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Seasonal Cashier Team Member Description:Provide fast, fun and friendly checkout service.|Resolve guest concerns in a positive, helpful manner.|Work as part of a team.|Know REDcardSM messaging and solicit guest registrations.| Requirements: Use excellent guest service...> Read More
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Retail Sales Associate / Photographer Part-time Retail Sales Associate / Photographer Have a passion for sales and photography? Picture yourself here Join the Flash Digital Portraits team as a photographer & sales professional. We'll teach you everything you'll need to know.> Read More
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Caregiver Caregivers and CNA's Needed Every day is SPECIAL when you are a Comfort Keeper. To learn more about this career opportunity, contact us at 864-573-2353, or apply online at http://ck690.ersp.biz... Applications are also accepted in our...> Read More
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Crew Member ( Entry Level Food Service / Customer Service ) Building the perfect burrito – and having fun doing it – is the first step to building your career at Chipotle. Sharpen your customer service and teamwork skills, learn to make great food, and get ready to grow. We are currently hiring for...> Read More
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148) Sales Representative (S) 100677 Base Pay + Comm.
Sales Representative (S) 100677 Base Pay + Comm. TruGreen is committed to Diversity and Inclusion. We encourage diverse candidates to apply to this position. An Equal Opportunity/ Affirmative Action Employer EOE of Minorities/Females/Vets/Disability Position Overview Generates revenue in...> Read More
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149) Warehouse Person (Loader) - Full-Time Evenings - Weekends Included
Warehouse Person (Loader) - Full-Time Evenings - Weekends Included This labor-intensive position is responsible for loading transport, bulk, and route trucks. Each type of loading requires strict attention to order accuracy with respect to loading outgoing trucks and unloading incoming trucks. Works from...> Read More
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Mid-level/Allied Healthcare Jobs Occupational Therapist SUCCESS THERAPY CAREERS Montoursville, PA ABOUT US REHABILITATION SERVICES THERAPIST built, owned and operated - rehabilitation company providing physical, occupational and speech therapy, contract, management and...> Read More
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Yerevan shooting: MP's son-in-law charged with attempted murder
May 21, 2014 - 19:15 AMT
PanARMENIAN.Net - The son-in-law of the Prosperous Armenia MP Karo Karapetyan, Gor Arakelyan, who opened fire near Vernissage open-air market on Saturday, May 17, has been charged with attempted murder and illegal possession of arms, the Armenian Police press service reported.
In an earlier statement, the Armenian Interior Ministry said the suspect was arrested May 18. The statement said the suspect turned in a Makarov pistol he said he had used.
The Saturday shooting near Vernissage open-air market left 5 injured. Norayr Harutyunyan and Valery Hovhannisyan, both aged 30; Myasnik Hayrapetyan, 26; Albina Badalyan 64 and Mnatsakan Sukiasyan 72, underwent surgeries in the Yerevan's Heratsi Hospital No. 1 and their lives are not threatened.
A criminal case has been instigated under Articles 235.1 and 258.4 of the Criminal Code of the Republic of Armenia. An investigation is underway.
Top stories
The report is designed to shape the debate around new global development goals that are to be finalized next year.
"I think it will be useful if we share the rich experience and the practical skills with the young generation," Ralph Yirikian said.
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The winners of each category split the prize money of about $1.1 mln. Each winner also receives a diploma and a gold medal.
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Stoltenberg: NATO war planes had to scramble 400 times this year "NATO does not possess or NATO doesn't sell military equipment, that is something the nations are doing or not doing," Stoltenberg said.
Muslim Brotherhood senior leader arrested in Egypt President Mohammed Morsi, who won office with Brotherhood backing, was ousted when the military retook power in 2013.
Jolie says wants to switch her focus to directing "I like being pushed mentally to have to learn so much and be a part of every single aspect of a production," she said.


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A method for gene shuffling to form a mutant or chimeric gene, the method comprising: (a) assigning one or more segments of one or more genes based on regions of encoded amino acid sequence; (b) amplifying the one or more assigned segments of the gene using primers specific for each segment; and (c) causing recombination of the one or more amplified segments to form a mutant or chimeric gene. An oligonucleotide primer suitable for use in gene shuffling, the primer having a non-degenerate core based on a segment or template of a gene to be amplified, and the core being flanked by both 5′ and 3′ degenerate ends.
1. A method for gene shuffling to form at least one mutant or chimeric gene, the method comprising:
amplifying at least a first segment of a first parental gene and a second segment of a second parental gene, wherein at least one first segment is amplified using a first primer comprising a non-degenerate core flanked by 5′ and 3′ degenerate ends, and wherein at least one second segment is amplified using a second primer comprising a non-degenerate core flanked by 5′ and 3′ degenerate ends, wherein the non-degenerate core of the first primer is complementary to the non-degenerate core of the second primer; and
causing recombination between at least one amplified first segment and at least one amplified second segment to form at least one mutant or chimeric gene.
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The present invention relates to methods for gene shuffling to form mutant or chimeric genes encoding useful gene products.
Stemmer (Stemmer, W. P. C. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389-391, 1994; Stemmer, W. P. C. DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc. Natl. Acad. USA 91, 10747-10751, 1994) has discussed the most effective methods to search sequence space in vitro to yield the greatest diversity of protein variants. Until recently, the most popular methods of creating combinatorial libraries are recursive strategies that seek to evolve sequences by the addition of point mutations. For in vitro evolution, inclusion of recombinant polymerase chain reaction PCR (gene shuffling) offers practical and theoretical advantages over simple recursive mutagenesis methods. It will rapidly fine tune the mutational load in several parts of the protein by recombining point mutations and wild-type sequences. The technique (and its variations) have been used to enhance enzyme activity, substrate specificity and stability. Family shuffling is usually achieved by fragmentation of the genes to be shuffled followed by PCR. This method relies on homologous recombination during the PCR reassembly step. Most methods require relatively high levels of sequence similarity between the genes to be shuffled as �cross-over points� appear to occur in these regions.
If sequence similarity is low between the input genes, the majority products tend to be the reassembled parental genes and extensive searches need to be carried out to find recombinants (Kikuchi, M., Ohnishi, K. & Harayama, S. Novel family shuffling methods for the in vitro evolution of enzymes. Gene 236, 159-167, 1999; Qstermeier, M., Shim, J. H. & Benkovic, S. J. A combinatorial approach to hybrid enzymes independent of DNA homology. Nat. Biotechnol. 17, 1205-1209, 1999.). Kichuchi et al (1999) have reported on methods for gene shuffling that make use of unique restriction enzyme sites in the sequences of the parental molecules and following cleavage, several PCR steps were carried out to amplify the recombinant genes, a process that allowed hybrid genes to be formed at high frequency. An entirely different procedure was proposed by Ostermeier et al (1999) that allowed the preparation of combinatorial fusion libraries by progressive truncation of coding sequences of the two parental sequences followed by ligation of the fragments and selection for enzyme activity. Either parent can be used to provide 5′ sequence for the hybrid gene. This procedure, termed iterative truncation for the creation of hybrid enzymes (ITCHY), can accommodate recombination between genes with as little as 50% sequence similarity and was found to give a wider range of crossovers compared with standard gene shuffling techniques.
