlas. Since DR cytoarchitecture is organized with respect to the midline, we sought to identify genes that were expressed in a pattern with respect to the midline, either enriched or depleted, rather than those that were homogenously expressed throughout the DR. Less than 10% of the screened genes for voltage-gated ion channels showed patterned expression within the DR. Identified genes included voltage-gated sodium channel beta subunits, potassium channels, P/Q-, N-type calcium channels, as well as the alpha2/delta-1 calcium channel. Several voltage-gated chloride channels were also identified, although these may function within intracellular compartments. Of the ligand-gated ion channels examined, 20% showed patterned expression. These consisted primarily of glutamate and GABA-A receptor subunits. The identified genes likely contribute to unique excitable properties of different groups of neurons in the DR and may include novel pharmacologic targets for affective disorders. PMID:22534482
Templin, J Scott; Bang, Sun Jung; Soiza-Reilly, Mariano; Berde, Charles B; Commons, Kathryn G
Cloning of the mouse cDNA encoding DNA topoisomerase I and chromosomal location of the gene.
The mouse cDNA encoding DNA topoisomerase I (TopoI) was cloned and the nucleotide sequence of 3512 bp was determined. The cDNA clone contained an open reading frame encoding a protein of 767 amino acids (aa), which is 2 aa longer than its human counterpart. Overall aa sequence homology between the mouse and human, and between the mouse and yeast (Saccharomyces cerevisiae) sequences was 96% and 42%, respectively. The mouse TopI gene was mapped at position 54.5 on chromosome 2 from linkage analyses of a three-point cross test with Geg, Ada, and a as marker genes. PMID:8096488
Koiwai, O; Yasui, Y; Sakai, Y; Watanabe, T; Ishii, K; Yanagihara, S; Andoh, T
Genome-wide identification of endothelial cell-enriched genes in the mouse embryo.
The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo-forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse. PMID:22535667
Takase, Haruka; Matsumoto, Ken; Yamadera, Rie; Kubota, Yoshiaki; Otsu, Ayaka; Suzuki, Rumiko; Ishitobi, Hiroyuki; Mochizuki, Hiromi; Kojima, Takahiro; Takano, Shingo; Uchida, Kazuhiko; Takahashi, Satoru; Ema, Masatsugu
Impact of cigarette smoke on the human and mouse lungs: a gene-expression comparison study.
Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases. PMID:24663285
Morissette, Mathieu C; Lamontagne, Maxime; B�rub�, Jean-Christophe; Gaschler, Gordon; Williams, Andrew; Yauk, Carole; Couture, Christian; Laviolette, Michel; Hogg, James C; Timens, Wim; Halappanavar, Sabina; Stampfli, Martin R; Boss�, Yohan
Impact of Cigarette Smoke on the Human and Mouse Lungs: A Gene-Expression Comparison Study
Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases.
Morissette, Mathieu C.; Lamontagne, Maxime; Berube, Jean-Christophe; Gaschler, Gordon; Williams, Andrew; Yauk, Carole; Couture, Christian; Laviolette, Michel; Hogg, James C.; Timens, Wim; Halappanavar, Sabina; Stampfli, Martin R.; Bosse, Yohan
Cloning and chromosomal mapping of the mouse and human genes encoding the orphan glucocorticoid-induced receptor (GPR83)
The mouse glucocorticoid-induced receptor (GIR) is an orphan G protein-coupled receptor highly expressed in brain and thymus (Harrigan et al., 1989; 1991). We have cloned the mouse GIR gene (Gpr83), determined its genomic organization and compared it with the human gene. The genomic organization of the gene is similar in both species although differences leading to specific splicing variants in
L. De Moerlooze; J. Williamson; F. Liners; J. Perret; M. Parmentier
The ?A-crystallin gene: Conserved features of the 5?-flanking regions in human, mouse, and chicken
Summary Approximately 2 kb of 5?-flanking sequences of the lens-specific ?A-crystallin genes from human and mouse are presented and compared with similar regions of the chicken gene. A repetitive element was found approximately 1 kb upstream from the coding sequences of the ?A-crystallin gene in all three species (Alu in human, B2 in mouse, and CR1 in chicken), suggesting that
Cynthia J. Jaworski; Ana B. Chepelinsky; Joram Piatigorsky
[Phenotype analysis and mutant gene location of ventral yellow mouse (VY(Slac))].
The ventri-yellow pigmentation mouse (temporarily named VY(Slac)) arose spontaneously in the C57BL/6J inbred mouse strain, found and bred by Shanghai SLAC Laboratory Animal Co., Ltd. VY(Slac) presented a special phenotype marked by yellow coat on the ventral surface of neck and trunk that was without melanin deposition but maintained a normal structure. The number of melanocytes in epidermis and melanin in hair follicle of the abdominal skin of the mutant mouse were less than that of their background strain, while there was no significant difference between the dorsal skins of the two strains. This mutant phenotype was inherited as single-gene dominant inheritance, confirmed by genetic experiment, and there was no significant difference between VY(Slac) and B(6) for other biological parameters such as weight, anatomic and histological structures of major organs and blood physiology. When the linkage relationship between the genomic DNA samples of F(2) 48 mice (VY(Slac)D(2)F(1)�D(2)) and mutant phenotype were evaluated, the mutant gene was confirmed on chromosome 2 near D2Mit229. New microsatellite and SNP markers were selected to amplify genomic DNA samples of 196 F(2) mice and the mutant gene was narrowed down to 5.3 Mb region between rs13476833 and rs27310903 on chromosome 2. The preliminary results of our phenotype analysis and gene location provides a solid basis for further identification of this mutant gene. PMID:22653857
Shi, Mei-Lian; Xu, Ping; Yin, Xiao-Shu; Yang, Wei-Wei; Gu, Mei-Er; Yu, Li-Ping; Liu, Gui-Jie; Wu, Bao-Jin
Deletion of the Parkin coregulated gene causes male sterility in the quakingviable mouse mutant
Quakingviable (qkv) is a recessive neurological mouse mutation with severe dysmyelination of the CNS and spermiogenesis failure. The molecular lesion in the qkv mutant is a deletion of ?1 Mb on mouse chromosome 17 that alters the expression of the qk gene in oligodendrocytes. Complementation analysis between the qkv mutation and qk mutant alleles generated through chemical mutagenesis showed that the male sterility is a distinctive feature of the qkv allele. This observation suggested that the sperm differentiation defect in qkv is due to the deletion of a gene(s) distinct from qk. Here, we demonstrate that the deletion of Pacrg is the cause of male sterility in the qkv mutant. Pacrg is the mouse homologue of the human PARKIN-coregulated gene (PACRG), which encodes for a protein whose biochemical function remains unclear. We show that Pacrg is highly expressed in the testes in both mice and humans. In addition, the expression pattern of Pacrg during spermiogenesis suggests that it plays a role in sperm differentiation. In support of this hypothesis, we show that transgenic expression of Pacrg in testes restores spermiogenesis and fertility in qkv males. This finding provides the first in vivo evidence, to our knowledge, for the function of Pacrg in a model organism. Immunolocalization experiments on isolated spermatozoa show that the Pacrg protein is present in mature sperm. Remarkably, the mammalian Pacrg protein shares significant sequence similarities with gene products from flagellated protozoans, suggesting that Pacrg may be necessary for proper flagellar formation in many organisms.
Lorenzetti, Diego; Bishop, Colin E.; Justice, Monica J.
The breast cancer-associated stromelysin-3 gene is expressed during mouse mammary gland apoptosis
We have cloned from a mouse placenta cDNA library a mouse homologue of the human stromelysin-3 (ST3) cDNA, which codes for a putative matrix metalloproteinase expressed in breast carcinomas. The ST3 protein is well conserved between humans and mice, and the pattern of ST3 gene expression is similar in both species, and shows expression in the placenta, in the uterus, and during limb bud morphogenesis. We show that the ST3 gene can also be expressed in the normal mouse mammary gland. ST3 gene expression was not detected during mammary growth, neither in virgin nor in pregnant mice, but was specifically observed during postlactating involution of the gland, an apoptotic process associated with intense extracellular matrix remodeling. ST3 transcripts were found in fibroblasts immediately surrounding degenerative ducts, suggesting that ST3 gene expression may be associated with the basement membrane dissolution, which occurs during mammary gland involution. Since the ST3 gene is also specifically expressed in fibroblastic cells surrounding invasive neoplastic cells of breast carcinomas, we suggest that ST3 is implicated in extracellular matrix remodeling processes common to mammary apoptosis and breast cancer progression.
Extraordinary Sequence Divergence at Tsga8, an X-linked Gene Involved in Mouse Spermiogenesis
The X chromosome plays an important role in both adaptive evolution and speciation. We used a molecular evolutionary screen of X-linked genes potentially involved in reproductive isolation in mice to identify putative targets of recurrent positive selection. We then sequenced five very rapidly evolving genes within and between several closely related species of mice in the genus Mus. All five genes were involved in male reproduction and four of the genes showed evidence of recurrent positive selection. The most remarkable evolutionary patterns were found at Testis-specific gene a8 (Tsga8), a spermatogenesis-specific gene expressed during postmeiotic chromatin condensation and nuclear transformation. Tsga8 was characterized by extremely high levels of insertion�deletion variation of an alanine-rich repetitive motif in natural populations of Mus domesticus and M. musculus, differing in length from the reference mouse genome by up to 89 amino acids (27% of the total protein length). This population-level variation was coupled with striking divergence in protein sequence and length between closely related mouse species. Although no clear orthologs had previously been described for Tsga8 in other mammalian species, we have identified a highly divergent hypothetical gene on the rat X chromosome that shares clear orthology with the 5? and 3? ends of Tsga8. Further inspection of this ortholog verified that it is expressed in rat testis and shares remarkable similarity with mouse Tsga8 across several general features of the protein sequence despite no conservation of nucleotide sequence across over 60% of the rat-coding domain. Overall, Tsga8 appears to be one of the most rapidly evolving genes to have been described in rodents. We discuss the potential evolutionary causes and functional implications of this extraordinary divergence and the possible contribution of Tsga8 and the other four genes we examined to reproductive isolation in mice.
Good, Jeffrey M.; Vanderpool, Dan; Smith, Kimberly L.; Nachman, Michael W.
Generation of general and tissue-specific gene knockout mouse models.
Knockout technology has established the functions of many genes affecting plasma lipid and lipoprotein levels and the development of atherosclerosis. However, many genes remain to be characterized. The ability to produce mice lacking whole-body expression of a given gene is still one of the most powerful techniques available for determining gene function. A complementary approach, underutilized yet vitally important to understanding lipoprotein metabolism, is the ability to create mice with gene deficiency only in a specific tissue. Liver, intestine, and macrophages are the major tissues and cells involved in lipoprotein metabolism and atherosclerosis, and additional tissues such as adipose tissue and brain are also of interest. Thus, feasible approaches to prepare general and tissue-specific gene knockout mouse models are necessary. Here, we describe our general procedure for generating whole-body knockout mice, using as an example the preparation of general (whole-body) phospholipid transfer protein (PLTP) gene knockout mice. We also describe several approaches to generating liver, intestine, and myeloid cell-specific tissue-specific knockout mice, using as an example the preparation of tissue-specific knockout mice for the subunit 2 of serine palmitoyltransferase (SPT), a key enzyme for sphingomyelin de novo synthesis. Bone marrow transplantation is an alternative means of creating myeloid cell-specific knockout mice. The general principles and techniques described here apply to the establishment of other gene knockout mouse models as well. The ability to manipulate gene expression in specific tissues as well as throughout the entire body of the mouse is anticipated to yield novel insights into lipid and lipoprotein metabolism and the development of atherosclerosis. PMID:23912991
Jiang, Xian-Cheng
Comparative DNA sequence analysis of mouse and human protocadherin gene clusters
The genomic organization of the human protocadherin alpha, beta, and gamma gene clusters (designated Pcdh{alpha} [gene symbol PCDHA], Pcdh{beta} [PCDHB], and Pcdh{gamma} [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large ''variable'' region exon, while the intracellular domains are encoded by three small ''constant'' region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdhi{alpha} and Pcdh{beta} gene clusters in the two species is virtually identical, whereas the mouse Pcdhi{beta} gene cluster is larger and contains more genes than the human Pcdh{beta} gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.
Wu, Qiang; Zhang, Theresa; Cheng, Jan-Fang; Kim, Youngwook; Grimwood, Jane; Schmutz, Jeremy; Dickson, Mark; Noonan, James P.; Zhang, Michael Q.; Myers, Richard M.; Maniatis, Tom
A new Pax gene, Pax-9, maps to mouse chromosome 12.
Members of the Pax gene family have recently been shown to play important roles in mouse embryogenesis. Of eight so far characterized Pax genes, three have been associated with mouse developmental mutants. Here we report the cloning of a new Pax gene, Pax-9. Most of the DNA sequence encoding the highly conserved paired domain has been determined and compared with previously known paired domains. This comparison classifies Pax-9 as a member of the same subgroup as Pax-1/undulated. By analysis of the segregation of a Pax-9 restriction fragment length polymorphism and a large number of simple sequence length polymorphisms in an interspecific C57BL/6 x Mus musculus mollosinus backcross, Pax-9 was mapped close to the D12Nds1 locus on the proximal part of Chromosome (Chr) 12. PMID:8358169
Wallin, J; Mizutani, Y; Imai, K; Miyashita, N; Moriwaki, K; Taniguchi, M; Koseki, H; Balling, R
The gene encoding protein kinase SEK1 maps to mouse chromosome 11 and human chromosome 17
We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human-rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouse Serk1 gene was mapped to chromosome 11, closely linked to D11Mit4, using genomic DNAs from a (C57BL/6J x Mus spretus)F{sub 1} x M, spretus backcross. 17 refs., 2 figs.
White, R.A. [Childrens Mercy Hospital, Kansas, MO (United States)] [Childrens Mercy Hospital, Kansas, MO (United States); Hughes, R.T.; Zon, L.I. [Childrens Hospital and Dana Farber Cancer Institute, Boston, MA (United States)] [and others] [Childrens Hospital and Dana Farber Cancer Institute, Boston, MA (United States); and others
Mapping of the ARIX homeodomain gene to mouse chromosome 7 and human chromosome 11q13
The recently described homeodomain protein ARIX is expressed specifically in noradreneric cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine {beta}-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouse Arix was positioned approximately 50 cM distal to the centromere of chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 11q13.3-q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.
