© 2008 Cottingham et al. The production, manipulation and rescue of a bacterial artificial chromosome clone of Vaccinia virus (VAC-BAC) in order to expedite construction of expression vectors and mutagenesis of the genome has been described (Domi & Moss, 2002, PNAS 99 12415-20). The genomic BAC clone was rescued back to infectious virus using a Fowlpox virus helper to supply transcriptional machinery. We apply here a similar approach to the attenuated strain Modified Vaccinia virus Ankara (MVA), now widely used as a safe non-replicating recombinant vaccine vector in mammals, including humans. Four apparently full-length, rescuable clones were obtained, which had indistinguishable immunogenicity in mice. One clone was shotgun sequenced and found to be identical to the parent. We employed GalK recombination-mediated genetic engineering (recombineering) of MVA-BAC to delete five selected viral genes. Deletion of C12L, A44L, A46R or B7R did not significantly affect CD8+ T cell immunogenicity in BALB/c
Thus far, the partial RFG8 cDNA sequence consists of 3673 bp and the corresponding amino acid sequence of 1081 amino acids (Fig. 3) ⇓ . Database searches using both cDNA and protein sequences revealed the following significant similarities: (a) the human bacterial artificial chromosome clone RG300C03 (human bacterial artificial chromosome library CITB-HS-A) mapped to chromosome 7q31.2 was detected showing several interrupted sequence similarities between 51 and 78%. We conclude that a RFG8-related gene on chromosome 7 may exist; (b) several EST clone sequences from mice, rats, and humans have been identified showing very high similarities to the RFG8 sequence (up to 99%), indicating that the same or a highly related gene is expressed in these species. Most of the human EST sequences cover 3′ RFG8 sequence regions, and some of them belong to clones that are mapped to chromosome 18 (e.g., cDNA clones NHTBCae15h12 and IMAGE:36907). Therefore, it cannot be excluded that they have sequences ...
Herpesvirus of turkey (HVT) is an alphaherpesvirus that is widely used as a live vaccine against Marek's disease because of its antigenic relationship with Marek's disease virus (MDV). In spite of a similar genome structure, HVT has several unique genes, the functions of which are not completely understood. As a first step in carrying out detailed analysis of the functions of the HVT genes, a full-length infectious bacterial artificial chromosome (BAC) clone of HVT was constructed. DNA from two independent BAC clones, upon transfection into chicken embryo fibroblasts, produced plaques similar to those produced by the wild-type virus. Viruses derived from the BAC clones were stable during in vitro passage, but showed differences in in vitro growth kinetics compared with the wild-type virus. Using a one-step mutagenesis protocol to delete the essential glycoprotein B gene from the HVT genome, followed by construction of the revertant virus, BAC clones of HVT were shown to be amenable to standard
Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder that affects men and women in equal numbers, but some epidemiological studies indicate there may be sex differences in disease progression. One of the early symptoms of HD is disruptions in the circadian timing system, but it is currently unknown whether sex is a factor in these alterations. Since sex differences in HD could provide important insights to understand cellular and molecular mechanism(s) and designing early intervention strategies, we used the bacterial artificial chromosome transgenic mouse model of HD (BACHD) to examine whether sex differences in circadian behavioral rhythms are detectable in an animal model of the disease. Similar to BACHD males, BACHD females display circadian disruptions at both 3 and 6 months of age; however, deficits to BACHD female mouse activity levels, rhythm precision, and behavioral fragmentation are either delayed or less severe relative to males. These sex differences are associated
Enterohemorrhagic E. coli (EHEC) O157:H7 can cause serious gastrointestinal and systemic disease in humans following direct or indirect exposure to the bacterium excreted in ruminant feces. The main colonization site of EHEC O157:H7 in cattle is the terminal rectum where the bacteria intimately attach to the epithelium and multiply in the intestinal mucus. This study aimed to identify genomic regions of EHEC O157:H7 that contribute to colonization and multiplication at this site. A bacterial artificial chromosome (BAC) library was generated from a derivative of the sequenced E. coli O157:H7 Sakai strain. The library contains 1152 clones averaging 150 kbp. To verify the library, clones containing a complete locus of enterocyte effacement (LEE) were identified by DNA hybridization. In line with a previous report, these did not confer a type III secretion capacity to the K-12 host strain. However, conjugation of one of the BAC clones into a strain containing a partial LEE deletion restored type III
The muscle-specific transcription factors Myf5 and Mrf4 are two of the four myogenic regulatory factors involved in the transcriptional cascade responsible for skeletal myogenesis in the vertebrate embryo. Myf5 is the first of these four genes to be expressed in the mouse. We have previously described discrete enhancers that drive Myf5 expression in epaxial and hypaxial somites, branchial arches and central nervous system, and argued that additional elements are required for proper expression (Summerbell, D., Ashby, P. R., Coutelle, O., Cox, D., Yee, S. P. and Rigby, P. W. J. (2000) Development 127, 3745-3757). We have now investigated the transcriptional regulation of both Myf5 and Mrf4 using bacterial artificial chromosome transgenesis. We show that a clone containing Myf5 and 140 kb of upstream sequences is sufficient to recapitulate the known expression patterns of both genes. Our results confirm and reinforce the conclusion of our earlier studies, that Myf5 expression is regulated ...
Citation: Murdoch, B., Fu, A., Meng, Y., Li, C., Hansen, C., Snelling, W.M., Moore, S.S. 2004. Assignment of the SIAT4A gene to bovine chromosome 14 by linkage mapping of an associated microsatellite. Animal Genetics 35:146-147. Interpretive Summary: A new DNA marker for the Sialyltransferase 4A (SIAT4A) gene was developed and mapped. The marker was developed from the CHORI-240 bacterial artificial chromosome library. Cattle from an Angus-based commercial seedstock line, and two USDA-MARC reference families were genotyped. The USDA-MARC reference family genotypes were used to map the marker onto cattle chromosome 14. Technical Abstract: CHORI-240 bovine bacterial artificial chromosome library high density filters were probed with gene-specific overgo primers for Sialyltransferase 4A (SIAT4A). All positive clones were confirmed by polymerase chain reaction (PCR) with different gene-specific primers. Subsequently the clones were digested with Sau3 AI and subcloned into the E. coli cloning vector ...