The present inventors recently isolated a gene coding for a thermophilic beta-xylanase that had improved performance in the bleaching of paper pulp. It was desired to investigate the possibility of obtaining mutant derivatives that had enhanced stability and an altered pH optimum. Experiments using error-prone PCR and mis-incorporation mutagenesis followed by gene shuffling allowed the identification of mutant genes that coded for a limited sample of the variations in sequence space but required extensive screening for their identification. Gene shuffling following DNAsel fragmentation of related genes (family shuffling) overwhelmingly gave wild type parental sequences as the major products. After several trials of methods designed to reduce the background, a technique was devised that allows shuffling of genes that differ widely in sequence similarity and G:C content and greatly reduces the appearance of wild type genes. Furthermore, the primer extension conditions may be modified to bias the resulting progeny genes towards any one (or more) of the parental input genes. The present inventors term this procedure Degenerate Oligonucleotide Gene Shuffling (DOGS) and note its compatibility with other recursive point mutation techniques.
In a general first aspect, the present invention provides a method for gene shuffling to form a mutant or chimeric gene product, the method comprising:
(a) assigning one or more segments of the gene;
(b) amplifying the one or more of the assigned segments of the gene; and
(c) causing recombination of the one or more amplified segments to form a mutant or chimeric gene.
In a second aspect, the present invention provides a method for forming a chimeric gene from two or more genes, the method comprising:
(a) assigning one or more segments of each gene;
(b) amplifying the one or more segments of the genes; and
(c) combining at least some of the amplified segments so as to form a chimeric gene.
Preferably, the two or more genes belong to the same gene family encoding the same functional protein. It will be appreciated, however, that recombinant genes may be prepared from genes belonging to different families.
Preferably, the segments are assigned based on regions of encoded amino acid sequence of the gene. More preferably, the gene segments are assigned based on regions of conserved amino acid sequence of the respective gene product.
Preferably, the one or more assigned segments of the gene are amplified using primers specific for each segment.
Preferably, the amplifying of the one or more segments of the genes is achieved by using degenerate primers to produce amplified segments with complementary ends corresponding to the degenerate primers.
Preferably, the amplification is by polymerase chain reaction (PCR).
In one preferred form, the amplified segments are mixed in defined ratios so as to alter the likelihood of recombination of a segment into the chimeric gene. In this manner, a gene segment of interest can be provided in a higher ratio than other amplified segments to ensure that a higher percentage of recombinants will contain the segment of interest.
Preferably, the chimeric gene is produced by overlap extension of the combined amplified segments and multiple copies of the gene are produced by PCR amplification.
After the segments have been assigned in the gene(s) and the PCR primers specific for the gene segment devised, each segment is amplified, usually separately, from the parent gene(s) of interest. In order to obtain chimeric or mutant genes, the amplified segments are combined and joined by overlap extension. The mutant genes can then be placed in suitable expression vectors known to the art and the gene product produced. The resultant mutant gene product can be assayed for functional activity and compared with the activity of the parent gene product. Examples of possible gene products include, but not limited to, enzymes, growth factors, inhibitors, antibodies, antigens, structural proteins, transport proteins, toxins, and the like.
An advantage of the present invention is that there is no need to cleave the gene by nucleases prior to amplification and recombination. As a result, the present invention results in higher yields of chimera production and thus low generation of wild-type recombinants.
The gene(s) to be shuffled can be mutated or altered by standard techniques prior to being processed by the present invention.
In a third aspect, the present invention provides a chimeric or mutant gene produced by the method according to the first or second aspects of the present invention.
In a fourth aspect, the present invention provides an oligonucleotide primers having a non-degenerate core flanked by both 5′ and 3′ degenerate ends. These primers are referred to herein as complementary degenerate-end primers (CDE primers), in the amplification of segments of a gene to produce chimeric genes.
Preferably the oligonucleotide primer is suitable for use in gene shuffling and has a non-degenerate core based on a segment or template of a gene to be amplified and the core flanked by both 5′ and 3′ degenerate ends.
The 3′ degenerate end gives each CDE primer their template-binding specificity, while the non-degenerate region acts as a stabilising clamp in subsequent rounds of the PCR. The 5′ degenerate end is not required to contribute to the binding efficiency of the CDE primer during PCR, however, it plays an important role in allowing efficient binding and subsequent overlap-extension of PCR products (amplified gene segments) generated using respectively, the forward or the reverse CDE primers.
The non-degenerate core of CDE primers is generally based upon the corresponding coding sequence of one gene, designated the parental gene for shuffling. This results in the formation of chimeric fragments which retain parental sequence at the points of segment overlap.
CDE primers allow efficient and specific amplification of portions (referred to herein as gene segments) of related but divergent genes.
The 5′ degenerate end of CDE primers ensures that separate PCR products generated with the respective forward or reverse complementary CDE primers can anneal equally well to initiate overlap extension, regardless of the parental origin of each segment.
The 3′ and 5′ degenerate ends of CDE primers would preferably be (though not limited to) 6-12 nucleotides in length, corresponding to 2-4 conserved amino acid residues. It will be appreciated that the length of the ends can vary depending on the gene or genes to be amplified.
The 3′ and 5′ degenerate ends of CDE primers should be of sufficient length to allow correct primer to template annealing in PCR amplification, or correct template to template annealing in overlap extension, and subsequent strand synthesis by a DNA polymerase.
Multiple (one or more) pairs of CDE primers allow the generation of consecutive PCR products (gene segments) with complementary ends suitable for overlap extension and PCR resulting in the generation of recombined segments.
CDE primers may also be used in combination with complementary degenerate primers that do not have a non-degenerate core to generate consecutive PCR products (gene segments) with complementary ends suitable for overlap extension and PCR resulting in the generation of recombined segments.
The mixing of segments amplified from related genes followed by overlap extension and PCR results in the efficient generation of chimeric gene fragments.
The non-degenerate core of each complementary CDE primer set may be (but does not have to be) based upon the gene designated the parental gene.
Gene segments amplified from related genes can be mixed in unequal amounts allowing control of the level of incorporation of each segment into resultant chimeric gene fragments.
In a fifth aspect, the present invention provides use of the primers according to the fourth aspect of the present invention to form mutant or chimeric genes.
In a sixth aspect, the present invention provides use of primers according to the fourth aspect of the present invention to incorporate mutations in genes. Preferably, degenerate 3′ portions of the primers can allow point mutations to be incorporated into the final reassembled gene even though all segments are from the same parent.
Improvement of the biochemical characteristics of enzymes has been aided materially by random mutagenesis techniques involving misincorporation mutagenesis and DNA shuffling which have allowed exploration of the sequence space for selected proteins. The shuffling techniques can be used on a collection of mutants of the same parental gene or related families of proteins can be shuffled to produce multiple mutants with enhanced gene products. One difficulty with the current shuffling procedures is the predominance of unshuffled molecules in the pool of mutants. The present inventors have devised a procedure for gene shuffling based on consensus primers and PCR primer extension that allows control of the particular segments of a family of genes that are shuffled and reduces the frequency of the parental, non-recombinant products. This procedure has the advantage of avoiding the use of nucleases or restriction enzymes for gene fragmentation prior to shuffling and allows the use of random mutagenesis of selected segments of the gene as part of the procedure. The use of the technique has been successfully demonstrated with a diverse family of beta-xylanase genes of widely different G:C contents that provide a simple activity assay for identifying mutants.
Throughout this specification, unless the context requires otherwise, the word �comprise�, or variations such as �comprises� or �comprising�, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.
In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following drawings and examples.
PCR Concepts and Methodologies
PCR methods and theory can be found in a number of texts and references known to the art. The following reference: PCR Primer�A Laboratory Manual. 1995. Editors, Dieffenbach C. W. & Dveksler, G. S. Cold Spring Harbor Laboratory Press, USA, is a good example and is incorporated herein by reference.