Johnson, K.R. [Jackson Lab., Bar Harbor, ME (United States)] [Jackson Lab., Bar Harbor, ME (United States); Smith, L.; Rhodes, J. [Oregon Health Sciences Univ., Portland, OR (United States)] [and others] [Oregon Health Sciences Univ., Portland, OR (United States); and others
An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse
Background We have used a sensitized ENU mutagenesis screen to produce mouse lines that carry mutations in genes required for epigenetic regulation. We call these lines Modifiers of murine metastable epialleles (Mommes). Results We report a basic molecular and phenotypic characterization for twenty of the Momme mouse lines, and in each case we also identify the causative mutation. Three of the lines carry a mutation in a novel epigenetic modifier, Rearranged L-myc fusion (Rlf), and one gene, Rap-interacting factor 1 (Rif1), has not previously been reported to be involved in transcriptional regulation in mammals. Many of the other lines are novel alleles of known epigenetic regulators. For two genes, Rlf and Widely-interspaced zinc finger (Wiz), we describe the first mouse mutants. All of the Momme mutants show some degree of homozygous embryonic lethality, emphasizing the importance of epigenetic processes. The penetrance of lethality is incomplete in a number of cases. Similarly, abnormalities in phenotype seen in the heterozygous individuals of some lines occur with incomplete penetrance. Conclusions Recent advances in sequencing enhance the power of sensitized mutagenesis screens to identify the function of previously uncharacterized factors and to discover additional functions for previously characterized proteins. The observation of incomplete penetrance of phenotypes in these inbred mutant mice, at various stages of development, is of interest. Overall, the Momme collection of mouse mutants provides a valuable resource for researchers across many disciplines.
Gene Entropy-Fractal Dimension Informatics with Application to Mouse-Human Translational Medicine
DNA informatics represented by Shannon entropy and fractal dimension have been used to form 2D maps of related genes in various mammals. The distance between points on these maps for corresponding mRNA sequences in different species is used to study evolution. By quantifying the similarity of genes between species, this distance might be indicated when studies on one species (mouse) would tend to be valid in the other (human). The hypothesis that a small distance from mouse to human could facilitate mouse to human translational medicine success is supported by the studied ESR-1, LMNA, Myc, and RNF4 sequences. ID1 and PLCZ1 have larger separation. The collinearity of displacement vectors is further analyzed with a regression model, and the ID1 result suggests a mouse-chimp-human translational medicine approach. Further inference was found in the tumor suppression gene, p53, with a new hypothesis of including the bovine PKM2 pathways for targeting the glycolysis preference in many types of cancerous cells, consistent with quantum metabolism models. The distance between mRNA and protein coding CDS is proposed as a measure of the pressure associated with noncoding processes. The Y-chromosome DYS14 in fetal micro chimerism that could offer protection from Alzheimer's disease is given as an example.
Holden, T.; Cheung, E.; Dehipawala, S.; Ye, J.; Tremberger, G.; Lieberman, D.; Cheung, T.
In vivo identification of a negative regulatory element in the mouse renin gene using direct gene transfer.
DBA/2J mouse contains two renin gene loci (Ren1d and Ren2d). Ren2d but not Ren1d is expressed in submandibular gland (SMG) while both are expressed in the kidney. Based on vitro studies, we have postulated that a negative regulatory element (NRE) in the renin gene promoter is involved in its tissue-specific expression. In this study, we examined the molecular mechanism at the in vivo level using direct gene transfer. Fragments of the Ren1d or Ren2d promoter were fused to a chloramphenicol acetyltransferase (CAT) gene expression vector. These constructs complexed in fusogenic liposomes were injected directly into the mouse SMG or intraarterially into the mouse kidney via the renal artery. The vector containing the CAT exhibited readily detectable in vivo expressions in both SMG and kidney. In the SMG, Ren1d fragment containing the NRE abolished CAT expression while deletion of the NRE restored CAT expression. The homologous fragment from the Ren2d promoter did not inhibit CAT expression while deletion of the 150-bp insertion resulted in the inhibition. Cotransfection of Ren1d construct with Ren1d-NRE oligonucleotides as transcriptional factor decoy restored CAT expression. Contrary to the SMG, transfection with Ren1d fragment-CAT construct or Ren2d fragment-CAT construct into the kidney resulted in similar levels of CAT expression. Interestingly, human c-myc NRE oligonucleotides which share homology with Ren1d-NRE competed effectively with these oligonucleotides for the regulation of Ren1d gene expression in vivo. This NRE sequence is also homologous to silencer elements found in multiple mammalian genes, suggesting the presence of a family of NRE/NRE binding proteins regulating expression of diverse genes. Images
Yamada, T; Horiuchi, M; Morishita, R; Zhang, L; Pratt, R E; Dzau, V J
Identification of candidate genes for human retinal degeneration loci using differentially expressed genes from mouse photoreceptor dystrophy models
Purpose Retinal degeneration (RD) is a complex mechanism that appears to involve many biologic processes including oxidative stress, apoptosis, and cellular remodeling. Currently there are 51 mapped, but not identified, RD human disease loci. Methods To assign possible disease genes to RD loci, we have used a comparative genomics procedure that incorporates microarray gene expression data of three independent mouse models for photoreceptor dystrophy (rd1, rd2, and constant light-damage in BALB/c mice), human ortholog data, and databases of known chromosomal locations involved in human RD. Immunohistochemistry and enzyme activity assays were used to further characterize a candidate gene product. Results Our analysis yielded candidate genes for four mapped, but unsolved, human chromosomal locations and confirmed two previously identified monogenic disease loci for human RD, thus validating our approach. PLA2G7 (phospholipase A2, group VII; PAF-AH, Lp-PLA2), a candidate for a dominant form macular dystrophy (Benign Concentric Annular Macular Dystrophy [BCMAD]), was selected for further study. The PLA2G7 enzyme is known to mediate breakdown of oxidatively damaged phospholipids, a contributor to oxidative stress in the retina. PLA2G7 protein was enriched in mouse photoreceptor inner and outer segments. In the rd1, rd2, and BALB/c mice exposed to constant light, retinal tissue activity levels, but not plasma levels, were significantly reduced at the onset of photoreceptor cell death. Conclusions We have shown that this comparative genomics approach verified existing RD genes as well as identified novel RD candidate genes. The results on the characterization of the PLA2G7 protein, one of the novel RD genes, suggests that retinal tissue PLA2G7 levels may constitute an important risk factor for BCMAD. In summary, this reverse mapping approach, using accepted mouse models of human disease and known human RD loci, may prove useful in identifying possible novel disease candidates for RD and may be applicable to other human diseases.
Demos, Christina; Bandyopadhyay, Mausumi
A mouse speciation gene encodes a meiotic histone H3 methyltransferase.
Speciation genes restrict gene flow between the incipient species and related taxa. Three decades ago, we mapped a mammalian speciation gene, hybrid sterility 1 (Hst1), in the intersubspecific hybrids of house mouse. Here, we identify this gene as Prdm9, encoding a histone H3 lysine 4 trimethyltransferase. We rescued infertility in male hybrids with bacterial artificial chromosomes carrying Prdm9 from a strain with the "fertility" Hst1(f) allele. Sterile hybrids display down-regulated microrchidia 2B (Morc2b) and fail to compartmentalize gammaH2AX into the pachynema sex (XY) body. These defects, seen also in Prdm9-null mutants, are rescued by the Prdm9 transgene. Identification of a vertebrate hybrid sterility gene reveals a role for epigenetics in speciation and opens a window to a hybrid sterility gene network. PMID:19074312
Mihola, Ondrej; Trachtulec, Zdenek; Vlcek, Cestmir; Schimenti, John C; Forejt, Jiri
Differential expression of sex-linked and autosomal germ-cell-specific genes during spermatogenesis in the mouse
We have examined expression during spermatogenesis in the mouse of three Y-linked genes, 11 X-linked genes and 22 autosomal genes, all previously shown to be germ-cell-specific and expressed in premeiotic spermatogonia, plus another 21 germ-cell-specific autosomal genes that initiate expression in meiotic sper- matocytes. Our data demonstrate that, like sex-linked housekeeping genes, germ-cell-specific sex-linked genes are subject to meiotic sex-chromosome
P. Jeremy Wang; David C. Page; John R. McCarrey
MicroRNA genes are frequently located near mouse cancer susceptibility loci
MicroRNAs (miRNAs) are short 19- to 24-nt RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. Abnormal expression of miRNAs has been observed in several human cancers, and furthermore, germ-line and somatic mutations in human miRNAs were recently identified in patients with chronic lymphocytic leukemia. Thus, human miRNAs can act as tumor suppressor genes or oncogenes, where mutations, deletions, or amplifications can underlie the development of certain types of leukemia. In addition, previous studies have shown that miRNA expression profiles can distinguish among human solid tumors from different organs. Because a single miRNA can simultaneously influence the expression of two or more protein-coding genes, we hypothesized that miRNAs could be candidate genes for cancer risk. Research in complex trait genetics has demonstrated that genetic background determines cancer susceptibility or resistance in various tissues, such as colon and lung, of different inbred mouse strains. We compared the genome positions of mouse tumor susceptibility loci with those of mouse miRNAs. Here, we report a statistically significant association between the chromosomal location of miRNAs and those of mouse cancer susceptibility loci that influence the development of solid tumors. Furthermore, we identified distinct patterns of flanking DNA sequences for several miRNAs located at or near susceptibility loci in inbred strains with different tumor susceptibilities. These data provide a catalog of miRNA genes in inbred strains that could represent genes involved in the development and penetrance of solid tumors.
Sevignani, Cinzia; Calin, George A.; Nnadi, Stephanie C.; Shimizu, Masayoshi; Davuluri, Ramana V.; Hyslop, Terry; Demant, Peter; Croce, Carlo M.; Siracusa, Linda D.
Peptidoglycan enhances transcriptional expression of CCAAT\\/enhancer-binding protein ? gene in mouse macrophages
Summary��Peptidoglycan-activated gene expression is mediated through various transcription factors including CCAAT\\/enhancer-binding\\u000a protein ? (C\\/EBP?). The purpose of the present study is to elucidate the mechanism of PGN-activated C\\/EBP? gene. PGN stimulated\\u000a C\\/EBP? protein and mRNA expression in mouse macrophages RAW 264.7 cells. Analysis of C\\/EBP? promoter activity by luciferase\\u000a reporter assay indicated that PGN-induced C\\/EBP? gene activation is partially mediated
Yu-Chiuan Huang; Wen-Chang Chang; Jyan-Gwo J. Su; Jheng-Liang Cai; Chun-Chia Chen; Jan-Jong Hung; Yi-Wen Liu
Metallothionein Protection of Cadmium Toxicity
The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-molecular weight, cysteine-rich protein called metallothionein (MT) in Cd toxicology. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases billiary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an association between human and rodent Cd exposure and prostate cancers, especially in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases were devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicology, with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is critical for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.
Klaassen, Curtis D.; Liu, Jie; Diwan, Bhalchandra A.
Metallothionein protection of cadmium toxicity
The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-molecular weight, cysteine-rich protein called metallothionein (MT) in Cd toxicology. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an association between human and rodent Cd exposure and prostate cancers, especially in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicology, with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is critical for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.
Klaassen, Curtis D. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160-7417 (United States)], E-mail: firstname.lastname@example.com; Liu, Jie [Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at NIEHS, Research Triangle Park, NC 27709 (United States); Diwan, Bhalchandra A. [Basic Science Program, SAIC-Frederick, Inc., NCI Frederick, MD (United States)
Gene transfer to the developing mouse inner ear by in vivo electroporation.
The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development(6). The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol(11). Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol. PMID:22781586
Wang, Lingyan; Jiang, Han; Brigande, John V
Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation
The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6. The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.
Wang, Lingyan; Jiang, Han; Brigande, John V.
Differential gene expression by Osterix knockdown in mouse chondrogenic ATDC5 cells.
Osterix (Osx) is a transcription factor required for osteoblast differentiation during intramembranous and endochondral ossification. Recently, several reports have described novel functions of Osx in chondrocyte differentiation. In an in vitro study, in which the effects of Osx gene silencing were examined in mouse chondrogenic ATDC5 cells, chondrocyte marker genes were found to be expressionally downregulated and chondrocyte differentiation reduced. On the other hand, in vivo studies based on chondrocyte-specific Osx knockouts demonstrated impaired endochondral bone formation with delayed chondrocyte differentiation and reduced cartilage matrix ossification. However, little is known about the mechanism or targets of Osx involved in the control of chondrocyte differentiation. Here, we attempted to high-density of Affymetrix GeneChip microarray to investigate global gene expression profile changes caused by Osx knockdown in ATDC5 chondrocytes. The mRNA expressions of 112 genes were significantly modified by Osx knockdown: 68 genes were upregulated and 44 genes downregulated. Functional categories of gene expression classified by gene ontology demonstrated that genes related to cell adhesion, development, and signal transduction were highly affected by Osx knockdown. The expressions of differential genes, such as Sfrp2, Sema3a, Nox4, Rgs4, Zfp521, Has2, Sox6, Scn2a1, Sirpa, and Thbs2, were validated by quantitative real-time PCR. This study shows that expression profiling can be used to identify genes that are transcriptionally modified following Osx knockdown and to reveal the molecular mechanism of chondrocyte differentiation regulated by Osx. PMID:23337593
Park, Seung-Yoon; Kim, Jung-Eun
Lessons on Conditional Gene Targeting in Mouse Adipose Tissue
Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ?2% of spermatozoa. Thus, different �adipocyte-specific� Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology.
