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Sample records for genomic bac library

  1. Genome-wide BAC-end sequencing of Cucumis melo using two BAC libraries

    PubMed Central

    2010-01-01

    Background Although melon (Cucumis melo L.) is an economically important fruit crop, no genome-wide sequence information is openly available at the current time. We therefore sequenced BAC-ends representing a total of 33,024 clones, half of them from a previously described melon BAC library generated with restriction endonucleases and the remainder from a new random-shear BAC library. Results We generated a total of 47,140 high-quality BAC-end sequences (BES), 91.7% of which were paired-BES. Both libraries were assembled independently and then cross-assembled to obtain a final set of 33,372 non-redundant, high-quality sequences. These were grouped into 6,411 contigs (4.5 Mb) and 26,961 non-assembled BES (14.4 Mb), representing ~4.2% of the melon genome. The sequences were used to screen genomic databases, identifying 7,198 simple sequence repeats (corresponding to one microsatellite every 2.6 kb) and 2,484 additional repeats of which 95.9% represented transposable elements. The sequences were also used to screen expressed sequence tag (EST) databases, revealing 11,372 BES that were homologous to ESTs. This suggests that ~30% of the melon genome consists of coding DNA. We observed regions of microsynteny between melon paired-BES and six other dicotyledonous plant genomes. Conclusion The analysis of nearly 50,000 BES from two complementary genomic libraries covered ~4.2% of the melon genome, providing insight into properties such as microsatellite and transposable element distribution, and the percentage of coding DNA. The observed synteny between melon paired-BES and six other plant genomes showed that useful comparative genomic data can be derived through large scale BAC-end sequencing by anchoring a small proportion of the melon genome to other sequenced genomes. PMID:21054843

  2. Construction of the BAC Library of Small Abalone (Haliotis diversicolor) for Gene Screening and Genome Characterization.

    PubMed

    Jiang, Likun; You, Weiwei; Zhang, Xiaojun; Xu, Jian; Jiang, Yanliang; Wang, Kai; Zhao, Zixia; Chen, Baohua; Zhao, Yunfeng; Mahboob, Shahid; Al-Ghanim, Khalid A; Ke, Caihuan; Xu, Peng

    2016-02-01

    The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33%. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor.

  3. Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research.

    PubMed

    Zhang, Hong-Bin; Scheuring, Chantel F; Zhang, Meiping; Zhang, Yang; Wu, Cheng-Cang; Dong, Jennifer J; Li, Yaning

    2012-02-16

    Large-insert BAC (bacterial artificial chromosome) and BIBAC (binary BAC) libraries are essential for modern genomics research for all organisms. We helped pioneer the BAC and BIBAC technologies, and by using them we have constructed hundreds of BAC and BIBAC libraries for different species of plants, animals, marine animals, insects, algae and microbes. These libraries have been used globally for different aspects of genomics research. Here we describe the procedure with the latest improvements that we have made and used for construction of BIBAC libraries. The procedure includes the preparation of BIBAC vectors, the preparation of clonable fragments of the desired size from the source DNA, the construction and transformation of BIBACs and, finally, the characterization and assembly of BIBAC libraries. We also specify the modifications necessary for construction of BAC libraries using the protocol. The entire protocol takes ∼7 d.

  4. The infectious BAC genomic DNA expression library: a high capacity vector system for functional genomics

    PubMed Central

    Lufino, Michele M. P.; Edser, Pauline A. H.; Quail, Michael A.; Rice, Stephen; Adams, David J.; Wade-Martins, Richard

    2016-01-01

    Gene dosage plays a critical role in a range of cellular phenotypes, yet most cellular expression systems use heterologous cDNA-based vectors which express proteins well above physiological levels. In contrast, genomic DNA expression vectors generate physiologically-relevant levels of gene expression by carrying the whole genomic DNA locus of a gene including its regulatory elements. Here we describe the first genomic DNA expression library generated using the high-capacity herpes simplex virus-1 amplicon technology to deliver bacterial artificial chromosomes (BACs) into cells by viral transduction. The infectious BAC (iBAC) library contains 184,320 clones with an average insert size of 134.5 kb. We show in a Chinese hamster ovary (CHO) disease model cell line and mouse embryonic stem (ES) cells that this library can be used for genetic rescue studies in a range of contexts including the physiological restoration of Ldlr deficiency, and viral receptor expression. The iBAC library represents an important new genetic analysis tool openly available to the research community. PMID:27353647

  5. Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries

    PubMed Central

    2011-01-01

    Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ≥ 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome

  6. Toward an Integrated BAC Library Resource for Genome Sequencing and Analysis

    SciTech Connect

    Simon, M. I.; Kim, U.-J.

    2002-02-26

    We developed a great deal of expertise in building large BAC libraries from a variety of DNA sources including humans, mice, corn, microorganisms, worms, and Arabidopsis. We greatly improved the technology for screening these libraries rapidly and for selecting appropriate BACs and mapping BACs to develop large overlapping contigs. We became involved in supplying BACs and BAC contigs to a variety of sequencing and mapping projects and we began to collaborate with Drs. Adams and Venter at TIGR and with Dr. Leroy Hood and his group at University of Washington to provide BACs for end sequencing and for mapping and sequencing of large fragments of chromosome 16. Together with Dr. Ian Dunham and his co-workers at the Sanger Center we completed the mapping and they completed the sequencing of the first human chromosome, chromosome 22. This was published in Nature in 1999 and our BAC contigs made a major contribution to this sequencing effort. Drs. Shizuya and Ding invented an automated highly accurate BAC mapping technique. We also developed long-term collaborations with Dr. Uli Weier at UCSF in the design of BAC probes for characterization of human tumors and specific chromosome deletions and breakpoints. Finally the contribution of our work to the human genome project has been recognized in the publication both by the international consortium and the NIH of a draft sequence of the human genome in Nature last year. Dr. Shizuya was acknowledged in the authorship of that landmark paper. Dr. Simon was also an author on the Venter/Adams Celera project sequencing the human genome that was published in Science last year.

  7. Development of genomic resources for the narrow-leafed lupin (Lupinus angustifolius): construction of a bacterial artificial chromosome (BAC) library and BAC-end sequencing

    PubMed Central

    2011-01-01

    Background Lupinus angustifolius L, also known as narrow-leafed lupin (NLL), is becoming an important grain legume crop that is valuable for sustainable farming and is becoming recognised as a potential human health food. Recent interest is being directed at NLL to improve grain production, disease and pest management and health benefits of the grain. However, studies have been hindered by a lack of extensive genomic resources for the species. Results A NLL BAC library was constructed consisting of 111,360 clones with an average insert size of 99.7 Kbp from cv Tanjil. The library has approximately 12 × genome coverage. Both ends of 9600 randomly selected BAC clones were sequenced to generate 13985 BAC end-sequences (BESs), covering approximately 1% of the NLL genome. These BESs permitted a preliminary characterisation of the NLL genome such as organisation and composition, with the BESs having approximately 39% G:C content, 16.6% repetitive DNA and 5.4% putative gene-encoding regions. From the BESs 9966 simple sequence repeat (SSR) motifs were identified and some of these are shown to be potential markers. Conclusions The NLL BAC library and BAC-end sequences are powerful resources for genetic and genomic research on lupin. These resources will provide a robust platform for future high-resolution mapping, map-based cloning, comparative genomics and assembly of whole-genome sequencing data for the species. PMID:22014081

  8. Comparative genomics of Lupinus angustifolius gene-rich regions: BAC library exploration, genetic mapping and cytogenetics

    PubMed Central

    2013-01-01

    Background The narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n = 40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome. Results The genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, five clones were chosen for sequencing. Within the sequences of clones that hybridized in FISH to a single-locus, two large GRRs were identified. The GRRs showed strong and conserved synteny to Glycine max duplicated genome regions, illustrated by both identical gene order and parallel orientation. In contrast, in the clones with dispersed FISH signals, more than one-third of sequences were transposable elements. Sequenced, single-locus clones were used to develop 12 genetic markers, increasing the number of L. angustifolius chromosomes linked to appropriate linkage groups by five pairs. Conclusions In general, probes originating from MFLP sequences can assist genome screening and gene discovery. However, such probes are not useful for positional cloning, because they tend to hybridize to numerous loci. GRRs identified in L. angustifolius contained a low number of interspersed repeats and had a high level of synteny to the genome of the model legume G. max. Our results showed that

  9. BAC libraries construction from the ancestral diploid genomes of the allotetraploid cultivated peanut

    PubMed Central

    Guimarães, Patricia M; Garsmeur, Olivier; Proite, Karina; Leal-Bertioli, Soraya CM; Seijo, Guilhermo; Chaine, Christian; Bertioli, David J; D'Hont, Angelique

    2008-01-01

    Background Cultivated peanut, Arachis hypogaea is an allotetraploid of recent origin, with an AABB genome. In common with many other polyploids, it seems that a severe genetic bottle-neck was imposed at the species origin, via hybridisation of two wild species and spontaneous chromosome duplication. Therefore, the study of the genome of peanut is hampered both by the crop's low genetic diversity and its polyploidy. In contrast to cultivated peanut, most wild Arachis species are diploid with high genetic diversity. The study of diploid Arachis genomes is therefore attractive, both to simplify the construction of genetic and physical maps, and for the isolation and characterization of wild alleles. The most probable wild ancestors of cultivated peanut are A. duranensis and A. ipaënsis with genome types AA and BB respectively. Results We constructed and characterized two large-insert libraries in Bacterial Artificial Chromosome (BAC) vector, one for each of the diploid ancestral species. The libraries (AA and BB) are respectively c. 7.4 and c. 5.3 genome equivalents with low organelle contamination and average insert sizes of 110 and 100 kb. Both libraries were used for the isolation of clones containing genetically mapped legume anchor markers (single copy genes), and resistance gene analogues. Conclusion These diploid BAC libraries are important tools for the isolation of wild alleles conferring resistances to biotic stresses, comparisons of orthologous regions of the AA and BB genomes with each other and with other legume species, and will facilitate the construction of a physical map. PMID:18230166

  10. Characterization of a BAC Library from Channel Catfish Ictalurus punctatus: Indications of High Rates of Evolution Among Teleost Genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The CHORI-212 bacterial artificial chromosome (BAC) library was constructed by cloning EcoRI/EcoRI partially digested DNA into the pTARBAC2.1 vector. The library has an average insert size of 161 kb, and provides 10.6-fold coverage of the channel catfish haploid genome. Screening of 32 genes using o...

  11. The 19 Genomes of Drosophila: A BAC Library Resource for Genus-Wide and Genome-Scale Comparative Evolutionary Research

    PubMed Central

    Song, Xiang; Goicoechea, Jose Luis; Ammiraju, Jetty S. S.; Luo, Meizhong; He, Ruifeng; Lin, Jinke; Lee, So-Jeong; Sisneros, Nicholas; Watts, Tom; Kudrna, David A.; Golser, Wolfgang; Ashley, Elizabeth; Collura, Kristi; Braidotti, Michele; Yu, Yeisoo; Matzkin, Luciano M.; McAllister, Bryant F.; Markow, Therese Ann; Wing, Rod A.

    2011-01-01

    The genus Drosophila has been the subject of intense comparative phylogenomics characterization to provide insights into genome evolution under diverse biological and ecological contexts and to functionally annotate the Drosophila melanogaster genome, a model system for animal and insect genetics. Recent sequencing of 11 additional Drosophila species from various divergence points of the genus is a first step in this direction. However, to fully reap the benefits of this resource, the Drosophila community is faced with two critical needs: i.e., the expansion of genomic resources from a much broader range of phylogenetic diversity and the development of additional resources to aid in finishing the existing draft genomes. To address these needs, we report the first synthesis of a comprehensive set of bacterial artificial chromosome (BAC) resources for 19 Drosophila species from all three subgenera. Ten libraries were derived from the exact source used to generate 10 of the 12 draft genomes, while the rest were generated from a strategically selected set of species on the basis of salient ecological and life history features and their phylogenetic positions. The majority of the new species have at least one sequenced reference genome for immediate comparative benefit. This 19-BAC library set was rigorously characterized and shown to have large insert sizes (125–168 kb), low nonrecombinant clone content (0.3–5.3%), and deep coverage (9.1–42.9×). Further, we demonstrated the utility of this BAC resource for generating physical maps of targeted loci, refining draft sequence assemblies and identifying potential genomic rearrangements across the phylogeny. PMID:21321134

  12. The 19 genomes of Drosophila: a BAC library resource for genus-wide and genome-scale comparative evolutionary research.

    PubMed

    Song, Xiang; Goicoechea, Jose Luis; Ammiraju, Jetty S S; Luo, Meizhong; He, Ruifeng; Lin, Jinke; Lee, So-Jeong; Sisneros, Nicholas; Watts, Tom; Kudrna, David A; Golser, Wolfgang; Ashley, Elizabeth; Collura, Kristi; Braidotti, Michele; Yu, Yeisoo; Matzkin, Luciano M; McAllister, Bryant F; Markow, Therese Ann; Wing, Rod A

    2011-04-01

    The genus Drosophila has been the subject of intense comparative phylogenomics characterization to provide insights into genome evolution under diverse biological and ecological contexts and to functionally annotate the Drosophila melanogaster genome, a model system for animal and insect genetics. Recent sequencing of 11 additional Drosophila species from various divergence points of the genus is a first step in this direction. However, to fully reap the benefits of this resource, the Drosophila community is faced with two critical needs: i.e., the expansion of genomic resources from a much broader range of phylogenetic diversity and the development of additional resources to aid in finishing the existing draft genomes. To address these needs, we report the first synthesis of a comprehensive set of bacterial artificial chromosome (BAC) resources for 19 Drosophila species from all three subgenera. Ten libraries were derived from the exact source used to generate 10 of the 12 draft genomes, while the rest were generated from a strategically selected set of species on the basis of salient ecological and life history features and their phylogenetic positions. The majority of the new species have at least one sequenced reference genome for immediate comparative benefit. This 19-BAC library set was rigorously characterized and shown to have large insert sizes (125-168 kb), low nonrecombinant clone content (0.3-5.3%), and deep coverage (9.1-42.9×). Further, we demonstrated the utility of this BAC resource for generating physical maps of targeted loci, refining draft sequence assemblies and identifying potential genomic rearrangements across the phylogeny.

  13. Human BAC library: construction and rapid screening.

    PubMed

    Asakawa, S; Abe, I; Kudoh, Y; Kishi, N; Wang, Y; Kubota, R; Kudoh, J; Kawasaki, K; Minoshima, S; Shimizu, N

    1997-05-20

    We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96,000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6 x 6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.

  14. Construction of BAC Libraries from Flow-Sorted Chromosomes.

    PubMed

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

    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 genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

  15. Adventures in the Enormous: A 1.8 Million Clone BAC Library for the 21.7 Gb Genome of Loblolly Pine

    PubMed Central

    Magbanua, Zenaida V.; Ozkan, Seval; Bartlett, Benjamin D.; Chouvarine, Philippe; Saski, Christopher A.; Liston, Aaron; Cronn, Richard C.; Nelson, C. Dana; Peterson, Daniel G.

    2011-01-01

    Loblolly pine (LP; Pinus taeda L.) is the most economically important tree in the U.S. and a cornerstone species in southeastern forests. However, genomics research on LP and other conifers has lagged behind studies on flowering plants due, in part, to the large size of conifer genomes. As a means to accelerate conifer genome research, we constructed a BAC library for the LP genotype 7-56. The LP BAC library consists of 1,824,768 individually-archived clones making it the largest single BAC library constructed to date, has a mean insert size of 96 kb, and affords 7.6X coverage of the 21.7 Gb LP genome. To demonstrate the efficacy of the library in gene isolation, we screened macroarrays with overgos designed from a pine EST anchored on LP chromosome 10. A positive BAC was sequenced and found to contain the expected full-length target gene, several gene-like regions, and both known and novel repeats. Macroarray analysis using the retrotransposon IFG-7 (the most abundant repeat in the sequenced BAC) as a probe indicates that IFG-7 is found in roughly 210,557 copies and constitutes about 5.8% or 1.26 Gb of LP nuclear DNA; this DNA quantity is eight times the Arabidopsis genome. In addition to its use in genome characterization and gene isolation as demonstrated herein, the BAC library should hasten whole genome sequencing of LP via next-generation sequencing strategies/technologies and facilitate improvement of trees through molecular breeding and genetic engineering. The library and associated products are distributed by the Clemson University Genomics Institute (www.genome.clemson.edu). PMID:21283709

  16. Physical Analysis of the Complex Rye (Secale cereale L.) Alt4 Aluminium (Aluminum) Tolerance Locus Using a Whole-Genome BAC Library of Rye cv. Blanco

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rye is a diploid crop species with many outstanding qualities, and is also important as a source of new traits for wheat and triticale improvement. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies. The library provides a 6 × genome ...

  17. Construction of a California condor BAC library and first-generation chicken-condor comparative physical map as an endangered species conservation genomics resource.

    PubMed

    Romanov, Michael N; Koriabine, Maxim; Nefedov, Mikhail; de Jong, Pieter J; Ryder, Oliver A

    2006-12-01

    To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing approximately 14-fold coverage of the presumed approximately 1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken-condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8x subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken-condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease.

  18. Construction of genome-wide physical BAC contigs using mapped cDNA as probes: Toward an integrated BAC library resource for genome sequencing and analysis. Annual report, July 1995--January 1997

    SciTech Connect

    Mitchell, S.C.; Bocskai, D.; Cao, Y.

    1997-12-31

    The goal of human genome project is to characterize and sequence entire genomes of human and several model organisms, thus providing complete sets of information on the entire structure of transcribed, regulatory and other functional regions for these organisms. In the past years, a number of useful genetic and physical markers on human and mouse genomes have been made available along with the advent of BAC library resources for these organisms. The advances in technology and resource development made it feasible to efficiently construct genome-wide physical BAC contigs for human and other genomes. Currently, over 30,000 mapped STSs and 27,000 mapped Unigenes are available for human genome mapping. ESTs and cDNAs are excellent resources for building contig maps for two reasons. Firstly, they exist in two alternative forms--as both sequence information for PCR primer pairs, and cDoreen genomic libraries efficiently for large number of DNA probes by combining over 100 cDNA probes in each hybridization. Second, the linkage and order of genes are rather conserved among human, mouse and other model organisms. Therefore, gene markers have advantages over random anonymous STSs in building maps for comparative genomic studies.