Mice carrying bacterial artificial chromosome (BAC) transgenes have become important tools for neuroscientists, providing a powerful means of dissecting complex neural circuits in the brain. Recently, it was reported that one popular line of these mice--mice possessing a BAC transgene with a D(2) dopamine receptor (Drd2) promoter construct coupled to an enhanced green fluorescent protein (eGFP) reporter--had abnormal striatal gene expression, physiology, and motor behavior. Unlike most of the work using BAC mice, this interesting study relied upon mice backcrossed on the outbred Swiss Webster (SW) strain that were homozygous for the Drd2-eGFP BAC transgene. The experiments reported here were conducted to determine whether mouse strain or zygosity was a factor in the reported abnormalities. As reported, SW mice were very sensitive to transgene expression. However, in more commonly used inbred strains of mice (C57BL/6, FVB/N) that were hemizygous for the transgene, the Drd2-eGFP BAC transgene did ...
In this communication, we report a novel strategy for the genetic manipulation of large viral DNA genomes. In a single step we cloned an infectious cytomegalovirus DNA as a bacterial artificial chromosome in E. coli and reconstituted virus progeny after transfection of the BAC plasmid into eukaryotic cells. This approach makes the CMV genome accessible to the genetic techniques established for E. coli. As an example for the power of the mutagenesis procedures, we performed a targeted insertion of four nucleotides into the 230-kb MCMV genome. In principle, any mutation (point mutations, insertions, and deletions) in any region of the genome can now be introduced using the described mutagenesis procedure. Moreover, other procedures, for example a random transposon mutagenesis of the CMV genome are conceivable. Multiple mutations can be introduced in consecutive rounds of mutagenesis without the need to reconstitute infectious viral intermediates. Construction of revertant genomes can be easily ...
The construction and analysis of metagenomic (microbial community) libraries has provided knowledge of the genetics and biochemistry of noncultivable inhabitants of soil and marine communities (4, 9, 27). We have followed the same technological approach to begin to investigate the metabolic structure of the bowel community and to detect, by a functional screen, enzymes encoded by the genomes of the gut microbiota of mice. Phylogeny of bacterial communities can also be investigated by screening metagenomic libraries for 16S rRNA genes. We did not pursue this option because a catalogue of the murine gut microbiota derived from PCR-amplified 16S rRNA genes has been provided by Salzman et al. (30) and because Béjà et al. (4) have reported that the qualitative phylogenetic representation obtained with a BAC library was in general agreement with previous reports about the recovery of PCR-amplified rRNA genes from a marine community.. As in the case of soil, the digesta contains compounds that ...
The fruit fly Drosophila melanogaster is a principal model organism in metazoan genetics and molecular biology. Here, we describe a BAC-based physical map of chromosomes 2 and 3 constructed as part of the effort to determine the D. melanogaster genome sequence (1). There are five chromosomes (X, 2,3, 4, and Y), and the second and third together account for ∼97 Mb of the ∼120-Mb euchromatic portion of the genome. Several clone-based physical maps have been described previously. Low-resolution yeast artificial chromosome maps of the genome have been produced by polytene chromosome in situ hybridization (2), and cosmid maps of regions of theX chromosome have been made by STS content and fingerprint mapping (3). The most complete previous map is the P1-based map by Kimmerly et al. (4) [also see (5)], constructed by polymerase chain reaction-based STS content mapping and polytene chromosome in situ hybridization. On chromosomes 2 and 3, it comprises 348 sets of contiguously overlapping clones ...
How does the genome encode instructions that guide embryonic development? Our research uses genes that are expressed during vertebrate development as systems for investigating this question. We have two long-term goals. The first is to shed light on regulatory events driving bone and cartilage development. This is relevant to understanding birth defects, osteoporosis and arthritis. The second is to locate and understand the function of long-range genomic sequences that control gene regulation. These sequences can act across hundreds of kilobases and are often well conserved. We study these elements using tools such as BAC (Bacterial Artificial Chromosome) transgenesis and genomic sequence comparisons. Currently, we are studying three BMP (Bone Morphogenetic Protein) family genes. All are transcribed in complex patterns during development. Precise regulation of these genes is controlled by multiple, distant cis-regulatory elements. Using transgenic assays in mice and zebrafish, we are charting
To align the developing chicken BAC contig physical maps with the existing linkage map, its necessary to identify BACs corresponding to the DNA-based markers on the latter. Chicken BAC libraries, derived from DNA of a single UCD001 inbred Red Jungle Fowl, have been generated by our collaborators. Characterization of the first BAC library based on BamHI partial digest fragments initially was done by filter hybridization with pools of labeled, PCR-amplified fragments based on marker or gene DNA sequences. Individual marker/BAC assignments were made by Southern hybridization of BAC DNA with individual marker probes and/or PCR analysis. In this manner, 31 markers from 9 linkage groups generated 71 BamHI BAC candidates (2.3 clones per locus). This approach is labor- and cost-intensive. Thus, we began using pools of overgo probes, that are complementary synthetic oligonucleotides extended in vitro to generate ~40 base pair, double-stranded DNA probes. Two BAC libraries were hybridized to pools of 36 ...
BACs are now being utilized to a greater extent in modelling genetic diseases, often alongside transgenic mice. BACs have been useful in this field as complex genes may have several regulatory sequences upstream of the encoding sequence, including various promoter sequences that will govern a genes expression level. BACs have been used to some degree of success with mice when studying neurological diseases such as Alzheimers disease or as in the case of aneuploidy associated with Down syndrome. There have also been instances when they have been used to study specific oncogenes associated with cancers. They are transferred over to these genetic disease models by electroporation/transformation, transfection with a suitable virus or microinjection. BACs can also be utilized to detect genes or large sequences of interest and then used to map them onto the human chromosome using BAC arrays. BACs are preferred for these kind of genetic studies because they accommodate much larger sequences without ...
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As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the ...