Lee, Kevin Y.; Russell, Steven J.; Ussar, Siegfried; Boucher, Jeremie; Vernochet, Cecile; Mori, Marcelo A.; Smyth, Graham; Rourk, Michael; Cederquist, Carly; Rosen, Evan D.; Kahn, Barbara B.; Kahn, C. Ronald
Global gene expression profile progression in Gaucher disease mouse models
Background Gaucher disease is caused by defective glucocerebrosidase activity and the consequent accumulation of glucosylceramide. The pathogenic pathways resulting from lipid laden macrophages (Gaucher cells) in visceral organs and their abnormal functions are obscure. Results To elucidate this pathogenic pathway, developmental global gene expression analyses were conducted in distinct Gba1 point-mutated mice (V394L/V394L and D409 V/null). About 0.9 to 3% of genes had altered expression patterns (? � 1.8 fold change), representing several categories, but particularly macrophage activation and immune response genes. Time course analyses (12 to 28 wk) of INF?-regulated pro-inflammatory (13) and IL-4-regulated anti-inflammatory (11) cytokine/mediator networks showed tissue differential profiles in the lung and liver of the Gba1 mutant mice, implying that the lipid-storage macrophages were not functionally inert. The time course alterations of the INF? and IL-4 pathways were similar, but varied in degree in these tissues and with the Gba1 mutation. Conclusions Biochemical and pathological analyses demonstrated direct relationships between the degree of tissue glucosylceramides and the gene expression profile alterations. These analyses implicate IFN?-regulated pro-inflammatory and IL-4-regulated anti-inflammatory networks in differential disease progression with implications for understanding the Gaucher disease course and pathophysiology.
Dynamics of Global Gene Expression Changes during Mouse Preimplantation Development
Understanding preimplantation development is important both for basic reproductive biology and for practical applications including regenerative medicine and livestock breeding. Global expression profiles revealed and characterized the distinctive patterns of maternal RNA degradation and zygotic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA); the second wave, named mid-preimplantation
Toshio Hamatani; Mark G. Carter; Alexei A. Sharov; Minoru S. H. Ko
Cloning and chromosomal mapping of the mouse and human genes encoding the orphan glucocorticoid-induced receptor (GPR83).
The mouse glucocorticoid-induced receptor (GIR) is an orphan G protein-coupled receptor highly expressed in brain and thymus (Harrigan et al., 1989; 1991). We have cloned the mouse GIR gene (Gpr83), determined its genomic organization and compared it with the human gene. The genomic organization of the gene is similar in both species although differences leading to specific splicing variants in the mouse have been found. Three introns interrupting the coding sequence are common to both mouse and human. A short sequence in the second intron of the mouse gene can be alternatively spliced in, leading to an insertion in the second intracellular loop of the receptor. This insertion constitutes an additional exon which is not present in the human genome. The human GIR polypeptide shares 89.5% and 91.5% identity with its mouse and dog orthologs respectively. Splice variants lacking the first extracellular loop and the third transmembrane domain have been found in human and mouse species. The receptor variants resulting from these minor transcripts are likely to be non functional. Comparative genetic mapping of the Gpr83 gene showed that it maps to regions of conserved synteny on mouse chromosome 9 (A2-3 region) and human chromosome 11 (q21 region). PMID:11060465
De Moerlooze, L; Williamson, J; Liners, F; Perret, J; Parmentier, M
The mouse homologue of the tuberin gene (TSC2) maps to a conserved synteny group between mouse chromosome 17 and human 16p13.3
The tuberous sclerosis gene (TSC2) on human chromosome 16p13.3 has recently been identified. Several markers from this region have previously been shown to be members of a conserved synteny group, in the mouse located on chromosome 17. The mouse region includes markers D17Lon1, D17Lon2, D17Lon3, and D17Lon4, which are linked to the α-globin pseudogene Hba-ps4 on chromosome 17, while the
P. G. Olsson; H. F. Sutherland; U. Nowicka; B. Korn; A. Poustka; A.-M. Frischauf
Identification of candidate cancer-causing genes in mouse brain tumors by retroviral tagging
Murine retroviruses may cause malignant tumors in mice by insertional mutagenesis of host genes. The use of retroviral tagging as a means of identifying cancer-causing genes has, however, almost entirely been restricted to hematopoietic tumors. The aim of this study was to develop a system allowing for the retroviral tagging of candidate genes in malignant brain tumors. Mouse gliomas were induced by a recombinant Moloney murine leukemia virus encoding platelet-derived growth factor (PDGF) B-chain. The underlying idea was that tumors evolve through a combination of PDGF-mediated autocrine growth stimulation and insertional mutagenesis of genes that cooperate with PDGF in gliomagenesis. Common insertion sites (loci that were tagged in more than one tumor) were identified by cloning and sequencing retroviral flanking segments, followed by blast searches of mouse genome databases. A number of candidate brain tumor loci (Btls) were identified. Several of these Btls correspond to known tumor-causing genes; these findings strongly support the underlying idea of our experimental approach. Other Btls harbor genes with a hitherto unproven role in transformation or oncogenesis. Our findings indicate that retroviral tagging with a growth factor-encoding virus may be a powerful means of identifying candidate tumor-causing genes in nonhematopoietic tumors.
Johansson, Fredrik K.; Brodd, Josefin; Eklof, Charlotta; Ferletta, Maria; Hesselager, Goran; Tiger, Carl-Fredrik; Uhrbom, Lene; Westermark, Bengt
The mouse (Mus musculus) T cell receptor alpha (TRA) and delta (TRD) variable genes.
'The Mouse (Mus musculus) T cell receptor alpha (TRA) and delta (TRD) variable genes' 'IMGT Locus in Focus' report provides the first complete list of the mouse TRAV and TRDV genes which span 1550 kb on chromosome 14 at 19.7 cM. The total number of TRAV genes per haploid genome is 98 belonging to 23 subgroups. This includes 10 TRAV/DV genes which belong to seven subgroups. The functional TRAV genomic repertoire comprises 72-82 TRAV (including 9-10 TRAV/DV) belonging to 19 subgroups. The total number of TRDV genes per haploid genome is 16 (including the 10 TRAV/DV) belonging to 12 subgroups. The functional TRDV genomic repertoire comprises 14-15 genes (5 TRDV and 9-10 TRAV/DV) belonging to 11-12 subgroups. The eight tables and three figures of this report are available at the IMGT Marie-Paule page of IMGT. The international ImMunoGeneTics information system (http://imgt.cines.fr) created by Marie-Paule Lefranc, Universit� Montpellier II, CNRS, France. PMID:12697305
Bosc, Nathalie; Lefranc, Marie-Paule
Cotransfer and phenotypic stabilisation of syntenic and asyntenic mink genes into mouse cells by chromosome-mediated gene transfer
By means of metaphase chromosomes, the genes for mink thymidine kinase (TK) and hypoxanthine-phosphoribosyltransferase (HPRT) were transferred to mutant mouse cells, LMTK-, A9 (HPRT-) and teratocarcinoma cells, PCC4-aza 1 (HPRT-). Eighteen colonies were isolated from LMTK- (series A), 9 from A9 (series B) and none from PCC4-aza 1. The transformed clones contained mink TK or HPRT. Analysis of syntenic markers
M. A. Sukoyan; N. M. Matveeva; N. D. Belyaev; S. D. Pack; A. A. Gradov; A. G. Shilov; N. S. Zhdanova; O. L. Serov
Metallothionein in human gingival amalgam tattoos
Amalgam tattoos occur when small particles of dental amalgam, composed largely of silver (Ag) and mercury (Hg), are inadvertently implanted into oral soft tissues during dental procedures. Metallothioneins (MTs) are ubiquitous, low molecular weight, cysteine-rich, metal-binding proteins that are inducible by many agents including metals and may be involved in the detoxification of toxic metals such as Hg. In this
John C Lau; Linda Jackson-Boeters; Tom D Daley; George P Wysocki; M. George Cherian
Mouse CD-RAP/MIA gene: structure, chromosomal localization, and expression in cartilage and chondrosarcoma.
A cDNA encoding a novel protein has been previously isolated from two independent sources: melanoma cell cultures and chondrocytes. The protein from human melanoma cell lines and tumors is called melanoma inhibitory activity (MIA) (Blesch et al. [1994] Cancer Res. 54:5695-5701) and the protein from primary bovine chondrocytes and cartilaginous tissues is called cartilage-derived retinoic acid-sensitive protein (CD-RAP) (Dietz and Sandell [1996] J. Biol. Chem. 271:3311-3316). In order to investigate the gene regulation and function of CD-RAP/MIA, the mouse gene locus was isolated and analyzed. Developmental expression was determined by in situ hybridization to mouse embryos. Expression was limited to cartilaginous tissues and was initiated with the advent of chondrogenesis, remaining abundant throughout development. The mouse gene was isolated and sequenced from a 129Sv library and sequenced directly from an additional strain, B6C3Fe. The mouse CD-RAP/MIA gene is 1.5 kbp and consists of four exons. The promoter sequence of the gene contains many potential regulatory domains including 8 basic helix-loop-helix protein-binding domains and an AT-rich domain, both motifs shown to be present in the cartilage-specific enhancer of the type II procollagen gene. Other potential cis-acting motifs include binding sites for GATA-1, NF-IL6, PEA3, w-elements, NF kappa B, Zeste and Sp1. The gene, called cdrap, was localized to the end of an arm of chromosome 7 at the same site as the transforming growth factor beta 1 (Tgf-beta 1) and the glucose phosphate isomerase 1 (Gpi 1) genes. Potential mouse mutants that mapped to the same region of chromosome 7 were identified. Two of the potential mutants with skeletal phenotypes were sequenced, pudgy (pu) and extra toes with spotting (XsJ); however, no mutations were found in the coding sequence. To determine whether CD-RAP/MIA is associated with tumors of cartilage, mRNAs from a variety of rodent tissues and cell lines were screened. Expression was detected in a rodent tumor, the Swarm rat chondrosarcoma and a chondrosarcoma cell line derived from it, but not in other tissues or tumors of non-cartilage origin. Immunolocalization revealed CD-RAP/MIA protein localized in cartilage only. These results show that the normal expression of CD-RAP/MIA is limited to cartilage; however, pathologically, it is expressed both in melanoma and chondrosarcoma. The restricted expression of CD-RAP/MIA may provide an opportunity to monitor cartilage metabolic activity as well as the tumor activity of melanoma and chondrosarcoma. PMID:9097023
Bosserhoff, A K; Kondo, S; Moser, M; Dietz, U H; Copeland, N G; Gilbert, D J; Jenkins, N A; Buettner, R; Sandell, L J
The gene encoding PBP74/CSA/motalin-1, a novel mouse hsp70, maps to mouse chromosome 18
The 70-kDa heat shock proteins (hsp70) function in folding of peptides and the assembly and disassembly of protein complexes. They are encoded by a multigene family comprising both heat-inducible and constitutively expressed genes. Different family members function in different organelles: hsp70 members such as hsp70 and hsc70 are present in the cytoplasm, BiP/GRP78 in the endoplasmic reticulum, and GRP75 in the mitochondria. PBP74/CSA/motalin-1 is a novel mouse hsp70 protein that was identified by three different groups. PBP74 was found to be a peptide-binding protein implicated in antigen processing. CSA is an antigen specific for the CM strain, and motalin-1 is a protein associated with cellular mortality. 10 refs., 1 fig.
Ohashi, Manabu; Oyanagi, Mitsuru; Kominami, Ryo [Niigata Univ. School of Medicine (Japan)] [and others
Light damage induced changes in mouse retinal gene expression
Oxidative stress plays a role in the light damage model of retinal degeneration as well as in age-related macular degeneration. The purpose of this study is to identify retinal genes induced by acute photo-oxidative stress, which may function as mediators of apoptosis or as survival factors. To accomplish this, Balb\\/c mice were exposed to bright cool white fluorescent light for
Lin Chen; Wayne Wu; Tzvete Dentchev; Yong Zeng; Jianhua Wang; Irena Tsui; John W. Tobias; Jean Bennett; Donald Baldwin; Joshua L. Dunaief
Mouse Modifier Genes in Mammary Tumorigenesis and Metastasis
Tumorigenesis and metastasis are complex multistep processes. In addition to the numerous somatic mutations that facilitate\\u000a cancer progression, there is abundant evidence that an individual�s genetic background not only contributes to overall cancer\\u000a risk, but also specifically influences metastatic potential. The handful of human susceptibility genes that have been identified\\u000a thus far do not fully account for hereditary cancer risk,
Scott F. Winter; Kent W. Hunter
MicroRNA gene expression in the mouse inner ear
MicroRNAs (miRNAs) are small non-coding RNAs that function through the RNA interference (RNAi) pathway and post-transcriptionally regulate gene expression in eukaryotic organisms. While miRNAs are known to affect cellular proliferation, differentiation, and morphological development, neither their expression nor roles in mammalian inner ear development have been characterized. We have investigated the extent of miRNA expression at various time points throughout
Michael D. Weston; Marsha L. Pierce; Sonia Rocha-Sanchez; Kirk W. Beisel; Garrett A. Soukup
The genetic mapping and gene structure of mouse paraoxonase/arylesterase
The physiological role of mammalian paraoxonase/arylesterase is unknown. However paraoxonase is an HDL-associated protein, and recent studies indicate that it may have anti-atherogenic functions. We describe the chromosomal localization and structure of the mouse paraoxonase gene (Pon1) to establish Pon1 as a candidate gene for genetically determined traits or pathological states in the mouse. The coding portion of Pon1 extends over approximately 25-26 kb and consists of nine exons and eight introns. We also present nucleotide sequences from the 5{prime}-flanking region of Pon1 containing numerous consensus sequences for DNA binding proteins. Haplotype analysis of 94 N2 progeny from an interspecific cross indicates that Pon1 is localized on proximal mouse chromosome 6 near D6Mit86. This assignment excludes Pon1 as a candidate for the atherosclerosis susceptibility genes Ath1, Ath2, and Ath3. However, Pon1 is a promising candidate for the remaining unmapped Ath genes. 29 refs., 3 figs., 2 tabs.
Sorenson, R.C.; Primo-Parmo, S.L.; La Du, B.N. [Univ of Michigan Medical School, Ann Arbor, MI (United States)] [and others] [Univ of Michigan Medical School, Ann Arbor, MI (United States); and others
Bisphenol A exposure modifies DNA methylation of imprint genes in mouse fetal germ cells.