  19. 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.

    PubMed

    Ammiraju, Jetty S S; Luo, Meizhong; Goicoechea, José L; Wang, Wenming; Kudrna, Dave; Mueller, Christopher; Talag, Jayson; Kim, HyeRan; Sisneros, Nicholas B; Blackmon, Barbara; Fang, Eric; Tomkins, Jeffery B; Brar, Darshan; MacKill, David; McCouch, Susan; Kurata, Nori; Lambert, Georgina; Galbraith, David W; Arumuganathan, K; Rao, Kiran; Walling, Jason G; Gill, Navdeep; Yu, Yeisoo; SanMiguel, Phillip; Soderlund, Carol; Jackson, Scott; Wing, Rod A

    2006-01-01

    Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.

  20. A HindIII BAC library construction of Mesobuthus martensii Karsch (Scorpiones:Buthidae): an important genetic resource for comparative genomics and phylogenetic analysis.

    PubMed

    Li, Songryong; Ma, Yibao; Jang, Shenghun; Wu, Yingliang; Liu, Hui; Cao, Zhijian; Li, Wenxin

    2009-12-01

    Scorpions are "living but sophisticated fossils" that have changed little in their morphology since their first appearance over the past 450 million years ago. To provide a genetic resource for understanding the evolution of scorpion genome and the relationships between scorpions and other organisms, we first determined the genome size of the scorpion Mesobuthus martensii Karsch (about 600 Mbp) in the order Scorpiones and constructed a HindIII BAC library of the male scorpion M. martensii Karsch from China. The BAC library consists of a total of 46,080 clones with an average insert size of 100 kb, providing a 7.7-fold coverage of the scorpion haploid genome size of 600 Mbp as revealed in this study. High-density colony hybridization-based library screening was performed using 18S-5.8S-28S rRNA gene that is one of the most commonly used phylogenetic markers. Both library screening and PCR identification results revealed six positive BAC clones which were overlapped, and formed a contig of approximately 120 kb covering the rDNA. BAC DNA sequencing analysis determined the complete sequence of M. martensii Karsch rDNA unit that has a total length of 8779 bp, including 1813 bp 18s rDNA, 157 bp 5.8s rDNA, 3823 bp 28s rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2. Interestingly, some tandem repeats are present in the rRNA intergenic sequence (IGS) and ITS1/2 regions. These results demonstrated that the BAC library of the scorpion M. martensii Karsch and the complete sequence of rDNA unit will provide important genetic resources and tools for comparative genomics and phylogenetic analysis.

  1. Physical analysis of the complex rye (Secale cereale L.) Alt4 aluminium (aluminum) tolerance locus using a whole-genome BAC library of rye cv. Blanco.

    PubMed

    Shi, B-J; Gustafson, J P; Button, J; Miyazaki, J; Pallotta, M; Gustafson, N; Zhou, H; Langridge, P; Collins, N C

    2009-08-01

    Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library's suitability for investigating Al tolerance genes. The library provides 6 x genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus.

  2. Preparation of high molecular weight gDNA and bacterial artificial chromosome (BAC) libraries in plants.

    PubMed

    Biradar, Siddanagouda S; Nie, Xiaojun; Feng, Kewei; Weining, Song

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable large-insert DNA libraries for physical mapping, positional cloning, comparative genomic analysis, complete genome sequencing, and evolutionary studies. Due to their stability and relative simplicity BAC libraries are most preferred over other approaches for cloning large genomic DNA fragments for large-insert libraries. Isolation of intact high molecular weight (HMW) DNA is a critical step underlying the success of large-insert genomic DNA library construction. It requires the isolation of purified nuclei, embedding them into LMP agarose plugs, restriction digestion of the plugs, and quite often size selection using PFGE and electro-elution of insert DNA. The construction of BAC libraries is complex and challenging for most molecular laboratories. To facilitate the construction of BAC libraries, we present a step-by-step protocol for isolation of HMW DNA and construction of plant BAC libraries.

  3. Library Resources for Bac End Sequencing. Final Technical Report

    SciTech Connect

    Pieter J. de Jong

    2000-10-01

    Studies directed towards the specific aims outlined for this research award are summarized. The RPCI II Human Bac Library has been expanded by the addition of 6.9-fold genomic coverage. This segment has been generated from a MBOI partial digest of the same anonymous donor DNA used for the rest of the library. A new cloning vector, pTARBAC1, has been constructed and used in the construction of RPCI-II segment 5. This new cloning vector provides a new strategy in identifying targeted genomic regions and will greatly facilitate a large-scale analysis for positional cloning. A new maleCS7BC/6J mouse BAC library has been constructed. RPCI-23 contain 576 plates (approx 210,000 clones) and represents approximately 11-fold coverage of the mouse genome.

  4. Generation of BAC-end sequences for rainbow trout genome analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For non-sequenced genomes, BAC end sequences (BES) provide a valuable sample of repetitive elements and gene content. Here we report the results of BAC end sequencing of just over half of the rainbow trout (Oncorhynchus mykiss) Swanson HindIII library. We sequenced 177,860 BAC ends that generated 17...

  5. Characterizing the walnut genome through analyses of BAC end sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots...

  6. End Sequencing and Finger Printing of Human & Mouse BAC Libraries

    SciTech Connect

    Fraser, C

    2005-09-27

    This project provided for continued end sequencing of existing and new BAC libraries constructed to support human sequencing as well as to initiate BAC end sequencing from the mouse BAC libraries constructed to support mouse sequencing. The clones, the sequences, and the fingerprints are now an available resource for the community at large. Research and development of new metaodologies for BAC end sequencing have reduced costs and increase throughput.

  7. Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries.

    PubMed

    Kakirde, Kavita S; Wild, Jadwiga; Godiska, Ronald; Mead, David A; Wiggins, Andrew G; Goodman, Robert M; Szybalski, Waclaw; Liles, Mark R

    2011-04-15

    Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The "Gram-negative shuttle BAC" vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication in diverse Gram-negative bacteria. As with other BAC vectors containing the oriV origin, this vector is capable of an arabinose-inducible increase in plasmid copy number. Resistance to both gentamicin and chloramphenicol is encoded on pGNS-BAC, permitting selection for the plasmid in diverse bacterial species. The oriT from an IncP plasmid was cloned into pGNS-BAC to enable conjugal transfer, thereby allowing both electroporation and conjugation of pGNS-BAC DNA into bacterial hosts. A soil metagenomic library was constructed in pGNS-BAC-1 (the first version of the vector, lacking gentamicin resistance and oriT), and recombinant clones were demonstrated to replicate in diverse Gram-negative hosts, including Escherichia coli, Pseudomonas spp., Salmonella enterica, Serratia marcescens, Vibrio vulnificus and Enterobacter nimipressuralis. This shuttle BAC vector can be utilized to clone genomic DNA from diverse sources, and then transfer it into diverse Gram-negative bacterial species to facilitate heterologous expression of recombinant pathways.

  8. Non-gridded library: a new approach for BAC (bacterial artificial chromosome) exploitation in hexaploid wheat (Triticum aestivum).

    PubMed

    Ma, Z; Weining, S; Sharp, P J; Liu, C

    2000-12-15

    The feasibility of exploiting non-gridded bacterial artificial chromosome (BAC) libraries and some major factors affecting the efficiency of handling such libraries were studied in hexaploid wheat. Even for a bacterial culture containing only 55% recombinants, some 2000 BAC clones with inserts ranging from 45 to 245 kb could be pooled. The pooled BAC clones could be amplified by culturing for up to 6 h without losing any target clones. These results imply that even for hexaploid wheat, which has an extremely large genome, some 250 pools are sufficient for a BAC library that should satisfy many research objectives. This non-gridded strategy would dramatically reduce the cost and make robotic equipment non-essential in exploiting BAC technology. To construct a representative library and to minimise clone competition, thawing and re-freezing ligation mixtures and bacterial cultures should be avoided in BAC library construction and application.

  9. BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.)

    PubMed Central

    2011-01-01

    Background 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 generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X. Conclusion BAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing. PMID:21595870

  10. Korean BAC library construction and characterization of HLA-DRA, HLA-DRB3.

    PubMed

    Park, Mi-Hyun; Lee, Hye-Ja; Bok, Jeong; Kim, Cheol-Hwan; Hong, Seong-Tshool; Park, Chan; Kimm, KuChan; Oh, Bermseok; Lee, Jong-Young

    2006-07-31

    A human bacterial artificial chromosome (BAC) library was constructed with high molecular weight DNA extracted from the blood of a male Korean. This Korean BAC library contains 100,224 clones of insert size ranging from 70 to 150 kb, with an average size of 86 kb, corresponding to a 2.9-fold redundancy of the genome. The average insert size was determined from 288 randomly selected BAC clones that were well distributed among all the chromosomes. We developed a pooling system and three-step PCR screen for the Korean BAC library to isolate desired BAC clones, and we confirmed its utility using primer pairs designed for one of the clones. The Korean BAC library and screening pools will allow PCR-based screening of the Korean genome for any gene of interest. We also determined the allele types of HLA-DRA and HLA-DRB3 of clone KB55453, located in the HLA class II region on chromosome 6p21.3. The HLA-DRA and DRB3 genes in this clone were identified as the DRA*010202 and DRB3*01010201 types, respectively. The haplotype found in this library will provide useful information in future human disease studies.

  11. Construction and characterization of human and mouse BAC libraries from sheared DNA

    SciTech Connect

    Shizuya, Hiroaki

    2002-08-23

    We have developed a new way to construct BAC libraries with small inserts using sheared DNA sources. Because of our use of the randomly sheared DNA as DNA sources, some regions of genome may be represented better in our libraries compared to the currently available and more conventional libraries constructed by enzymatic partial digestion. B263 We have developed a new fingerprinting method useful for physical mapping by large insert clones, in particular by BACs. It is based on four-color fluorescent labeling of fragments generated by combination of a type II and a type IIS restriction enzyme.

  12. Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

    SciTech Connect

    Jang Songhun; Zhou Fang; Xia Laixin; Zhao Wei; Cheng Hanhua . E-mail: hhcheng@whu.edu.cn; Zhou Rongjia . E-mail: rjzhou@whu.edu.cn

    2006-09-22

    A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes.

  13. A BAC based physical map and genome survey of the rice false smut fungus Villosiclava virens

    PubMed Central

    2013-01-01

    Background Rice false smut caused by Villosiclava virens is a devastating fungal disease that spreads in major rice-growing regions throughout the world. However, the genomic information for this fungal pathogen is limited and the pathogenic mechanism of this disease is still not clear. To facilitate genetic, molecular and genomic studies of this fungal pathogen, we constructed the first BAC-based physical map and performed the first genome survey for this species. Results High molecular weight genomic DNA was isolated from young mycelia of the Villosiclava virens strain UV-8b and a high-quality, large-insert and deep-coverage Bacterial Artificial Chromosome (BAC) library was constructed with the restriction enzyme HindIII. The BAC library consisted of 5,760 clones, which covers 22.7-fold of the UV-8b genome, with an average insert size of 140 kb and an empty clone rate of lower than 1%. BAC fingerprinting generated successful fingerprints for 2,290 BAC clones. Using the fingerprints, a whole genome-wide BAC physical map was constructed that contained 194 contigs (2,035 clones) spanning 51.2 Mb in physical length. Bidirectional-end sequencing of 4,512 BAC clones generated 6,560 high quality BAC end sequences (BESs), with a total length of 3,030,658 bp, representing 8.54% of the genome sequence. Analysis of the BESs revealed general genome information, including 51.52% GC content, 22.51% repetitive sequences, 376.12/Mb simple sequence repeat (SSR) density and approximately 36.01% coding regions. Sequence comparisons to other available fungal genome sequences through BESs showed high similarities to Metarhizium anisopliae, Trichoderma reesei, Nectria haematococca and Cordyceps militaris, which were generally in agreement with the 18S rRNA gene analysis results. Conclusion This study provides the first BAC-based physical map and genome information for the important rice fungal pathogen Villosiclava virens. The BAC clones, physical map and genome information will

  14. Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome.

    PubMed

    Krzywinski, Martin; Wallis, John; Gösele, Claudia; Bosdet, Ian; Chiu, Readman; Graves, Tina; Hummel, Oliver; Layman, Dan; Mathewson, Carrie; Wye, Natasja; Zhu, Baoli; Albracht, Derek; Asano, Jennifer; Barber, Sarah; Brown-John, Mabel; Chan, Susanna; Chand, Steve; Cloutier, Alison; Davito, Jonathon; Fjell, Chris; Gaige, Tony; Ganten, Detlev; Girn, Noreen; Guggenheimer, Kurtis; Himmelbauer, Heinz; Kreitler, Thomas; Leach, Stephen; Lee, Darlene; Lehrach, Hans; Mayo, Michael; Mead, Kelly; Olson, Teika; Pandoh, Pawan; Prabhu, Anna-Liisa; Shin, Heesun; Tänzer, Simone; Thompson, Jason; Tsai, Miranda; Walker, Jason; Yang, George; Sekhon, Mandeep; Hillier, LaDeana; Zimdahl, Heike; Marziali, Andre; Osoegawa, Kazutoyo; Zhao, Shaying; Siddiqui, Asim; de Jong, Pieter J; Warren, Wes; Mardis, Elaine; McPherson, John D; Wilson, Richard; Hübner, Norbert; Jones, Steven; Marra, Marco; Schein, Jacqueline

    2004-04-01

    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 rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

  15. Versatile P(acman) BAC Libraries for Transgenesis Studies in Drosophila melanogaster

    SciTech Connect

    Venken, Koen J.T.; Carlson, Joseph W.; Schulze, Karen L.; Pan, Hongling; He, Yuchun; Spokony, Rebecca; Wan, Kenneth H.; Koriabine, Maxim; de Jong, Pieter J.; White, Kevin P.; Bellen, Hugo J.; Hoskins, Roger A.

    2009-04-21

    We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95percent of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using Phi C31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns.

  16. Versatile P(acman) BAC Libraries for Transgenesis Studies in Drosophila melanogaster

    PubMed Central

    Venken, Koen J. T.; Carlson, Joseph W.; Schulze, Karen L.; Pan, Hongling; He, Yuchun; Spokony, Rebecca; Wan, Kenneth H.; Koriabine, Maxim; de Jong, Pieter J.; White, Kevin P.; Bellen, Hugo J.; Hoskins, Roger A.

    2009-01-01

    We constructed Drosophila melanogaster BAC libraries with 21-kb and 83-kb inserts in the P(acman) system. Clones representing 12-fold coverage and encompassing more than 95% of annotated genes were mapped onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using ΦC31 integrase to rescue mutations. They can be modified through recombineering, for example to incorporate protein tags and assess expression patterns. PMID:19465919

  17. A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization.

    PubMed

    Chung, Yeun-Jun; Jonkers, Jos; Kitson, Hannah; Fiegler, Heike; Humphray, Sean; Scott, Carol; Hunt, Sarah; Yu, Yuejin; Nishijima, Ichiko; Velds, Arno; Holstege, Henne; Carter, Nigel; Bradley, Allan

    2004-01-01

    Microarray-based comparative genomic hybridization (CGH) has become a powerful method for the genome-wide detection of chromosomal imbalances. Although BAC microarrays have been used for mouse CGH studies, the resolving power of these analyses was limited because high-density whole-genome mouse BAC microarrays were not available. We therefore developed a mouse BAC microarray containing 2803 unique BAC clones from mouse genomic libraries at 1-Mb intervals. For the general amplification of BAC clone DNA prior to spotting, we designed a set of three novel degenerate oligonucleotide-primed (DOP) PCR primers that preferentially amplify mouse genomic sequences while minimizing unwanted amplification of contaminating Escherichia coli DNA. The resulting 3K mouse BAC microarrays reproducibly identified DNA copy number alterations in cell lines and primary tumors, such as single-copy deletions, regional amplifications, and aneuploidy.

  18. Sequencing the Pig Genome Using a Mapped BAC by BAC Approach

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have generated a highly contiguous physical map covering >98% of the pig genome in just 176 contigs. The map is localised to the genome through integration with the UIUC RH map as well BAC end sequence alignments to the human genome. Over 265k HindIII restriction digest fingerprints totalling 1...

  19. A BAC-based physical map of the Drosophila buzzatii genome

    SciTech Connect

    Gonzalez, Josefa; Nefedov, Michael; Bosdet, Ian; Casals, Ferran; Calvete, Oriol; Delprat, Alejandra; Shin, Heesun; Chiu, Readman; Mathewson, Carrie; Wye, Natasja; Hoskins, Roger A.; Schein, JacquelineE.; de Jong, Pieter; Ruiz, Alfredo

    2005-03-18

    Large-insert genomic libraries facilitate cloning of large genomic regions, allow the construction of clone-based physical maps and provide useful resources for sequencing entire genomes. Drosophilabuzzatii is a representative species of the repleta group in the Drosophila subgenus, which is being widely used as a model in studies of genome evolution, ecological adaptation and speciation. We constructed a Bacterial Artificial Chromosome (BAC) genomic library of D. buzzatii using the shuttle vector pTARBAC2.1. The library comprises 18,353 clones with an average insert size of 152 kb and a {approx}18X expected representation of the D. buzzatii euchromatic genome. We screened the entire library with six euchromatic gene probes and estimated the actual genome representation to be {approx}23X. In addition, we fingerprinted by restriction digestion and agarose gel electrophoresis a sample of 9,555 clones, and assembled them using Finger Printed Contigs (FPC) software and manual editing into 345 contigs (mean of 26 clones per contig) and 670singletons. Finally, we anchored 181 large contigs (containing 7,788clones) to the D. buzzatii salivary gland polytene chromosomes by in situ hybridization of 427 representative clones. The BAC library and a database with all the information regarding the high coverage BAC-based physical map described in this paper are available to the research community.

  20. piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice.

    PubMed

    Xu, Chunlong; Qi, Xiaolan; Du, Xuguang; Zou, Huiying; Gao, Fei; Feng, Tao; Lu, Hengxing; Li, Shenglan; An, Xiaomeng; Zhang, Lijun; Wu, Yuanyuan; Liu, Ying; Li, Ning; Capecchi, Mario R; Wu, Sen

    2017-01-24

    CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.

  1. Construction and characterization of a bacterial artificial chromosome (BAC) library of Pacific white shrimp, Litopenaeus vannamei.