Source of Genes
Family 11 xylanase genes were available from the following bacterial strains: Dictyoglomus thermophilum Rt46B.1 xynB (Morris, D. D. Gibbs, D. D., Chin, C. W., Koh, M. H., Wong, K. K. Y., Allison, R. W., Nelson, P. J. & Bergquist, P. L. Cloning of the xynB gene from Dictyoglomus thermophilum strain Rt46B.1 and action of the gene-product on kraft pulp. Appl. Environ. Microbiol. 64,1759-1765, 1998); Clostridium stercorarium xynB (Sakka, K., Kojima, Y., Kondo, T., Karita, S., Ohmiya, K. & Shimada, K. Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A: identification of catalytic and cellulose binding domains. Biosci. Biotechnol. Biochem. 57, 273-277, 1993); Bacillus sp. V1-4 (Yang, V. W., Zhuang, Z., Elegir, G. & Jeffries, T. W. Alkaline-active xylanase produced by an alkaliphilic Bacillus sp isolated from kraft pulp. J. Industrial Microbiol. 15, 434-441, 1995); Caldicellulosiruptor sp. Rt69B.1 xynD (Morris, D. D., Gibbs, M. D., Ford, M., Thomas, J. & Bergquist, P. L. Family 10 and 11 xylanase genes from Caldicellulosiruptor isolate Rt69B.1. Extremophiles 3, 103-111, 1999); Clostridium thermocellum xynV (Fernandes A. C., Fontes C. M., Gilbert H. J., Hazlewood G. P., Fernandes T. H., Ferreira L. M. Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes. Biochem. J. 342, 105-110, 1999) and Streptomyces roseiscleroticus xyl3 (Elegir, G., Szakacs, G. & Jeffries, T. W. Purification, characterization and substrate specificity of multiple xylanases from Streptomyces sp strain B-12-2. Appl. Environ. Microbiol. 60, 2609-2615, 1994). Four of these xylanases were from thermophiles and coded for enzymes that had high temperature optima and the Bacillus and Streptomyces xylanase genes coded for mesophilic proteins that performed well in pulp bleaching tests.
Degenerate-end Complementary Primer Pairs for Efficient PCR Amplification of Gene Segments and Overlap-extension of Adjacent Segments
The nucleotide sequences of the genes were aligned and degenerate consensus primers were designed based on the conserved amino acid motifs found in all of the genes. The genes coding for the xylanases could be divided into eight fragments on the basis of alignment of the conserved regions (see
In some cases, the degenerate oligonucleotides were designed using a modification of the CODEHOP method of Rose et al 1998 (Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S. McCallum, C. M. & Henikoff, S. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 26, 1628-1635, 1998). These primers consisted of a non-degenerate central core flanked by degenerate ends of 6-7 nucleotides. All of the segments for each gene were amplified with the consensus degenerate oligonucleotides and individually gel-purified from the PCR mixtures. PCR conditions for this step were: one cycle of 95� C. for 1 min.; then 35 cycles 95� C. (denaturation), 30 sec; annealing at 35� C., 20 sec; and extension at 72� C., 40 sec. with a final incubation at 72� C. for 5 min. using Life Technologies Platinum Pfx polymerase.
Overlap Extension
The segments of each gene after gel purification were mixed in the appropriate ratio to give the desired level of chimerisation. For example, using the six candidate genes G1-G6, where G1 is the D. thermophilum Rt46B.1 gene, and deciding that this sequence should predominate in the shuffled progeny, the pooled PCR segments for each gene were mixed in the ration of 8.75 G1 to 1:1:1:1:1 to give chimeras with 5/8 segments from Rt46B.1 xynB. Fifty to 100 ng of mixed segments were then used as templates for overlap extension (Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., & Pease, L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59, 1989) using the following conditions: one cycle of 95� C. for 1 min; then 35 cycles 95� C. (denaturation), 30 sec; annealing at 35� C., 20 sec; and extension at 72� C., 40 sec. And a final incubation at 72� C. for 5 min using Life Technologies Platinum Pfx polymerase.
Chimera Amplification
Chimeric fragments were regenerated into complete genes by using the overlap-extended products (50-100 ng) as a template for PCR using the common flanking nested 5′- and 3′-primers under the following conditions: one cycle of 95� C. for 1 min. to activate the enzyme; then 20 cycles 95� C. (denaturation), 30 sec; annealing at 50� C., 20 sec; extension at 72� C., 40 sec. and a final incubation at 72� C. for 5 min using Life Technologies Inc Platinum Pfx DNA polymerase.
Cloning of Shuffled Products
DOGS PCR products were digested with the restriction enzymes BamHI and HindIII and ligated to pBSII KS- (Stratgene, Calif.) which had been digested with the same restriction enzymes, and treated with Shrimp alkaline phosphatase (Boehringer Mannheim). The ligated DNA was transformed into E. Coli strain DH5-alpha and plated onto plates containing ampicillin, X-gal and IPTG according to the vector manufacturers instructions. Individual colonies were picked and patched in duplicate onto new plates and screen for the expression of xylanase activity by the substrate overlay/Congo Red staining method as described in Morris et al 1999.
Enzyme Assays for Xylanase Activity
Methods for thermostability, pH optimum, etc, assays can be found in Morris et al 1999.
Results and Discussion
An overview of the DOGS technique is given in
Complementary degenerate-end primers for PCR and overlap-extension are shown in
This procedure was developed after finding a high frequency of parental non-shuffled products in early experiments using the established technique with DNAsel fragmentation introduced by Stemmer (1994a). This observation made extensive screening necessary for the isolation of candidate chimeric genes, particularly when distantly related genes are used in family shuffling. The background of parental molecules may also have been a consequence of the difficulty of completely removing undigested, full-length material during the gel purification of the fragments. The restriction enzyme approach by Kikuchi et al (1999) has the merits of simplicity and the wide range of restriction enzymes available should lower the frequency of regeneration of wild-type genes. In use, however, the method does not obtain the desired number or frequency of altered gene products. It was considered that it may be important to control the input of parental genes into the shuffling procedure and the present inventors demonstrated the rarity with which chimeras arose when genes with limited sequence similarity were used as parents. A description of the products from experiments entailing shuffling the six xylanase genes, using xynB from Rt46B.1 as the major input DNA is described below.
Degenerate-end Complementary Primer Pairs for Efficient PCR Amplification of Gene Segments and Overlap-extension of Adjacent Segments
The most commonly used strategy to isolate distantly-related sequences by PCR has been to design degenerate primers which bind to highly conserved regions of DNA sequence. The difficulty with this method is that as the primer degeneracy increases to accommodate more divergent genes, the number of primer molecules in a PCR that can correctly prime synthesis drops, and these primers may be used up in the first few cycles of the reaction. Non-specific amplification may then occur because of the abundance of primers that do not participate in amplification of the targeted gene, and so are available to prime non-specific synthesis, especially as low stringency annealing conditions are usually needed to detect mismatched homologs.
Rose et al. (1998) have described a strategy that overcomes problems of degenerate methods for primer design called Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP). CODEHOP primers consist of a relatively short 3′ degenerate end and a 5′ non-degenerate consensus clamp. Reducing the length of the 3′ core to a minimum decreases the total number of individual primers in the degenerate primer pool. Hybridization of the 3′ degenerate end with the target template is stabilized by the 5′ non-degenerate consensus clamp, which allows higher annealing temperatures without increasing the degeneracy of the pool. Although potential mismatches may occur between the 5′ consensus clamp of the primer and the target sequence during the initial PCR cycles, they are situated away from the 3′ hydroxyl extension site, and so mismatches between the primer and the target are less disruptive to priming of polymerase extension. Further amplification of primed PCR products during subsequent rounds of primer hybridization and extension is enhanced by the sequence similarity of all primers in the pool; this potentially allows utilization of all primers in the reaction.