The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategi …
Regulatory elements controlling gene expression are often found in separate, sometimes remote, regions around gene loci. Artificial chromosomes offer a means to capture all regulatory elements for study of gene regulation in a near-correct genomic context. The smooth muscle calponin gene (CNN1) encodes for a multifunctional protein involved in signaling, contractile force generation, and growth regulation. While CNN1s physiology has been studied extensively, its transcriptional regulation has proven to be intractable to conventional in vivo assays. Four evolutionarily-conserved serum response factor (SRF) binding sites (called CArG boxes) are present in the first intron of CNN1 and appear to be required for full transcriptional competence of CNN1 in cultured smooth muscle cells (SMC). To assess the functionality of CNN1 CArG elements in vivo, we exploited a 103-kb bacterial artificial chromosome (BAC) containing the human CNN1 locus shown previously to completely recapitulate endogenous mouse ...
Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the
References. Ammiraju, J.S.S.; Luo, M.; Goicoechea, J.L.; Wang, W.; Kudrna, D.; Mueller, C.; Talag, J.; Kim, H.; Sisneros N.B.; Blackmon, B.; Fang, E.; Tomkins, J.B.; Brar, D.; Mackill, D.; MacCouch, S.; Kurata, N.; Lambert, G.; Galbraith, D.W.; Arumuganathan, K.; Rao, K.; Walling, J.G.; Gill, N.; Yu, Y.; Sanmiguel, P.; Soderlund, C.; Jackson, S.; Wing, R.A. 2006. The Oryza bacterial artificial chromosome library resource: construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza. Genome Research 16: 140-147. [ Links ] Azevedo, C.F.; Resende, M.D.V.; Silva, F.F.; Viana, J.M.S.; Valente, M.S.F.; Resende Junior, M.F.R.; Muñoz, P. 2015. Ridge, Lasso and Bayesian additive-dominance genomic models. BMC Genetics 16: 105. [ Links ] Bennewitz, J.; Meuwissen, T.H.E. 2010. The distribution of QTL additive and dominance effects in porcine F2 crosses. Journal of Animal Breeding and Genetics 127: 171-179. [ Links ] De los Campos, G.; ...
BELARMINO, Luis C. et al. Mining plant genome browsers as a means for efficient connection of physical, genetic and cytogenetic mapping: an example using soybean. Genet. Mol. Biol. [online]. 2012, vol.35, n.1, suppl.1, pp.335-347. ISSN 1415-4757. http://dx.doi.org/10.1590/S1415-47572012000200015.. Physical maps are important tools to uncover general chromosome structure as well as to compare different plant lineages and species, helping to elucidate genome structure, evolution and possibilities regarding synteny and colinearity. The increasing production of sequence data has opened an opportunity to link information from mapping studies to the underlying sequences. Genome browsers are invaluable platforms that provide access to these sequences, including tools for genome analysis, allowing the integration of multivariate information, and thus aiding to explain the emergence of complex genomes. The present work presents a tutorial regarding the use of genome browsers to develop targeted physical ...
Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to
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The wheat genome sequence is an essential tool for advanced genomic research and improvements. The generation of a high-quality wheat genome sequence is challenging due to its complex 17 Gb polyploid genome. To overcome these difficulties, sequencing through the construction of BAC-based physical maps of individual chromosomes is employed by the wheat genomics community. Here, we present the construction of the first comprehensive physical map of chromosome 1BS, and illustrate its unique gene space organization and evolution. Fingerprinted BAC clones were assembled into 57 long scaffolds, anchored and ordered with 2,438 markers, covering 83% of chromosome 1BS. The BAC-based chromosome 1BS physical map and gene order of the orthologous regions of model grass species were consistent, providing strong support for the reliability of the chromosome 1BS assembly. The gene space for chromosome 1BS spans the entire length of the chromosome arm, with 76% of the genes organized in small gene islands, accompanied
Citation: Ma, J., Cannon, S.B., Jackson, S.A., Shoemaker, R.C. 2010. Soybean Comparative Genomics. In: Bilyeu, K., Ratnaparkhe, M.B., and Kole, C., editors. Genetics, Genomics, and Breeding of Soybean. Routledge, New York. p. 245-262. Interpretive Summary: Technical Abstract: The soybean (Glycine max L. Merr.) has developed into a reference species complete with a full set of genomic resources. Several Bacterial Artificial Chromosome libraries have been produced and physical maps have been assembled in genotypes representing both Northern and Southern germplasm. High throughput sequencing has already been applied to transcript profiling . A very large Expressed Sequence Tag (EST) collection has been developed. These ESTs have been evaluated to demonstrate that the soybean genome has undergone at least two rounds of large-scale duplication events. Soybean has one of the most dense molecular maps of any crop species. Molecular markers include RFLPs, SSRs and SNPs. The GeneChip by Affymetrix is ...
Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp). Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar Haruna Nijo. The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome
The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.
This chapter provides a broad overview of many applications of plasmids for genetic analysis, primarily in bacteria. Ever since DNA sequencing became accessible to most research laboratories, reverse genetic analysis has become a standard experimental approach to study bacterial gene function. Similar suicide vectors have also been used for nontargeted insertional mutagenesis by cloning random chromosomal DNA fragments into the plasmid. The use of suicide vectors also allows for easy identification of the insertion mutations. Plasmids that utilize different combinations of double-counter selective markers have been used for diverse applications, including the search for extremely rare suppressor mutations of essential Escherichia coli genes, and to improve the efficiency of allelic exchange on bacterial artificial chromosomes (BACs). Although temperature-sensitive vectors represent the majority of conditionally replicating plasmids, other plasmids that exhibit conditional replication have been described
Identifying enhancers in genomes is crucial for the understanding of the complexity and mechanisms of gene regulation [50]. Traditionally, regulatory regions have been identified and mapped by cloning candidate sequences upstream of a minimal promoter fused to a reporter gene and testing their transcriptional activity in cell lines or in transgenic organisms. Although more laborious and expensive to make, transgenic animals have the great advantage of providing complete spatio-temporal expression information simultaneously in all tissues and cell types of an overall healthy animal. More and more laboratories map enhancers using 100-200 kb bacterial artificial chromosomes, which allow for the testing of regulatory regions in a context that better resembles the endogenous one, compared with small constructs [51]. Detailed studies using these methods have determined that genes involved in embryonic development are controlled by several enhancers, like the example of the mouse Shh gene mentioned in ...