    PubMed

    Zhang, Xiaojun; Zhang, Yang; Scheuring, Chantel; Zhang, Hong-Bin; Huan, Pin; Wang, Bing; Liu, Chengzhang; Li, Fuhua; Liu, Bin; Xiang, Jianhai

    2010-04-01

    The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species.

  2. BACs as tools for the study of genomic imprinting.

    PubMed

    Tunster, S J; Van De Pette, M; John, R M

    2011-01-01

    Genomic imprinting in mammals results in the expression of genes from only one parental allele. Imprinting occurs as a consequence of epigenetic marks set down either in the father's or the mother's germ line and affects a very specific category of mammalian gene. A greater understanding of this distinctive phenomenon can be gained from studies using large genomic clones, called bacterial artificial chromosomes (BACs). Here, we review the important applications of BACs to imprinting research, covering physical mapping studies and the use of BACs as transgenes in mice to study gene expression patterns, to identify imprinting centres, and to isolate the consequences of altered gene dosage. We also highlight the significant and unique advantages that rapid BAC engineering brings to genomic imprinting research.

  3. Construction and characterisation of a BAC library made from field specimens of the onchocerciasis vector Simulium squamosum (Diptera: Simuliidae).

    PubMed

    Crainey, J L; Hurst, J; Wilson, M D; Hall, A; Post, R J

    2010-10-01

    A Bacterial Artificial Chromosome (BAC) library was made from wild-caught Simulium squamosum, which is an important vector of human onchocerciasis. The library is composed of 12,288 BACs, with an average insert size of 128 kb, and is expected to contain ~1.54 GB of cloned DNA. Random BAC-end sequencing generated over 95 kb of DNA sequence data from which putative S. squamosum gene sequences and novel repetitive DNA families were identified, including DNA transposons, retrotransposons and simple sequence repeats (SSRs). The sequence survey also provided evidence of DNA of microbial origin, and dissection of sample blackflies indicated that some of those used to prepare the library were likely to be parasitized by the mermithid Isomermis lairdi. Hybridisations with a set of three independent blackfly single-copy genes and two Wolbachia genes suggest that the library provides around 13-fold coverage of the S. squamosum genome and about 12-fold coverage of its Wolbachia endosymbiont.

  4. Construction and characterization of two Citrus BAC libraries and identification of clones containing the phytoene synthase gene.

    PubMed

    Baig, M N R; Yu, An; Guo, Wenwu; Deng, Xiuxin

    2009-05-01

    Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 2.5.1.32)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

  5. BAC library development, and clone characterization for dormancy-responsive DREB4A, DAM, and FT from leafy spurge (Euphorbia esula L.) identifies differential splicing and conserved promoter motifs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed two leafy spurge BAC libraries that together represent approximately 5X coverage of the leafy spurge genome. The BAC libraries have an average insert size of approximately 143 kb, and copies of the library and filters for hybridization-based screening are publicly available through the ...

  6. Cross-species bacterial artificial chromosome (BAC) library screening via overgo-based hybridization and BAC-contig mapping of a yield enhancement quantitative trait locus (QTL) yld1.1 in the Malaysian wild rice Oryza rufipogon.

    PubMed

    Song, Beng-Kah; Nadarajah, Kalaivani; Romanov, Michael N; Ratnam, Wickneswari

    2005-01-01

    The construction of BAC-contig physical maps is an important step towards a partial or ultimate genome sequence analysis. Here, we describe our initial efforts to apply an overgo approach to screen a BAC library of the Malaysian wild rice species, Oryza rufipogon. Overgo design is based on repetitive element masking and sequence uniqueness, and uses short probes (approximately 40 bp), making this method highly efficient and specific. Pairs of 24-bp oligos that contain an 8-bp overlap were developed from the publicly available genomic sequences of the cultivated rice, O. sativa, to generate 20 overgo probes for a 1-Mb region that encompasses a yield enhancement QTL yld1.1 in O. rufipogon. The advantages of a high similarity in melting temperature, hybridization kinetics and specific activities of overgos further enabled a pooling strategy for library screening by filter hybridization. Two pools of ten overgos each were hybridized to high-density filters representing the O. rufipogon genomic BAC library. These screening tests succeeded in providing 69 PCR-verified positive hits from a total of 23,040 BAC clones of the entire O. rufipogon library. A minimal tilling path of clones was generated to contribute to a fully covered BAC-contig map of the targeted 1-Mb region. The developed protocol for overgo design based on O. sativa sequences as a comparative genomic framework, and the pooled overgo hybridization screening technique are suitable means for high-resolution physical mapping and the identification of BAC candidates for sequencing.

  7. Construction and application of a bacterial artificial chromosome (BAC) library of Prunus armeniaca L. for the identification of clones linked to the self-incompatibility locus.

    PubMed

    Vilanova, S; Romero, C; Abernathy, D; Abbott, A G; Burgos, L; Llacer, G; Badenes, M L

    2003-08-01

    To facilitate gene discovery in the Rosaceae, a bacterial artificial chromosome (BAC) library was constructed using high-molecular-weight (HMW) DNA from apricot leaves (Prunus armeniaca L.). The library contains 101,376 clones (264, 384-well plates) with an average insert size of 64 kb, equivalent to 22-fold genome coverage. In the first application of this library, high-density filters were screened for self-incompatibility genes using apricot DNA probes. Eight positive BAC clones were detected and fingerprinted to determine clone relationships and assemble contigs. These results demonstrate the suitability of this library for gene identification and physical mapping of the apricot genome.

  8. Construction of a quinoa (Chenopodium quinoa Willd.) BAC library and its use in identifying genes encoding seed storage proteins.

    PubMed

    Stevens, M R; Coleman, C E; Parkinson, S E; Maughan, P J; Zhang, H-B; Balzotti, M R; Kooyman, D L; Arumuganathan, K; Bonifacio, A; Fairbanks, D J; Jellen, E N; Stevens, J J

    2006-05-01

    Quinoa (Chenopodium quinoa Willd.) is adapted to the harsh environments of the Andean Altiplano region. Its seeds have a well-balanced amino acid composition and exceptionally high protein content with respect to human nutrition. Quinoa grain is a staple in the diet of some of the most impoverished people in the world. The plant is an allotetraploid displaying disomic inheritance (2n=4x=36) with a di-haploid genome of 967 Mbp (megabase pair), or 2C=2.01 pg. We constructed two quinoa BAC libraries using BamHI (26,880 clones) and EcoRI (48,000 clones) restriction endonucleases. Cloned inserts in the BamHI library average 113 kb (kilobase) with approximately 2% of the clones lacking inserts, whereas cloned inserts in the EcoRI library average 130 kb and approximately 1% lack inserts. Three plastid genes used as probes of high-density arrayed blots of 73,728 BACs identified approximately 2.8% of the clones as containing plastid DNA inserts. We estimate that the combined quinoa libraries represent at least 9.0 di-haploid nuclear genome equivalents. An average of 12.2 positive clones per probe were identified with 13 quinoa single-copy ESTs as probes of the high-density arrayed blots, suggesting that the estimate of 9.0x coverage of the genome is conservative. Utility of the BAC libraries for gene identification was demonstrated by probing the library with a partial sequence of the 11S globulin seed storage protein gene and identifying multiple positive clones. The presence of the 11S globulin gene in four of the clones was verified by direct comparison with quinoa genomic DNA on a Southern blot. Besides serving as a useful tool for gene identification, the quinoa BAC libraries will be an important resource for physical mapping of the quinoa genome.

  9. Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones.

    PubMed

    Wild, Jadwiga; Hradecna, Zdenka; Szybalski, Waclaw

    2002-09-01

    The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ~100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P(araBAD) promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome

  10. Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

    PubMed

    Ye, Qing; He, Ke; Wu, Shao-Ying; Wan, Qiu-Hong

    2012-01-01

    The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

  11. Isolation of a 97-kb Minimal Essential MHC B Locus from a New Reverse-4D BAC Library of the Golden Pheasant

    PubMed Central

    Wu, Shao-Ying; Wan, Qiu-Hong

    2012-01-01

    The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC. PMID:22403630

  12. Structural analysis of the eukaryotic initiation factor 4E gene controlling potyvirus resistance in pepper: exploitation of a BAC library.

    PubMed

    Ruffel, Sandrine; Caranta, Carole; Palloix, Alain; Lefebvre, Véronique; Caboche, Michel; Bendahmane, Abdelhafid

    2004-09-01

    The pvr2 locus in pepper codes for a eukaryotic translation initiation factor 4E (eIF4E) gene that confers resistance to viruses belonging to the potyvirus genus. In this work, we describe the isolation and characterisation of the genomic sequence carrying the pvr2 locus. A Bacterial Artificial Chromosome (BAC) library that consisted of 239,232 clones with an average insert size of 123 kilobases (kb) was constructed from a Capsicum annuum line with the pvr2(+) allele for susceptibility to potato virus Y (PVY) and tobacco etch virus (TEV). Based on a polymerase chain reaction (PCR) screen with single-copy markers, three to seven positive BAC clones per markers were identified, indicating that the BAC library is suitable for pepper genome analysis. To determine the genomic organization of the pepper eIF4E gene, the library was screened with primers designed from the cDNA sequence and four positive BAC clones carrying the pvr2 locus were identified. A 7-kb DNA fragment containing the complete eIF4E gene was sub-cloned from the positive BAC clones and analysed. The eIF4E gene is organised into five exons and four introns and showed a strictly conserved exon/intron structure with eIF4E genes from Arabidopsis thaliana and rice. Moreover, the splice sites between plant exons 1/2 and 2/3 are conserved among eukaryotes including human, Drosophila and yeast. Several potential binding sites for MADS box transcription factors within the 5' flanking region of eIF4E genes from the three plant species were also predicted.

  13. Bac clones generated from sheared dna

    SciTech Connect

    Osoegawa, Kazutoyo; Vessere, Gery M.; Shu, Chung Li; Hoskins,Roger A.; Abad, Jose P.; de Pablos, Beatriz; Villasante, Alfredo; deJong, Pieter J.

    2006-08-09

    BAC libraries generated from restriction-digested genomic DNA display representational bias and lack some sequences. To facilitate completion of genome projects, procedures have been developed to create BACs from DNA physically sheared to create fragments extending up to 200kb. The DNA fragments were repaired to create blunt ends and ligated to a new BAC vector. This approach has been tested by generating BAC libraries from Drosophila DNA, with insert lengths of 50 kb to 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences of one library to the D. melanogaster genome sequence. The utility of ''sheared'' libraries was demonstrated by closure of a previous clone gap and by isolation of clones from telomeric regions, which were notably absent from previous Drosophila BAC libraries.

  14. Genomic libraries: I. Construction and screening of fosmid genomic libraries.

    PubMed

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Large insert genome libraries have been a core resource required to sequence genomes, analyze haplotypes, and aid gene discovery. While next generation sequencing technologies are revolutionizing the field of genomics, traditional genome libraries will still be required for accurate genome assembly. Their utility is also being extended to functional studies for understanding DNA regulatory elements. Here, we present a detailed method for constructing genomic fosmid libraries, testing for common contaminants, gridding the library to nylon membranes, then hybridizing the library membranes with a radiolabeled probe to identify corresponding genomic clones. While this chapter focuses on fosmid libraries, many of these steps can also be applied to bacterial artificial chromosome libraries.

  15. BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

    NASA Astrophysics Data System (ADS)

    Zhao, Cui; Zhang, Xiaojun; Liu, Chengzhang; Huan, Pin; Li, Fuhua; Xiang, Jianhai; Huang, Chao

    2012-05-01

    Little is known about the genome of Pacific white shrimp ( Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 pairedends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

  16. A genome-wide BAC end-sequence survey of sugarcane elucidates genome composition, and identifies BACs covering much of the euchromatin.

    PubMed

    Kim, Changsoo; Lee, Tae-Ho; Compton, Rosana O; Robertson, Jon S; Pierce, Gary J; Paterson, Andrew H

    2013-01-01

    BAC-end sequences (BESs) of hybrid sugarcane cultivar R570 are presented. A total of 66,990 informative BESs were obtained from 43,874 BAC clones. Similarity search using a variety of public databases revealed that 13.5 and 42.8 % of BESs match known gene-coding and repeat regions, respectively. That 11.7 % of BESs are still unmatched to any nucleotide sequences in the current public databases despite the fact that a close relative, sorghum, is fully sequenced, indicates that there may be many sugarcane-specific or lineage-specific sequences. We found 1,742 simple sequence repeat motifs in 1,585 BESs, spanning 27,383 bp in length. As simple sequence repeat markers derived from BESs have some advantages over randomly generated markers, these may be particularly useful for comparing BAC-based physical maps with genetic maps. BES and overgo hybridization information was used for anchoring sugarcane BAC clones to the sorghum genome sequence. While sorghum and sugarcane have extensive similarity in terms of genomic structure, only 2,789 BACs (6.4 %) could be confidently anchored to the sorghum genome at the stringent threshold of having both-end information (BESs or overgos) within 300 Kb. This relatively low rate of anchoring may have been caused in part by small- or large-scale genomic rearrangements in the Saccharum genus after two rounds of whole genome duplication since its divergence from the sorghum lineage about 7.8 million years ago. Limiting consideration to only low-copy matches, 1,245 BACs were placed to 1,503 locations, covering ~198 Mb of the sorghum genome or about 78 % of the estimated 252 Mb of euchromatin. BESs and their analyses presented here may provide an early profile of the sugarcane genome as well as a basis for BAC-by-BAC sequencing of much of the basic gene set of sugarcane.

  17. Development of four phylogenetically-arrayed BAC libraries and sequence of the APA locus in Phaseolus vulgaris.

    PubMed

    Kami, James; Poncet, Valérie; Geffroy, Valérie; Gepts, Paul

    2006-04-01

    The APA family of seed proteins consists of three subfamilies, in evolutionary order of hypothesized appearance: phytohaemagglutinins (PHA), alpha-amylase inhibitors (alphaAI), and arcelins (ARL). The APA family plays a defensive role against mammalian and insect seed predation in common bean (Phaseolus vulgaris L.). The main locus (APA) for this gene family is situated on linkage group B4. In order to elucidate the pattern of duplication and diversification at this locus, we developed a BAC library in each of four different Phaseolus genotypes that represent presumptive steps in the evolutionary diversification of the APA family. Specifically, BAC libraries were established in one P. lunatus (cv. 'Henderson: PHA+ alphaAI- ARL-) and three P. vulgaris accessions (presumed ancestral wild G21245 from northern Peru: PHA+ alphaAI+ ARL-; Mesoamerican wild G02771: PHA+ alphaAI+ ARL+; and Mesoamerican breeding line BAT93: PHA+ alphaAI+ ARL-). The libraries were constructed after HindIII digestion of high molecular weight DNA, obtained with a novel nuclei isolation procedure. The frequency of empty or cpDNA-sequence-containing clones in all libraries is low (generally <1%). The Henderson, G21245, and G02771 libraries have a 10x genome coverage, whereas the BAT93 library has a 20x coverage to allow further, more detailed genomic analysis of the bean genome. The complete sequence of a 155 kbp-insert clone of the G02771 library revealed six sequences belonging to the APA gene family, including members of the three subfamilies, as hypothesized. The different subfamilies were interspersed with retrotransposon sequences. In addition, other sequences were identified with similarity to chloroplast DNA, a dehydrin gene, and the Arabidopsis flowering D locus. Linkage between the dehydrin gene and the D1711 RFLP marker identifies a potential syntenic region between parts of common bean linkage group B4 and cowpea linkage group 2.

  18. Construction and analysis of Siberian tiger bacterial artificial chromosome library with approximately 6.5-fold genome equivalent coverage.

    PubMed

    Liu, Changqing; Bai, Chunyu; Guo, Yu; Liu, Dan; Lu, Taofeng; Li, Xiangchen; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

    2014-03-07

    Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger.

  19. A FISH approach for mapping the human genome using Bacterial Artificial Chromosomes (BACs)

    SciTech Connect

    Hubert, R.S.; Chen, X.N.; Mitchell, S.

    1994-09-01

    As the Human Genome Project progresses, large insert cloning vectors such as BACs, P1, and P1 Artificial Chromosomes (PACs) will be required to complement the YAC mapping efforts. The value of the BAC vector for physical mapping lies in the stability of the inserts, the lack of chimerism, the length of inserts (up to 300 kb), the ability to obtain large amounts of pure clone DNA and the ease of BAC manipulation. These features helped us design two approaches for generating physical mapping reagents for human genetic studies. The first approach is a whole genome strategy in which randomly selected BACs are mapped, using FISH, to specific chromosomal bands. To date, 700 BACs have been mapped to single chromosome bands at a resolution of 2-5 Mb in addition to BACs mapped to 14 different centromeres. These BACs represent more than 90 Mb of the genome and include >70% of all human chromosome bands at the 350-band level. These data revealed that >97% of the BACs were non-chimeric and have a genomic distribution covering most gaps in the existing YAC map with excellent coverage of gene-rich regions. In the second approach, we used YACs to identify BACs on chromosome 21. A 1.5 Mb contig between D21S339 and D21S220 nears completion within the Down syndrome congenital heart disease (DS-CHD) region. Seventeen BACs ranging in size from 80 kb to 240 kb were ordered using 14 STSs with FISH confirmation. We have also used 40 YACs spanning 21q to identify, on average, >1 BAC/Mb to provide molecular cytogenetic reagents and anchor points for further mapping. The contig generated on chromosome 21 will be helpful in isolating the genes for DS-CHD. The physical mapping reagents generated using the whole genome approach will provide cytogenetic markers and mapped genomic fragments that will facilitate positional cloning efforts and the identification of genes within most chromosomal bands.

  20. Development of cell lines from the sheep used to construct the CHORI-243 ovine BAC library

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two cell lines, designated MARC.OVSM and MARC.OKF, were initiated from the aorta and kidney, respectively, obtained from the Texel ram used to make the CHORI-243 Ovine BAC library. These cell lines have been submitted to the NIA Aging Cell Repository at the Coriell Cell Respositories, Camden, NJ, U...