A modification of the CODEHOP oligonucleotide design technique described and utilised by the present inventors allows efficient amplification of multiple overlapping segments of related genes, and subsequent overlap-extension of adjacent segments from different genes resulting in the formation of useful chimeric gene fragments or products.
The new technique entails the design of complementary pairs of primers. Each primer has a non-degenerate core flanked by both 5′ and 3′ degenerate ends, referred to herein as complementary degenerate-end primers (CDE primers). As with the CODEHOP primers, the 3′ degenerate end gives each CDE primer its template-binding specificity, while the non-degenerate region acts as a stabilising clamp in subsequent rounds of the PCR amplification. The 5′ degenerate end is not required to contribute to the binding efficiency of the CDE primer during PCR, however, it plays an important role in allowing efficient binding and subsequent overlap-extension of PCR products (gene segments) generated using respectively, the forward or the reverse CDE primers.
The non-degenerate core of CDE primers is generally based upon the corresponding coding sequence of one gene, designated the parental gene for shuffling. This results in the formation of chimeric fragments which retain parental sequence at the points of segment overlap. An example of the design strategy for making complementary oligonucleotide pairs suitable for the amplification of gene segments from related genes, and for the subsequent overlap extension of segments to generate chimeric genes, is shown in
Shuffling with D. thermophilum Rt46B.1 xynB as the Major Parent
Three gene shuffling libraries were generated, each with differing ratios of input DNAs. Libraries R1, R2 and R3 were generated by mixing equal amounts of each of the major parent gene segments with other gene segments in the ratios shown in Table 2. The ratios used were calculated to give on average 5/8, 6/8 and 7/8 major parent segments per gene, for the respective R1, R2 and R3 libraries.
A total of 100 colonies from each library were screened for the expression of xylanase activity. Seventy xylanase-positive transformants and 30 xylanase-negative transformants were identified and the plasmid insert of each was sequenced and the resulting data compared to the parental sequences. It was possible to assign the origin of each segment in a recombinant from the sequence data, as shown in
What is striking about
A further experiment was carried out in which the output ratios for all segments were 1:1:1:1:1:1. A total of 20 chimeras were sequenced and on average only one segment per chimera was derived from D. thermophilium xynB6. This result indicated that the xynB6-derived degenerate clamp of CDE primers did not bias recombination toward reformation of the parental gene.
Xylanase Assays on Shuffled Gene Products
Results of gene shuffling experiments of xylanase genes are shown in
The Use of Nested Primer Sets for Gene Shuffling and Mis-incorporation Mutagenesis
In the method described, gene-specific primer sets (forward and reverse) with gene-specific 3′ ends and common 5′-overhanging ends were designed for the genes selected for DNA shuffling. Forward and reverse nested primers were designed to be substantially identical to the 5′-overhanging ends of the gene-specific primers. Each gene was initially PCR amplified using the gene-specific primer sets. Each PCR product could then be used in subsequent PCR amplification using the common nested primer set, or for amplification of individual gene segments (see
The use of nested primers assists in ensuring that the end (5′ and 3′) segments of genes can be amplified, and that following overlap extension PCR, chimeric genes can be amplified equally well regardless of which genes contributed to the end (5′ and 3′) segments.
The use of nested primers assists in ensuring that any mis-incorporation mutagenesis procedure is capable of changing possible codons within the gene to be mutated.
The DOGS procedure described above demonstrates that it is possible to shuffle members of a gene family that are not particularly closely related and still obtain chimeric molecules at high frequency so that comprehensive and time consuming screens are not necessary, in comparison with prior art methods. The recombination frequencies can be controlled by altering the segment input ratios so that shuffling of particular fragments can be enhanced. Accordingly, the procedure allows domain-swapping experiments to be conducted with relative ease, replacing previous methods relying on suitable restriction enzyme sites. It is evident that PCR-induced misincorporation or error-prone mutagenesis can be incorporated as part of the procedure to introduce even more diversity into the products. In this respect, it lends itself to random mutagenesis of individual segments to assist in fine-tuning of the encoded enzyme product. It gives the investigator control over the extent and nature of specific segment mutagenesis by introducing a DNA polymerase without proof-reading activity at an appropriate step in the procedure. In addition, it is clear from the design of the degenerate primers and the results that altered nucleotide sequences will be generated even using a high fidelity DNA polymerase because of the mismatches inherent in the primers. Even greater mis-incorporation mutagenesis can be generated by employing a polymerase without proof-reading activity in the amplification and primer extension steps.
The use of CDE primers has allowed the reliable PCR amplification and shuffling of equivalent gene segments from a diverse range of genes with low overall sequence homology. The CDE primers used ranged from 23-29 nucleotides in length. The CDE primers used in this study represent may not represent the maximum degree of degeneracy tolerated in order for segment amplification and subsequent overlap extension to work reliably. Accordingly, the length of the primers can vary depending on the source of the parent gene or genes and the segments to be amplified.
The experiments described above have used only a single round of chimera formation. Clearly, more diversity can be introduced to allow exploration of the sequence space by additional rounds of DOGS. The procedure lends itself to combination with other gene shuffling and combinatorial mutagenesis techniques.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
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A.C.T Lighting rolls back MA Pricing to 2007 Levels
Due to an unexpected increase in the value of the U.S. Dollar against the Euro, A.C.T Lighting is able to roll back all MA Lighting pricing to the October, 2007 levels. This new pricing is effective immediately, and good on all orders shipped between now and the end of calendar year 2008. We thank our customers for their understanding during Dollar's decline, and we are pleased to offer this pricing relief now.
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Friday, 28 November 2014
Last updated 4 hours ago
Mar 11 2008 | 9:57am ET
While most hedge funds haven't had a lot to cheer about in the New Year, a few are proving their worth in a difficult market.
New York-based Paulson & Co. said that all three of its top strategies have posted a positive return in the first two months of 2008. And while the returns are not on the level of the spectacular triple-digit figures the firm posted last year—it began unwinding its subprime mortgage shorts late in the year—with all but two hedge fund strategies in the red through February, according to Hedge Fund Research, investors are unlikely to complain.
The firm's flagship merger arbitrage strategy is up between 4.3% and 9%, according to a Friday letter to investors. Its credit strategy returned more than 8% over that time, Reuters reports, and its event-driven strategy is up between 5.5% and 10%.
Meanwhile, Brevan Howard Asset Management is making its case for investing in its planned second listed feeder fund. The firm's $17 billion flagship global macro Master Fund is up an eye-popping 17.1% through February.
London-based Brevan Howard plans to launch the BH Global Fund on the London Stock Exchange. The firm hopes to raise about US$500 million for the fund, which will invest in other Brevan funds, with as much as 50% in the booming Master fund.
Winton Capital Management investors are also smiling: Its Futures Fund rose 7.9% last month, and is up 12% year-to-date. The US$5.4 billion fund enjoyed "renewed strengths in the commodities markets, with our long positions in grain, precious metal and energy sectors all making positive contributions," the firm said in a letter to investors. It also credited the continued decline in the U.S. dollar.
Nov 24 2014 | 12:41pm ET
Neil Azous is the founder and managing member of Rareview Macro, an advisory firm...
Nov 26 2014 | 11:40am ET
Citadel Investment Group's Kenneth Griffin is making clear to his estranged wife...
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Peer-to-peer lending was invented with the individual investor in mind. But despite...