The expression of genes from genomic loci can be relatively complex, utilizing exonic, intronic and flanking sequences to regulate tissue and developmental specificity. Infectious bacterial artificial chromosomes (iBACs) have been shown to deliver and express large genomic loci (up to 135 kb) into primary cells for functional analyses. The delivery of large genomic DNA inserts allows the expression of complex loci and of multiple splice variants. Herein, we demonstrate for the first time that an iBAC will deliver and correctly express in human glioma cells the entire CDKN2A/CDKN2B genomic region, which encodes for at least three important cell-cycle regulatory proteins (p16(INK4a), p14(ARF) and p15(INK4b)). Two of these proteins are expressed from overlapping genes, utilizing alternative splicing and promoter usage. The delivered locus expresses each gene at physiological levels and cellular responses (apoptosis versus growth arrest) occur dependent on cellular p53 status, as expected. The work further
Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide an …
This track shows the location of fluorescent in situ hybridization (FISH)-mapped clones along the assembly sequence. The locations of these clones were obtained from the NCBI Human BAC Resource here. Earlier versions of this track obtained this information directly from the paper Cheung, et al. (2001). More information about the BAC clones, including how they may be obtained, can be found at the Human BAC Resource and the Clone Registry web sites hosted by NCBI. To view Clone Registry information for a clone, click on the clone name at the top of the details page for that item.. ...
This track shows the location of fluorescent in situ hybridization (FISH)-mapped clones along the assembly sequence. The locations of these clones were obtained from the NCBI Human BAC Resource here. Earlier versions of this track obtained this information directly from the paper Cheung, et al. (2001). More information about the BAC clones, including how they may be obtained, can be found at the Human BAC Resource and the Clone Registry web sites hosted by NCBI. To view Clone Registry information for a clone, click on the clone name at the top of the details page for that item.. ...
Breaking (shearing) DNA by Alkali Hydrolysis Suitable for BAC clones before labelling using random-primer (random priming, oligolabeling) labeling (which is best and most random with shorter fragments than the full-length BACs; autoclaving of syringing the DNA requires too much volume). 300 ng DNA + 6.5 ul 4M NaOH made up to 155 ul, final NaOH 200 mM Mix and incubate room temperature 20min Add 65 ul 5M ammonium acetate, 550 ul ice cold 100% ethanol (total volume 770ul) Allow to precipitate (e.g. 2 hr), centrifuge as usual and redissolve are required. Trude Schwarzacher September 2004. ...
Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium‐mediated transformation
Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium‐mediated transformation
2. Transfer 50ul of culture to a 50ml flask with 50ml 2XYT + CM (12.5ug/ml), and incubate/shake for 14-16 hours at 250rpm. (14 hour incubation best ...
Fungi are prolific producers of secondary metabolites (SMs) and have been producing 45% of bioactive molecules from all microbial sources since the turn of the century (Bérdy, 2012). Conservative estimates suggest that there are more than 5 million fungal species (Blackwell, 2011), of which fewer than 5% have been described and less than 1% are available in the worlds culture collections (Colwell, 1997). In addition, each of these fungal genomes may harbor 50 or more different SM gene clusters (Norberg et al., 2013). Fungal secondary metabolite (SM) gene cluster sequences available for characterization far outstrip our current ability to characterize each cluster, as most of the fungi are not genetically amendable. To address this post-genomic SM characterization gridlock, we have demonstrated a new technology that generates unbiased shuttle bacterial artificial chromosome (BAC) or fungal artificial chromosome (FAC) libraries, pooled FAC next-gen sequences to rapidly capture all the SM gene ...
Thanks again, perneseblue! For yesterdays experiment, I used SW102 cells (modified E. Coli from NCI-Frederick for recombineering) directly from glycerol stock (40% final concentration of glycerol). I didnt specifically prepare the bacteria in the regular way people make electrocompetent cells (by washing sequentially with 10% glycerol) because they are electrocompetent cells already. I used 0.2 cm cuvette (thats the only one i can find in the lab), with capacitance of 25uF, and i got ~3.5 millisecond time constant and an arcing, w/o beeping ...
In an attempt to establish a method that allows for scalable generation of recombineered BACs and transgenic lines, we have implemented a procedure comprising three distinct main phases: (1) in silico design of the experiment and selection of an appropriate BAC (see http://www.hubrecht.eu/research/schulte-merker/protocols.html); (2) BAC recombineering (Fig. 1); and (3) the actual generation of the transgenic line. In using this method, we were particularly interested in addressing whether there is a negative correlation between BAC size and the rate of transgenesis, whether the recombineered BACs show sufficiently widespread expression to make them immediately useful for transient experiments (without the need to generate a transgenic line), and whether position effects are observable among different lines generated from the same BAC.. In previous experiments (Spoorendonk et al., 2008; Hogan et al., 2009), we have used I-SceI-mediated transgenesis (Grabher et al., 2004; Kimura et al., 2006) to ...
Targeted disruption of Paip2a. A PCR fragment amplified with a primer set of Paip2a 5′ Fwd and Rev (Supplemental Table 1) was used as a probe to isolate genomic BAC DNA clone 103F10 from the 129/Sv mouse BAC genomic library RPCI-22. The targeting vector was constructed by recombination (55), and routine cloning methods were employed with a 10.5-kbp mouse Paip2a genomic fragment from clone 103F10, as illustrated in Figure 2A. The final targeting fragment was excised from its cloning vector backbone by NotI and electroporated into R1 ES cells. Southern blot analysis was performed with two probes corresponding to the 5′ and 3′ sequences outside the targeting region, as indicated in Figure 2A. To generate the 5′ and 3′ probes, the primer sets (Paip2a 5′ Fwd and Rev for the 5′ probe and Paip2a 3′ Fwd and Rev for the 3′ probe) listed in Supplemental Table 1 were used. After the LoxP-flanked Neo cassette was eliminated by subsequent transient transfection with a cre recombinase ...
Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.
Many functional studies require the transfection of bacterial artificial chromosome (BAC) clones into mammalian cells. Because most BAC vectors do not have a mammalian selection marker or do not have...
Department of Genetics, Harvard Medical School, Boston, MA, USA.. A method for construction of bacterial artificial chromosome (BAC) contigs from a yeast artificial chromosome (YAC) physical map is described. An approximately 2 Mb contig, consisting of two large BAC contigs linked by a small YAC, has been assembled in the region around 80 cM of Arabidopsis thaliana chromosome 2. Clones from this contig will facilitate gene isolation in the region and can be used directly as substrates for DNA sequencing.. MeSH Terms ...