  1. Complete genomic sequence and an infectious BAC clone of feline herpesvirus-1 (FHV-1).

    PubMed

    Tai, S H Sheldon; Niikura, Masahiro; Cheng, Hans H; Kruger, John M; Wise, Annabel G; Maes, Roger K

    2010-06-05

    Infection with feline herpesvirus-1 (FHV-1) is a major cause of upper respiratory and ocular diseases in Felidae. We report the first complete genomic sequence of FHV-1, as well as the construction and characterization of a bacterial artificial chromosome (BAC) clone of FHV-1, which contains the entire FHV-1 genome and has the BAC vector inserted at the left end of U(L). Complete genomic sequences were derived from both the FHV-1 BAC clone and purified virion DNA. The FHV-1 genome is 135,797bp in size with an overall G+C content of 45%. A total of 78 open reading frames were predicted, encoding 74 distinct proteins. The gene arrangement is collinear with that of most sequenced varicelloviruses. The virus regenerated from the BAC was very similar to the parental C-27 strain in vitro in terms of plaque morphology and growth characteristics and highly virulent in cats in a preliminary in vivo study.

  2. Association and in silico assignment of sequences from turkey BACs.

    PubMed

    Reed, Kent M; Faile, Gretchen M; Kreuth, Stacy B; Chaves, Lee D; Sullivan, Laura M

    2008-01-01

    Bacterial artificial chromosomes (BACs) provide an important resource in genetic mapping. An initial set of BACs corresponding to microsatellite markers in the turkey (Meleagris gallopavo) was isolated from the CHORI-260 turkey BAC library. The selected markers were distributed on both macro- and microchromosomes and included a genetically unlinked marker. End sequences were obtained for a subset of the recovered BACs and compared to the chicken whole genome sequence. Close association of the turkey BAC-end sequences and original marker sequences was generally conserved in the chicken genome. Gene content of the turkey BACs is predicted from the comparative sequence alignments.

  3. A bacterial artificial chromosome (BAC) genomic approach reveals partial clustering of the furanocoumarin pathway genes in parsnip.

    PubMed

    Roselli, Sandro; Olry, Alexandre; Vautrin, Sonia; Coriton, Olivier; Ritchie, Dave; Galati, Gianni; Navrot, Nicolas; Krieger, Célia; Vialart, Guilhem; Bergès, Hélène; Bourgaud, Frédéric; Hehn, Alain

    2017-03-01

    Furanocoumarins are specialized metabolites that are involved in the defense of plants against phytophagous insects. The molecular and functional characterization of the genes involved in their biosynthetic pathway is only partially complete. Many recent reports have described gene clusters responsible for the biosynthesis of specialized metabolites in plants. To investigate possible co-localization of the genes involved in the furanocoumarin pathway, we sequenced parsnip BAC clones spanning two different gene loci. We found that two genes previously identified in this pathway, CYP71AJ3 and CYP71AJ4, were located on the same BAC, whereas a third gene, PsPT1, belonged to a different BAC clone. Chromosome mapping using fluorescence in situ hybridization (FISH) indicated that PsPT1 and the CYP71AJ3-CYP71AJ4 clusters are located on two different chromosomes. Sequencing the BAC clone harboring PsPT1 led to the identification of a gene encoding an Fe(II) α-ketoglutarate-dependent dioxygenase (PsDIOX) situated in the neighborhood of PsPT1 and confirmed the occurrence of a second gene cluster involved in the furanocoumarin pathway. This enzyme metabolizes p-coumaroyl CoA, leading exclusively to the synthesis of umbelliferone, an important intermediate compound in furanocoumarin synthesis. This work provides an insight into the genomic organization of genes from the furanocoumarin biosynthesis pathway organized in more than one gene cluster. It also confirms that the screening of a genomic library and the sequencing of BAC clones represent a valuable tool to identify genes involved in biosynthetic pathways dedicated to specialized metabolite synthesis.

  4. Construction and characterization of bacterial artificial chromosomes (BACs) containing herpes simplex virus full-length genomes.

    PubMed

    Nagel, Claus-Henning; Pohlmann, Anja; Sodeik, Beate

    2014-01-01

    Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models.

  5. The complexity of Rhipicephalus (Boophilus) microplus genome characterised through detailed analysis of two BAC clones

    PubMed Central

    2011-01-01

    Background Rhipicephalus (Boophilus) microplus (Rmi) a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crustaceans. Model arthropod genomes such as Drosophila and Anopheles are too taxonomically distant for a reference in tick genomic sequence analysis. This study focuses on the de-novo assembly of two R. microplus BAC sequences from the understudied R microplus genome. Based on available R. microplus sequenced resources and comparative analysis, tick genomic structure and functional predictions identify complex gene structures and genomic targets expressed during tick-cattle interaction. Results In our BAC analyses we have assembled, using the correct positioning of BAC end sequences and transcript sequences, two challenging genomic regions. Cot DNA fractions compared to the BAC sequences confirmed a highly repetitive BAC sequence BM-012-E08 and a low repetitive BAC sequence BM-005-G14 which was gene rich and contained short interspersed elements (SINEs). Based directly on the BAC and Cot data comparisons, the genome wide frequency of the SINE Ruka element was estimated. Using a conservative approach to the assembly of the highly repetitive BM-012-E08, the sequence was de-convoluted into three repeat units, each unit containing an 18S, 5.8S and 28S ribosomal RNA (rRNA) encoding gene sequence (rDNA), related internal transcribed spacer and complex intergenic region. In the low repetitive BM-005-G14, a novel gene complex was found between to 2 genes on the same strand. Nested in the second intron of a large 9 Kb papilin gene was a helicase gene. This helicase overlapped in two exonic regions with the papilin. Both these genes were shown expressed in different tick life stage important in ectoparasite interaction with the host. Tick specific sequence differences were also

  6. BAC-pool 454-sequencing: A rapid and efficient approach to sequence complex tetraploid cotton genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New and emerging next generation sequencing technologies have been promising in reducing sequencing costs, but not significantly for complex polyploid plant genomes such as cotton. Large and highly repetitive genome of G. hirsutum (~2.5GB) is less amenable and cost-intensive with traditional BAC-by...

  7. The nuclear genome of Brachypodium distachyon: analysis of BAC end sequences.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Due in part to its small genome (~350 Mb), Brachypodium distachyon is emerging as a model system for temperate grasses, including important crops like wheat and barley. We present the analysis of 10.9% of the Brachypodium genome based on 64,696 BAC end sequences (BES). Analysis of repeat DNA content...

  8. Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library

    PubMed Central

    Zhou, Qiongqiong; Sun, Wenjuan; Liu, Xiyan; Wang, Xiliang; Xiao, Yuncai; Bi, Dingren; Yin, Jingdong; Shi, Deshi

    2016-01-01

    Aim Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing. Methods After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis. Results Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression. Significance This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes

  9. Chromosomal mapping of canine-derived BAC clones to the red fox and American mink genomes.

    PubMed

    Kukekova, Anna V; Vorobieva, Nadegda V; Beklemisheva, Violetta R; Johnson, Jennifer L; Temnykh, Svetlana V; Yudkin, Dmitry V; Trut, Lyudmila N; Andre, Catherine; Galibert, Francis; Aguirre, Gustavo D; Acland, Gregory M; Graphodatsky, Alexander S

    2009-01-01

    High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene-containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations.

  10. Chromosome arm-specific BAC end sequences permit comparative analysis of homoeologous chromosomes and genomes of polyploid wheat

    PubMed Central

    2012-01-01

    Background Bread wheat, one of the world’s staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat. Results The end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359 bp of high quality sequence from 17,591 BAC-ends with an average length of 626 bp. The sequence represents 3.2% of t3AS with an average DNA sequence read every 19 kb. Overall, 79% of the sequence consisted of repetitive elements, 1.38% as coding regions (estimated 2,850 genes) and another 19% of unknown origin. Comparative sequence analysis suggested that 70-77% of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, gypsy/sabrina (12.4%) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4 kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39 kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695

  11. Mobilization of giant piggyBac transposons in the mouse genome.

    PubMed

    Li, Meng Amy; Turner, Daniel J; Ning, Zemin; Yusa, Kosuke; Liang, Qi; Eckert, Sabine; Rad, Lena; Fitzgerald, Tomas W; Craig, Nancy L; Bradley, Allan

    2011-12-01

    The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.

  12. Recombining overlapping BACs into single large BACs.

    PubMed

    Kotzamanis, George; Kotsinas, Athanassios

    2015-01-01

    BAC clones containing the entire genomic region of a gene including the long-range regulatory elements are very useful for gene functional analysis. However, large genes often span more than the insert of a BAC clone, and single BACs covering the entire region of interest are not available. Here, we describe a general system for linking two or more overlapping BACs into a single clone. Two rounds of homologous recombination are used. In the first, the BAC inserts are subcloned into the pBACLink vectors. In the second, the two BACs are combined together. Multiple BACs in a contig can be combined by alternating use of the pBACLInk vectors, resulting in several BAC clones containing as much of the genomic region of a gene as required. Such BACs can then be used in gene expression studies and/or gene therapy applications.

  13. Identification of an extensive gene cluster among a family of PPOs in Trifolium pratense L. (red clover) using a large insert BAC library

    PubMed Central

    2009-01-01

    Background Polyphenol oxidase (PPO) activity in plants is a trait with potential economic, agricultural and environmental impact. In relation to the food industry, PPO-induced browning causes unacceptable discolouration in fruit and vegetables: from an agriculture perspective, PPO can protect plants against pathogens and environmental stress, improve ruminant growth by increasing nitrogen absorption and decreasing nitrogen loss to the environment through the animal's urine. The high PPO legume, red clover, has a significant economic and environmental role in sustaining low-input organic and conventional farms. Molecular markers for a range of important agricultural traits are being developed for red clover and improved knowledge of PPO genes and their structure will facilitate molecular breeding. Results A bacterial artificial chromosome (BAC) library comprising 26,016 BAC clones with an average 135 Kb insert size, was constructed from Trifolium pratense L. (red clover), a diploid legume with a haploid genome size of 440–637 Mb. Library coverage of 6–8 genome equivalents ensured good representation of genes: the library was screened for polyphenol oxidase (PPO) genes. Two single copy PPO genes, PPO4 and PPO5, were identified to add to a family of three, previously reported, paralogous genes (PPO1–PPO3). Multiple PPO1 copies were identified and characterised revealing a subfamily comprising three variants PPO1/2, PPO1/4 and PPO1/5. Six PPO genes clustered within the genome: four separate BAC clones could be assembled onto a predicted 190–510 Kb single BAC contig. Conclusion A PPO gene family in red clover resides as a cluster of at least 6 genes. Three of these genes have high homology, suggesting a more recent evolutionary event. This PPO cluster covers a longer region of the genome than clusters detected in rice or previously reported in tomato. Full-length coding sequences from PPO4, PPO5, PPO1/5 and PPO1/4 will facilitate functional studies and provide

  14. Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells.

    PubMed

    Cheung, Wing; Kotzamanis, George; Abdulrazzak, Hassan; Goussard, Sylvie; Kaname, Tadashi; Kotsinas, Athanassios; Gorgoulis, Vassilis G; Grillot-Courvalin, Catherine; Huxley, Clare

    2012-01-01

    Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.

  15. A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library

    PubMed Central

    Liles, Mark R.; Manske, Brian F.; Bintrim, Scott B.; Handelsman, Jo; Goodman, Robert M.

    2003-01-01

    We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, α-Proteobacteria, and CFB groups; greater representation among the β- and γ-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division. PMID:12732537

  16. BAC-end sequence-based SNP mining in Allotetraploid Cotton (Gossypium) utilizing re-sequencing data, phylogenetic inferences and perspectives for genetic mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A bacterial artificial chromosome (BAC) library and BAC-end sequences for Gossypium hirsutum L. have recently been developed. Here we report on genomic-based genome-wide SNP mining utilizing re-sequencing data with a BAC-end sequence reference for twelve G. hirsutum L. lines, one G. barbadense L. li...

  17. Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

    PubMed

    Muñoz-Amatriaín, María; Lonardi, Stefano; Luo, MingCheng; Madishetty, Kavitha; Svensson, Jan T; Moscou, Matthew J; Wanamaker, Steve; Jiang, Tao; Kleinhofs, Andris; Muehlbauer, Gary J; Wise, Roger P; Stein, Nils; Ma, Yaqin; Rodriguez, Edmundo; Kudrna, Dave; Bhat, Prasanna R; Chao, Shiaoman; Condamine, Pascal; Heinen, Shane; Resnik, Josh; Wing, Rod; Witt, Heather N; Alpert, Matthew; Beccuti, Marco; Bozdag, Serdar; Cordero, Francesca; Mirebrahim, Hamid; Ounit, Rachid; Wu, Yonghui; You, Frank; Zheng, Jie; Simková, Hana; Dolezel, Jaroslav; Grimwood, Jane; Schmutz, Jeremy; Duma, Denisa; Altschmied, Lothar; Blake, Tom; Bregitzer, Phil; Cooper, Laurel; Dilbirligi, Muharrem; Falk, Anders; Feiz, Leila; Graner, Andreas; Gustafson, Perry; Hayes, Patrick M; Lemaux, Peggy; Mammadov, Jafar; Close, Timothy J

    2015-10-01

    Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

  18. Construction of a genome-wide human BAC-Unigene resource. Final progress report, 1989--1996

    SciTech Connect

    Lim, C.S.; Xu, R.X.; Wang, M.

    1996-12-31

    Currently, over 30,000 mapped STSs and 27,000 mapped Unigenes (non-redundant, unigene sets of cDNA representing EST clusters) are available for human alone. A total of 44,000 Unigene cDNA clones have been supplied by Research Genetics. Unigenes, or cDNAs are excellent resource for map building for two reasons. Firstly, they exist in two alternative forms -- as both sequence information for PCR primer pairs, and cDNA clones -- thus making library screening by colony hybridization as well as pooled library PCR possible. The authors have developed an efficient and robust procedure to screen genomic libraries with large number of DNA probes. Secondly, the linkage and order of expressed sequences, or genes are highly conserved among human, mouse and other mammalian species. Therefore, mapping with cDNA markers rather than random anonymous STSs will greatly facilitate comparative, evolutionary studies as well as physical map building. They have currently deconvoluted over 10,000 Unigene probes against a 4X coverage human BAC clones from the approved library D by high density colony hybridization method. 10,000 batches of Unigenes are arrayed in an imaginary 100 X 100 matrix from which 100 row pools and 100 column pools are obtained. Library filters are hybridized with pooled probes, thus reducing the number of hybridization required for addressing the positives for each Unigene from 10,000 to 200. Details on the experimental scheme as well as daily progress report is posted on the Web site (http://www.tree.caltech.edu).

  19. Construction of a llama bacterial artificial chromosome library with approximately 9-fold genome equivalent coverage.

    PubMed

    Airmet, K W; Hinckley, J D; Tree, L T; Moss, M; Blumell, S; Ulicny, K; Gustafson, A K; Weed, M; Theodosis, R; Lehnardt, M; Genho, J; Stevens, M R; Kooyman, D L

    2012-01-01

    The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be 2.4 × 10⁹ bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama.

  20. Sequencing of a QTL-rich region of the Theobroma cacao genome using pooled BACs and the identification of trait specific candidate genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: BAC-based physical maps provide for sequencing across an entire genome or selected sub-genome regions of biological interest. Using the minimum tiling path as a guide, it is possible to select specific BAC clones from prioritized genome sections such as a genetically defined QTL interv...

  1. A BAC pooling strategy combined with PCR-based screenings in a large, highly repetitive genome enables integration of the maize genetic and physical maps

    PubMed Central

    Yim, Young-Sun; Moak, Patricia; Sanchez-Villeda, Hector; Musket, Theresa A; Close, Pamela; Klein, Patricia E; Mullet, John E; McMullen, Michael D; Fang, Zheiwei; Schaeffer, Mary L; Gardiner, Jack M; Coe, Edward H; Davis, Georgia L

    2007-01-01

    Background Molecular markers serve three important functions in physical map assembly. First, they provide anchor points to genetic maps facilitating functional genomic studies. Second, they reduce the overlap required for BAC contig assembly from 80 to 50 percent. Finally, they validate assemblies based solely on BAC fingerprints. We employed a six-dimensional BAC pooling strategy in combination with a high-throughput PCR-based screening method to anchor the maize genetic and physical maps. Results A total of 110,592 maize BAC clones (~ 6x haploid genome equivalents) were pooled into six different matrices, each containing 48 pools of BAC DNA. The quality of the BAC DNA pools and their utility for identifying BACs containing target genomic sequences was tested using 254 PCR-based STS markers. Five types of PCR-based STS markers were screened to assess potential uses for the BAC pools. An average of 4.68 BAC clones were identified per marker analyzed. These results were integrated with BAC fingerprint data generated by the Arizona Genomics Institute (AGI) and the Arizona Genomics Computational Laboratory (AGCoL) to assemble the BAC contigs using the FingerPrinted Contigs (FPC) software and contribute to the construction and anchoring of the physical map. A total of 234 markers (92.5%) anchored BAC contigs to their genetic map positions. The results can be viewed on the integrated map of maize [1,2]. Conclusion This BAC pooling strategy is a rapid, cost effective method for genome assembly and anchoring. The requirement for six replicate positive amplifications makes this a robust method for use in large genomes with high amounts of repetitive DNA such as maize. This strategy can be used to physically map duplicate loci, provide order information for loci in a small genetic interval or with no genetic recombination, and loci with conflicting hybridization-based information. PMID:17291341

  2. BAC-pool sequencing and analysis confirms growth-associated QTLs in the Asian seabass genome

    PubMed Central

    Shen, Xueyan; Ngoh, Si Yan; Thevasagayam, Natascha May; Prakki, Sai Rama Sridatta; Bhandare, Pranjali; Tan, Andy Wee Kiat; Tan, Gui Quan; Singh, Siddharth; Phua, Norman Chun Han; Vij, Shubha; Orbán, László

    2016-01-01

    The Asian seabass is an important marine food fish that has been cultured for several decades in Asia Pacific. However, the lack of a high quality reference genome has hampered efforts to improve its selective breeding. A 3D BAC pool set generated in this study was screened using 22 SSR markers located on linkage group 2 which contains a growth-related QTL region. Seventy-two clones corresponding to 22 FPC contigs were sequenced by Illumina MiSeq technology. We co-assembled the MiSeq-derived scaffolds from each FPC contig with error-corrected PacBio reads, resulting in 187 sequences covering 9.7 Mb. Eleven genes annotated within this region were found to be potentially associated with growth and their tissue-specific expression was investigated. Correlation analysis demonstrated that SNPs in ctsb, skp1 and ppp2ca can be potentially used as markers for selecting fast-growing fingerlings. Conserved syntenies between seabass LG2 and five other teleosts were identified. This study i) provided a 10 Mb targeted genome assembly; ii) demonstrated NGS of BAC pools as a potential approach for mining candidates underlying QTLs of this species; iii) detected eleven genes potentially responsible for growth in the QTL region; and iv) identified useful SNP markers for selective breeding programs of Asian seabass. PMID:27821852

  3. Integrated cytogenetic BAC map of the genome of the gray, short-tailed opossum, Monodelphis domestica.

    PubMed

    Duke, S E; Samollow, P B; Mauceli, E; Lindblad-Toh, K; Breen, M

    2007-01-01

    The generation of high-quality genome assemblies for numerous species is advancing at a rapid pace. As the number of genome assemblies increases, so does our ability to investigate genome relationships and their contributions to unraveling complex biological, evolutionary, and biomedical processes. A key process in the generation of a genome assembly is to determine and verify the precise physical location and order of the large sequence blocks (scaffolds) that result from the assembly. For organisms of relatively recent common ancestry this process may be achieved largely through comparative sequence alignment. However, as the evolutionary distance between species lengthens, the use of comparative sequence alignment becomes increasingly less reliable. Simultaneous cytogenetic mapping, using multicolor fluorescence in-situ hybridization (FISH) analysis, offers an alternative means to define the cytogenetic location and relative order of DNA sequences, thereby anchoring the genome sequence to the karyotype. In this article we report the molecular cytogenetic locations of 415 bacterial artificial chromosome (BAC) clones that served to anchor sequence scaffolds of the gray, short-tailed opossum (Monodelphis domestica) to its karyotype, which enabled accurate integration of these regions into the genome assembly.