Nov 4 2014 | 9:45am ET
Data management is important to every business, but for hedge funds, it is critical. FINalternatives recently asked Peter Sanchez, CEO of Northern Trust Hedge Fund Services, how fund managers can deal with the demands of managing data while at the same time remain transparent and abide by operational best practices. Read more…
Oct 22 2014 | 9:46am ET
We are always looking for guest articles from hedge fund managers and buy-side firms.
If you are interested in submitting a contributed piece for possible publication on FINalternatives, please take a look at the specs. Read more…
Reg NMS created a huge bifurcation in equity markets and while much of what has followed has been positive, in terms of lower fees and greater liquidity, many traders would like to see the market come...
Senator Paul sat down in the debut series of the Alpha Pages Interview to discuss the broken tax code, regulation surrounding Bitcoin, and his plans for the 2016 Presidential election.


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No right turn at Graphite India junction towards Brookfield from K R Puram is offered as one of the solutions Ask any resident of Whitefield about... ...http://www.bangaloremirror.com/bangalore/others/Graphite-India-junction-K-R-Puram-techies-and-expatsITPL-road-Brookefield-Road-Hope-Farm-junction-Hoodi-Road-Kundalahalli-junctionBBMP-BWSSB/articleshow/44806913.cms
This list contains the shortlisted startups for the IBM GEP India SmartCamp. The previous list contained 15 of the total selected startup. This... ...http://feedproxy.google.com/~r/pluggd/~3/rTahxmObJEE/
The company won the award due to new business initiatives such as training services, managed services, business analytics, and cloud computing... ...http://www.crn.in/crn/news/298135/value-systems-systems-integrator-south
Benchmark share indices ended flat on Friday but logged their sixth straight week of gains, the longest since the seven-week rally from January... ...http://www.rediff.com/money/report/how-the-markets-fared-on-sep-19/20140919.htm
Markets have turned choppy after investors booked profits at higher levels. Although , the US Federal Reserve's decision to keep US interest rates... ...http://www.business-standard.com/article/markets/markets-choppy-nifty-seeks-strong-support-at-8-100-114091900251_1.html


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RadioShack – Why Work For Us?
Why RadioShack?
RadioShack has created a loyal following of customers through our associates' expertise and collaborative spirit with consumers, celebrating the longstanding idea that "two heads are better than one." In a marketplace that is continuously flooded with new technology, it is this mantra that differentiates RadioShack from other retailers.
At the new RadioShack, "It Can Be Done, When We Do It Together." Beyond our great product assortment and price match guarantee, RadioShack fills a void for consumers who need advice to bring a technology idea to life.
RadioShack is the neighborhood technology resource with more than 4,300 U.S. company-owned stores and 30,000 knowledgeable associates. The company continues to roll out dozens of concept stores and almost all stores are getting a brighter, bolder, fresh new look. The stores serve as the collaboration center for consumers looking to bring a tech idea to life, and Do It Together-inspired projects will be featured in Idea Center kiosks in some stores.
Customers consistently praise our associates for their helpful and knowledgeable contributions to their shopping experience. Our curated product assortment, from personal and home technology and power supply to mobility, music, sound and digital fitness, enables consumers to find the right product to suit their lifestyle.
Adding Value. When you shop at RadioShack, you get unparalleled, value-added services in addition to expert advice.
- With our Price Match Guarantee, there's no need to shop around. We'll match advertised prices on identical products up to 30 days after purchase, in-store only. Visit your local RadioShack for details.
- Trade & Save enables consumers to trade in unwanted electronics for instant savings on their next purchase - while keeping old devices out of landfills.
- Our "IT Happens" protection plan offers an unmatched in-store claim process for qualifying products priced less than $300 - among many other benefits.
It's a new way of interacting with customers and helping them find what they need. It's about creating a new shopping experience that's truly interactive and engaging. And it's our people who will make this happen.
We're looking for people who appreciate technology and its powers of connectivity, entertainment, and ability to unify. We need people who can break down confusing jargon to the average person and explain how our products can improve their lives. This is how we separate ourselves from the pack.
If this sounds like something that's perfect for you, you just might be Shack material.
We can provide the training you'll need to turn your special talents into a rewarding career.
Jobs at The Shack come in many forms. With more than 30,000 employees, we rely on a vast team of innovative thinkers to help us continue to grow in the ever–evolving world of consumer electronics. We take pride in our corporate culture, which is built around helping our employees reach their full potential. We need people who are excited about being presented with new opportunities each
day–maybe people like you.
We realize location plays a pivotal role in your decision to accept a position. We have a great home in the Dallas/Fort Worth metroplex, and together, our two cities create one of the most vibrant and diverse communities in the country. You'll find family–friendly cosmopolitan urban living, world–class dining and many celebrated museums, and if you're into sports, well, you already know.
If this sounds like it might be right for you, you could be Shack material.
OPPORTUNITIES are available in many of the job categories listed below.
Business/Strategic Development
Communications/Public Relations
Customer Service
Dealer Franchise
Human Resources
Information Systems
Inside Sales
Loss Prevention
Real Estate
Supply Chain/Logistics
We're making your RadioShack better with new signage, products, and offers! Head to your local RadioShack to check them out. Share a photo of YOUR favorite new feature with #MyNewRadioShack to get featured at MyNewRadioShack!
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RadioShack Reviews
"Great for learning new customer service skills "
- Comp & Benefits
- Work/Life Balance
- Senior Management
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I have been working at RadioShack part-time (less than an year)Pros
Great if you like to work with the public must be able to learn quickly and efficiently. The benefits are good and you get spiffs and commission to balance out your pay check.Cons
just be ready to put in some long daysRecommendsPositive OutlookApproves of CEO
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I applied online. The process took a week – interviewed at RadioShack.Interview Details
I applied online and then had a follow up phone call to come in and do an in-store interview. Most questions were very basic. They wanted to know my background, what were my strengths/weaknesses. Why would I make a good employee. The usual stuff. I was only looking for a part-time position while in college.Interview Questions
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- What would I do to help RadioShack succeed? Answer Question
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Let us know if we're missing any workplace or industry recognition – Add an award
Additional Info
RadioShack (NYSE: RSH) is a leading national retailer of innovative mobile technology products and services, as well as products related to personal and home technology and power supply needs. RadioShack® offers consumers a targeted assortment of wireless phones and other electronic products and services from leading national brands, exclusive private brands and major wireless carriers, all within a comfortable and convenient shopping environment. RadioShack employs about 30,000 knowledgeable and helpful sales experts globally. RadioShack's retail network includes more... More


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At another panel at the Time Warner Summit, TechPresident co-founder Andrew Rasiej, talking with his fellow panelists about the recent coverage given to the Bradley Effect, made a prediction. "What we're going to see is the Obama Effect," he said: Obama over-performing because of people who secretly wanted to vote for him, but were afraid to say so.
The Kicker
06:42 PM - October 14, 2008
The Obama Effect?
At another panel at the Time Warner Summit, TechPresident co-founder Andrew Rasiej, talking with his fellow panelists about the recent coverage given to the Bradley Effect, made a prediction. "What we're going to see is the Obama Effect," he said: Obama over-performing because of people who secretly wanted to vote for him, but were afraid to say so.
- Comments Post a Comment
The up coming "Obama Affect" might also have somethign to do with the 100,000's of fraudulent voters ACORN is putting on the voter rolls.
I'm so entertained at the ACORN bogeyman the right has created. First off, fraudulent registrations are being caught. That's why elections offices confirm registration information.
Second, even if someone manages to REGISTER a bogus voter, it's not voter fraud until someone actually casts a vote using that fraudulent registration. (Hint: Mickey Mouse can't vote, even if he's on a voter roll, which he's not.)