The DNA construct for simultaneous generation of a null and a conditional allele for Zfp521 was made using recombineering, essentially as described previously (Liu et al., 2003; Warming et al., 2006). First, a retrieval vector was prepared by three-way ligation of mini-homology arms into pBlight-TK (a plasmid backbone containing the thymidine kinase from HSV) for counter-selection in embryonic stem cells using Ganciclovir (Warming et al., 2006). The homology arms were amplified from a mouse BAC containing the Zfp521 genomic region using the primers listed below. The BAC (CITB 454L20) was identified by screening a 129-based BAC library (CJ7 embryonic stem cell DNA, CITB, Research Genetics/Invitrogen), and BAC DNA was prepared using the BAC Nucleobond kit (Takara Bio Inc., BD). All primers were from Integrated DNA Technologies, and all PCR reactions were performed using Expand High Fidelity (Roche): 5′ retrieval F, 5′-AATAAAGGATCCGTGCTCCAGGCACTATAGAT-3′; 5′ retrieval R, ...
The kits utilize a novel strategy of cloning end-repaired, randomly sheared DNA instead of the conventional approach of cloning fragments generated by partial restriction endonuclease digestion. First, genomic DNA is sheared by passing it through a syringe needle. The sheared DNA is end-repaired to generate 5-phosphorylated blunt ends and size-selected using a low melting point agarose gel. The size-selected DNA is then ligated into the supplied linearized and dephosphorylated pWEB-TNCTM or pWEBTM Cosmid Vector, packaged using ultra-high efficiency MaxPlaxTM Lambda Packaging Extracts (,109 pfu/μg for phage lambda) and plated on phage T1-resistant EPI100TM-T1R E. coli plating cells, all included in the kit. The result is a complete and unbiased primary cosmid library ...
The kits utilize a novel strategy of cloning end-repaired, randomly sheared DNA instead of the conventional approach of cloning fragments generated by partial restriction endonuclease digestion. First, genomic DNA is sheared by passing it through a syringe needle. The sheared DNA is end-repaired to generate 5-phosphorylated blunt ends and size-selected using a low melting point agarose gel. The size-selected DNA is then ligated into the supplied linearized and dephosphorylated pWEB-TNCTM or pWEBTM Cosmid Vector, packaged using ultra-high efficiency MaxPlaxTM Lambda Packaging Extracts (,109 pfu/μg for phage lambda) and plated on phage T1-resistant EPI100TM-T1R E. coli plating cells, all included in the kit. The result is a complete and unbiased primary cosmid library ...
Genomic sequence contigs for unfinished chromosomes are assembled and laid out based largely on the clone tiling path. However, the tiling paths do not specify the orientation of the clone sequences or how they should be joined; therefore, data on the alignment of the input genomic sequences to each other and to other sequences are also used to guide the assembly. Genomic sequences that augment the initial set of genomic contigs based on the tiling path clones are also incorporated ...
Background Crocodilians (Order Crocodylia) are an ancient vertebrate group of tremendous ecological, social, and evolutionary importance. They are the only extant reptilian members of Archosauria, a monophyletic group that also includes birds, dinosaurs, and pterosaurs. Consequently, crocodilian genomes represent a gateway through which the molecular evolution of avian lineages can be explored. To facilitate comparative genomics within Crocodylia and between crocodilians and other archosaurs, we have constructed a bacterial artificial chromosome (BAC) library for the Australian saltwater crocodile, Crocodylus porosus. This is the first BAC library for a crocodile and only the second BAC resource for a crocodilian. Results The C. porosus BAC library consists of 101,760 individually archived clones stored in 384-well microtiter plates. Not I digestion of random clones indicates an average insert size of 102 kb. Based on a genome size estimate of 2778 Mb, the library affords 3.7 fold (3.7×) coverage of
OPPORTUNITY TO PROPOSE ORGANISMS FOR BAC LIBRARY CONSTRUCTION Release Date: December 19, 2001 NOTICE: NOT-HG-02-004 National Human Genome Research Institute Annual Submission Dates: February 10, June 10 and October 10 Over the past several years, the bacterial artificial chromosome (BAC) has emerged as the vector system of choice for the construction of the large- insert chromosomal DNA libraries that are needed in genomic studies. Because BAC clones are relatively large and appear to faithfully represent an organisms genome, the BAC system will also be the vehicle of choice for the isolation of targeted regions of genomic DNA from additional organisms being used in specific biological studies, a variety of mouse strains, and even from individual humans. With the increasing interest in genomic approaches to biological research, the demand for new BAC libraries is expected to increase rapidly in the next several years. To meet the need to increase the number of available BAC libraries, NHGRI, ...
TY - JOUR. T1 - 1-Mb resolution array-based comparative genomic hybridization using a BAC clone set optimized for cancer gene analysis. AU - Greshock, Joel. AU - Naylor, Tara L.. AU - Margolin, Adam. AU - Diskin, Sharon. AU - Cleaver, Stephen H.. AU - Futreal, P. Andrew. AU - deJong, Pieter J.. AU - Zhao, Shaying. AU - Liebman, Michael. AU - Weber, Barbara L.. PY - 2004/1/1. Y1 - 2004/1/1. N2 - Array-based comparative genomic hybridization (aCGH) is a recently developed tool for genome-wide determination of DNA copy number alterations. This technology has tremendous potential for disease-gene discovery in cancer and developmental disorders as well as numerous other applications. However, widespread utilization of aCGH has been limited by the lack of well characterized, high-resolution clone sets optimized for consistent performance in aCGH assays and specifically designed analytic software. We have assembled a set of ∼4100 publicly available human bacterial artificial chromosome (BAC) clones ...
To explore CR1 distribution in C. porosus, macroarrays were screened with CR1b-derived overgos or a combination of CR1a and CR1b overgos. Comparison of the CR1b- and CR1a/b-probed macroarrays revealed that the CR1a and CR1b subfamilies are not distinguishable in our assay, i.e., virtually no differences in hybridization pattern and intensity were observed when comparing the CR1b and CR1a/CR1b filters (data not shown). It was clear, however, that elements similar to CR1a and b are fairly abundant in C. porosus. Examination of one-quarter of the CR1a/b-probed macroarray (Figure 3A) indicates that 8.9% of clones show hybridization to the CR1a/b overgos while CR1b overgo hybridization to a filter stamped with the contents of a single 384-well plate from the BAC library suggests that 12.8% of clones (49 of 384) are positive for the CR1b overgo (Figure 4). Densitometric analysis of the macroarray reveals that there is a six-fold variation in positive clone hybridization intensity. If the lightest, but ...