  4. Localization and Characterization of 170 BAC-derived clones and mapping of Ninety-Four Microsatellites in the Hessian Fly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ninety-four microsatellites from enriched genomic libraries of Hessian fly (Mayetiola destructor (Say)) were localized to 170 cognate clones in a Hessian fly bacterial artificial chromosome (BAC) library. These microsatellite-positive BAC clones were physically mapped to polytene chromosomes by fl...

  5. Whole-Genome Sequencing: Manual Library Preparation.

    PubMed

    Mardis, Elaine; McCombie, W Richard

    2017-01-03

    This protocol describes a manual approach for the preparation of genomic DNA libraries suitable for Illumina sequencing. Genomic DNA fragments produced by shearing by sonication are ligated to adaptors and amplified by polymerase chain reaction (PCR). The amplified DNA, separated by size and gel-purified, is suitable for use as template in whole-genome sequencing.

  6. Construction and utility of 10-kb libraries for efficient clone-gap closure for rice genome sequencing.

    PubMed

    Yang, Tae-Jin; Yu, Yeisoo; Nah, Gyoungju; Atkins, Michael; Lee, Seunghee; Frisch, David A; Wing, Rod A

    2003-08-01

    Rice is an important crop and a model system for monocot genomics, and is a target for whole genome sequencing by the International Rice Genome Sequencing Project (IRGSP). The IRGSP is using a clone by clone approach to sequence rice based on minimum tiles of BAC or PAC clones. For chromosomes 10 and 3 we are using an integrated physical map based on two fingerprinted and end-sequenced BAC libraries to identifying a minimum tiling path of clones. In this study we constructed and tested two rice genomic libraries with an average insert size of 10 kb (10-kb library) to support the gap closure and finishing phases of the rice genome sequencing project. The HaeIII library contains 166,752 clones covering approximately 4.6x rice genome equivalents with an average insert size of 10.5 kb. The Sau3AI library contains 138,960 clones covering 4.2x genome equivalents with an average insert size of 11.6 kb. Both libraries were gridded in duplicate onto 11 high-density filters in a 5 x 5 pattern to facilitate screening by hybridization. The libraries contain an unbiased coverage of the rice genome with less than 5% contamination by clones containing organelle DNA or no insert. An efficient method was developed, consisting of pooled overgo hybridization, the selection of 10-kb gap spanning clones using end sequences, transposon sequencing and utilization of in silico draft sequence, to close relatively small gaps between sequenced BAC clones. Using this method we were able to close a majority of the gaps (up to approximately 50 kb) identified during the finishing phase of chromosome-10 sequencing. This method represents a useful way to close clone gaps and thus to complete the entire rice genome.

  7. A first generation physical map of the medaka genome in BACs essential for positional cloning and clone-by-clone based genomic sequencing.

    PubMed

    Khorasani, Maryam Zadeh; Hennig, Steffen; Imre, Gabriele; Asakawa, Shuichi; Palczewski, Stefanie; Berger, Anja; Hori, Hiroshi; Naruse, Kiyoshi; Mitani, Hiroshi; Shima, Akihiro; Lehrach, Hans; Wittbrodt, Jochen; Kondoh, Hisato; Shimizu, Nobuyoshi; Himmelbauer, Heinz

    2004-07-01

    In order to realize the full potential of the medaka as a model system for developmental biology and genetics, characterized genomic resources need to be established, culminating in the sequence of the medaka genome. To facilitate the map-based cloning of genes underlying induced mutations and to provide templates for clone-based genomic sequencing, we have created a first-generation physical map of the medaka genome in bacterial artificial chromosome (BAC) clones. In particular, we exploited the synteny to the closely related genome of the pufferfish, Takifugu rubripes, by marker content mapping. As a first step, we clustered 103,144 public medaka EST sequences to obtain a set of 21,121 non-redundant sequence entities. Avoiding oversampling of gene-dense regions, 11,254 of EST clusters were successfully matched against the draft sequence of the fugu genome, and 2363 genes were selected for the BAC map project. We designed 35mer oligonucleotide probes from the selected genes and hybridized them against 64,500 BAC clones of strains Cab and Hd-rR, representing 14-fold coverage of the medaka genome. Our data set is further supplemented with 437 results generated from PCR-amplified inserts of medaka cDNA clones and BAC end-fragment markers. Our current, edited, first generation medaka BAC map consists of 902 map segments that cover about 74% of the medaka genome. The map contains 2721 markers. Of these, 2534 are from expressed sequences, equivalent to a non-redundant set of 2328 loci. The 934 markers (724 different) are anchored to the medaka genetic map. Thus, genetic map assignments provide immediate access to underlying clones and contigs, simplifying molecular access to candidate gene regions and their characterization.

  8. High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6.

    PubMed

    Szinay, Dóra; Chang, Song-Bin; Khrustaleva, Ludmila; Peters, Sander; Schijlen, Elio; Bai, Yuling; Stiekema, Willem J; van Ham, Roeland C H J; de Jong, Hans; Klein Lankhorst, René M

    2008-11-01

    Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.

  9. Rapid detection of genomic imbalances using micro-arrays consisting of pooled BACs covering all human chromosome arms.

    PubMed

    Knijnenburg, Jeroen; van der Burg, Marja; Nilsson, Philomeen; Ploos van Amstel, Hans Kristian; Tanke, Hans; Szuhai, Károly

    2005-10-12

    A strategy is presented to select, pool and spot human BAC clones on an array in such a way that each spot contains five well performing BAC clones, covering one chromosome arm. A mini-array of 240 spots was prepared representing all human chromosome arms in a 5-fold as well as some controls, and used for comparative genomic hybridization (CGH) of 10 cell lines with aneusomies frequently found in clinical cytogenetics and oncology. Spot-to-spot variation within five replicates was below 6% and all expected abnormalities were detected 100% correctly. Sensitivity was such that replacing one BAC clone in a given spot of five by a BAC clone from another chromosome, thus resulting in a change in ratio of 20%, was reproducibly detected. Incubation time of the mini-array was varied and the fluorescently labelled target DNA was diluted. Typically, aneusomies could be detected using 30 ng of non-amplified random primed labelled DNA amounts in a 4 h hybridization reaction. Potential application of these mini-arrays for genomic profiling of disseminated tumour cells or of blastomeres for preimplantation genetic diagnosis, using specially designed DNA amplification methods, are discussed.

  10. Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

    PubMed Central

    Xu, Min; Wang, Yemin; Zhao, Zhilong; Gao, Guixi; Huang, Sheng-Xiong; Kang, Qianjin; He, Xinyi; Lin, Shuangjun; Pang, Xiuhua; Deng, Zixin

    2016-01-01

    ABSTRACT Genome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries in Streptomyces spp. We demonstrate mining from a strain of Streptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate host Streptomyces lividans SBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic from S. rochei. This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways. IMPORTANCE Microbial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites from Streptomyces. The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including

  11. Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept

    PubMed Central

    Maughan, Peter J.; Smith, Scott M.; Raney, Joshua A.

    2012-01-01

    Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooled Amaranthus hypochondriacus BAC library. Ninety-six SNP loci, spanning the length of A. hypochondriacus linkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of ~$0.05 per reaction. PMID:22910714

  12. Generating a transgenic mouse line stably expressing human MHC surface antigen from a HAC carrying multiple genomic BACs.

    PubMed

    Hasegawa, Yoshinori; Ishikura, Tomoyuki; Hasegawa, Takanori; Watanabe, Takashi; Suzuki, Junpei; Nakayama, Manabu; Okamura, Yoshiaki; Okazaki, Tuneko; Koseki, Haruhiko; Ohara, Osamu; Ikeno, Masashi; Masumoto, Hiroshi

    2015-03-01

    The human artificial chromosome (HAC) vector is a promising tool to improve the problematic suppression and position effects of transgene expression frequently seen in transgenic cells and animals produced by conventional plasmid or viral vectors. We generated transgenic mice maintaining a single HAC vector carrying two genomic bacterial artificial chromosomes (BACs) from human HLA-DR loci (DRA and DRB1). Both transgenes on the HAC in transgenic mice exhibited tissue-specific expression in kidney, liver, lung, spleen, lymph node, bone marrow, and thymus cells in RT-PCR analysis. Stable functional expression of a cell surface HLA-DR marker from both transgenes, DRA and DRB1 on the HAC, was detected by flow cytometric analysis of splenocytes and maintained through at least eight filial generations. These results indicate that the de novo HAC system can allow us to manipulate multiple BAC transgenes with coordinated expression as a surface antigen through the generation of transgenic animals.

  13. Recombineering linear BACs.

    PubMed

    Chen, Qingwen; Narayanan, Kumaran

    2015-01-01

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.

  14. BAC-derived diagnostic markers for sex determination in asparagus.

    PubMed

    Jamsari, A; Nitz, I; Reamon-Büttner, S M; Jung, C

    2004-04-01

    A HindIII BAC (bacterial artificial chromosome) library of asparagus ( Asparagus officinalis L.) was established from a single male plant homozygous for the male flowering gene ( MM). The library represents approximately 5.5 haploid genome equivalents with an average insert size of 82 kb. A subset of the library (2.6 haploid genome equivalents) was arranged into DNA pools. Using nine sex-linked amplified fragment length polymorphism (AFLP) and two sequence-tagged site (STS) markers, 13 different BAC clones were identified from this part of the library. The BACs were arranged into a first-generation physical map around the sex locus. Four PCR-derived markers were developed from the BAC ends, one of which could be scored in a co-dominant way. Using a mapping population of 802 plants we mapped the BAC-derived markers to the same position close to the M gene as the corresponding AFLP and STS markers. The markers are useful for further chromosome walking studies and as diagnostic markers for selecting male plants homozygous for the M gene.

  15. Construction and characterization of a bacterial artificial chromosome library for hexaploid wheat line 92R137

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was compos...

  16. Recent transposition of yabusame, a novel piggyBac-like transposable element in the genome of the silkworm, Bombyx mori.

    PubMed

    Daimon, Takaaki; Mitsuhiro, Masao; Katsuma, Susumu; Abe, Hiroaki; Mita, Kazuei; Shimada, Toru

    2010-08-01

    On the W chromosome of the silkworm, Bombyx mori, we found a novel piggyBac-like DNA transposon that potentially encodes an intact transposase (610 amino acid residues), which is flanked by 16-bp perfect inverted terminal repeats and a duplicated TTAA target site. Interestingly, we also identified another intact copy of this transposon on an autosome (chromosome 21), which showed 99.6% identity in the DNA sequence of the transposase (99.3% amino acid identity). These features raised the possibility that this novel piggyBac-like DNA transposon, designated as yabusame, may retain transposition activity. Here we report the identification and characterization of yabusame transposons from the silkworm. We cloned the full length of the yabusame transposon on the W chromosome (yabusame-W) and its autosomal copy (yabusame-1). Southern blot analysis showed that there are interstrain polymorphisms in yabusame elements for their insertion sites and copy number. We also found strong evidence for the recent transposition of yabusame elements in the silkworm genome. Although our in vitro excision assays suggested that the transposition activity of yabusame-1 and yabusame-W has been lost almost entirely, our data will lead to a greater understanding of the characteristics of piggyBac superfamily elements.

  17. Counter-selection recombineering of the baculovirus genome: a strategy for seamless modification of repeat-containing BACs.

    PubMed

    Westenberg, Marcel; Soedling, Helen M; Mann, Derek A; Nicholson, Linda J; Dolphin, Colin T

    2010-09-01

    Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.

  18. The Oryza map alignment project: Construction, alignment and analysis of 12 BAC fingerprint/end sequence framework physical maps that represent the 10 genome types of genus Oryza

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Oryza Map Alignment Project (OMAP) provides the first comprehensive experimental system for understanding the evolution, physiology and biochemistry of a full genus in plants or animals. We have constructed twelve deep-coverage BAC libraries that are representative of both diploid and tetraploid...

  19. Large insert environmental genomic library production.

    PubMed

    Taupp, Marcus; Lee, Sangwon; Hawley, Alyse; Yang, Jinshu; Hallam, Steven J

    2009-09-23

    The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties

  20. Human artificial chromosome assembly by transposon-based retrofitting of genomic BACs with synthetic alpha-satellite arrays.

    PubMed

    Basu, Joydeep; Willard, Huntington F; Stromberg, Gregory

    2007-01-01

    The development of methodologies for the rapid assembly of synthetic alpha-satellite arrays recapitulating the higher-order periodic organization of native human centromeres permits the systematic investigation of the significance of primary sequence and sequence organization in centromere function. Synthetic arrays with defined mutations affecting sequence and/or organization may be evaluated in a de novo human artificial chromosome assay. This unit describes strategies for the assembly of custom built alpha-satellite arrays containing any desired mutation as well as strategies for the construction and manipulation of alpha satellite-based transposons. Transposons permit the rapid and reliable retrofitting of any genomic bacterial artificial chromosome (BAC) with synthetic alpha-satellite arrays and other functional components, thereby facilitating conversion into BAC-based human artificial chromosome vectors. These techniques permit identification and optimization of the critical parameters underlying the unique ability of alpha-satellite DNA to facilitate de novo centromere assembly, and they will establish the foundation for the next generation of human artificial chromosome vectors.

  1. A Comparative BAC Map for the Gilthead Sea Bream (Sparus aurata L.)

    PubMed Central

    Kuhl, Heiner; Sarropoulou, Elena; Tine, Mbaye; Kotoulas, Georgios; Magoulas, Antonios; Reinhardt, Richard

    2011-01-01

    This study presents the first comparative BAC map of the gilthead sea bream (Sparus aurata), a highly valuated marine aquaculture fish species in the Mediterranean. High-throughput end sequencing of a BAC library yielded 92,468 reads (60.6 Mbp). Comparative mapping was achieved by anchoring BAC end sequences to the three-spined stickleback (Gasterosteus aculeatus) genome. BACs that were consistently ordered along the stickleback chromosomes accounted for 14,265 clones. A fraction of 5,249 BACs constituted a minimal tiling path that covers 73.5% of the stickleback chromosomes and 70.2% of the genes that have been annotated. The N50 size of 1,485 “BACtigs” consisting of redundant BACs is 337,253 bp. The largest BACtig covers 2.15 Mbp in the stickleback genome. According to the insert size distribution of mapped BACs the sea bream genome is 1.71-fold larger than the stickleback genome. These results represent a valuable tool to researchers in the field and may support future projects to elucidate the whole sea bream genome. PMID:21049003

  2. Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening

    PubMed Central

    Lane, Andrew B.; Strzelecka, Magdalena; Ettinger, Andreas; Grenfell, Andrew W.; Wittmann, Torsten; Heald, Rebecca

    2015-01-01

    Summary CRISPR-based technologies have emerged as powerful tools to alter genomes and mark chromosomal loci, but an inexpensive method for generating large numbers of RNA guides for whole genome screening and labeling is lacking. Using a method that permits library construction from any source of DNA, we generated guide libraries that label repetitive loci or a single chromosomal locus in Xenopus egg extracts and show that a complex library can target the E. coli genome at high frequency. PMID:26212133

  3. Physical mapping of black spot disease resistance/susceptibility-related genome regions in Japanese pear (Pyrus pyrifolia) by BAC-FISH.

    PubMed

    Yamamoto, Masashi; Terakami, Shingo; Takada, Norio; Yamamoto, Toshiya

    2016-06-01

    Black spot disease, caused by Alternaria alternata Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. In the present study, the locations of black spot disease resistance/susceptibility-related genome regions were studied by fluorescence in situ hybridization using BAC clone (BAC-FISH) on Japanese pear (Pyrus pyrifolia (Burm. f.) Nakai) chromosomes. Root tips of self-pollinated seedlings of 'Osa Gold' were used as materials. Chromosome samples were prepared by the enzymatic maceration and air-drying method. The BAC clone adjacent to the black spot disease-related gene was labeled as a probe for FISH analysis. Black spot disease-related genome regions were detected in telomeric positions of two medium size chromosomes. These two sites and six telomeric 18S-5.8S-25S rDNA sites were located on different chromosomes as determined from the results of multi-color FISH. The effectiveness of the physical mapping of useful genes on pear chromosomes achieved by the BAC-FISH method was unequivocally demonstrated.

  4. Chromosome region-specific libraries for human genome analysis. Final progress report, 1 March 1991--28 February 1994

    SciTech Connect

    Kao, F.T.