My prediction: Of the 1+ million voters registered by ACORN, fewer than 20 will be identified trying to vote illegally on election day. Those votes will appropriately get tossed out.
Net result: Zero voter fraud, but an excuse for a few nutcases on the right to claim Obama stole an election he's likely (as of today) to win by a million or more votes.
Why won't journalists ask Bill Cosby the tough questions? - Sexual assault charges are hardly ever mentioned to the TV star
Knoxville's alt-weekly wasn't losing money. It got shut down anyway - Scripps pulls the plug on Metro Pulse in favor of an entertainment supplement for the local daily
Chuck Todd's Obama book says more about the author than it does about the president - 'The Stranger' underscores a broader problem with the way we cover politics
Beware labeling Pope Francis a liberal - Political boxes like liberal/conservative and evolutionist/creationist miss his real significance
The Virginian-Pilot produces a breakthrough investigation amid layoffs - Reporter John Holland discusses how the story came together, newsroom cutbacks, and colleagues who cover for each other
Why won't journalists ask Bill Cosby the tough questions? (17)
Serial creators don't know what will happen to Adnan Syed (11)
Knoxville's alt-weekly wasn't losing money. It got shut down anyway (10)
Beware labeling Pope Francis a liberal (8)
Email blasts from CJR writers and editors
Which media outlets use 'illegal immigrant'? (Fusion)
"[I]n spite of all the good reasons not to use the phrase, it is still very easy to find in the US press, even in headlines"
Jack Shafer on losing his job (Capital New York)
"Right now, my immediate plan is to go to work as a lay therapist at The Intercept to bring the healing there so John Cook and Matt Taibbi can return. I have great interpersonal skills."
On outlets that continue to call unknown drone victims 'militants' (The Intercept)
"Like the US drone program itself, this deceitful media practice continues unabated"
UNITY's convention is no more (All Digitocracy)
"The organization's board of directors decided that UNITY will no longer host the quadrennial conferences for which it had become known"
Greg Marx discusses democracy and news with Tom Rosenstiel of the American Press Institute
CJR's Guide to Online News Startups
ACEsTooHigh.com – Reporting on the science, education, and policy surrounding childhood trauma
Who Owns What
The Business of Digital Journalism
A report from the Columbia University Graduate School of Journalism
Questions and exercises for journalism students.
Should all journalists be on Twitter? – Reasons to take up or forgo the 140-character platform
Is it ever okay to name victims? – Editors question the policy of omitting information in sexual assault and domestic violence cases


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Dramatic scenes have unfolded in central Queensland where a teenager has been rescued from raging floodwaters, as the remnants of ex-tropical cyclone Oswald lash the region.
The 14-year-old boy was found clinging to a tree in floodwaters in Rockhampton this morning.
Six swift water rescue crews rushed to the scene and managed to reach the teen.
As the boy grabbed hold of a tree, and climbed towards other rescuers waiting on a bank, the man who'd saved him was swept away.
He went under a nearby bridge but was able to pull himself up onto a bank. He was taken away in an ambulance but is believed to be fine.
Dozens of stranded people have been rescued from homes and cars overnight and this morning as the state struggled to cope with the deluge.
More than 70 roads, including major highways, have been cut by floodwaters since Oswald was downgraded to a storm after crossing the Cape York Peninsula's west coast on Tuesday.
Rockhampton was suffering its wettest day in 60 years and councillor Bill Ludwig said many parts of the city were under water, and the city was also experiencing ''very destructive winds''.
''They're saying this is not a cyclone but it's not far off it,'' Ludwig told ABC TV.
He said he was not going to venture outside the building where he was on Friday morning, while a large corrugated sheet of iron was flapping from another building nearby.
A search was underway for two fisherman whose boat started taking on water off Rockhampton.
The skipper of the 38-foot fishing vessel made the call early this morning.
He told authorities he was in Casuarina Passage.
Rough weather conditions prevented authorities from launching an immediate search, police said.
Air and sea searches began this morning, but heavy rain and high winds are hampering the operation.
The ABC said planes were unable to land at Rockhampton because of the 90kmh winds, heavy rain and poor visibility, while many roads are inaccessible.
In the flood-hit Rockhampton suburb of Frenchville, a house has been hit by a large landslide.
The home was still standing but debris was piled up against the two-storey dwelling, the Department of Community Safety said.
At Yeppoon, northeast of Rockhampton, more than 10 homes have flooded and people were sandbagging other properties in a bid to save them, the ABC reported.
"We've had some flash flooding in low lying areas, and those low lying areas are all still cut," one Yeppoon resident told the broadcaster.
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Killer bear photos recovered from victim's phone
EU calls for 5000 doctors to fight Ebola
Call for world leaders to eliminate nuclear weapons
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Rugby World Cup 2015, history will be made
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Wallabies staff damage Dublin dressing room
Internet shares gift of Prince George photo
Top Gear star reveals head injury's dark years
Sutton departure: 'It's a win for women'
Phillip Smith could be released from prison


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Winnipeg Free Press - PRINT EDITION
Voting power to the people
Elections Manitoba spreads word on need to cast a ballot
To a poor inner-city neighbourhood struggling with a laundry list of tangled issues, Angela Chalmers arrived bearing a strong and simple message: "You need to get out and vote."
On Wednesday, Chalmers, the education co-ordinator for Elections Manitoba, spoke to about 30 students at the University of Manitoba's social work facility on Selkirk Avenue. It was the first in a planned series of workshops to boost the lagging North End vote -- and Chalmers was pumped.
"If we can... impact just a few of these people, then they can be powerful ambassadors," raved Chalmers, after delivering an energetic 40-minute presentation on the how-tos of voting.
Elections can be dry, but Chalmers' presentation cut right to the nuts and bolts of daily life. If you take a shower, Chalmers said, you need the city's water. If you need the bus to get around, that's a city issue, too. She talked about how to vote -- yes, you can take your kids with you -- and how to gather information about candidates. "Bringing it down to the personal level is what needs to happen," Chalmers explained.
"People who live in these areas are directly affected by the choices councillors are making. A lot of people take the bus, a lot of people have kids in daycares. They're almost more impacted than people in other areas."
And yet, they vote the least. In 2006, only 28 per cent of eligible voters cast a ballot in Mynarski, the lowest turn-out in all of the city's 15 wards.
"Ninety per cent of people in this area are just trying to keep their head above water," sighed Roanna Hepburn, who works at the School of Inner-City Social Work and helped arrange Wednesday's workshop.
"People around here have a very hard life and they don't stop to think about how they can improve it by voting."
That's especially true this election since the Mynarski race is wide open after Harry Lazarenko retired after 30 years following a brain aneurysm last May. Six candidates are vying for his council seat and organizers hope the open race could drive more voters to the polls.
Enter North End Votes. An initiative of a group of north Winnipeg resident associations, NEV is working to get people more engaged in this election. They organized a September Mynarski council candidate forum, have a mayoral debate planned for Oct. 18 and are planning four more voting 101 sessions before election day on Oct. 27, which will also be hosted by Chalmers. (Anyone interested in attending can email firstname.lastname@example.com for details.)
If the response to the first workshop is any indication, all these efforts might boost turnout. Jamie Monkman, 27, was among the students watching Chalmers' presentation. She lives in the Point Douglas ward, which also struggled with low turnout in 2006 and hasn't voted for a while, she said.
The last time Monkman voted, she took an "eeny, meeny, miney mo" approach to a slate of candidates who hadn't taken any information to her door. Now, she's inspired to take a different approach. "(Chalmers) made some important points," Monkman said. "I never thought of making a list of things that are important in my community. I'll probably go online and take a look at their ideas (this time)."