Bisphenol A (BPA) is an estrogenic environmental toxin widely used for the production of plastics. Human frequent exposure to this chemical has been proposed to be a potential public health risk. The objective of this study was to assess the effects of BPA on DNA methylation of imprinting genes in fetal mouse germ cell. Pregnant mice were treated with BPA at doses of 0, 40, 80 and 160 ?g BPA/kg body weight/day from 0.5 day post coitum. DNA methylation of imprinting genes, Igf2r, Peg3 and H19, was decreased with the increase of BPA concentration in fetal mouse germ cells (p < 0.01).The relative mRNA levels of Nobox were lower in BPA-treated group compared to control (BPA free) in female fetal germ cells, but in male fetal germ cells, a significant higher in Nobox expression was observed in BPA-treated group compared to control. Decreased mRNA expression of specific meiotic genes including Stimulated by Stra8 and Dazl were obtained in the female fetal germ cells. In conclusion, BPA exposure can affect the DNA methylation of imprinting genes in fetal mouse germ cells. PMID:22699882
Zhang, Xi-Feng; Zhang, Lian-Jun; Feng, Yan-Ni; Chen, Bo; Feng, Yan-Min; Liang, Gui-Jin; Li, Lan; Shen, Wei
Nlrp2, a Maternal Effect Gene Required for Early Embryonic Development in the Mouse
Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (?118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse.
Peng, Hui; Chang, Bohao; Lu, Chenglong; Su, Jianmin; Wu, Yongyan; Lv, Pin; Wang, Yongsheng; Liu, Jun; Zhang, Bowei; Quan, Fusheng; Guo, Zekun; Zhang, Yong
Nlrp2, a maternal effect gene required for early embryonic development in the mouse.
Maternal effect genes encode proteins that are produced during oogenesis and play an essential role during early embryogenesis. Genetic ablation of such genes in oocytes can result in female subfertility or infertility. Here we report a newly identified maternal effect gene, Nlrp2, which plays a role in early embryogenesis in the mouse. Nlrp2 mRNAs and their proteins (?118 KDa) are expressed in oocytes and granulosa cells during folliculogenesis. The transcripts show a striking decline in early preimplantation embryos before zygotic genome activation, but the proteins remain present through to the blastocyst stage. Immunogold electron microscopy revealed that the NLRP2 protein is located in the cytoplasm, nucleus and close to nuclear pores in the oocytes, as well as in the surrounding granulosa cells. Using RNA interference, we knocked down Nlrp2 transcription specifically in mouse germinal vesicle oocytes. The knockdown oocytes could progress through the metaphase of meiosis I and emit the first polar body. However, the development of parthenogenetic embryos derived from Nlrp2 knockdown oocytes mainly blocked at the 2-cell stage. The maternal depletion of Nlrp2 in zygotes led to early embryonic arrest. In addition, overexpression of Nlrp2 in zygotes appears to lead to normal development, but increases blastomere apoptosis in blastocysts. These results provide the first evidence that Nlrp2 is a member of the mammalian maternal effect genes and required for early embryonic development in the mouse. PMID:22295082
Peng, Hui; Chang, Bohao; Lu, Chenglong; Su, Jianmin; Wu, Yongyan; Lv, Pin; Wang, Yongsheng; Liu, Jun; Zhang, Bowei; Quan, Fusheng; Guo, Zekun; Zhang, Yong
Biodegradable polycation and plasmid DNA multilayer film for prolonged gene delivery to mouse osteoblasts.
Sustained release of functional plasmid DNA from the surfaces of materials which support cell adhesion for tissue formation could have a significant impact on gene therapy and tissue engineering. We report here layer-by-layer assembled multilayer film from a degradable cationic poly(2-aminoethyl propylene phosphate) and plasmid DNA encoding for enhanced green fluorescent protein (EGFP) for mouse osteoblast cell adhesion and prolonged gene delivery. Multilayer film growth was monitored by UV spectrophotometry and intensity of absorbance at 260 nm related to incorporated DNA increased in an exponential manner with increase the number of deposited polymer and plasmid layers. It degraded upon incubation in phosphate-buffered saline (PBS) at 37 degrees C and sustained the release of bioactive plasmid DNA up to 2 months. The multilayer film facilitated initial mouse osteoblast cell adhesion onto the surface and enhanced cellular alkaline phosphatase activity and calcium accumulation. It sustained delivering transcriptional active DNA to mouse osteoblast cells cultured on the film, and directly prolonged gene expression in the presence of serum without any exogenous transfection agent. This biodegradable multilayer assembly is promising for the local and sustained delivery of plasmid DNA and such a layer-by-layer system suggests an alternative method for plasmid DNA incorporation which may be useful for surface modification of implanted materials or scaffold for gene therapy and tissue regeneration. PMID:17997482
Lu, Zhen-Zhen; Wu, Juan; Sun, Tian-Meng; Ji, Jing; Yan, Li-Feng; Wang, Jun
The Cd6 gene as a permissive locus for targeted transgenesis in the mouse.
The introduction of a transgene into the genome through homologous recombination or sequence-specific enzymatic modification is a key technique for producing transgenic mice. The Rosa26 gene has been widely used to produce transgenic mice because the gene is transcriptionally active in various cell types and, at many developmental stages, is permissive for constitutive expression of integrated transgenes, and is dispensable for normal development. However, permissive loci other than Rosa26 are needed to generate mice that harbor multiple transgenes for complex studies. Here, we identified the Cd6 locus on mouse chromosome 19 as a transgene integration site in a transgenic mouse strain showing widespread reporter expression. Using this locus, we generated a knock-in mouse line that harbors a CAG promoter-driven reporter transgene, and found that the homozygous transgenic mice are viable and fertile, although transgene insertion disrupted Cd6 gene expression. The transgene on the Cd6 locus expressed reporter genes extensively throughout embryos, neonates, and adults. Combined with the Cre/loxP binary system, blood and lymphatic endothelial cell-specific reporter expression from the transgenic locus was achieved. These results suggest that Cd6 is valuable as an alternative site for targeted transgenesis. PMID:24700560
Ichise, Hirotake; Ichise, Taeko; Sasanuma, Hiroki; Yoshida, Nobuaki
A family of highly diverse human and mouse genes structurally links leukocyte FcR, gp42 and PECAM-1
. A group of genes encoding proteins structurally related to the leukocyte Fc receptors (FcRs) and termed the IFGP family was identified in human and mouse. Sequences of four human and two mouse cDNAs predict proteins differing by domain composition. One of the mouse cDNAs encodes a secreted protein, which, in addition to four immunoglobulin (Ig)-like domains, contains a scavenger
Sergei V. Guselnikov; Svetlana A. Ershova; Ludmila V. Mechetina; Alexander M. Najakshin; Olga Y. Volkova; Boris Y. Alabyev; Alexander V. Taranin
This book contains 13 chapters. Some of the titles are: Gene Transfer and Expression of Mammalian Cell Receptors; Mapping Endocrine Genes with Sorted Human Chromosomes; Structure, Function, Hormonal Regulation of Steroidogenic Enzyme Genes; Molecular Analysis of Steroid Hormone Action Using the Human Metallothionein Genes as a Model.
Lau, Y.F.
Mouse in utero electroporation: controlled spatiotemporal gene transfection.
In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses. PMID:21860382
Matsui, Asuka; Yoshida, Aya C; Kubota, Mayumi; Ogawa, Masaharu; Shimogori, Tomomi
Cadmium-binding protein (metallothionein) in carp.
When carp (Cyprinus carpio) were exposed to 5 and 30 ppm Cd in the water, the contents of Cd-binding protein, which has low molecular weight, increased in the hepatopancreas, kidney, gills and gastrointestinal tract with the duration of exposure. This Cd-binding protein was purified from hepatopancreas, kidney, gills, and spleen of carp administered 2 mg/kg Cd (as CdCl2), intraperitoneally for 6 days. Two Cd-binding proteins were separated by DEAE-Sephadex A-25 column chromatography. These proteins had Cd-mercaptide bond, high cysteine contents (ca. 29-34%), but no aromatic amino acids or histidine. From these characteristics the Cd-binding proteins were identified as metallothionein. By using antiserum obtained from a rabbit to which carp hepatopancreas MT-II had been administered, immunological characteristics between hepatopancreas MT-I, II and kidney MT-II were studied, and a slight difference in antigenic determinant was observed among them. By immunological staining techniques with horseradish peroxidase, the localization of metallothionein was investigated. In the nontreated group, metallothionein was present in the acinar cells of hepatopancreas and renal convoluted tubules. In the Cd-treated group (2 mg/kg IP daily for 3 days), metallothionein was present in the nuclei, sinusoids, and extracellular space of hepatopancreas, in addition to the acinar cells. Carp were bred in 1 ppm Cd, 5 ppm Zn solution, and tap water for 14 days, following transfer to 15 ppm Cd solution, respectively. The survival ratio was the highest in the Zn group followed by Cd-treated and control groups. The metallothionein contents increased in hepatopancreas and kidney in the order: Zn greater than Cd greater than control group. Images FIGURE 5. FIGURE 6.
Kito, H; Ose, Y; Sato, T
Gene expression profiling in the striatum of inbred mouse strains with distinct opioid-related phenotypes
Background Mouse strains with a contrasting response to morphine provide a unique model for studying the genetically determined diversity of sensitivity to opioid reward, tolerance and dependence. Four inbred strains selected for this study exhibit the most distinct opioid-related phenotypes. C57BL/6J and DBA/2J mice show remarkable differences in morphine-induced antinociception, self-administration and locomotor activity. 129P3/J mice display low morphine tolerance and dependence in contrast to high sensitivity to precipitated withdrawal observed in SWR/J and C57BL/6J strains. In this study, we attempted to investigate the relationships between genetic background and basal gene expression profile in the striatum, a brain region involved in the mechanism of opioid action. Results Gene expression was studied by Affymetrix Mouse Genome 430v2.0 arrays with probes for over 39.000 transcripts. Analysis of variance with the control for false discovery rate (q < 0.01) revealed inter-strain variation in the expression of ~3% of the analyzed transcripts. A combination of three methods of array pre-processing was used to compile a list of ranked transcripts covered by 1528 probe-sets significantly different between the mouse strains under comparison. Using Gene Ontology analysis, over-represented patterns of genes associated with cytoskeleton and involved in synaptic transmission were identified. Differential expression of several genes with relevant neurobiological function (e.g. GABA-A receptor alpha subunits) was validated by quantitative RT-PCR. Analysis of correlations between gene expression and behavioural data revealed connection between the level of mRNA for K homology domain containing, RNA binding, signal transduction associated 1 (Khdrbs1) and ATPase Na+/K+ alpha2 subunit (Atp1a2) with morphine self-administration and analgesic effects, respectively. Finally, the examination of transcript structure demonstrated a possible inter-strain variability of expressed mRNA forms as for example the catechol-O-methyltransferase (Comt) gene. Conclusion The presented study led to the recognition of differences in the gene expression that may account for distinct phenotypes. Moreover, results indicate strong contribution of genetic background to differences in gene transcription in the mouse striatum. The genes identified in this work constitute promising candidates for further animal studies and for translational genetic studies in the field of addictive and analgesic properties of opioids.
Korostynski, Michal; Kaminska-Chowaniec, Dorota; Piechota, Marcin; Przewlocki, Ryszard
Positions of pluripotency genes and hepatocyte-specific genes in the nucleus before and after mouse ES cell differentiation.
Spatial positioning of genes in the cell nucleus plays an important role in the regulation of genomic functions. Evidence for changes in gene positioning associated with transcriptional activity has been reported. However, our understanding of this phenomenon is still quite limited. We examined how pluripotency genes and hepatocyte-specific genes behave during the differentiation of mouse embryonic stem (ES) cells into hepatocytes, by targeting the loci of the Klf4, Nanog, Oct4, Sox2, Cyp7?1, Pck1, Tat, and Tdo2 genes, and using three-dimensional fluorescence in situ hybridization analyses. We found that each gene has a distinctly inherent localization profile in the ES cell nucleus. During differentiation, the Klf4, Nanog, Oct4, Cyp7?1, Pck1, and Tat loci shifted toward the nuclear center, while the Sox2 and Tdo2 loci shifted toward the periphery. The Klf4, Nanog, Oct4, and Tdo2 seem to prefer the outer regions, rather than the inner regions, when they are active. We also found that the radial positioning of the focused genes in the hepatocyte cell nucleus was highly correlated with the local GC content and the gene density of the surrounding region, but not with gene activity. PMID:24737423
Udagawa, K; Ohyama, T
The Mechanism of Expansion and the Volatility it created in Three Pheromone Gene Clusters in the Mouse (Mus musculus) Genome
Three families of proteinaceous pheromones have been described in the house mouse: androgen-binding proteins (ABPs), exocrine gland�secreting peptides (ESPs), and major urinary proteins (MUPs), each of which is thought to communicate different information. All three are encoded by large gene clusters in different regions of the mouse genome, clusters that have expanded dramatically during mouse evolutionary history. We report copy number variation among the most recently duplicated Abp genes, which suggests substantial volatility in this gene region. It appears that groups of these genes behave as low copy repeats (LCRs), duplicating as relatively large blocks of genes by nonallelic homologous recombination. An analysis of gene conversion suggested that it did not contribute to the very low or absent divergence among the paralogs duplicated in this way. We evaluated the ESP and MUP gene regions for signs of the LCR pattern but could find no compelling evidence for duplication of gene blocks of any significant size. Assessment of the entire Abp gene region with the Mouse Paralogy Browser supported the conclusion that substantial volatility has occurred there. This was especially evident when comparing strains with all or part of the Mus musculus musculus or Mus musculus castaneus Abp region. No particularly remarkable volatility was observed in the other two gene families, and we discuss the significance of this in light of the various roles proposed for the three families of mouse proteinaceous pheromones.
Laukaitis, Christina M.