    1994-04-01

    The objectives of this grant proposal include (1) development of a chromosome microdissection and PCR-mediated microcloning technology, (2) application of this microtechnology to the construction of region-specific libraries for human genome analysis. During this grant period, the authors have successfully developed this microtechnology and have applied it to the construction of microdissection libraries for the following chromosome regions: a whole chromosome 21 (21E), 2 region-specific libraries for the long arm of chromosome 2, 2q35-q37 (2Q1) and 2q33-q35 (2Q2), and 4 region-specific libraries for the entire short arm of chromosome 2, 2p23-p25 (2P1), 2p21-p23 (2P2), 2p14-p16 (wP3) and 2p11-p13 (2P4). In addition, 20--40 unique sequence microclones have been isolated and characterized for genomic studies. These region-specific libraries and the single-copy microclones from the library have been used as valuable resources for (1) isolating microsatellite probes in linkage analysis to further refine the disease locus; (2) isolating corresponding clones with large inserts, e.g. YAC, BAC, P1, cosmid and phage, to facilitate construction of contigs for high resolution physical mapping; and (3) isolating region-specific cDNA clones for use as candidate genes. These libraries are being deposited in the American Type Culture Collection (ATCC) for general distribution.

  5. A general method to modify BACs to generate large recombinant DNA fragments.

    PubMed

    Shen, Wei; Huang, Yue; Tang, Yi; Liu, De-Pei; Liang, Chih-Chuan

    2005-11-01

    Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA fragment containing the inverted human alpha-globin genes (theta, alpha1, alpha2, and zeta) from BAC191K2 and the locus control region (LCR) of human beta-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

  6. Genomic libraries: II. Subcloning, sequencing, and assembling large-insert genomic DNA clones.

    PubMed

    Quail, Mike A; Matthews, Lucy; Sims, Sarah; Lloyd, Christine; Beasley, Helen; Baxter, Simon W

    2011-01-01

    Sequencing large insert clones to completion is useful for characterizing specific genomic regions, identifying haplotypes, and closing gaps in whole genome sequencing projects. Despite being a standard technique in molecular laboratories, DNA sequencing using the Sanger method can be highly problematic when complex secondary structures or sequence repeats are encountered in genomic clones. Here, we describe methods to isolate DNA from a large insert clone (fosmid or BAC), subclone the sample, and sequence the region to the highest industry standard. Troubleshooting solutions for sequencing difficult templates are discussed.

  7. Complete Genomic Sequence and an Infectious BAC Clone of Feline Herpesvirus-1 (FHV-1)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feline herpesvirus type 1 (FHV-1) is classified under the genus Varicellovirus within the Alphaherpesvirinae subfamily, and is a major cause of upper respiratory infection in cats. In this report, we present the first complete genomic sequence of FHV-1, as well as a bacterial artificial chromosome (...

  8. The complexity of Rhipicephalus (Boophilus) microplus genome characterised through detailed analysis of two BAC clones

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Rhipicephalus (Boophilus) microplus (Rmi) a major cattle ectoparasite and tick borne disease vector, impacts on animal welfare and industry productivity. In arthropod research there is an absence of a complete Chelicerate genome, which includes ticks, mites, spiders, scorpions and crusta...

  9. Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

    PubMed

    Wang, Gang; Yang, Luhan; Grishin, Dennis; Rios, Xavier; Ye, Lillian Y; Hu, Yong; Li, Kai; Zhang, Donghui; Church, George M; Pu, William T

    2017-01-01

    Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

  10. Integrated karyotyping of sorghum by in situ hybridization of landed BACs.

    PubMed

    Kim, Jeong-Soon; Childs, Kevin L; Islam-Faridi, M Nurul; Menz, Monica A; Klein, Robert R; Klein, Patricia E; Price, H James; Mullet, John E; Stelly, David M

    2002-04-01

    The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum (Sorghum bicolor (L.) Moench), the genome of which contains ca. 780 Mbp spread across n = 10 chromosomes. Our approach relies on fluorescence in situ hybridization (FISH) and integrated structural genomic resources, including large-insert genomic clones in bacterial artificial chromosome (BAC) libraries. To develop robust FISH probes, we selected sorghum BACs by association with molecular markers that map near the ends of linkage groups, in regions inferred to be high in recombination. Overall, we selected 22 BACs that encompass the 10 linkage groups. As a prelude to development of a multiprobe FISH cocktail, we evaluated BAC-derived probes individually and in small groups. Biotin- and digoxygenin-labeled probes were made directly from the BAC clones and hybridized in situ to chromosomes without using suppressive unlabelled C0t-1 DNA. Based on FISH-signal strength and the relative degree of background signal, we judged 19 BAC-derived probes to be satisfactory. Based on their relative position, and collective association with all 10 linkage groups, we chose 17 of the 19 BACs to develop a 17-locus probe cocktail for dual-color detection. FISH of the cocktail allowed simultaneous identification of all 10 chromosomes. The results indicate that linkage and physical maps of sorghum allow facile selection of BAC clones according to position and FISH-signal quality. This capability will enable development of a high-quality molecular cytogenetic map and an integrated genomics system for sorghum, without need of chromosome flow sorting or microdissection. Moreover, transgeneric FISH experiments suggest that the sorghum system might be applicable to other Gramineae.

  11. Adaptation of a commercial robot for genome library replication

    SciTech Connect

    Uber, D.C.; Searles, W.L.

    1994-01-01

    This report describes tools and fixtures developed at the Human Genome Center at Lawrence Berkeley Laboratory for the Hewlett-Packard ORCA{trademark} (Optimized Robot for Chemical Analysis) to replicate large genome libraries. Photographs and engineering drawings of the various custom-designed components are included.

  12. Construction, characterization and FISH mapping of a bacterial artificial chromosome library of Chinese pangolin (Manis pentadactyla).

    PubMed

    Che, J; Wang, J; Su, W; Ye, J; Wang, Y; Nie, W; Yang, F

    2008-01-01

    Chinese pangolins as a representative species in the order Pholidota have highly specified morphological characters and occupy an important place in the mammalian phylogenetic tree. To obtain genomic data for this species, we have constructed a bacterial artificial chromosome (BAC) library of Chinese pangolin. The library contains 208,272 clones with an average insert size of 122.1 kb and represents approximately eight times the Chinese pangolin haploid genome (if we assume that the Chinese pangolins have a genome size similar to human). One hundred and twenty randomly-selected BAC clones were mapped onto Chinese pangolin chromosomes by fluorescence in situ hybridization (FISH), showing a largely unbiased chromosomal distribution. Several clones containing repetitive DNA and ribosomal DNA genes were also found. The BAC library and FISH mapped BAC clones are useful resources for comparative genomics and cytogenetics of mammals and in particular, the ongoing genome sequencing project of Chinese pangolins.

  13. An anchored framework BAC map of mouse chromosome 11 assembled using multiplex oligonucleotide hybridization.

    PubMed

    Cai, W W; Reneker, J; Chow, C W; Vaishnav, M; Bradley, A

    1998-12-15

    Despite abundant library resources for many organisms, physical mapping of these organisms has been seriously limited due to lack of efficient library screening techniques. We have developed a highly efficient strategy for large-scale screening of genomic libraries based on multiplex oligonucleotide hybridization on high-density genomic filters. We have applied this strategy to generate a bacterial artificial chromosome (BAC) anchored map of mouse chromosome 11. Using the MIT mouse SSLP data, 320 pairs of oligonucleotide probes were designed with an "overgo" computer program that selects new primer sequences that avoid the microsatellite repeat. BACs identified by these probes are automatically anchored to the chromosome. Ninety-two percent of the probes identified positive clones from a 5.9-fold coverage mouse BAC library with an average of 7 positive clones per marker. An average of 4.2 clones was confirmed for 204 markers by PCR. Our data show that a large number of clones can be efficiently isolated from a large genomic library using this strategy with minimal effort. This strategy will have wide application for large-scale mapping and sequencing of human and other large genomes.

  14. Gene enrichment in plant genomic shotgun libraries.

    PubMed

    Rabinowicz, Pablo D; McCombie, W Richard; Martienssen, Robert A

    2003-04-01

    The Arabidopsis genome (about 130 Mbp) has been completely sequenced; whereas a draft sequence of the rice genome (about 430 Mbp) is now available and the sequencing of this genome will be completed in the near future. The much larger genomes of several important crop species, such as wheat (about 16,000 Mbp) or maize (about 2500 Mbp), may not be fully sequenced with current technology. Instead, sequencing-analysis strategies are being developed to obtain sequencing and mapping information selectively for the genic fraction (gene space) of complex plant genomes.

  15. Antigen discovery using whole-genome phage display libraries.

    PubMed

    Beghetto, Elisa; Gargano, Nicola

    2013-01-01

    In the last two decades phage display technology has been used for investigating complex biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical cloning and expression system, has also been exploited for generating display libraries of small peptides and protein domains. More recently, large cDNA and whole-genome lambda-display libraries of human pathogens have been generated for the discovery of new antigens for biomedical applications. Here, we describe the construction of a whole-genome library of a common pathogen-Streptococcus pneumoniae-and the use of this library for the molecular dissection of the human B-cell response against bacterial infection and colonization.

  16. Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains

    PubMed Central

    Nelson, William; Luo, Meizhong; Ma, Jianxin; Estep, Matt; Estill, James; He, Ruifeng; Talag, Jayson; Sisneros, Nicholas; Kudrna, David; Kim, HyeRan; Ammiraju, Jetty SS; Collura, Kristi; Bharti, Arvind K; Messing, Joachim; Wing, Rod A; SanMiguel, Phillip; Bennetzen, Jeffrey L; Soderlund, Carol

    2008-01-01

    Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely

  17. BAC-End Microsatellites from Intra and Inter-Genic Regions of the Common Bean Genome and Their Correlation with Cytogenetic Features

    PubMed Central

    Blair, Matthew Wohlgemuth; Córdoba, Juana Marcela; Muñóz, Claritza; Yuyó, Deissy K.

    2014-01-01

    Highly polymorphic markers such as simple sequence repeats (SSRs) or microsatellites are very useful for genetic mapping. In this study novel SSRs were identified in BAC-end sequences (BES) from non-contigged, non-overlapping bacterial artificial clones (BACs) in common bean (Phaseolus vulgaris L.). These so called “singleton” BACs were from the G19833 Andean gene pool physical map and the new BES-SSR markers were used for the saturation of the inter-gene pool, DOR364×G19833 genetic map. A total of 899 SSR loci were found among the singleton BES, but only 346 loci corresponded to the single di- or tri-nucleotide motifs that were likely to be polymorphic (ATT or AG motifs, principally) and useful for primer design and individual marker mapping. When these novel SSR markers were evaluated in the DOR364×G19833 population parents, 136 markers revealed polymorphism and 106 were mapped. Genetic mapping resulted in a map length of 2291 cM with an average distance between markers of 5.2 cM. The new genetic map was compared to the most recent cytogenetic analysis of common bean chromosomes. We found that the new singleton BES-SSR were helpful in filling peri-centromeric spaces on the cytogenetic map. Short genetic distances between some new singleton-derived BES-SSR markers was common showing suppressed recombination in these regions compared to other parts of the genome. The correlation of singleton-derived SSR marker distribution with other cytogenetic features of the bean genome is discussed. PMID:25254501

  18. A bacterial artificial chromosome library for soybean PI 437654 and identification of clones associated with cyst nematode resistance.

    PubMed

    Tomkins, J P; Mahalingam, R; Smith, H; Goicoechea, J L; Knap, H T; Wing, R A

    1999-09-01

    We have constructed a soybean bacterial artificial chromosome (BAC) library using the plant introduction (PI) 437654. The library contains 73 728 clones stored in 192 384-well microtiter plates. A random sampling of 230 BACs indicated an average insert size of 136 kb with a range of 20 to 325 kb, and less than 4% of the clones do not contain inserts. Ninety percent of BAC clones in the library have an average insert size greater than 100 kb. Based on a genome size of 1115 Mb, library coverage is 9 haploid genome equivalents. Screening the BAC library colony filters with cpDNA sequences showed that contamination of the genomic library with chloroplast clones was low (1.85%). Library screening with three genomic RFLP probes linked to soybean cyst nematode (SCN) resistance genes resulted in an average of 18 hits per probe (range 7 to 30). Two separate pools of forward and reverse suppression subtractive cDNAs obtained from SCN-infected and uninfected roots of PI437654 were hybridized to the BAC library filters. The 488 BACs identified from positive signals were fingerprinted and analyzed using FPC software (version 4.0) resulting in 85 different contigs. Contigs were grouped and analyzed in three categories: (1) contigs of BAC clones which hybridized to forward subtracted cDNAs, (2) contigs of BAC clones which hybridized to reverse subtracted cDNAs, and (3) contigs of BAC clones which hybridized to both forward and reverse subtracted cDNAs. This protocol provides an estimate of the number of genomic regions involved in early resistance response to a pathogenic attack.

  19. Screening of an E. coli O157:H7 Bacterial Artificial Chromosome Library by Comparative Genomic Hybridization to Identify Genomic Regions Contributing to Growth in Bovine Gastrointestinal Mucus and Epithelial Cell Colonization

    PubMed Central

    Bai, Jianing; McAteer, Sean P.; Paxton, Edith; Mahajan, Arvind; Gally, David L.; Tree, Jai J.

    2011-01-01

    Enterohemorrhagic E. coli (EHEC) O157:H7 can cause serious gastrointestinal and systemic disease in humans following direct or indirect exposure to ruminant feces containing the bacterium. 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 (T3S) capacity to the K-12 host strain. However, conjugation of one of the BAC clones into a strain containing a partial LEE deletion restored T3S. Three hundred eighty-four clones from the library were subjected to two different selective screens; one involved three rounds of adherence assays to bovine primary rectal epithelial cells while the other competed the clones over three rounds of growth in bovine rectal mucus. The input strain DNA was then compared with the selected strains using comparative genomic hybridization (CGH) on an E. coli microarray. The adherence assay enriched for pO157 DNA indicating the importance of this plasmid for colonization of rectal epithelial cells. The mucus assay enriched for multiple regions involved in carbohydrate utilization, including hexuronate uptake, indicating that these regions provide a competitive growth advantage in bovine mucus. This BAC-CGH approach provides a positive selection screen that complements negative selection transposon-based screens. As demonstrated, this may be of particular use for identifying genes with redundant functions such as adhesion and carbon

  20. The Atlas Genome Assembly System

    PubMed Central

    Havlak, Paul; Chen, Rui; Durbin, K. James; Egan, Amy; Ren, Yanru; Song, Xing-Zhi; Weinstock, George M.; Gibbs, Richard A.

    2004-01-01

    Atlas is a suite of programs developed for assembly of genomes by a “combined approach” that uses DNA sequence reads from both BACs and whole-genome shotgun (WGS) libraries. The BAC clones afford advantages of localized assembly with reduced computational load, and provide a robust method for dealing with repeated sequences. Inclusion of WGS sequences facilitates use of different clone insert sizes and reduces data production costs. A core function of Atlas software is recruitment of WGS sequences into appropriate BACs based on sequence overlaps. Because construction of consensus sequences is from local assembly of these reads, only small (<0.1%) units of the genome are assembled at a time. Once assembled, each BAC is used to derive a genomic layout. This “sequence-based” growth of the genome map has greater precision than with non-sequence-based methods. Use of BACs allows correction of artifacts due to repeats at each stage of the process. This is aided by ancillary data such as BAC fingerprint, other genomic maps, and syntenic relations with other genomes. Atlas was used to assemble a draft DNA sequence of the rat genome; its major components including overlapper and split-scaffold are also being used in pure WGS projects. PMID:15060016

  1. Variant discovery and breakpoint region prediction for studying the human 22q11.2 deletion using BAC clone and whole genome sequencing analysis.

    PubMed

    Guo, Xingyi; Delio, Maria; Haque, Nousin; Castellanos, Raquel; Hestand, Matthew S; Vermeesch, Joris R; Morrow, Bernice E; Zheng, Deyou

    2016-09-01

    Velo-cardio-facial syndrome/DiGeorge syndrome/22q11.2 deletion syndrome (22q11.2DS) is caused by meiotic non-allelic homologous recombination events between flanking low copy repeats termed LCR22A and LCR22D, resulting in a 3 million base pair (Mb) deletion. Due to their complex structure, large size and high sequence identity, genetic variation within LCR22s among different individuals has not been well characterized. In this study, we sequenced 13 BAC clones derived from LCR22A/D and aligned them with 15 previously available BAC sequences to create a new genetic variation map. The thousands of variants identified by this analysis were not uniformly distributed in the two LCR22s. Moreover, shared single nucleotide variants between LCR22A and LCR22D were enriched in the Breakpoint Cluster Region pseudogene (BCRP) block, suggesting the existence of a possible recombination hotspot there. Interestingly, breakpoints for atypical 22q11.2 rearrangements have previously been located to BCRPs To further explore this finding, we carried out in-depth analyses of whole genome sequence (WGS) data from two unrelated probands harbouring a de novo 3Mb 22q11.2 deletion and their normal parents. By focusing primarily on WGS reads uniquely mapped to LCR22A, using the variation map from our BAC analysis to help resolve allele ambiguity, and by performing PCR analysis, we infer that the deletion breakpoints were most likely located near or within the BCRP module. In summary, we found a high degree of sequence variation in LCR22A and LCR22D and a potential recombination breakpoint near or within the BCRP block, providing a starting point for future breakpoint mapping using additional trios.

  2. A Multiway Analysis for Identifying High Integrity Bovine BACs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In large genomics projects involving many different types of analyses of bacterial artificial chromosomes (BACs), such as fingerprinting, end sequencing (BES) and full BAC sequencing there are many opportunities for the identities of BACs to become confused. However, by comparing the results from t...

  3. Construction and characterization of two bacterial artificial chromosome libraries of grass carp.

    PubMed

    Jang, Songhun; Liu, Hang; Su, Jianguo; Dong, Feng; Xiong, Feng; Liao, Lanjie; Wang, Yaping; Zhu, Zuoyan

    2010-06-01

    Bacterial artificial chromosome (BAC) library is an important tool in genomic research. We constructed two libraries from the genomic DNA of grass carp (Ctenopharyngodon idellus) as a crucial part of the grass carp genome project. The libraries were constructed in the EcoRI and HindIII sites of the vector CopyControl pCC1BAC. The EcoRI library comprised 53,000 positive clones, and approximately 99.94% of the clones contained grass carp nuclear DNA inserts (average size, 139.7 kb) covering 7.4 x haploid genome equivalents and 2% empty clones. Similarly, the HindIII library comprised 52,216 clones with approximately 99.82% probability of finding any genomic fragments containing single-copy genes; the average insert size was 121.5 kb with 2.8% insert-empty clones, thus providing genome coverage of 6.3 x haploid genome equivalents of grass carp. We selected gene-specific probes for screening the target gene clones in the HindIII library. In all, we obtained 31 positive clones, which were identified for every gene, with an average of 6.2 BAC clones per gene probe. Thus, we succeeded in constructing the desired BAC libraries, which should provide an important foundation for future physical mapping and whole-genome sequencing in grass carp.