Republished from the Winnipeg Free Press print edition October 8, 2010 B2
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Eric Fisher delivered an RBI single in the bottom of the eighth inning to lift the Arkansas Razorbacks to a 2-1 victory over the No. 28 ranked Alabama Crimson Tide on Saturday afternoon from Baum Stadium.
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Get up close and personal with these new EA Sports UFC screens
EA Sports recently added a few names to the EA Sports UFC roster, joined by some in-game shots of fighters from the neck up. The fighters playable in the game and seen in the included gallery are former light heavyweight champ Forrest Griffin, "The Korean Zombie" Chan Sung Jung, Costas Philippou, M...
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Think BR: When is 0.03% a big number?
By Tim Elkington, brandrepublic.com, Wednesday, 17 October 2012 08:00AM
Why have consumer goods brands massively increased their digital display spend, asks Tim Elkington, director of research and strategy, IAB.
IAB/PwC Digital Adspend results for H1 2012 included an impressive range of figures - a total half year value of £2.6 billion, an increase of 43% for online video advertising, a growth rate of 132% for online mobile advertising and £591m spent on digital display advertising in a six month period - all against a backdrop of GDP decline.
In among all these numbers, there was a much smaller, but much more significant figure - 0.03%. This slither of a percentage represents the difference in market share of digital display spend between consumer goods and financial advertisers.
Financial advertisers accounted for 15.75% of digital display spend, but significantly consumer goods advertisers accounted for 15.78% - making them the largest digital display advertisers.
The rise of consumer goods advertisers has been meteoric. In the first half of 2009 consumer goods advertisers accounted for 9% of display spend and this has almost doubled to 15.78% (let's call it 16%) by the first half of 2012.
But what's behind this change, why have consumer goods brands so massively increased their digital display spend?
Advertising online used to be about direct response, in the form of click-throughs from banner ads. The more click-throughs you got the better - and campaigns were assessed by using the click-through rate.
This easy accountability made online straight forward to use for direct response advertisers - it was easy to tell if your activity had worked by using simple click-through metrics. But what about brands - like FMCG advertisers - with campaigns that weren't interested in immediate response; brands that wanted to drive traditional brand metrics like favourability and purchase consideration?
For a long time even those campaigns without direct response objectives were assessed using click through metrics. Advertisers were happy using what had become a familiar, though inappropriate, metric.
Though as click-through rates declined over time, partly due to the proliferation of online advertising, some came to the conclusion that online advertising was no longer working.
However the smarter advertisers asked the obvious question - just because someone hasn't clicked on my ad does that mean that it hasn't worked, what about the positive branding effect of being exposed to the ad?
Over time it became clearer that actually online ads worked a lot like offline ads. Just like seeing a great TV, magazine or outdoor ad, exposure to an online ad could have a positive effect on brand metrics, even if no click-through occurred.
The most exciting thing was that these learnings coincided with an explosion of creative formats for online display advertising. Advertisers were no longer bound by the restricting dimensions of the banner and could employ real creativity engaging with much larger formats including the custom background half page, and billboard - as explored in the IAB's Size Matters research.
Those consumer goods advertisers used to preparing creative for TV even found that - preferably with some tailoring - this creative could also be used around the growing area of video content online.
So the increase in the use of digital display advertising by consumer goods brands reflects the changing nature of display advertising online and on mobile.
What was previously a direct response channel is evolving to become a rich branding medium in which brands can ignore less relevant metrics, take advantage of the creative opportunities offered by new larger ad formats and enjoy the branding benefits that digital display has to offer.
Tim Elkington, director of research and strategy, IAB
This article was first published on brandrepublic.com
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From Dirty Slums 2 w/ Mick Boogie, due next Tuesday.
From Slum Village and Mick Boogie's Dirty Slums 2 mixtape, coming January 29th.
SV – which now pretty much consists of T3 – takes a more rugged approach for this song from the Dirty Slums 2 mixtape, hitting Jan. 29th.
Final mixtape release of Halloween is from Slum Village's T3 and company. T3 (Slum Village) – "3illa Madness" (Mixtape)
Another one from DJ Booth / Event Electronics Music Series, produced by Focus. Download
Taken from T3′s upcoming Violator Radio mixtape w/ DJ Scrap Dirty and DJ Supa, which hits on Halloween. LISTEN: T3 (Slum Village) – "Dirty Mit" T3 (Slum Village) – "Dirty Mit"
From Music of the Misinterpreted (Deluxe Edition), which hits 11.11.11. LISTEN: Focus – "The Hard Way" (feat. T3 + Joe Scudda)
From the upcoming DJ Scrap Dirty + DJ Supa Violator Radio Mixtape.
From T3′s digital EP, 3illa, hitting June 21st. Young RJ on the track.
Don't mind the pic of the Black Milk and young Young RJ at HipHopSite in 2004. Whutchu know about B.R. Gunna? Another fat Shout to FWMJ's Rappers I Know. LISTEN: Young RJ – "Back 2 the Studio" (feat. T3, Illa J & Vice Verse) (MP3) Young RJ – "Back 2 The Studio" (feat. T3, Illa [cont.]
T3 dismisses recent talks of beefs and break-ups of Slum Village and some of the motivations behind the tracks on Villa Manifesto.
Looks like Villa Manifesto will be the last Slum Village LP. Being that two of the original members have passed, it seems kind of an obvious conclusion, but Twitter blew up today with #ripslumvillage posts, but something isn't right with the remaining members – obviously some behind the scenes drama going on, judging from the [cont.]
Onra steps up with "The One" (feat. T3 from Slum Village) from the album "Long Distance", available now on All City Records. Video directed by Donal Thornton.
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Dr. Roland Sabates, MD
Ophthalmology, Center For Outpatient Surgery
- 6675 Holmes Rd Fl 2
Kansas City, MO 64131
- 4 Office Locations
- (816) 276-7850
Dr. Roland Sabates has been practicing medicine for 38 years. Dr. Sabates graduated from the University of Kansas School of Medicine in 1976. He practices medicine in Kansas City, MO and 3 other locations and specializes in Ophthalmology. Dr. Sabates is affiliated with Research Medical Center Brookside Campus, Research Medical Center, Saint Joseph Medical Center and Belton Regional Medical Center. He speaks English and Spanish.
Gender: Male
Years in Practice: 38 Years
Languages Spoken: English, Spanish
Medical School: University of Kansas School of Medicine
Graduation Date: 1976
Hospital Affiliations: Research Medical Center Brookside Campus, Research Medical Center, Saint Joseph Medical Center, Belton Regional Medical Center
Specialties: Ophthalmology
*This physician has multiple office locations. See Appointments for details.


text:
SleepyHead - Read CPAP data with this tool - Windows 8 Downloads
SleepyHead 0.9.3 Beta
SleepyHead was developed as a powerful and interactive viewer for CPAP machine data. CPAP machines record a great deal of data about the sleep patterns of patients.
SleepyHead 0.9.3 Beta - Key details
|Open Source
|File Size:
|Jun 7, 2013
|Total: 39 | This Week: 0
|CPAP viewer, CPAP analyzer, analyze CPAP, CPAP, sleep, analyzer
|Mark Watkins
|Author URL:
Windows 8 Downloads - SleepyHead awards
SleepyHead - Full description
SleepyHead was developed as a powerful and interactive viewer for CPAP machine data. CPAP machines record a great deal of data about the sleep patterns of patients.
This data is often saved on a smart card that the patient can take along to the doctor or respiratory therapist's office. Some machines report the data directly via wireless modem.