TY - JOUR. T1 - Construction and characterization of a yeast artificial chromosome library containing 1.5 equivalents of human chromosome 21. AU - Potier, M. C.. AU - Kuo, W. L.. AU - Dutriaux, A.. AU - Gray, J.. AU - Goedert, M.. PY - 1992/10. Y1 - 1992/10. N2 - A library of yeast artificaial chromosomes (YACs) was constructed from a human/hamster somatic cell hybrid containing human chromosome 21 (q11-qter). Cells were embedded in agarose, and the DNA was partially digested with EcoRI, released into solution by agarase treatment of the agarose plugs, ligated into pYAC4, and transferred into yeast. Doule screening of the yeast transformants with human and hamster genomic DNA allowed the selection of clones hybridizing only with human DNA. The library consists of 321 clones, amounting to 1.5 equivalents (61 Mb) of chromosome 21. The mean YAC size calculated from 178 clones is 190 ± 100 kb. Screening of the library with eight sequence-tagged sites gave six positives. Among 21 YACs tested by in ...
Although second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place. Result: Five new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (HindIII, EcoRI, MboI and BstXI ) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also ...
Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a
TY - JOUR. T1 - Global genomic diversity of Oryza sativa varieties revealed by comparative physical mapping. AU - Wang, Xiaoming. AU - Kudrna, David A.. AU - Pan, Yonglong. AU - Wang, Hao. AU - Liu, Lin. AU - Lin, Haiyan. AU - Zhang, Jianwei. AU - Song, Xiang. AU - Goicoechea, Jose Luis. AU - Wing, Rod A.. AU - Zhang, Qifa. AU - Luo, Meizhong. PY - 2014/4. Y1 - 2014/4. N2 - Bacterial artificial chromosome (BAC) physical maps embedding a large number of BAC end sequences (BESs) were generated for Oryza sativa ssp. indica varieties Minghui 63 (MH63) and Zhenshan 97 (ZS97) and were compared with the genome sequences of O. sativa spp. japonica cv. Nipponbare and O. sativa ssp. indica cv. 93-11. The comparisons exhibited substantial diversities in terms of large structural variations and small substitutions and indels. Genome-wide BAC-sized and contig-sized structural variations were detected, and the shared variations were analyzed. In the expansion regions of the Nipponbare reference sequence, in ...
Cultivated peanut is an allotetraploid with two nuclear genomic components, AA and BB. Although it is generally agreed that these component genomes are derived from diploid wild ancestors, the exact species involved has been a matter of some research and discussion. Although the evidence is not completely clear cut, analysis of data from molecular markers, cytogenetics, morphology and geographical distributions support that A. duranensis and A. ipaënsis are the direct ancestors of cultivated peanut [8, 30].. Genomic in situ hybridization (GISH) of A. hypogaea metaphase chromosomes with total genomic DNA from the AA genome of A. duranensis and the BB genome of A. ipaënsis allowed a clear differentiation of the A and B chromosomes. Firstly, this observation reinforces the evidence of the close relationship between the genomes of A. duranensis, A. ipaënsis and cultivated peanut. Secondly, since GISH relies largely on the hybridization of repetitive sequences, it also indicates that A. duranensis ...
In drug users, drug-related cues alone can induce dopamine release in the dorsal striatum. Instructive cues activate inputs to the striatum from both dopaminergic and cholinergic neurons, which are thought to work together to support motor learning and motivated behaviors. Imbalances in these neuromodulatory influences can impair normal action selection and might thus contribute to pathologically repetitive and compulsive behaviors such as drug addiction. Dopamine and acetylcholine can have either antagonistic or synergistic effects on behavior, depending on the state of the animal and the receptor signaling systems at play. Semi-synchronized activation of cholinergic interneurons in the dorsal striatum drives dopamine release via presynaptic nicotinic acetylcholine receptors located on dopamine terminals. Nicotinic receptor blockade is known to diminish abnormal repetitive behaviors (stereotypies) induced by psychomotor stimulants. By contrast, blockade of postsynaptic acetylcholine muscarinic
Medical definition of bacterial artificial chromosome: a genetically engineered bacterial chromosome that is used as a vector to clone DNA segments, …
Chromosome segregation and cell division are essential, highly ordered processes that depend on numerous protein complexes. Results from recent RNA interference screens indicate that the identity and composition of these protein complexes is incompletely understood. Using gene tagging on bacterial artificial chromosomes, protein localization, and tandem-affinity purification-mass spectrometry, the MitoCheck consortium has analyzed about 100 human protein complexes, many of which had not or had only incompletely been characterized. This work has led to the discovery of previously unknown, evolutionarily conserved subunits of the anaphase-promoting complex and the gamma-tubulin ring complex--large complexes that are essential for spindle assembly and chromosome segregation. The approaches we describe here are generally applicable to high-throughput follow-up analyses of phenotypic screens in mammalian cells ...
Schambra, U.B.,Lauder, J.M., Connelly, B., Centeno, A., Rosen G., and Williams, R.W. http://epmba.org/. Electronic Prenatal Mouse Brain Atlas. 2007 to present. Schambra, U.B., Mackensen, G.B., Stafford Smith, M., Haines, D.E., and Schwinn, D.A. (2005). Neuron Specific a-Adrenergic Receptor Expression in Human Cerebellum: Implications for Emerging Cerebellar Roles in Neurologic Disease. Neuroscience 135:507-523. Gong, S., Zheng, C., Doughty, M.L., Losos, K., Didkovsky, N., Schambra, U.B., Nowak, N.J., Joyner, A., Leblanc, G., Hatten, M.E., Heintz, N. (2003) A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917-925. Stafford Smith, M., Schambra, U. B., Wilson, K. H., Page, S. O., and Schwinn, D. A. (1999). a1-Adrenergic receptors in human spinal cord: Specific localized expression of mRNA encoding a1-adrenergic receptor subtypes at four distinct levels. Molecular Brain Research 63, 254-261. Stafford Smith, M., Schambra, U.B., Wilson, K.H., ...