Progesterone Receptor-Induced Gene Expression in Primary Mouse Granulosa Cell Cultures1
The progesterone receptor (PGR) is induced by luteinizing hormone (LH) in granulosa cells of preovulatory follicles, and the PGR-A isoform is essential for ovulation based on the phenotypes of Pgr isoform-specific knockout mice. Although several genes regulated by PGR-A in vivo have been identified, whether these genes are primary targets of PGR-A or if their expression also depends on other signaling molecules that are induced by the LH surge has not been resolved. Therefore, to identify genes that are either induced or repressed by PGR in the absence of LH-mediated signaling cascades, we infected primary cultures of mouse granulosa cells with either PGR-A or PGR-B adenoviral vectors without or with R-5020 as a PGR ligand. Total RNA was extracted from infected cells at 16 h and analyzed by Affymetrix Mouse 430 2.0 microarrays. PGR-A in the presence or absence of ligand significantly induced approximately 50 genes 2-fold or more (local pooled error test at P ? 0.01). Fewer and different genes were induced by PGR-B in the absence of ligand. Edn1, Apoa1, and Cited1 were primarily regulated by PGR-A as verified by additional RT-PCR analyses, suppression by the PGR antagonist RU486, and the lack of induction by protein kinase A, protein kinase C, or epidermal growth factor (EGF)-like factors pathways. PGR regulation of these genes was confirmed further by gene expression analyses in hormonally primed Pgr mutant mouse ovaries. Because Edn1, Apoa1, and Cited1 are known to regulate angiogenesis, PGR may affect the neovascularization of follicles that is initiated with ovulation.
Sriraman, Venkataraman; Sinha, Mala; Richards, JoAnne S.
Molecular cloning and chromosomal mapping of the mouse cyclin-dependent kinase 5 gene
Cyclin-dependent kinase 5 (Cdk5) is predominantly expressed in neurons. In vitro, Cdk5 purified from the nervous tissue phosphorylates both high-molecular-weight neurofilament and microtubule-associated tau. The mouse gene encoding Cdk5 (Cdk5) was found to be 5 kb in length and divided into 12 exons. All of the exon-intron junctions matched the expected consensus sequence with the exception of the splice junction for intron 9, which has AT and AC dinucleotides instead of the usual GT and AG bordering sequence. In the 5{prime}-flanking region of mouse Cdk5, several putative promoter elements were present, including AP1, Sp1, PuF, and TATA motifs. A metal regulatory element was also identified at position -207 to -201. Nucleotide sequence analysis of mouse Cdk5 showed high identity to the homologues of other vertebrate species, indicating that this kinase is highly conserved during evolution. Mouse Cdk5 was mapped to the centromeric region of mouse chromosome 5. 20 refs., 2 figs., 1 tab.
Ohshima, Toshio; Nagle, J.W.; Brady, R.O.; Kozak, C.A. [National Institutes of Health, Bethesda, MD (United States)] [and others] [National Institutes of Health, Bethesda, MD (United States); and others
A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts.
Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, ? that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, ? induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. ? treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching "[Formula: see text]" the complex transcriptional gene network. PMID:22848790
Pandian, Ganesh N; Nakano, Yusuke; Sato, Shinsuke; Morinaga, Hironobu; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi
A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts
NASA Astrophysics Data System (ADS)
Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, ? that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, ? induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. ? treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching ``'' the complex transcriptional gene network.
Pandian, Ganesh N.; Nakano, Yusuke; Sato, Shinsuke; Morinaga, Hironobu; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi
Expression of the mouse serum albumin gene introduced into differentiated and dedifferentiated rat hepatoma cells.
A 23-kilobase-pair segment of DNA containing the entire mouse serum albumin gene as well as 2.2 kilobase pairs of 5' and 4.3 kilobase pairs of 3' flanking sequences has been introduced into pSV2dhfr, a plasmid in which expression of the mouse dihydrofolate reductase cDNA is under the control of simian virus 40 sequences. This vector, pSV2dhfr-alb, was used to transfect differentiated and variant dedifferentiated rat hepatoma cells. Nine independent clones of transfected differentiated cells secrete considerable amounts of mouse albumin, while the expression of the normal rat albumin is the same as in nontransfected cells. In contrast, only small amounts of mouse and rat albumin are produced by transfected dedifferentiated cells. The amounts of albumin mRNA present in the cells are consistent with the amounts of albumin produced. These results show that a transfected gene can be regulated in a fashion consistent with the overall differentiation profile of the cell. Images
Deschatrette, J; Fougere-Deschatrette, C; Corcos, L; Schimke, R T
Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse.
The osteochondrodysplasias are a genetically heterogeneous group of disorders affecting skeletal development, linear growth and the maintenance of cartilage and bone. We have studied a large inbred Pakistani family with a distinct form of recessively inherited spondyloepimetaphyseal dysplasia (SEMD) and mapped a gene associated with this dwarfing condition to chromosome 10q23-24, a region syntenic with the locus for the brachymorphic mutation on mouse chromosome 19. We identified two orthologous genes, ATPSK2 and Atpsk2, encoding novel ATP sulfurylase/APS kinase orthologues in the respective regions of the human and mouse genomes. We characterized a nonsense mutation in ATPSK2 in the SEMD family and a missense mutation in the region of Atpsk2 encoding the APS kinase activity in the brachymorphic mouse. ATP sulfurylase/APS kinase catalyses the metabolic activation of inorganic sulfate to PAPS, the universal donor for post-translational protein sulfation in all cell types. The cartilage-specificity of the human and mouse phenotypes provides further evidence of the critical role of sulfate activation in the maturation of cartilage extracellular matrix molecules and the effect of defects in this process on the architecture of cartilage and skeletogenesis. PMID:9771708
Faiyaz ul Haque, M; King, L M; Krakow, D; Cantor, R M; Rusiniak, M E; Swank, R T; Superti-Furga, A; Haque, S; Abbas, H; Ahmad, W; Ahmad, M; Cohn, D H
Gene order is conserved within the human chromosome 21 linkage group on mouse chromosome 10
One hundred progeny from each of two intersubspecific mouse backcrosses were used to construct a comparative genetic map of a region of mouse chromosome 10 (MMU10) that is homologous to the distal tip of the long arm of human chromosome 21 (HSA21). The analysis included five genes and three simple sequence repeat markers, two of which flanked the HSA21-homologous cluster on either side. Analysis of 200 backcross progeny detected at least one crossover between each pair of adjacent genes and demonstrated that the proximal to distal orientation of the cluster was reversed between human and mouse. The order was determined to be Fyn-1-D10Mit20-S100b-Col6a1-Itgb2-Pfk1/D10Mit7-D10Mit11. Comparative mapping supports the order of corresponding markers on HSA21 determined using pulsed-field gel electrophoresis and radiation hybrid line data. However, sequence tagged site content mapping of human yeast artificial chromosomes (YACs) yielded conflicting data on the relative positions of human COL6A1 and S100B on HSA21. This discrepancy was resolved here by demonstrating that several key YACs used in the human contig analysis were mistyped for S100B. The murine map reported here provides a scaffold for construction of physical maps and yeast artificial chromosome contigs that will be useful in the development of mouse models for the study of Down syndrome. 28 refs., 4 figs., 2 tabs.
Irving, N.G.; Cabin, D.E.; Swanson, D.A.; Reeves, R.H. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States))
Mouse pseudouridine synthase 1: gene structure and alternative splicing of pre-mRNA.
Evidence for the alternative splicing of the message for mouse pseudouridine synthase 1 (mPus1p) was found when several expressed sequence tag clones were completely sequenced. The genomic DNA for the MPUS1 gene (6.9 kb) was cloned from a mouse genomic library; the gene contains seven exons, of which three are alternatively spliced. In addition, one of the internal exons (exon VI) is unusually large. RNase protection analysis confirmed that several alternatively spliced messages were present in mouse tissues and cells in culture. A Western blot of total cellular protein from mouse tissues and cultured cells was reacted with an antibody specific for mPus1p; at least three proteins were detected. One protein corresponds to the predicted molecular mass of mPus1p (44 kDa) and is the most abundant. The two other isoforms, one 2 kDa larger and one 7 kDa smaller than mPus1p, were differentially expressed. The cDNA species for the three isoforms were cloned into expression plasmids; the proteins were synthesized in vitro and tested for pseudouridine synthase activity. The two isoforms, one containing an insert of 18 amino acids in a region of the enzyme assumed to be critical for activity, and the other, which has a deletion of the protein coding potential of two exons, were both inactive on tRNA substrates that mPus1p modifies. PMID:11085940
Chen, J; Patton, J R
Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations
We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies. 43 refs., 6 figs., 1 tab.
Moore, R.C.; Redhead, N.J.; Selfridge, J. [Univ. of Edinburgh (United Kingdom)] [and others] [Univ. of Edinburgh (United Kingdom); and others
Identification of Novel SHOX Target Genes in the Developing Limb Using a Transgenic Mouse Model
Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, L�ri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development.
Beiser, Katja U.; Glaser, Anne; Kleinschmidt, Kerstin; Scholl, Isabell; Roth, Ralph; Li, Li; Gretz, Norbert; Mechtersheimer, Gunhild; Karperien, Marcel; Marchini, Antonio; Richter, Wiltrud; Rappold, Gudrun A.
AAV-mediated gene therapy in mouse models of recessive retinal degeneration
In recent years, more and more mutant genes that cause retinal diseases have been detected. At the same time, many naturally occurring mouse models of retinal degeneration have also been found, which show similar changes to human retinal diseases. These, together with improved viral vector quality allow more and more traditionally incurable inherited retinal disorders to become potential candidates for gene therapy. Currently, the most common vehicle to deliver the therapeutic gene into target retinal cells is the adeno-associated viral vector (AAV). Following delivery to the immuno-priviledged subretinal space, AAV-vectors can efficiently target both retinal pigment epithelium and photoreceptor cells, the origin of most retinal degenerations. This review focuses on the AAV-based gene therapy in mouse models of recessive retinal degenerations, especially those in which delivery of the correct copy of the wild-type gene has led to significant beneficial effects on visual function, as determined by morphological, biochemical, electroretinographic and behavioral analysis. The past studies in animal models and ongoing successful LCA2 clinical trials, predict a bright future for AAV gene replacement treatment for inherited recessive retinal diseases.
Pang, Ji-jing; Lei, Lei; Dai, Xufeng; Shi, Wei; Liu, Xuan; Dinculescu, Astra; McDowell, J. Hugh
Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain
NASA Astrophysics Data System (ADS)
Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.
Ramesh, Govindarajan; Wu, Honglu
Subretinal injection of gene therapy vectors and stem cells in the perinatal mouse eye.
The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.(1) Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.(2) These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.(3-4) Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.(5-8) In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue. PMID:23207897
Wert, Katherine J; Skeie, Jessica M; Davis, Richard J; Tsang, Stephen H; Mahajan, Vinit B
A conditional knockout resource for the genome-wide study of mouse gene function
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
Skarnes, William C.; Rosen, Barry; West, Anthony P.; Koutsourakis, Manousos; Bushell, Wendy; Iyer, Vivek; Mujica, Alejandro O.; Thomas, Mark; Harrow, Jennifer; Cox, Tony; Jackson, David; Severin, Jessica; Biggs, Patrick; Fu, Jun; Nefedov, Michael; de Jong, Pieter J.; Stewart, A. Francis; Bradley, Allan
Glutamate is the main excitatory neurotransmitter in both the peripheral and central auditory systems. Changes of glutamate and glutamate-related genes with age may be an important factor in the pathogenesis of age-related hearing loss - presbycusis. In this study, changes in glutamate-related mRNA gene expression in the CBA mouse inferior colliculus with age and hearing loss were examined and correlations were sought between these changes and functional hearing measures, such as the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs). Gene expression of 68 glutamate-related genes was investigated using both genechip microarray and real-time PCR (qPCR) molecular techniques for four different age/hearing loss CBA mouse subject groups. Two genes showed consistent differences between groups for both the genechip and qPCR. Pyrroline-5-carboxylate synthetase enzyme (Pycs) showed down-regulation with age and a high-affinity glutamate transporter (Slc1a3) showed up-regulation with age and hearing loss. Since Pycs plays a role in converting glutamate to proline, its deficiency in old age may lead to both glutamate increases and proline deficiencies in the auditory midbrain, playing a role in the subsequent inducement of glutamate toxicity and loss of proline neuroprotective effects. The up-regulation of Slc1a3 gene expression may reflect a cellular compensatory mechanism to protect against age-related glutamate or calcium excitoxicity.
Tadros, Sherif F.; D'Souza, Mary; Zettel, Martha L.; Zhu, XiaoXia; Frisina, Robert D.
Extensive compensatory cis-trans regulation in the evolution of mouse gene expression.
Gene expression levels are thought to diverge primarily via regulatory mutations in trans within species, and in cis between species. To test this hypothesis in mammals we used RNA-sequencing to measure gene expression divergence between C57BL/6J and CAST/EiJ mouse strains and allele-specific expression in their F1 progeny. We identified 535 genes with parent-of-origin specific expression patterns, although few of these showed full allelic silencing. This suggests that the number of imprinted genes in a typical mouse somatic tissue is relatively small. In the set of nonimprinted genes, 32% showed evidence of divergent expression between the two strains. Of these, 2% could be attributed purely to variants acting in trans, while 43% were attributable only to variants acting in cis. The genes with expression divergence driven by changes in trans showed significantly higher sequence constraint than genes where the divergence was explained by variants acting in cis. The remaining genes with divergent patterns of expression (55%) were regulated by a combination of variants acting in cis and variants acting in trans. Intriguingly, the changes in expression induced by the cis and trans variants were in opposite directions more frequently than expected by chance, implying that compensatory regulation to stabilize gene expression levels is widespread. We propose that expression levels of genes regulated by this mechanism are fine-tuned by cis variants that arise following regulatory changes in trans, suggesting that many cis variants are not the primary targets of natural selection. PMID:22919075
Goncalves, Angela; Leigh-Brown, Sarah; Thybert, David; Stefflova, Klara; Turro, Ernest; Flicek, Paul; Brazma, Alvis; Odom, Duncan T; Marioni, John C
Homologs of genes expressed in Caenorhabditis elegans GABAergic neurons are also found in the developing mouse forebrain
BACKGROUND: In an effort to identify genes that specify the mammalian forebrain, we used a comparative approach to identify mouse homologs of transcription factors expressed in developing Caenorhabditis elegans GABAergic neurons. A cell-specific microarray profiling study revealed a set of transcription factors that are highly expressed in embryonic C. elegans GABAergic neurons. RESULTS: Bioinformatic analyses identified mouse protein homologs of
Elizabeth AD Hammock; Kathie L Eagleson; Susan Barlow; Laurie R Earls; David M Miller; Pat Levitt
Meta-Analysis of Differentiating Mouse Embryonic Stem Cell Gene Expression Kinetics Reveals Early Change of a Small Gene Set
Stem cell differentiation involves critical changes in gene expression. Identification of these should provide endpoints useful for optimizing stem cell propagation as well as potential clues about mechanisms governing stem cell maintenance. Here we describe the results of a new meta-analysis methodology applied to multiple gene expression datasets from three mouse embryonic stem cell (ESC) lines obtained at specific time points during the course of their differentiation into various lineages. We developed methods to identify genes with expression changes that correlated with the altered frequency of functionally defined, undifferentiated ESC in culture. In each dataset, we computed a novel statistical confidence measure for every gene which captured the certainty that a particular gene exhibited an expression pattern of interest within that dataset. This permitted a joint analysis of the datasets, despite the different experimental designs. Using a ranking scheme that favored genes exhibiting patterns of interest, we focused on the top 88 genes whose expression was consistently changed when ESC were induced to differentiate. Seven of these (103728_at, 8430410A17Rik, Klf2, Nr0b1, Sox2, Tcl1, and Zfp42) showed a rapid decrease in expression concurrent with a decrease in frequency of undifferentiated cells and remained predictive when evaluated in additional maintenance and differentiating protocols. Through a novel meta-analysis, this study identifies a small set of genes whose expression is useful for identifying changes in stem cell frequencies in cultures of mouse ESC. The methods and findings have broader applicability to understanding the regulation of self-renewal of other stem cell types.