SleepyHead for Windows 8 - Post your review
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text:
Letter: Cannabis on trial
Monday 16 November 1998
Further clinical trials, which the Government requires, are unnecessary because if there was a debilitating side-effect then 2000 years of medical usage would have made it apparent ("Cannabis is `safer than drinking' ", 13 November). Anecdotal and historic evidence proves the superiority of herbal cannabis in the treatment of MS, epilepsy and many other diseases.
Cannabis treatment is witheld because the pharmaceutical companies will not make money from a common plant. If cannabis is God's gift to the sick then it is our duty to make sure they can legally obtain it.
In the past cannabis has been targeted as a menace and a threat to society; now the media should champion the cause of those who undergo persecution because of a quirk in the law.
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Hi Guys! Groovin' to your songs, especially my FAVE - SERENITY...I'd love to make a FB Fan page for you...let me know...K? Cheers ~Linda
Great tunes y'all we are diggin' here at the coop!.......dp
hi there and thanks for sharing...FEEL FREE to visit us whenever you'd like some more SEXY FUNNY MUSIC to refresh your soul.KEEP HAVING FUN and may the GODS be with you too
Thanks for join us! STAY WARDEN!
I'd like to welcome you to my Reverb Nation family... come check out my demo tracks at:
Thanks for being a fan, I'm your fan as well Lets be fans of fb too:)
Feel free to drop your fb link on my page and I will like you back.
Have a beautiful day.
We've got a feeling that 2012 is going to be a crazy good year! Hold on tight! Here's to a safe and happy New Year's Celebration!
"Official Facebook Page & New Official Video(Holes)" ---> http://www.facebook.com/pages/Amorphead/104336263339 & http://www.youtube.com/watch?v=5AAZ5Bxr2AI&feature=channel_video_title
Great work...Rock on...
Killer tunes!


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Canyon Del Oro Football
Canyon del Oro 40, Marana 12: Thursday's work pays off Friday
CDO's Isaac Chacon is stopped just short of the end zone by Marana's Domenic Holiday. Chacon scored four times and rushed for 154 yards.
Canyon del Oro 40, Marana 12: Thursday's work pays off Friday
A Hail Mary pass in the fourth quarter slips through the hands of Marana's Jeffery Carranza, and neither teammate Eric Huerta nor CDO's Mike Khawam can grab the loose ball.
Canyon del Oro 40, Marana 12: Thursday's work pays off Friday
Tommy Sawyer (36), who had a 7-yard TD for the Dorados, grabs Marana QB Ben Honea for a first-quarter sack.
Canyon del Oro 40, Marana 12: Thursday's work pays off Friday
Canyon del Oro's defense spends practice every Thursday doing almost nothing but stripping ball carriers and catching tipped passes.
Canyon del Oro 36, Flowing Wells 15: CDO breaks away in second half, beats Cabs for first win
Canyon del Oro receiver Tommy Sawyer grabs the ball away from Flowing Wells defender Cameron Holt for a third-quarter touchdown, Sawyer's second of the game.
Canyon del Oro 36, Flowing Wells 15: CDO breaks away in second half, beats Cabs for first win
Canyon del Oro dug itself into a hole in the first month of the season, losing its first four games.
Gilbert Williams Field 27, Canyon del Oro 23
In Gilbert, CDO (0-4) held a 16-13 halftime lead before Williams Field answered with 14 third-quarter points for the win. CDO running back Isaac Chacon accounted for 118 of the Dorados' 297 total offensive yards.
The Nighthawks' night
Ironwood Ridge running back Anthony Braunreiter slips through the grip of Canyon del Oro defenders in the second quarter. Braunreiter, a senior, finished with 12 carries and two catches for 111 yards.
The Nighthawks' night
Ironwood Ridge defenders Jake Matthews (6), John Robert Klass (5), Cris Thorson (7) and Thomas Gutierrez (9) swarm over Canyon del Oro's Isaac Chacon in the second quarter.
I-Ridge scores early, often to hand Dorados worst defeat since '99
Ironwood Ridge turned a bitter rivalry into a blowout Friday night. The Nighthawks jumped out to a 29-0 lead by halftime on their way to a 36-0 win over Canyon del Oro in the season opener for both teams. It was CDO's worst loss since 1999.
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March 1, 2014 6:34:36 PM
By DAVID MILLER
Special to The Dispatch
STARKVILLE -- Hit open shots, finish at the rim, and defend.
The Starkville High boys basketball team had excelled in those areas this season and were a win away from returning to the Class 6A State Tournament in Jackson. Then, Grenada High, a team the Yellowjackets had beaten twice this season, showed up to The Beehive and did everything better.
It was that simple, according to coach Greg Carter, who watched his team cap a "sluggish month of February" with a 61-43 loss in the 6A North State semis Friday night.
"We're sitting there talking about an opportunity to play for a state tournament berth," Carter said. "When you do some of the things we did tonight, you don't deserve to go. The transition points, not being able to make open shots and finish at the basket -- we didn't do it."
Despite pulling down nearly 20 offensive rebounds and forcing 6-foot-10 Grenada center Joniah White into first half foul trouble, the Jackets trailed by as many as 15 in the first half. Starkville (24-5) was held scoreless for nearly the first four minutes of the second quarter.
But when White had to sit due to two fouls at the 6-minute mark of the second quarter, Starkville mounted a furious rally behind put-backs and contested layups by Raphael Leonard, who scored 14 points, and Dontavious Self. Starkville cut Grenada's lead to 31-26 and went into the break down by eight points.
"When we went back in the locker room, down by nine, coach told us we still had a chance, to defend and rebound," Self said.
But with White back in the game and manning the paint, Starkville opened the second half on a three-minute scoring drought. White registered three blocks and a pair of buckets to spark a 10-0 run that effectively wiped the Jackets out of the game.
"Knowing they got a 6-10, 6-11 kid back there, you do tend to rush shots a litlte bit," Carter said. "But we have to make those shots."
"We just had a bad night on offense, Self said. "Nothing was going in."
Grenada was led by Tyrin Jones, who scored 15 of his 22 points in the first half. Trey Spencer and LaCurtis Allen each scored 10 points.
"Jones, we couldn't guard him the whole night," Carter said. "Spencer and Allen both scored their average. You let those three guys score like that, they'll have a chance to win every one of them."
Adam Minichino is the Sports Editor for The Commercial Dispatch.
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VCCP follows Mediacom with its own naked calendar
By Staff, campaignlive.co.uk, Thursday, 11 February 2010 08:00AM
Hot on the heels of Mediacom's naked calendar, VCCP has also stripped off in the name of charity.
But are some of those featured more Photoshop than hotshop?
- January - Barney Ware (account director)
- February - Allan Ward (digital producer) and Belinda Norris (Mac operator)
- March - Luis Marmolejo (handyman)
- April - Victoria Brattle (head of HR)
- May - Anthony Borkett and Chris Green (project managers)
- June - Jessica Hill (HR Assistant), Lauren Park (office admin), Shannon Singh (account co-ordinator) and Anna Chittenden (account co-ordinator)
- July - Mark Orbine (creative director)
- August - Tom Lakeman and Stu Hogg (account managers)
- September - Catherine Melligan (PA) and Matt Ramsden (account manager)
- October - Bidisha Datta, Cydney Chadwick, Flo Mapes and Charlie Davies (reception)
- November - Richard Connell, Ed Kaye and Alex Mavor (creatives)
- December - Kirsty Shore (PA) and Michael Sugden (managing director)
Now you've seen the best assets of both VCCP and Mediacom, which agency model gets your vote?
This article was first published on campaignlive.co.uk
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