  4. The first report of a Pelecaniformes defensin cluster: Characterization of β-defensin genes in the crested ibis based on BAC libraries

    PubMed Central

    Lan, Hong; Chen, Hui; Chen, Li-Cheng; Wang, Bei-Bing; Sun, Li; Ma, Mei-Ying; Fang, Sheng-Guo; Wan, Qiu-Hong

    2014-01-01

    Defensins play a key role in the innate immunity of various organisms. Detailed genomic studies of the defensin cluster have only been reported in a limited number of birds. Herein, we present the first characterization of defensins in a Pelecaniformes species, the crested ibis (Nipponia nippon), which is one of the most endangered birds in the world. We constructed bacterial artificial chromosome libraries, including a 4D-PCR library and a reverse-4D library, which provide at least 40 equivalents of this rare bird's genome. A cluster including 14 β-defensin loci within 129 kb was assigned to chromosome 3 by FISH, and one gene duplication of AvBD1 was found. The ibis defensin genes are characterized by multiform gene organization ranging from two to four exons through extensive exon fusion. Splicing signal variations and alternative splice variants were also found. Comparative analysis of four bird species identified one common and multiple species-specific duplications, which might be associated with high GC content. Evolutionary analysis revealed birth-and-death mode and purifying selection for avian defensin evolution, resulting in different defensin gene numbers among bird species and functional conservation within orthologous genes, respectively. Additionally, we propose various directions for further research on genetic conservation in the crested ibis. PMID:25372018

  5. Whole-Genome Sequencing: Automated, Nonindexed Library Preparation.

    PubMed

    Mardis, Elaine; McCombie, W Richard

    2017-03-01

    This protocol describes an automated procedure for constructing a nonindexed Illumina DNA library and relies on the use of a CyBi-SELMA automated pipetting machine, the Covaris E210 shearing instrument, and the epMotion 5075. With this method, genomic DNA fragments are produced by sonication, using high-frequency acoustic energy to shear DNA. Here, double-stranded DNA is fragmented when exposed to the energy of adaptive focused acoustic shearing (AFA). The resulting DNA fragments are ligated to adaptors, amplified by polymerase chain reaction (PCR), and subjected to size selection using magnetic beads. The product is suitable for use as template in whole-genome sequencing.

  6. Whole-Genome Sequencing: Automated, Indexed Library Preparation.

    PubMed

    Mardis, Elaine; McCombie, W Richard

    2017-03-01

    This protocol describes an automated procedure for constructing an indexed Illumina DNA library. With this method, genomic DNA fragments are produced by sonication, using high-frequency acoustic energy to shear DNA. Double-stranded DNA (dsDNA) will fragment when exposed to the energy of adaptive focused acoustic shearing (AFA). The resulting DNA fragments are ligated to adaptors, amplified by polymer chain reaction (PCR), and subjected to size selection using magnetic beads. The product is suitable for use as template in whole-genome sequencing.

  7. Multiplexed direct genomic selection (MDiGS): a pooled BAC capture approach for highly accurate CNV and SNP/INDEL detection.

    PubMed

    Alvarado, David M; Yang, Ping; Druley, Todd E; Lovett, Michael; Gurnett, Christina A

    2014-06-01

    Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ∼ 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest.

  8. MultiBac turns sweet

    PubMed Central

    Palmberger, Dieter; Klausberger, Miriam; Berger, Imre; Grabherr, Reingard

    2013-01-01

    The baculovirus/insect cell system has proven to be a powerful tool for the expression of eukaryotic proteins. Therapeutics, especially in the field of vaccinology, are often composed of several different protein subunits. Conventional baculoviral expression schemes largely lack efficient strategies for simultaneous multi-gene expression. The MultiBac technology which is based on an engineered genome of Autographa californica nuclear polyhedrosis virus in combination with specially designed transfer vectors is an elegant way for flexible generation of multi-subunit proteins in insect cells. Yet, the glycosylation pattern of insect cell-derived products is not favorable for many applications. Therefore, a modified version of MultiBac, SweetBac, was generated allowing for a flexible glycosylation of target proteins in insect cells. Beyond the SweetBac technology MultiBac can further be designed for bridging the gap between cell engineering and transient modulation of host genes for improved and product tailored expression of recombinant proteins. PMID:23018636

  9. Herpesvirus BACs: past, present, and future.

    PubMed

    Warden, Charles; Tang, Qiyi; Zhu, Hua

    2011-01-01

    The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy.

  10. Construction and targeted retrieval of specific clone from a non-gridded soybean bacterial artificial chromosome library.

    PubMed

    Xia, Zhengjun; Wu, Hongyan; Watanabe, Satoshi; Harada, Kyuya

    2014-01-01

    Although a post-genomic era is emerging for many plants, the bacterial artificial chromosome (BAC) library is still a valuable tool for genomic studies and preservation of precious genetic resources. Construction of non-gridded BAC libraries would dramatically reduce cost and save storage space. A non-gridded BAC library composed of approximately 96,000 insert-containing clones in 80 pools with an average insert size of 75 kb was constructed. This library represented 5.2 genome equivalents. We successfully developed a unique procedure to retrieve positive clones from the non-gridded pools. With this retrieving protocol, the non-gridded library system can be adapted to different species and to serve various research needs.

  11. Oligonucleotide Arrays vs. Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

    PubMed Central

    Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers. PMID:25415307

  12. Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

    PubMed

    Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

  13. Chromosome region-specific libraries for human genome analysis

    SciTech Connect

    Kao, Fa-Ten.

    1991-01-01

    We have made important progress since the beginning of the current grant year. We have further developed the microdissection and PCR- assisted microcloning techniques using the linker-adaptor method. We have critically evaluated the microdissection libraries constructed by this microtechnology and proved that they are of high quality. We further demonstrated that these microdissection clones are useful in identifying corresponding YAC clones for a thousand-fold expansion of the genomic coverage and for contig construction. We are also improving the technique of cloning the dissected fragments in test tube by the TDT method. We are applying both of these PCR cloning technique to human chromosomes 2 and 5 to construct region-specific libraries for physical mapping purposes of LLNL and LANL. Finally, we are exploring efficient procedures to use unique sequence microclones to isolate cDNA clones from defined chromosomal regions as valuable resources for identifying expressed gene sequences in the human genome. We believe that we are making important progress under the auspices of this DOE human genome program grant and we will continue to make significant contributions in the coming year. 4 refs., 4 figs.

  14. Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes

    SciTech Connect

    McNeal, Joel R.; Leebens-Mack, James H.; Arumuganathan, K.; Kuehl, Jennifer V.; Boore, Jeffrey L.; dePamphilis, Claude W.

    2005-08-26

    Organellar genome sequences provide numerous phylogenetic markers and yield insight into organellar function and molecular evolution. These genomes are much smaller in size than their nuclear counterparts; thus, their complete sequencing is much less expensive than total nuclear genome sequencing, making broader phylogenetic sampling feasible. However, for some organisms it is challenging to isolate plastid DNA for sequencing using standard methods. To overcome these difficulties, we constructed partial genomic libraries from total DNA preparations of two heterotrophic and two autotrophic angiosperm species using fosmid vectors. We then used macroarray screening to isolate clones containing large fragments of plastid DNA. A minimum tiling path of clones comprising the entire genome sequence of each plastid was selected, and these clones were shotgun-sequenced and assembled into complete genomes. Although this method worked well for both heterotrophic and autotrophic plants, nuclear genome size had a dramatic effect on the proportion of screened clones containing plastid DNA and, consequently, the overall number of clones that must be screened to ensure full plastid genome coverage. This technique makes it possible to determine complete plastid genome sequences for organisms that defy other available organellar genome sequencing methods, especially those for which limited amounts of tissue are available.

  15. Toward a Molecular Cytogenetic Map for Cultivated Sunflower (Helianthus annuus L.) by Landed BAC/BIBAC Clones

    PubMed Central

    Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

    2013-01-01

    Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (∼100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC- fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)−derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources. PMID:23316437

  16. Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones.

    PubMed

    Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

    2013-01-01

    Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (~100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)-derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.

  17. Sequencing of 15,622 gene-bearing BACs clarifies the gene-dense regions of the barley genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework....

  18. The development and characterisation of a bacterial artificial chromosome library for Fragaria vesca

    PubMed Central

    Bonet, Julio; Girona, Elena Lopez; Sargent, Daniel J; Muñoz-Torres, Monica C; Monfort, Amparo; Abbott, Albert G; Arús, Pere; Simpson, David W; Davik, Jahn

    2009-01-01

    Background The cultivated strawberry Fragaria ×ananassa is one of the most economically-important soft-fruit species. Few structural genomic resources have been reported for Fragaria and there exists an urgent need for the development of physical mapping resources for the genus. The first stage in the development of a physical map for Fragaria is the construction and characterisation of a high molecular weight bacterial artificial chromosome (BAC) library. Methods A BAC library, consisting of 18,432 clones was constructed from Fragaria vesca f. semperflorens accession 'Ali Baba'. BAC DNA from individual library clones was pooled to create a PCR-based screening assay for the library, whereby individual clones could be identified with just 34 PCR reactions. These pools were used to screen the BAC library and anchor individual clones to the diploid Fragaria reference map (FV×FN). Findings Clones from the BAC library developed contained an average insert size of 85 kb, representing over seven genome equivalents. The pools and superpools developed were used to identify a set of BAC clones containing 70 molecular markers previously mapped to the diploid Fragaria FV×FN reference map. The number of positive colonies identified for each marker suggests the library represents between 4× and 10× coverage of the diploid Fragaria genome, which is in accordance with the estimate of library coverage based on average insert size. Conclusion This BAC library will be used for the construction of a physical map for F. vesca and the superpools will permit physical anchoring of molecular markers using PCR. PMID:19772672

  19. Sequencing, annotation and comparative analysis of nine BACs of giant panda (Ailuropoda melanoleuca).

    PubMed

    Zheng, Yang; Cai, Jing; Li, JianWen; Li, Bo; Lin, RunMao; Tian, Feng; Wang, XiaoLing; Wang, Jun

    2010-01-01

    A 10-fold BAC library for giant panda was constructed and nine BACs were selected to generate finish sequences. These BACs could be used as a validation resource for the de novo assembly accuracy of the whole genome shotgun sequencing reads of giant panda newly generated by the Illumina GA sequencing technology. Complete sanger sequencing, assembly, annotation and comparative analysis were carried out on the selected BACs of a joint length 878 kb. Homologue search and de novo prediction methods were used to annotate genes and repeats. Twelve protein coding genes were predicted, seven of which could be functionally annotated. The seven genes have an average gene size of about 41 kb, an average coding size of about 1.2 kb and an average exon number of 6 per gene. Besides, seven tRNA genes were found. About 27 percent of the BAC sequence is composed of repeats. A phylogenetic tree was constructed using neighbor-join algorithm across five species, including giant panda, human, dog, cat and mouse, which reconfirms dog as the most related species to giant panda. Our results provide detailed sequence and structure information for new genes and repeats of giant panda, which will be helpful for further studies on the giant panda.

  20. Development of bacterial artificial chromosome library resources for parasitoid Hymenoptera (Nasonia vitripennis and Nasonia giraulti: Pteromalidae).

    PubMed

    Muñoz-Torres, M C; Saski, C; Blackmon, B; Romero-Severson, J; Werren, J H

    2010-02-01

    The species of the genus Nasonia possess qualities that make them excellent candidates for genetic and genomic studies. To increase the wealth of genomic resources for the genus we constructed publicly available bacterial artificial chromosome (BAC) libraries for Nasonia vitripennis and Nasonia giraulti. Libraries have 36 864 clones each, empty-vector contents of approximately 2% and average insert sizes of 113.1 and 97.7 Kb, respectively, representing 12 and 11 genome equivalents. The N. vitripennis library was used for genome sequence assembly and in efforts at positional cloning of a developmental gene. The genome assembly of N. vitripennis is currently composed on 6181 un-joined scaffolds. These BAC libraries can be used to identify and close regions between scaffolds of the genome assemblies of both species.

  1. Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries

    SciTech Connect

    Branscomb, E.; Slezak, T.; Pae, R.; Carrano, A.V. ); Galas, D.; Waterman, M. )

    1990-01-01

    The authors present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Their primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, they adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called contigs.' These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.

  2. Exploring the genome of the salt-marsh Spartina maritima (Poaceae, Chloridoideae) through BAC end sequence analysis.

    PubMed

    Ferreira de Carvalho, J; Chelaifa, H; Boutte, J; Poulain, J; Couloux, A; Wincker, P; Bellec, A; Fourment, J; Bergès, H; Salmon, A; Ainouche, M

    2013-12-01

    Spartina species play an important ecological role on salt marshes. Spartina maritima is an Old-World species distributed along the European and North-African Atlantic coasts. This hexaploid species (2n = 6x = 60, 2C = 3,700 Mb) hybridized with different Spartina species introduced from the American coasts, which resulted in the formation of new invasive hybrids and allopolyploids. Thus, S. maritima raises evolutionary and ecological interests. However, genomic information is dramatically lacking in this genus. In an effort to develop genomic resources, we analysed 40,641 high-quality bacterial artificial chromosome-end sequences (BESs), representing 26.7 Mb of the S. maritima genome. BESs were searched for sequence homology against known databases. A fraction of 16.91% of the BESs represents known repeats including a majority of long terminal repeat (LTR) retrotransposons (13.67%). Non-LTR retrotransposons represent 0.75%, DNA transposons 0.99%, whereas small RNA, simple repeats and low-complexity sequences account for 1.38% of the analysed BESs. In addition, 4,285 simple sequence repeats were detected. Using the coding sequence database of Sorghum bicolor, 6,809 BESs found homology accounting for 17.1% of all BESs. Comparative genomics with related genera reveals that the microsynteny is better conserved with S. bicolor compared to other sequenced Poaceae, where 37.6% of the paired matching BESs are correctly orientated on the chromosomes. We did not observe large macrosyntenic rearrangements using the mapping strategy employed. However, some regions appeared to have experienced rearrangements when comparing Spartina to Sorghum and to Oryza. This work represents the first overview of S. maritima genome regarding the respective coding and repetitive components. The syntenic relationships with other grass genomes examined here help clarifying evolution in Poaceae, S. maritima being a part of the poorly-known Chloridoideae sub-family.

  3. A BAC-based physical map of the Hessian fly (Mayetiola destructor) genome anchored to polytene chromosomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Hessian fly (Mayetiola destructor) is an important insect pest of wheat and an experimental organism for studies of plant-insect interactions. It has tractable genetics, polytene chromosomes, a relatively small genome (158 Mb), and shares a gene-for-gene relationship with wheat. To improve its...

  4. A library of TAL effector nucleases spanning the human genome.

    PubMed

    Kim, Yongsub; Kweon, Jiyeon; Kim, Annie; Chon, Jae Kyung; Yoo, Ji Yeon; Kim, Hye Joo; Kim, Sojung; Lee, Choongil; Jeong, Euihwan; Chung, Eugene; Kim, Doyoung; Lee, Mi Seon; Go, Eun Mi; Song, Hye Jung; Kim, Hwangbeom; Cho, Namjin; Bang, Duhee; Kim, Seokjoong; Kim, Jin-Soo

    2013-03-01

    Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

  5. Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone.

    PubMed

    McGregor, Alistair; Liu, Fenyong; Schleiss, Mark R

    2004-05-01

    We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.

  6. Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives.

    PubMed

    Wang, Chao; Shi, Xue; Liu, Lin; Li, Haiyan; Ammiraju, Jetty S S; Kudrna, David A; Xiong, Wentao; Wang, Hao; Dai, Zhaozhao; Zheng, Yonglian; Lai, Jinsheng; Jin, Weiwei; Messing, Joachim; Bennetzen, Jeffrey L; Wing, Rod A; Luo, Meizhong

    2013-11-01

    Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

  7. DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones.

    PubMed

    Fiegler, Heike; Carr, Philippa; Douglas, Eleanor J; Burford, Deborah C; Hunt, Sarah; Scott, Carol E; Smith, James; Vetrie, David; Gorman, Patricia; Tomlinson, Ian P M; Carter, Nigel P

    2003-04-01

    We have designed DOP-PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP-PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome-specific clones spotted on microarrays by use of DNA amplified from flow-sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1-Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet.

  8. Bacterial Artificial Chromosome Libraries of Pulse Crops: Characteristics and Applications

    PubMed Central

    Yu, Kangfu

    2012-01-01

    Pulse crops are considered minor on a global scale despite their nutritional value for human consumption. Therefore, they are relatively less extensively studied in comparison with the major crops. The need to improve pulse crop production and quality will increase with the increasing global demand for food security and people's awareness of nutritious food. The improvement of pulse crops will require fully utilizing all their genetic resources. Bacterial artificial chromosome (BAC) libraries of pulse crops are essential genomic resources that have the potential to accelerate gene discovery and enhance molecular breeding in these crops. Here, we review the availability, characteristics, applications, and potential applications of the BAC libraries of pulse crops. PMID:21811383

  9. Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.

    PubMed

    Le Paslier, M C; Pierce, R J; Merlin, F; Hirai, H; Wu, W; Williams, D L; Johnston, D; LoVerde, P T; Le Paslier, D

    2000-04-15

    A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.

  10. Physical mapping of a large plant genome using global high-information-content-fingerprinting: the distal region of the wheat ancestor Aegilops tauschii chromosome 3DS.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of wheat genome, Aegilops tauschii, is used as a resource for wheat...

  11. Physical mapping of a large plant genome using global high-information content fingerprinting: a distal region of wheat chromosome 3DS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of wheat. We report the use of the Ae. tauschii, the diploid ancestor of the wheat D genome, for the construction of t...

  12. Human genome libraries. Final progress report, February 1, 1994--August 31, 1997

    SciTech Connect

    Kao, Fa-Ten

    1998-01-01

    The goal of this program is to use a novel technology of chromosome microdissection and microcloning to construct chromosome region-specific libraries as resources for various human genome program studies. Region specific libraries have been constructed for the entire human chromosomes 2 and 18.