Glover, Clive H; Marin, Michael; Eaves, Connie J; Helgason, Cheryl D; Piret, James M; Bryan, Jennifer
A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development
The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.
Laig-Webster, M.; Lim, M.E.; Chehab, F.F. [Univ. of California, San Francisco, CA (United States)
cDNA cloning and nucleotide sequence comparison of Chinese hamster metallothionein I and II mRNAs.
Polyadenylated RNA was extracted from a cadmium resistant Chinese hamster (CHO) cell line, enriched for metal-induced, abundant RNA sequences and cloned as double-stranded cDNA in the plasmid pBR322. Two cDNA clones, pCHMT1 and pCHMT2, encoding two Chinese hamster isometallothioneins were identified, and the nucleotide sequence of each insert was determined. The two Chinese hamster metallothioneins show nucleotide sequence homologies of 80% in the protein coding region and approximately 35% in both the 5' and 3' untranslated regions. Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far. Estimated nucleotide substitution rates derived from interspecies comparisons were used to calculate a metallothionein gene duplication time of 45 to 120 million years ago.
Griffith, B B; Walters, R A; Enger, M D; Hildebrand, C E; Griffith, J K
Undermethylation of structural gene sequences in extraembryonic lineages of the mouse.
The first two lineages to differentiate in the mouse embryo are the trophectoderm and primitive endoderm, which give rise to various extraembryonic structures only. Previous work has shown that all derivatives of these two lineages share the property of undermethylation of repetitive DNA sequences, both satellite and dispersed. Here we show that this undermethylation is not a peculiarity of these repetitive elements but is also a feature of structural gene sequences within both lineages. alpha-Fetoprotein, albumin, and major urinary protein gene sequences all showed extensive undermethylation at MspI restriction sites in extraembryonic lineages, which did not correlate with their expression in these tissues. The same sequences were heavily methylated in embryonic tissues as early as 7.5 days of development. There are, therefore, major global differences in DNA methylation between the earliest cell lineages to be established in the mouse embryo. The significance of these differences for cellular commitment events remains to be elucidated. PMID:2428685
Rossant, J; Sanford, J P; Chapman, V M; Andrews, G K
Transcriptome-Wide Identification of Novel Imprinted Genes in Neonatal Mouse Brain
Imprinted genes display differential allelic expression in a manner that depends on the sex of the transmitting parent. The degree of imprinting is often tissue-specific and/or developmental stage-specific, and may be altered in some diseases including cancer. Here we applied Illumina/Solexa sequencing of the transcriptomes of reciprocal F1 mouse neonatal brains and identified 26 genes with parent-of-origin dependent differential allelic expression. Allele-specific Pyrosequencing verified 17 of them, including three novel imprinted genes. The known and novel imprinted genes all are found in proximity to previously reported differentially methylated regions (DMRs). Ten genes known to be imprinted in placenta had sufficient expression levels to attain a read depth that provided statistical power to detect imprinting, and yet all were consistent with non-imprinting in our transcript count data for neonatal brain. Three closely linked and reciprocally imprinted gene pairs were also discovered, and their pattern of expression suggests transcriptional interference. Despite the coverage of more than 5000 genes, this scan only identified three novel imprinted refseq genes in neonatal brain, suggesting that this tissue is nearly exhaustively characterized. This approach has the potential to yield an complete catalog of imprinted genes after application to multiple tissues and developmental stages, shedding light on the mechanism, bioinformatic prediction, and evolution of imprinted genes and diseases associated with genomic imprinting.
Wang, Xu; Sun, Qi; McGrath, Sean D.; Mardis, Elaine R.; Soloway, Paul D.; Clark, Andrew G.
Quantitative trait loci, genes, and polymorphisms that regulate bone mineral density in mouse
This is an in silico analysis of data available from genome-wide scans. Through analysis of QTL, genes and polymorphisms that regulate BMD, we identified 82 BMD QTL, 191 BMD-associated (BMDA) genes, and 83 genes containing known BMD-associated polymorphisms (BMDAP). The catalogue of all BMDA/BMDAP genes and relevant literatures are provided. In total, there are substantially more BMDA/BMDAP genes in regions of the genome where QTL have been identified than in non-QTL regions. Among 191 BMDA genes and 83 BMDAP genes, 133 and 58 are localized in QTL region, respectively. The difference was still noticeable for the chromosome distribution of these genes between QTL and non-QTL regions. These results have allowed us to generate an integrative profile of QTL, genes, polymorphisms that determine BMD. These data could facilitate more rapid and comprehensive identification of causal genes underlying the determination of BMD in mouse and provide new insights into how BMD is regulated in humans.
Xiong, Qing; Jiao, Yan; Hasty, Karen A.; Canale, S. Terry; Stuart, John M.; Beamer, Wesley G.; Deng, Hong-Wen; Baylink, David; Gu, Weikuan
Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos
Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.
Hermann, Mario; Maeder, Morgan L.; Rector, Kyle; Ruiz, Joseph; Becher, Burkhard; Burki, Kurt; Khayter, Cyd; Aguzzi, Adriano; Joung, J. Keith
Chromosomal localization of Cdx2, a murine homologue of the Drosophila gene caudal, to mouse chromosome 5
This report describes the localization of the Cdx2 gene to mouse chromosome 5 using the method of interspecific backcross analysis. This homeobox gene acts as a transcription factor and is the murine homologue of the Drosophila caudal gene. 14 refs., 1 fig.
Chawengsaksophak, K.; Beck, F. [Univ. of Melbourne, Victoria (Australia)] [Univ. of Melbourne, Victoria (Australia)
Identification, characterization and metagenome analysis of oocyte-specific genes organized in clusters in the mouse genome
BACKGROUND: Genes specifically expressed in the oocyte play key roles in oogenesis, ovarian folliculogenesis, fertilization and\\/or early embryonic development. In an attempt to identify novel oocyte-specific genes in the mouse, we have used an in silico subtraction methodology, and we have focused our attention on genes that are organized in genomic clusters. RESULTS: In the present work, five clusters have
Am�lie Paillisson; S�bastien Dad�; Isabelle Callebaut; Martine Bontoux; Rozenn Dalbi�s-Tran; Daniel Vaiman; Philippe Monget
Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes
Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat
Oliver H. Tam; Alexei A. Aravin; Paula Stein; Angelique Girard; Elizabeth P. Murchison; Sihem Cheloufi; Emily Hodges; Martin Anger; Ravi Sachidanandam; Richard M. Schultz; Gregory J. Hannon
Gene expression profile of amyloid beta protein-injected mouse model for Alzheimer disease
Aim:To investigate the gene expression profile changes in the cerebral cortex of mice injected icv with amyloid beta-protein (A?) fragment 25-35 using cDNA microarray.Methods:Balb\\/c mice were randomly divided into a control group and A?-treated group. The Morris water maze test was performed to detect the peffect of A?-injection on the learning and memory of mice. Atlas Mouse 1.2 Expression Arrays
Ling-na Kong; Ping-ping Zuo; Liang Mu; Yan-yong Liu; Nan Yang
Effect of Interleukin1 Receptor Antagonist Gene Deletion on Male Mouse Fertility
Members of the IL-1 family are pleiotropic cytokines that are involved in inflammation, immuno- regulation, and other homeostatic functions in the body. IL-1, IL-1, and the IL-1 antagonistic molecule(IL-1receptorantagonist(IL-1Ra))arepresentinthetestisundernormalhomeostasis,and they further increase upon infection\\/inflammation. In the present study, we examined the effect of IL-1 Ra gene deletion on male mouse fertility. Male mice (wild type (WT) and IL-1 Ra knockout
Masaood Ganaiem; Mahmoud AbuElhija; Eitan Lunenfeld; Nataly Cherniy; Neomi Weisze; Sarit Bar-Sheshet Itach; Haiem Breitbart; Ronnie Apte; Mahmoud Huleihel
The sparse fur mouse as a model for gene therapy in ornithine carbamoyltransferase deficiency.
The sparse fur (spf/Y) mouse was evaluated as a model for studying gene therapy in ornithine carbamoyltransferase deficiency (OCTD), the most common inborn error of urea synthesis. Previous studies have defined a number of biochemical characteristics of this animal model that are analogous to the human disease: OCTD in liver, elevated ammonium and glutamine, low citrulline and arginine in plasma, elevated urinary orotic acid excretion, neurochemical alterations and responsiveness to alternative pathway therapy. In this study, metabolic flux, survival, behavior and learning of these animals were examined in preparation for a trial of gene therapy. We found that, as has been previously reported, OCT activity in liver ranged from 10 to 20% of control. Yet, stable isotope studies using 15N ammonium chloride to follow ureagenesis in vivo showed 55% of normal urea synthetic capacity. This suggests that partial correction with gene therapy may be sufficient to normalize urea synthesis. Although it has been suggested that liver OCTD and its consequent metabolic effects normalize without treatment by adulthood in the spf/Y mouse, we did not find this to be the case. We documented that the spf/Y mouse had a markedly decreased lifespan (< 10% of normal) and remained runted throughout life. In terms of behavior, the spf/Y mice had evidence of decreased learning in a passive avoidance task that was not attributable to alterations in activity. These clearly definable metabolic and behavioral abnormalities suggest that the spf/Y mouse should prove a useful model for studying the efficacy of gene therapy in OCTD. PMID:8750014
Batshaw, M L; Yudkoff, M; McLaughlin, B A; Gorry, E; Anegawa, N J; Smith, I A; Hyman, S L; Robinson, M B
Mouse Small eye results from mutations in a paired-like homeobox-containing gene
SMALL eye (Sey) in mouse is a semidominant mutation which in the homozygous condition results in the complete lack of eyes and nasal primordia. On the basis of comparative mapping studies and on phenotypic similarities, Sey has been suggested to be homologous to congenital aniridia (lack of iris) in human1,2. A candidate gene for the aniridia (AN) locus at 11p13
Robert E. Hill; Jack Favor; Brigid L. M. Hogan; Carl C. T. Ton; Grady F. Saunders; Isabel M. Hanson; Jane Prosser; Tim Jordan; Nicholas D. Hastie; Veronica Van Heyningen
Altered expression of Autism-associated genes in the brain of Fragile X mouse model.
Autism is a severe neurodevelopmental disorder, which typically emerges in early childhood. Most cases of autism have not been linked to mutations in a specific gene, and the etioloty of the disorder remains to be established [S.S. Moy, J.J. Nadler, T.R. Magnuson, J.N. Crawley, Mouse models of autism spectrum disorders: the challenge for behavioral genetics, Am. J. Med. Genet. 142 (2006) 40-51]. Fragile X syndrome is caused by mutation in the FMR1 gene and is characterized by mental retardation, physical abnormalities, and, in most case, autistic-like behavior [R.J. Hagerman, A.W. Jackson, A. Levitas, B. Rimland, M. Braden, An analysis of autism in fifty males with the Fragile X syndrome, Am. J. Med. Genet. 23 (1986) 359-374, C.E. Bakker, C. Verheij, R. Willemsen, R. van der Helm, F. Oerlemans, M. Vermeij, A. Bygrave, A.T. Hoogeveen, B.A. Oostra, E. Reyniers, K. De Boulle, R. D'Hooge, P. Cras, D. van Velzen, G. Nagels, J.J. Marti, P. De Deyn, J.K. Darby, P.J. Willems, Fmr1 knockout mice: a model to study Fragile X mental retardation, Cell 78 (1994) 23-33]. The FMR1 knockout (KO) mouse is one of the best characterized animal models for human disorders associated with autism [S.S. Moy, J.J. Nadler, T.R. Magnuson, J.N. Crawley, Mouse models of autism spectrum disorders: the challenge for behavioral genetics, Am. J. Med. Genet. 142 (2006) 40-51]. We have used real-time PCR to investigate changes in expression levels of three genes: WNT2, MECP2, and FMR1 in different brain regions of Fagile X mice and litter mate controls. We found major changes in the expression pattern for the three genes examined. FMR1, MECP2, and WNT2 expression were drastically down regulated in the Fragile X mouse brain. PMID:19138667
Zhang, Aiying; Shen, Chang-Hui; Ma, Shuang Yong; Ke, Yang; El Idrissi, Abdeslem
Preclinical Evaluation of Gene Therapy for NF2 Lesions in Mouse Models Using Amplicon Vectors and Prodrug Activation.
National Technical Information Service (NTIS)
These studies were designed to characterize tumors in mouse models of NF2 and to evaluate vector mediated therapy. Magnetic resonance, bioluminescence and near-infrared imaging were used in monitoring changes in tumor volume and in tracking gene delivery ...
X. O. Breakefield
Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene.