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Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from...
The past decade witnessed the wide spread use of clones from BAC libraries of numerous organisms for functional studies. The large insert DNA size and easy maneuverability of that DNA in bacteria has contributed to the growing popularity of BACs in transgenic animal studies. The realization that many control elements of genes important during vertebrate development are actually located at large distances along the DNA from the coding sequences of the gene have made BACs increasingly indispensable for studies of developmentally regulated genes using transgenic animals. A different area of interest arose from the same attractive features of BACs, and relates to their use as vectors for cloning the very large genomes of several DNA viruses. Faithful propagation and easy mutational analyses of the BAC-viral DNA in bacteria allowed rapid assignment of function(s) to the numerous open reading frames in the viral genome when that BAC-viral DNA was reintroduced into permissive hosts for a productive ...
Genome analysis of soybean (Glycine max L.) has been complicated by its paleo-autopolyploid nature and conserved homeologous regions. Landmarks of expressed sequence tags (ESTs) located within a minimum tile path (MTP) of contiguous (contig) bacterial artificial chromosome (BAC) clones or radiation hybrid set can identify stress and defense related gene rich regions in the genome. A physical map of about 2,800 contigs and MTPs of 8,064 BAC clones encompass the soybean genome. That genome is being sequenced by whole genome shotgun methods so that reliable estimates of gene family size and gene locations will provide a useful tool for finishing. The aims here were to develop methods to anchor plant defense- and stress-related gene paralogues on the MTP derived from the soybean physical map, to identify gene rich regions and to correlate those with QTL for disease resistance. The probes included 143 ESTs from a root library selected by subtractive hybridization from a multiply disease resistant soybean
Empty clones: 1.33 %. Hybridization with labeled total DV92 DNA showed that all the other BAC clones have wheat DNA. 85% of the Not I fragments showed strong hybridization signal suggesting the presence of repetitive sequences.. ...
A major innovation of the Human Genome Project has been the development of BACs, which harbor large, stable genomic fragments of DNA (50). BACs have been the workhorses in the sequencing of the human genome (12, 13, 53). In addition, BACs have been used to complement genetic mutations leading to gene identification (2, 43) and to pinpoint distal regulatory elements that confer cell-restricted gene expression in transgenic mice (37). The latter approach has been of particular interest inasmuch as many muscle-restricted transcription units are governed bycis-acting elements that may reside tens of kilobases away from the core promoter region (9, 47, 57). Defining such distal elements through small phage genomic clones can be time consuming and may have untoward effects on the expression of the reporter transgene (see below). In this report we used a BAC harboring the hSM-Calp transcription unit to show first that the expression of hSM-Calp exhibited similar regulatory expression in an in vitro ...
Rosalind M John is Professor of Developmental Epigenetics and Head of the Biomedicine Division within the School of Biosciences at Cardiff University. She received her PhD from Imperial College, University of London and trained at University of San Francisco California (UCSF) and Stanford, USA, and Cambridge University. She has a ,20 year track record in the epigenetics of fetal and placental development using animal models to study the relevance of genomic imprinting, and how gene dosage may be influenced by environmental factors mediating short and life long phenotypic outcomes. She is an expert in the generation of BAC transgenic mice (Phlda2, Cdkn1c and Ascl2) and the use of loss-of-function models (Cdkn1c, Phlda2 and Peg3) to gain insight in the relevance of controlled gene dosage. Her group have reported phenotypes affecting fetal growth, placental development, metabolism, adult behaviour and, most recently, maternal behaviour in response to placental endocrine dysfunction. Professor John ...
Laboratoire de Phytopathologie Moleculaire, URA CNRS 1128, Universite Paris-sud, Orsay, France.. A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial EcoRI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. ...
After all this is verified, we perform mass transformations, plating and pick. We use the following robotic platforms: G3 (Genomic Solutions), QPIX2 (Genetix) and MegaPIX (Genetix). The robots do blue/white selection and pick white clones into 384-well plates of glycerol storage solution. The plates of clones are grown overnight (~18 hours), and then we determine the exact plate and well position for all wells that did not grow (we call these missed wells).. We re-grid a subset of the clones onto 22cm x 22cm agar plates with XGAL+IPTG+Antibiotics and allow the colonies to grow 18 hours, then incubate for several days at a specific temp to allow any blue clones to be very obvious. We then count the blue clones and enter into a database. Example of Custom Mouse BAC Library QC Data There are several sheets in this file, including one that tracks if any pins on the robot are going bad. Your team will get a similar Excel file when the library is completed. The combined missed wells and blue ...
Image shows a z-series of a mouse ES cell carrying a multi-copy alpha-globin BAC transgene array. Relative distribution of the BAC sequences, identif...
Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.
My own research interests which map onto these targets are flowering time, leaf senescence and how plant chemistry affects the conversion efficiency of energy crops. Research tools that have or are being developed include bacterial artificial chromosome (BAC) libraries, genetic, trait and physical maps, the establishment of forward mutation populations, the exploitation of syntenic relationships to associate genotype to phenotype, and the development of high throughput virtual phenotyping methods including the use of infrared spectroscopy for cell wall chemistry.. ...
My own research interests which map onto these targets are flowering time, leaf senescence and how plant chemistry affects the conversion efficiency of energy crops. Research tools that have or are being developed include bacterial artificial chromosome (BAC) libraries, genetic, trait and physical maps, the establishment of forward mutation populations, the exploitation of syntenic relationships to associate genotype to phenotype, and the development of high throughput virtual phenotyping methods including the use of infrared spectroscopy for cell wall chemistry.. ...
Now that weve learned a bit about F factors, you might imagine how a cloning vector could be created that was based on an F factor origin of replication. We call such engineered F plasmids BACs or Bacterial Artificial Chromosomes. BACs are capable of carrying approximately 200 kbp of inserted DNA sequence, and the F factor origin of replication maintains their level at approximately one copy per cell. Of course, we neednt stop there! We can also use YACs which are Yeast Artificial Chromosomes, and depend on being able to replicate and be maintained in Saccharomyces cerevisiae. YACs can carry approximately 500 kbp of foreign DNA, though they are often criticized due to the problem of natural recombination in the host.. Handling DNA of this size is a real problem, as I have mentioned before, due to the potential for shearing. The way this is solved is to embed the cells from which a library is going to be made, in low melting point agarose. The cells can be lysed in the agarose, simply be ...