The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression. Images
Ash, J; Ke, Y; Korb, M; Johnson, L F
Identification of Pax6-Dependent Gene Regulatory Networks in the Mouse Lens
Lineage-specific DNA-binding transcription factors regulate development by activating and repressing particular set of genes required for the acquisition of a specific cell type. Pax6 is a paired domain and homeodomain-containing transcription factor essential for development of central nervous, olfactory and visual systems, as well as endocrine pancreas. Haploinsufficiency of Pax6 results in perturbed lens development and homeostasis. Loss-of-function of Pax6 is incompatible with lens lineage formation and results in abnormal telencephalic development. Using DNA microarrays, we have identified 559 genes expressed differentially between 1-day old mouse Pax6 heterozygous and wild type lenses. Of these, 178 (31.8%) were similarly increased and decreased in Pax6 homozygous embryonic telencephalon [Holm PC, Mader MT, Haubst N, Wizenmann A, Sigvardsson M, G�tz M (2007) Loss- and gain-of-function analyses reveals targets of Pax6 in the developing mouse telencephalon. Mol Cell Neurosci 34: 99�119]. In contrast, 381 (68.2%) genes were differently regulated between the lens and embryonic telencephalon. Differential expression of nine genes implicated in lens development and homeostasis: Cspg2, Igfbp5, Mab21l2, Nrf2f, Olfm3, Spag5, Spock1, Spon1 and Tgfb2, was confirmed by quantitative RT-PCR, with five of these genes: Cspg2, Mab21l2, Olfm3, Spag5 and Tgfb2, identified as candidate direct Pax6 target genes by quantitative chromatin immunoprecipitation (qChIP). In Mab21l2 and Tgfb2 promoter regions, twelve putative individual Pax6-binding sites were tested by electrophoretic mobility shift assays (EMSAs) with recombinant Pax6 proteins. This led to the identification of two and three sites in the respective Mab21l2 and Tgfb2 promoter regions identified by qChIPs. Collectively, the present studies represent an integrative genome-wide approach to identify downstream networks controlled by Pax6 that control mouse lens and forebrain development.
Wolf, Louise V.; Yang, Ying; Wang, Jinhua; Xie, Qing; Braunger, Barbara; Tamm, Ernst R.; Zavadil, Jiri; Cvekl, Ales
Cloning and structural analysis of cDNA and the gene for mouse transcription factor UBF.
The gene and protein structure of the mouse UBF (mUBF), a transcription factor for mouse ribosomal RNA gene, have been determined by cDNA and genomic clones. The unique mUBF gene consists of 21 exons spanning over 13 kb. Two mRNAs coding for mUBF1 and mUBF2 having 765 a.a. and 728 a.a., respectively, are produced by an alternative splicing of exon 8. It specifies 37 amino acids constituting a part of the regions homologous to high mobility group proteins (HMG box 2). A human UBF (hUBF) cDNA obtained by polymerase chain reaction also indicates the presence of two kinds of mRNAs, the shorter form lacking the same region as mUBF2. Comparison of the cDNAs from hUBF and mUBF revealed an unusual conservation of nucleotide sequence in the 3'-terminal non-coding region. We examined the relative amounts of expression of mUBF1 and mUBF2. The eight tissues studied contained both molecular species, although mUBF2 was the predominant form of UBF. The mRNA of mUBF1 was expressed one half of the mUBF2 in quiescent mouse fibroblasts but reached the same amount in growing state. Images
Hisatake, K; Nishimura, T; Maeda, Y; Hanada, K; Song, C Z; Muramatsu, M
Imaging gene delivery in a mouse model of congenital neuronal ceroid lipofuscinosis
Adeno-associated virus (AAV) mediated gene replacement for lysosomal disorders have been spurred by the ability of some serotypes to efficiently transduce neurons in the brain and by the ability of lysosomal enzymes to cross-correct among cells. Here, we explored enzyme replacement therapy in a knock-out mouse model of congenital neuronal ceroid lipofuscinosis (NCL), the most severe of the NCLs in humans. The missing protease in this disorder, cathepsin D (CathD) has high levels in the central nervous system (CNS). This enzyme has the potential advantage for assessing experimental therapy in that it can be imaged using a near-infrared fluorescence (NIRF) probe activated by CathD. Injections of an AAV2/rh8 vector encoding mouse cathepsin D (mCathD) into both cerebral ventricles and peritoneum of newborn knock-out mice resulted in a significant increase in lifespan. Successful delivery of active CathD by the AAV2/rh8-mCathD vector was verified by NIRF imaging of mouse embryonic fibroblasts (MEFs) from knock-out mice in culture, as well as by ex vivo NIRF imaging of brain and liver after gene transfer. These studies support the potential effectiveness and imaging evaluation of enzyme replacement therapy to the brain and other organs in CathD null mice via AAV-mediated gene delivery in neonatal animals.
Pike, Lisa S.; Tannous, Bakhos A.; Deliolanis, Nikolaos C.; Hsich, Gary; Morse, Danielle; Tung, Ching-Hsuan; Sena-Esteves, Miguel; Breakefield, Xandra O.
Metallothionein rescues hypoxia-inducible factor-1 transcriptional activity in cardiomyocytes under diabetic conditions
Metallothionein (MT) is effective in the prevention of diabetic cardiomyopathy, and hypoxia-inducible factor-1 (HIF-1) is known to control vascular endothelial growth factor (VEGF) gene expression and regulate angiogenesis in diabetic hearts. We examined whether or not MT affects HIF-1 activity in the heart of diabetic mice and in the cardiac cells cultured in high glucose (HG) media. Diabetes was induced
Wenke Feng; Yuehui Wang; Lu Cai; Y. James Kang
Testis-Specific Expression of a Metallothionein I-Driven Transgene Correlates with Undermethylation of the Locus in Testicular DNA
Mice carrying a chimeric transgene of the human testis-specific lactate dehydrogenase cDNA driven by mouse metallothionein I promoter have been reported to express the transgene in a testis-specific manner in six founder lines. To study the mechanism by which this testis-specific expression is mediated, we have examined genomic placement, expression pattern, and methylation status of the transgene. Our results indicate
Kourosh Salehi-Ashtiani; Robert J. Widrow; Clement L. Markert; Erwin Goldberg
A Comparative Study of Mouse Hepatic and Intestinal Gene Expression Profiles under PPAR? Knockout by Gene Set Enrichment Analysis
Gene expression profiling of PPAR? has been used in several studies, but fewer studies went further to identify the tissue-specific pathways or genes involved in PPAR? activation in genome-wide. Here, we employed and applied gene set enrichment analysis to two microarray datasets both PPAR? related respectively in mouse liver and intestine. We suggested that the regulatory mechanism of PPAR? activation by WY14643 in mouse small intestine is more complicated than in liver due to more involved pathways. Several pathways were cancer-related such as pancreatic cancer and small cell lung cancer, which indicated that PPAR? may have an important role in prevention of cancer development. 12 PPAR? dependent pathways and 4 PPAR? independent pathways were identified highly common in both liver and intestine of mice. Most of them were metabolism related, such as fatty acid metabolism, tryptophan metabolism, pyruvate metabolism with regard to PPAR? regulation but gluconeogenesis and propanoate metabolism independent of PPAR? regulation. Keratan sulfate biosynthesis, the pathway of regulation of actin cytoskeleton, the pathways associated with prostate cancer and small cell lung cancer were not identified as hepatic PPAR? independent but as WY14643 dependent ones in intestinal study. We also provided some novel hepatic tissue-specific marker genes.
He, Kan; Wang, Qishan; Yang, Yumei; Wang, Minghui; Pan, Yuchun
Zinc and low-dose of cadmium protect sertoli cells against toxic-dose of cadmium: The role of metallothionein.
Background: The impact of cadmium (Cd) on male infertility may be related to the interaction with metal-binding proteins known as metallothioneins (Mts). Trace elements like zinc (Zn) have protective effects on testicular damage induced by Cd. Objective: We determined the effect of Zn and low-dose Cd pre-treatment on the expression of Mt1 and Mt2 genes on testicular Sertoli cells. Materials and Methods: The cultured TM4 mouse sertoli cells were treated with 50 ?M ZnSO4 (Zn pre-treated group; ZnPG), 2 ?M CdCl2 (Cd pre-treated group; CdPG), or distilled water (DW pre-treated group; DWPG). After 18 hour, all of these groups were exposed to 100 ?M CdCl2 for different periods of time (1, 2, 3, and 6 hours). There was also a control group for all three groups, which was treated only with distilled water (without Cd or Zn pre-treatment). Cellular viability, Zn and Cd concentrations and gene expression were assessed by MTT, atomic absorption spectrometry and real time PCR methods, respectively. Results: The expression of Mt1 and Mt2 genes in ZnPG, CdPG, and DWPG was greater than the control group (p=0.02 and p=0.01, respectively). Cd concentrations in CdPG and DWPG were greater than the control group (p=0.00). Expression of both genes in ZnPG and CdPG increased after 3 hours of treatment and Cd concentration decreased simultaneously, which was more obvious in ZnPG. Conclusion: Zn and short term low-dose Cd pre-treatment might reduce the adverse effects of Cd by increasing expression of Mts genes in Sertoli cells. The protective effect of Zn was stronger than Cd. PMID:24639783
Kheradmand, Fatemeh; Nourmohammadi, Issa; Ahmadi-Faghih, Mohamad Amin; Firoozrai, Mohsen; Modarressi, Mohammad Hossein
898. Liver-Directed, AAV and Lentivirus-Mediated Gene Therapy in the Phenylketonuria Mouse Model Pah-enu2
A somatic gene therapy method for phenylketonuria (PKU) in a PKU-mouse model that carries the Pah-enu2 allele was developed. The Pah-enu2 allele is a mutant Pah gene, coding for a phenylalanine hydroxylase (PAH), with a single amino acid exchange in exon 7 giving rise to an inactive enzyme and hyperphenylalaninemia. The Pah-enu2 mouse, generously supplied by Dr. MacDonald (Wichita State
Zhaobing Ding; Panco Georgiev; Beat Thony
Mouse NRH:quinone oxidoreductase (NQO2): cloning of cDNA and gene- and tissue-specific expression.
The mouse NQO2 cDNA and gene with flanking regions were cloned and sequenced. Analysis of the primary structure of the mouse NQO2 protein revealed the presence of glycosylation, myristylation, protein kinase C and caseine kinase II phosphorylation sites. These sites are conserved in the human NQO2 protein. The mouse NQO2 gene promoter contains several important cis-elements, including the antioxidant response element (ARE), the xenobiotic response element (XRE), and an Sp1 binding site. Northern analysis of eight mouse tissues indicated wide variations in the expression of the NQO2 and NQO1 genes. NQO2 gene expression was higher in liver and testis compared with the NQO1 gene, which was highest in the heart. NQO1 gene expression was undetectable in the testis. Mouse kidney showed significantly higher expression levels of NQO1 compared with NQO2. Brain, spleen, lung, and skeletal muscle showed undetectable levels of NQO2 and NQO1 gene expression. NQO2 activity followed a more or less similar pattern of tissue-specific expression as NQO2 RNA. Interestingly, the NQO2 activity remained unchanged in the NQO1-/-mice tissues compared with NQO1+/+ mice, with the exception of the liver. The livers from NQO1-/-mice showed a 45% increase in NQO2 activity compared with the NQO1+/+ mice. The mouse NQO2 cDNA was subcloned into the pMT2 eukaryotic expression vector which, upon transfection in monkey kidney COS1 cells, produced a significant increase in NQO2 activity. Deletion of 54 amino acids from the N-terminus of the mouse NQO2 protein resulted in the loss of NQO2 expression and activity in transfected COS1 cells. This indicates that deletion of exon(s) encoding the N-terminus of NQO2 from the endogenous gene in mouse embryonic (ES) stem cells should result in NQO2-null mice. PMID:10903442
Long, D J; Jaiswal, A K
Genetic and Molecular Basis of QTL of Diabetes in Mouse: Genes and Polymorphisms
A systematic study has been conducted of all available reports in PubMed and OMIM (Online Mendelian Inheritance in Man) to examine the genetic and molecular basis of quantitative genetic loci (QTL) of diabetes with the main focus on genes and polymorphisms. The major question is, What can the QTL tell us? Specifically, we want to know whether those genome regions differ from other regions in terms of genes relevant to diabetes. Which genes are within those QTL regions, and, among them, which genes have already been linked to diabetes? whether more polymorphisms have been associated with diabetes in the QTL regions than in the non-QTL regions. Our search revealed a total of 9038 genes from 26 type 1 diabetes QTL, which cover 667,096,006 bp of the mouse genomic sequence. On one hand, a large number of candidate genes are in each of these QTL; on the other hand, we found that some obvious candidate genes of QTL have not yet been investigated. Thus, the comprehensive search of candidate genes for known QTL may provide unexpected benefit for identifying QTL genes for diabetes.
Gao, Peng; Jiao, Yan; Xiong, Qing; Wang, Cong-Yi; Gerling, Ivan; Gu, Weikuan
Genome-wide expression profiling and bioinformatics analysis of diurnally regulated genes in the mouse prefrontal cortex
Background The prefrontal cortex is important in regulating sleep and mood. Diurnally regulated genes in the prefrontal cortex may be controlled by the circadian system, by sleep:wake states, or by cellular metabolism or environmental responses. Bioinformatics analysis of these genes will provide insights into a wide-range of pathways that are involved in the pathophysiology of sleep disorders and psychiatric disorders with sleep disturbances. Results We examined gene expression in the mouse prefrontal cortex at four time points during a 24 hour (12 hour light:12 hour dark) cycle using microarrays, and identified 3,890 transcripts corresponding to 2,927 genes with diurnally regulated expression patterns. We show that 16% of the genes identified in our study are orthologs of identified clock, clock controlled or sleep/wakefulness induced genes in the mouse liver and suprachiasmatic nucleus, rat cortex and cerebellum, or Drosophila head. The diurnal expression patterns were confirmed for 16 out of 18 genes in an independent set of RNA samples. The diurnal genes fall into eight temporal categories with distinct functional attributes, as assessed by Gene Ontology classification and analysis of enriched transcription factor binding sites. Conclusion Our analysis demonstrates that approximately 10% of transcripts have diurnally regulated expression patterns in the mouse prefrontal cortex. Functional annotation of these genes will be important for the selection of candidate genes for behavioral mutants in the mouse and for genetic studies of disorders associated with anomalies in the sleep:wake cycle and circadian rhythm.
Yang, Shuzhang; Wang, Kai; Valladares, Otto; Hannenhalli, Sridhar; Bucan, Maja
Localization of a human homolog of the mouse pericentrin gene (PCNT) to chromosome 21qter
Exon trapping was used to identify portions of genes from cosmid DNA of a human chromosome 21-specific library LL21NC02-Q. More than 650 potential exons have been cloned and characterized to date. Among these, 3 trapped {open_quotes}exons{close_quotes} showed strong homology to different regions of the cDNA for the mouse pericentrin (Pcnt) gene, indicating that these 3 exons are portions of a human homolog of the mouse pericentrin gene. With PCR amplification, Southern blot analysis, and FISH, we have mapped this presumed human pericentrin gene (PCNT) to the long arm of chromosome 21 between marker PFKL and 21qter. Pericentrin is a conserved protein component of the filamentous matrix of the centrosome involved in the initial establishment of the organized microtubule array. No candidate hereditary disorder for pericentrin deficiency/abnormality has yet been mapped in the most distal region of 21q; in addition the role of triplication of the pericentrin gene in the pathophysiology or etiology of trisomy 21 is currently unknown. 16 refs., 3 figs.
Chen, Haiming [Univ. of Geneva Medical School (Switzerland)] [Univ. of Geneva Medical School (Switzerland); Gos, A.; Morris, M.A. [Cantonal Hospital, Geneva (Switzerland)] [and others] [Cantonal Hospital, Geneva (Switzerland); and others
Modeling human lymphoid precursor cell gene therapy in the SCID-hu mouse.
Gene therapy of human T-lymphocyte disorders, including acquired immunodeficiency syndrome (AIDS), would be greatly facilitated by the development of an in vivo system in which transduced human hematopoietic stem cells can be used to reconstitute the T-lymphoid compartment. Here we use the SCID-hu mouse as a recipient for human CD34+ hematopoietic progenitor cells transduced in vitro with a retroviral vector carrying the neomycin resistance gene (neoR). The transduced cells engraft and reconstitute the lymphoid compartments of the human thymus implant with as few as 5 x 10(4) CD34+ cells. The neoR gene was expressed at low levels in human thymocytes and there was no apparent effect on thymocyte differentiation as a result of vector transduction. Thus, this SCID-hu mouse system is the first in vivo model showing human thymopoiesis after transduction of exogenous vectors, and should allow preclinical testing of gene therapeutic reagents designed to function in human cells of the T-lymphoid lineage. Because human immunodeficiency virus type 1 infection induces depletion of human thymocytes in SCID-hu mice, this system may be particularly valuable in evaluating efficacy of gene therapies to combat AIDS. PMID:7520766
Akkina, R K; Rosenblatt, J D; Campbell, A G; Chen, I S; Zack, J A
Activity-dependent genes in mouse olfactory sensory neurons.
Activity-dependent survival of olfactory sensory neurons (OSNs) may allow animals to tune their olfactory systems to match their odor environment. Activity-dependent genes should play important roles in this process, motivating experiments to identify them. Both unilateral naris occlusion of mice for 6 days and genetic silencing of OSNs decreased S100A5, Lrrc3b, Kirrel2, Slc17a6, Rasgrp4, Pcp4l1, Plcxd3, and Kcnn2 while increasing Kirrel3. Naris occlusion also decreased Eml5, Ptprn, and Nphs1. OSN number was unchanged and stress-response mRNAs were unaffected after 6 days of naris occlusion. This leaves odor stimulation as the most likely cause of differential abundance of these mRNAs, but through a mechanism that is slow or indirect for most because 30-40 min of odor stimulation increased only 3 of 11 mRNAs decreased by naris occlusion: S100A5, Lrrc3b, and Kirrel2. Odorant receptor (OR) mRNAs were significantly more variable than the average mRNA, consistent with difficulty in reliably detecting changes in these mRNAs after 6 days of naris occlusion. One OR mRNA, Olfr855, was consistently decreased, however. These results suggest that the latency from the cessation of odor stimulation to effects on activity-dependent OSN survival must be a week or more in juvenile mice. PMID:24692514
Fischl, Adrian M; Heron, Paula M; Stromberg, Arnold J; McClintock, Timothy S
Chromosomal mapping of the peroneal muscular atrophy (pma) gene in the mouse.
We conducted chromosomal mapping of the pma gene that is a causative gene in the peroneal muscular atrophy mouse, which shows a club foot at birth and unusual gait due to a dropped foot in the adult. Linkage analyses using backcross progeny revealed a significant linkage between the pma gene and three microsatellite markers, D5Mit263 at 73 cM, D5Mit141 and D5Mit97 at 74 cM on Chr 5. The gene order was determined as follows: centromere-D5Mit263-[2.65 cM]-D5Mit141-[2.56 cM]-pma-[5.13 cM]-D5Mit97-telomere. PMID:14625412
Katoh, Hideki; Watanabe, Yuka; Ebukuro, Michi; Muguruma, Kaori; Takabayashi, Shuji; Shiroishi, Toshihiko
Gene expression profiling analysis of deoxynivalenol-induced inhibition of mouse thymic epithelial cell proliferation.
Deoxynivalenol (DON) is a mycotoxin produced as a secondary metabolite by fungal species. It has been shown that DON has serious toxic effects on many kinds of immune cells. However, the toxic effects on thymic epithelial cells were poorly understood. The purpose of this study is to investigate the gene expression differences for the DON-induced inhibition on the proliferation of mouse thymic epithelial cell line 1 (MTEC1). After the experiments of cell viability, morphological investigation and cell cycle analysis, microarray analysis was carried out. The differentially expressed genes belong to a variety of functional categories, including genes involved in metabolic process, cell cycle, oxidation-reduction process and apoptosis. Our results provide molecular insights into the gene expression differences of DON-induced toxic effects and suggest that p53 signaling pathway may play an important role in the inhibition of MTEC1 cell proliferation. PMID:23827195
Li, Daotong; Ye, Yaqiong; Deng, Li; Ma, Haoran; Fan, Xiaolong; Zhang, Yuan; Yan, Haikuo; Deng, Xianbo; Li, Yugu; Ma, Yongjiang
The mouse mammary tumor virus envelope gene product is required for superantigen presentation to T cells
Transgenic mice expressing either the mouse mammary tumor virus (MMTV) superantigen gene (sag) alone or in combination with the viral envelope genes (env) (LEL), or all of the viral genes (gag, pol, env, and sag) (HYB PRO), deleted V beta 14+ T cells from their immune repertoire. However, only LEL or HYB PRO transgenic antigen-presenting cells were capable of stimulating a proliferative response from nontransgenic primary T cells or interleukin 2 production from a V beta 15-bearing T cell hybridoma. These T cell responses could be inhibited by a monospecific antibody directed against the MMTV gp52 cell surface glycoprotein. These results indicate that the MMTV gp52 gene product participates in the presentation of superantigen to T cells, resulting in their stimulation, a requisite step in the MMTV infection pathway. Thus, gp52 could play a role in the transfer of virus between different subsets of lymphocytes.
Cloning and characterization of the mouse glucokinase gene locus and identification of distal liver-specific DNase I hypersensitive sites
We cloned and characterized an 83-kb fragment of mouse genomic DNA containing the entire glucokinase (GK) gene. The 11 exons of the gene span a total distance of 49 kb, with exons 1{beta} and 1L being separated by 35 kb. A total of 25,266 bp of DNA sequence information was determined: from {approximately}-9.2 to {approximately}+15 kb (24,195 bp), relative to the hepatocyte transcription start site, and from -335 to -736 bp (1071 bp), relative to the transcription start site in {beta} cells. These sequences revealed that mouse GK is >94% identical to rat and human GK. Mouse hepatic GK mRNA is regulated by fasting and refeeding, as also occurs in the rat. Alignment of the upstream and downstream promoter regions of the mouse, rat, and human genes revealed several evolutionarily conserved regions that may contribute to transcriptional regulation. However, fusion gene studies in transgenic mice indicate that the conserved regions near the transcription start site in hepatocytes are themselves not sufficient for position-independent expression in liver. Analysis of the chromatin structure of a 48-kb region of the mouse gene using DNase I revealed eight liver-specific hypersensitive sites whose locations ranged from 0.1 to 36 kb upstream of the liver transcription start site. The availability of a single, contiguous DNA fragment containing the entire mouse GK gene should allow further studies of cell-specific expression of GK to be performed. 46 refs., 8 figs.
Postic, C.; Niswender, K.D.; Shelton, K.D.; Pettepher, C.C.; Granner, D.K.; Magnuson, M.A. [Vanderbilt Univ., Nashville, TN (United States)] [and others
Identification of novel striatal genes by expression profiling in adult mouse brain.
Large-scale transcriptome analysis in the brain is a powerful approach to identify novel genes of potential interest toward understanding cerebral organization and function. We utilized the microarray technology to measure expression levels of about 24,000 genes and expressed sequence tags in mouse hippocampus, frontal cortex and striatum. Using expression profile obtained from whole brain as a reference, we categorized the genes into groups of genes either enriched in, or restricted to, one of the three areas of interest. We found enriched genes for each target area. Further, we identified 14 genes in the category of genes restricted to the striatum, among which were the orphan G protein-coupled receptor GPR88 and retinoic acid receptor-beta. These two genes were already reported to be selectively expressed in the striatum, thus validating our experimental approach. We selected 6 striatal-restricted genes, as well as 10 striatal-enriched candidates, that were previously undescribed. We analyzed their expression by in situ hybridization analysis in the brain, and quantitative RT-PCR in both brain and peripheral organs. Two of these unknown genes displayed a notable expression pattern. The striatal-restricted gene H3076B11 shows uniform expression throughout and uniquely in the striatum, representing a genuine striatal marker. The striatal-enriched gene 4833421E05Rik is preferentially expressed in the rostral striatum, and is also abundant in kidney, liver and lung. These two genes may contribute to some of the many striatal-controlled behaviors, including initiation of movement, habit formation, or reward and motivation. PMID:17395390
Ghate, A; Befort, K; Becker, J A J; Filliol, D; Bole-Feysot, C; Demebele, D; Jost, B; Koch, M; Kieffer, B L
Animal models of melanoma: a somatic cell gene delivery mouse model allows rapid evaluation of genes implicated in human melanoma
The increasing incidence and mortality associated with advanced stages of melanoma are cause for concern. Few treatment options are available for advanced melanoma and the 5-year survival rate is less than 15%. Targeted therapies may revolutionize melanoma treatment by providing less toxic and more effective strategies. However, maximizing effectiveness requires further understanding of the molecular alterations that drive tumor formation, progression, and maintenance, as well as elucidating the mechanisms of resistance. Several different genetic alterations identified in human melanoma have been recapitulated in mice. This review outlines recent progress made in the development of mouse models of melanoma and summarizes what these findings reveal about the human disease. We begin with a discussion of traditional models and conclude with the recently developed RCAS/TVA somatic cell gene delivery mouse model of melanoma.
McKinney, Andrea J.; Holmen, Sheri L.
Comprehensive transcriptional profiling of prion infection in mouse models reveals networks of responsive genes
Background Prion infection results in progressive neurodegeneration of the central nervous system invariably resulting in death. The pathological effects of prion diseases in the brain are morphologically well defined, such as gliosis, vacuolation, and the accumulation of disease-specific protease-resistant prion protein (PrPSc). However, the underlying molecular events that lead to the death of neurons are poorly characterised. Results In this study cDNA microarrays were used to profile gene expression changes in the brains of two different strains of mice infected with three strains of mouse-adapted scrapie. Extensive data was collected and analyzed, from which we identified a core group of 349 prion-related genes (PRGs) that consistently showed altered expression in mouse models. Gene ontology analysis assigned many of the up-regulated genes to functional groups associated with one of the primary neuropathological features of prion diseases, astrocytosis and gliosis; protein synthesis, inflammation, cell proliferation and lipid metabolism. Using a computational tool, Ingenuity Pathway Analysis (IPA), we were able to build networks of interacting genes from the PRG list. The regulatory cytokine TGFB1, involved in modulating the inflammatory response, was identified as the outstanding interaction partner for many of the PRGs. The majority of genes expressed in neurons were down-regulated; a number of these were involved in regulatory pathways including synapse function, calcium signalling, long-term potentiation and ERK/MAPK signalling. Two down-regulated genes coding for the transcription regulators, EGR1 and CREB1, were also identified as central to interacting networks of genes; these factors are often used as markers of neuronal activity and their deregulation could be key to loss of neuronal function. Conclusion These data provides a comprehensive list of genes that are consistently differentially expressed in multiple scrapie infected mouse models. Building networks of interactions between these genes provides a means to understand the complex interplay in the brain during neurodegeneration. Resolving the key regulatory and signaling events that underlie prion pathogenesis will provide targets for the design of novel therapies and the elucidation of biomarkers.
Sorensen, Garrett; Medina, Sarah; Parchaliuk, Debra; Phillipson, Clark; Robertson, Catherine; Booth, Stephanie A
Gene expression profile of mouse fibroblasts exposed to a biodegradable iron alloy for stents.
Iron-based materials could constitute an interesting option for cardiovascular biodegradable stent applications due to their superior ductility compared to their counterparts - magnesium alloys. Since the predicted degradation rate of pure iron is considered slow, manganese (35% w/w), an alloying element for iron, was explored to counteract this problem through the powder metallurgy process (Fe-35 Mn). However, manganese presents a high cytotoxic potential; thus its effect on cells must first be established. Here, we established the gene expression profile of mouse 3T3 fibroblasts exposed to Fe-35 Mn degradation products in order to better understand cell response to potentially cytotoxic degradable metallic material (DMM). Mouse 3T3 cells were exposed to degradation products eluting through tissue culture insert filter (3 ?m pore size) containing cytostatic amounts of 3.25 mg ml(-1) of Fe-35 Mn powder, 0.25 mg ml(-1) of pure Mn powder or 5 mg ml(-1) of pure iron powder for 2