Description: Cloned unidirectionally. Primer: Oligo dT. Average insert size 2.2 kb. Constructed by Life Technologies. Note: this is a Xenopus Gene Collection library ...
Tumor formation requires several genetic and epigenetic changes (hits) that occur stochastically in a given tissue. Cooperating hits are positively selected and give rise to progressed tumors. Cooperation of hits in tumors is a crucial issue in cancer therapy. Currently, cooperation is addressed in combinatorial mouse models that harbour several genetic changes (presence of oncogenic transgenes and/or loss of tumor suppressor genes). However, this approach is biased and does not mimic the evolution process of cancer that selects the most potent cooperating hits. Being aware of these drawbacks, we are establishing Multi-Hit mouse models that are based on the Cre/loxP technology and harbour Multi-Hit BAC transgenes that allow the randomized introduction of several hits in a given tissue. Currently, we employ this approach to identify the requirement and cooperations between the MAPK, Ral and Pi3K pathways activated by oncogenic Ras in different cancers.. ...
Tumor formation requires several genetic and epigenetic changes (hits) that occur stochastically in a given tissue. Cooperating hits are positively selected and give rise to progressed tumors. Cooperation of hits in tumors is a crucial issue in cancer therapy. Currently, cooperation is addressed in combinatorial mouse models that harbour several genetic changes (presence of oncogenic transgenes and/or loss of tumor suppressor genes). However, this approach is biased and does not mimic the evolution process of cancer that selects the most potent cooperating hits. Being aware of these drawbacks, we are establishing Multi-Hit mouse models that are based on the Cre/loxP technology and harbour Multi-Hit BAC transgenes that allow the randomized introduction of several hits in a given tissue. Currently, we employ this approach to identify the requirement and cooperations between the MAPK, Ral and Pi3K pathways activated by oncogenic Ras in different cancers.. ...
Finds sub-sequence or patterns in the sequence and highlights the matching region. The tool works with standard single letter nucleotide or protein codes including ambiguities and can match Prosite patterns in protein sequences ...
Investigating and assigning gene functions of herpesviruses is a process, which profits from consistent technical innovation. Cloning of bacterial artificial chromosomes encoding herpesvirus genomes permits nearly unlimited possibilities in the construction of genetically modified viruses. Targeted or randomized screening approaches allow rapid identification of essential viral proteins. Nevertheless, mapping of essential genes reveals only limited insight into function. The usage of dominant-negative (DN) proteins has been the tool of choice to dissect functions of proteins during the viral life cycle. DN proteins also facilitate the analysis of host-virus interactions. Finally, DNs serve as starting-point for design of new antiviral strategies.
The chromosome-based HPP aims to expand our understanding of the human proteome with a focus on expanding the understanding of each and every gene on each chromosome. For the most complete and up-to-date information available, visit the C-HPP portal.. ...
Mapping resources The group discussed the benefits of construction of a database of restriction fragment fingerprints for a BAC library. The value of this database will be to reduce the redundancy of the clone sets with which the mappers have to work, thereby simplifying the mapping problem. End sequences from the fingerprinted clones would be very valuable as an additional data set. There is no evidence that fingerprints are a true measure of clone fidelity. The issue was raised as to whether further investment should be made in generating and mapping additional random STSs to assist in long-range mapping. It was agreed that additional markers will be needed but that the most useful ones will be those generated from the ends of contigs, rather than random markers. There was some agreement that there may be a need for rapid RH mapping of such directed markers in a year or so. More importantly, there was general agreement that a long range mapping plan is needed. ...
Complete information for ALPK2 gene (Protein Coding), Alpha Kinase 2, including: function, proteins, disorders, pathways, orthologs, and expression. GeneCards - The Human Gene Compendium
利用者は、研究成果の公表にあたって寄託者の指定する文献を引用する(Genes Genet. Syst. 83, 245-256, 2008)。ショウジョウバエBACクローンを用いた研究成果の公表に当たっては、ナショナルバイオリソースプロジェクト「ショウジョウバエ」に対する謝辞の表明を必要とする ...
We have used massively parallel paired end sequencing strategies to reconstruct the genomic landscape of 24 breast cancer genomes, through the identification and characterization of 2166 somatically acquired genomic rearrangements. These studies have revealed considerable complexity in the patterns of structural variation, identified novel fusion genes and unveiled new insights into the complex structure of amplicons. ...
bdv sorry. It is for option 1. The mapping of contigs to a reference using BLAT (BLAT/BLAST section). From BLAT generate the syntenylist (baiscally the orientation layout of contigs) which you can use subsequently to Join (you find it in the Artemis/MUMmer section) in an artificial chromosome using spacers and/or linker providing an EMBL layout file as well for inspecting in for instance ACT or Artemis of Sanger.. ...
R. Beck, G90(1). Grass silage; country of origin unknown. Type strain. Taxonomy/description (3239, 3767, 38511). Sequence accession no. whole genome shotgun sequence: AYZB00000000. Murein: A11.31 (3239). (Medium 11, 30°C ...
R.S. Tanner; 37AN3*. Soil; USA. Type strain. Taxonomy/description (8502). Sequence accession no. 16S rRNA gene: AJ306612, whole genome shotgun sequence: FNQH00000000. Murein: A11.31 (8502). (Medium 92, 28°C ...
Screenlist: File Name: lactation_fetish 198.avi | Duration: 21min 47s | File Size: 217 MB | Resolution: 416x320 Download File: lactation_fetish 198.rar
Ive done quite a bit of TOPO cloning and, if it works, is easy, fast and efficient. I did have a problem when trying to subclone a large insert (9.5 kb) as it would always be in the wrong orientation. TOPO is supposed to be a 50/50 for orientation but sometimes (especially with large inserts) it can take on a tertiary structure which inhibits the TOPO ligation. Only way to find out is to try it!. ...
The value listed under genome equivalents is for the amplified library. Coverage was calculated by dividing the number of independent recombinants by the genome equivalents of the unamplified library. This value is 18 ...
The value listed under genome equivalents is for the amplified library. Coverage was calculated by dividing the number of independent recombinants by the genome equivalents of the unamplified library. This value is 34 ...