  13. A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.

    PubMed

    Cotta-de-Almeida, Vinicius; Schonhoff, Susan; Shibata, Tomoyuki; Leiter, Andrew; Snapper, Scott B

    2003-09-01

    Generating knockout mice is still an expensive and highly time-consuming process. Target construct generation, the first labor-intensive step in this process, requires the manipulation of large fragments of DNA and numerous, and often cumbersome, cloning steps. Here we show the development of a rapid approach for generating targeting constructs that capitalizes on efficient homologous recombination between linear DNA fragments and circular plasmids in Escherichia coli ("recombineering"), the availability of bacterial artificial chromosomes (BACs), and the accessibility of the sequence of the mouse genome. Employing recombineering, we demonstrate with only 1-2 template plasmids, short homologies (40-50bp) between donor and target DNA, and one subcloning step that we can efficiently manipulate BACs in situ to generate a complicated targeting vector. This procedure avoids the need to construct or screen genomic libraries and permits the generation of most standard, conditional, or knock-in targeting vectors, often within two weeks.

  14. Characterization of the genome of bald cypress

    PubMed Central

    2011-01-01

    Background Bald cypress (Taxodium distichum var. distichum) is a coniferous tree of tremendous ecological and economic importance. It is a member of the family Cupressaceae which also includes cypresses, redwoods, sequoias, thujas, and junipers. While the bald cypress genome is more than three times the size of the human genome, its 1C DNA content is amongst the smallest of any conifer. To learn more about the genome of bald cypress and gain insight into the evolution of Cupressaceae genomes, we performed a Cot analysis and used Cot filtration to study Taxodium DNA. Additionally, we constructed a 6.7 genome-equivalent BAC library that we screened with known Taxodium genes and select repeats. Results The bald cypress genome is composed of 90% repetitive DNA with most sequences being found in low to mid copy numbers. The most abundant repeats are found in fewer than 25,000 copies per genome. Approximately 7.4% of the genome is single/low-copy DNA (i.e., sequences found in 1 to 5 copies). Sequencing of highly repetitive Cot clones indicates that most Taxodium repeats are highly diverged from previously characterized plant repeat sequences. The bald cypress BAC library consists of 606,336 clones (average insert size of 113 kb) and collectively provides 6.7-fold genome equivalent coverage of the bald cypress genome. Macroarray screening with known genes produced, on average, about 1.5 positive clones per probe per genome-equivalent. Library screening with Cot-1 DNA revealed that approximately 83% of BAC clones contain repetitive sequences iterated 103 to 104 times per genome. Conclusions The BAC library for bald cypress is the first to be generated for a conifer species outside of the family Pinaceae. The Taxodium BAC library was shown to be useful in gene isolation and genome characterization and should be an important tool in gymnosperm comparative genomics, physical mapping, genome sequencing, and gene/polymorphism discovery. The single/low-copy (SL) component of

  15. Constructing gene-enriched plant genomic libraries using methylation filtration technology.

    PubMed

    Rabinowicz, Pablo D

    2003-01-01

    Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive. For this reason, gene targeted partial genomic sequencing becomes a realistic option. The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration. This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated. Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered. To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA. Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

  16. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution.

    PubMed

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-04-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the "natural" AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid-DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid-genome identity correlation. Overall, our observations demonstrate that production of "natural" AAVs results in low capsid mosaicism and high capsid-genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype-genotype correlation.

  17. High Capsid–Genome Correlation Facilitates Creation of AAV Libraries for Directed Evolution

    PubMed Central

    Nonnenmacher, Mathieu; van Bakel, Harm; Hajjar, Roger J; Weber, Thomas

    2015-01-01

    Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the “natural” AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid–DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid–genome identity correlation. Overall, our observations demonstrate that production of “natural” AAVs results in low capsid mosaicism and high capsid–genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype–genotype correlation. PMID:25586687

  18. Acc homoeoloci and the evolution of wheat genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We analyzed the DNA sequences of BACs from many wheat libraries containing the Acc-1 and Acc-2 loci, encoding the plastid and cytosolic forms of the enzyme acetyl-CoA carboxylase, to gain understanding of the evolution of these genes and the origin of the three genomes in modern hexaploid wheat. Mor...

  19. A second generation integrated map of the rainbow trout (Oncorhynchus mykiss) genome: analysis of synteny with model fish genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this paper we generated DNA fingerprints and end sequences from bacterial artificial chromosomes (BACs) from two new libraries to improve the first generation integrated physical and genetic map of the rainbow trout (Oncorhynchus mykiss) genome. The current version of the physical map is compose...

  20. Chromosome region-specific libraries for human genome analysis

    SciTech Connect

    Kao, Fa-Ten.

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  1. Integration of the draft sequence and physical map as a framework for genomic research in soybean (Glycine max (L.) Merr.) and wild soybean (Glycine soja Sieb. and Zucc.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean is a model for the legume research community due to its importance as a crop, a well populated genetic map, and the availability of a genome sequence. Even though a whole genome shotgun sequence and Bacterial Artificial Chromosome (BAC) libraries are available, a high-resolution chromosome-b...

  2. Use of BAC clones as standardized reagents for Marek’s disease virus research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cloning of the Marek’s disease virus (MDV) genome as an infectious bacterial artificial chromosome (BAC) clone have led to major advances through our ability to study individual gene function by making precise insertions and deletions in the viral genome. We believe that MDV BAC clones will repl...

  3. Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents

    SciTech Connect

    Albertsen, H.M.; Abderrahim, H.; Cann, H.M.; Dausset, J.; Le Paslier, D.; Cohen, D. )

    1990-06-01

    Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, the authors performed a series of preliminary experiments aimed at developing a suitable protocol. They found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable size variations. A protocol was developed for preparative electrophoretic enrichment of high molecular mass human DNA fragments from partial restriction digests and ligation with the YAC vector in agarose. A YAC library has been constructed from large fragments of DNA from an Epstein-Barr virus-transformed human lymphoblastoid cell line. The library presently contains 50,000 clones, 95% of which are greater than 250 kilobase pairs in size. The mean YAC size of the library, calculated from 132 randomly isolated clones, is 430 kilobase pairs. The library thus contains the equivalent of approximately seven haploid human genomes.

  4. Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats

    PubMed Central

    Jung, Chris J.; Ménoret, Séverine; Brusselle, Lucas; Tesson, Laurent; Usal, Claire; Chenouard, Vanessa; Remy, Séverine; Ouisse, Laure-Hélène; Poirier, Nicolas; Vanhove, Bernard; de Jong, Pieter J.; Anegon, Ignacio

    2016-01-01

    BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications. PMID:27530248

  5. Scalable whole-genome single-cell library preparation without preamplification.

    PubMed

    Zahn, Hans; Steif, Adi; Laks, Emma; Eirew, Peter; VanInsberghe, Michael; Shah, Sohrab P; Aparicio, Samuel; Hansen, Carl L

    2017-02-01

    Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

  6. Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

    PubMed

    Coyne, C J; McClendon, M T; Walling, J G; Timmerman-Vaughan, G M; Murray, S; Meksem, K; Lightfoot, D A; Shultz, J L; Keller, K E; Martin, R R; Inglis, D A; Rajesh, P N; McPhee, K E; Weeden, N F; Grusak, M A; Li, C-M; Storlie, E W

    2007-09-01

    Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

  7. Democratizing Human Genome Project Information: A Model Program for Education, Information and Debate in Public Libraries.

    ERIC Educational Resources Information Center

    Pollack, Miriam

    The "Mapping the Human Genome" project demonstrated that librarians can help whomever they serve in accessing information resources in the areas of biological and health information, whether it is the scientists who are developing the information or a member of the public who is using the information. Public libraries can guide library…

  8. BAC-End Sequence-Based SNP Mining in Allotetraploid Cotton (Gossypium) Utilizing Resequencing Data, Phylogenetic Inferences, and Perspectives for Genetic Mapping.

    PubMed

    Hulse-Kemp, Amanda M; Ashrafi, Hamid; Stoffel, Kevin; Zheng, Xiuting; Saski, Christopher A; Scheffler, Brian E; Fang, David D; Chen, Z Jeffrey; Van Deynze, Allen; Stelly, David M

    2015-04-09

    A bacterial artificial chromosome library and BAC-end sequences for cultivated cotton (Gossypium hirsutum L.) have recently been developed. This report presents genome-wide single nucleotide polymorphism (SNP) mining utilizing resequencing data with BAC-end sequences as a reference by alignment of 12 G. hirsutum L. lines, one G. barbadense L. line, and one G. longicalyx Hutch and Lee line. A total of 132,262 intraspecific SNPs have been developed for G. hirsutum, whereas 223,138 and 470,631 interspecific SNPs have been developed for G. barbadense and G. longicalyx, respectively. Using a set of interspecific SNPs, 11 randomly selected and 77 SNPs that are putatively associated with the homeologous chromosome pair 12 and 26, we mapped 77 SNPs into two linkage groups representing these chromosomes, spanning a total of 236.2 cM in an interspecific F2 population (G. barbadense 3-79 × G. hirsutum TM-1). The mapping results validated the approach for reliably producing large numbers of both intraspecific and interspecific SNPs aligned to BAC-ends. This will allow for future construction of high-density integrated physical and genetic maps for cotton and other complex polyploid genomes. The methods developed will allow for future Gossypium resequencing data to be automatically genotyped for identified SNPs along the BAC-end sequence reference for anchoring sequence assemblies and comparative studies.

  9. Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique.

    PubMed

    Hahn, Gabriele; Rose, Dietlind; Wagner, Markus; Rhiel, Sylvia; McVoy, Michael A

    2003-03-01

    The 230-kb human cytomegalovirus genome is among the largest of the known viruses. Experiments to determine the genetic determinants of attenuation, pathogenesis, and tissue tropism are underway; however, a lack of complete sequence data for multiple strains and substantial problems with genetic instability during in vitro propagation create serious complications for such studies. For example, recent findings suggest that common laboratory strains Towne and AD169 passaged in cultured human fibroblasts are missing up to 15 kb of genetic information relative to clinical isolates. To establish standard, genetically stable genomes that can be sequenced, disseminated, and repeatedly reconstituted to produce virus stocks, we have undertaken to clone two variants of Towne, designated Towne(long) and Towne(short) (referred to as TownevarRIT3) (A., Proc. Natl. Acad. Sci. USA 98, 7829-7834), and the pathogenic strain Toledo into bacterial artificial chromosomes (BACs). We further demonstrate the ease with which mutagenesis can be achieved by deleting 13.5 kb from the Toledo genome using a PCR-based technique.

  10. Synthesis of an arrayed sgRNA library targeting the human genome

    PubMed Central

    Schmidt, Tobias; Schmid-Burgk, Jonathan L.; Hornung, Veit

    2015-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested. PMID:26446710

  11. Sequence composition of BAC clones and SSR markers mapped to Upland cotton chromosomes 11 and 21 targeting resistance to soil-borne pathogens

    PubMed Central

    Wang, Congli; Ulloa, Mauricio; Shi, Xinyi; Yuan, Xiaohui; Saski, Christopher; Yu, John Z.; Roberts, Philip A.

    2015-01-01

    Genetic and physical framework mapping in cotton (Gossypium spp.) were used to discover putative gene sequences involved in resistance to common soil-borne pathogens. Chromosome (Chr) 11 and its homoeologous Chr 21 of Upland cotton (G. hirsutum) are foci for discovery of resistance (R) or pathogen-induced R (PR) genes underlying QTLs involved in response to root-knot nematode (Meloidogyne incognita), reniform nematode (Rotylenchulus reniformis), Fusarium wilt (Fusarium oxysporum f.sp. vasinfectum), Verticillium wilt (Verticillium dahliae), and black root rot (Thielaviopsis basicola). Simple sequence repeat (SSR) markers and bacterial artificial chromosome (BAC) clones from a BAC library developed from the Upland cotton Acala Maxxa were mapped on Chr 11 and Chr 21. DNA sequence through Gene Ontology (GO) of 99 of 256 Chr 11 and 109 of 239 Chr 21 previously mapped SSRs revealed response elements to internal and external stimulus, stress, signaling process, and cell death. The reconciliation between genetic and physical mapping of gene annotations from new DNA sequences of 20 BAC clones revealed 467 (Chr 11) and 285 (Chr 21) G. hirsutum putative coding sequences, plus 146 (Chr 11) and 98 (Chr 21) predicted genes. GO functional profiling of Unigenes uncovered genes involved in different metabolic functions and stress response elements (SRE). Our results revealed that Chrs 11 and 21 harbor resistance gene rich genomic regions. Sequence comparisons with the ancestral diploid D5 (G. raimondii), A2 (G. arboreum) and domesticated tetraploid TM-1 AD1 (G. hirsutum) genomes revealed abundance of transposable elements and confirmed the richness of resistance gene motifs in these chromosomes. The sequence information of SSR markers and BAC clones and the genetic mapping of BAC clones provide enhanced genetic and physical frameworks of resistance gene-rich regions of the cotton genome, thereby aiding discovery of R and PR genes and breeding for resistance to cotton diseases. PMID

  12. Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4+ T Cells

    PubMed Central

    Gogol-Döring, Andreas; Ammar, Ismahen; Gupta, Saumyashree; Bunse, Mario; Miskey, Csaba; Chen, Wei; Uckert, Wolfgang; Schulz, Thomas F; Izsvák, Zsuzsanna; Ivics, Zoltán

    2016-01-01

    The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We analyzed the genome-wide distributions of Sleeping Beauty (SB) and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4+ T cells with respect to a panel of 40 chromatin states. The distribution of SB transposon insertions displayed the least deviation from random, while the PB transposon and the MLV retrovirus showed unexpected parallels across all chromatin states. Both MLV and PB insertions are enriched at transcriptional start sites (TSSs) and co-localize with BRD4-associated sites. We demonstrate physical interaction between the PB transposase and bromodomain and extraterminal domain proteins (including BRD4), suggesting convergent evolution of a tethering mechanism that directs integrating genetic elements into TSSs. We detect unequal biases across the four systems with respect to targeting genes whose deregulation has been previously linked to serious adverse events in gene therapy clinical trials. The SB transposon has the highest theoretical chance of targeting a safe harbor locus in the human genome. The data underscore the significance of vector choice to reduce the mutagenic load on cells in clinical applications. PMID:26755332

  13. Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

    PubMed Central

    Ruberti, I; Fragapane, P; Pierandrei-Amaldi, P; Beccari, E; Amaldi, F; Bozzoni, I

    1982-01-01

    Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved. Images PMID:6296782

  14. A new age in functional genomics using CRISPR/Cas9 in arrayed library screening

    PubMed Central

    Agrotis, Alexander; Ketteler, Robin

    2015-01-01

    CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening. PMID:26442115

  15. A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.

    PubMed

    Agrotis, Alexander; Ketteler, Robin

    2015-01-01

    CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

  16. Sequencing the maize genome.

    PubMed

    Martienssen, Robert A; Rabinowicz, Pablo D; O'Shaughnessy, Andrew; McCombie, W Richard

    2004-04-01

    Sequencing of complex genomes can be accomplished by enriching shotgun libraries for genes. In maize, gene-enrichment by copy-number normalization (high C(0)t) and methylation filtration (MF) have been used to generate up to two-fold coverage of the gene-space with less than 1 million sequencing reads. Simulations using sequenced bacterial artificial chromosome (BAC) clones predict that 5x coverage of gene-rich regions, accompanied by less than 1x coverage of subclones from BAC contigs, will generate high-quality mapped sequence that meets the needs of geneticists while accommodating unusually high levels of structural polymorphism. By sequencing several inbred strains, we propose a strategy for capturing this polymorphism to investigate hybrid vigor or heterosis.

  17. A Computational Solution to Automatically Map Metabolite Libraries in the Context of Genome Scale Metabolic Networks.

    PubMed

    Merlet, Benjamin; Paulhe, Nils; Vinson, Florence; Frainay, Clément; Chazalviel, Maxime; Poupin, Nathalie; Gloaguen, Yoann; Giacomoni, Franck; Jourdan, Fabien

    2016-01-01

    This article describes a generic programmatic method for mapping chemical compound libraries on organism-specific metabolic networks from various databases (KEGG, BioCyc) and flat file formats (SBML and Matlab files). We show how this pipeline was successfully applied to decipher the coverage of chemical libraries set up by two metabolomics facilities MetaboHub (French National infrastructure for metabolomics and fluxomics) and Glasgow Polyomics (GP) on the metabolic networks available in the MetExplore web server. The present generic protocol is designed to formalize and reduce the volume of information transfer between the library and the network database. Matching of metabolites between libraries and metabolic networks is based on InChIs or InChIKeys and therefore requires that these identifiers are specified in both libraries and networks. In addition to providing covering statistics, this pipeline also allows the visualization of mapping results in the context of metabolic networks. In order to achieve this goal, we tackled issues on programmatic interaction between two servers, improvement of metabolite annotation in metabolic networks and automatic loading of a mapping in genome scale metabolic network analysis tool MetExplore. It is important to note that this mapping can also be performed on a single or a selection of organisms of interest and is thus not limited to large facilities.

  18. A Computational Solution to Automatically Map Metabolite Libraries in the Context of Genome Scale Metabolic Networks

    PubMed Central

    Merlet, Benjamin; Paulhe, Nils; Vinson, Florence; Frainay, Clément; Chazalviel, Maxime; Poupin, Nathalie; Gloaguen, Yoann; Giacomoni, Franck; Jourdan, Fabien

    2016-01-01

    This article describes a generic programmatic method for mapping chemical compound libraries on organism-specific metabolic networks from various databases (KEGG, BioCyc) and flat file formats (SBML and Matlab files). We show how this pipeline was successfully applied to decipher the coverage of chemical libraries set up by two metabolomics facilities MetaboHub (French National infrastructure for metabolomics and fluxomics) and Glasgow Polyomics (GP) on the metabolic networks available in the MetExplore web server. The present generic protocol is designed to formalize and reduce the volume of information transfer between the library and the network database. Matching of metabolites between libraries and metabolic networks is based on InChIs or InChIKeys and therefore requires that these identifiers are specified in both libraries and networks. In addition to providing covering statistics, this pipeline also allows the visualization of mapping results in the context of metabolic networks. In order to achieve this goal, we tackled issues on programmatic interaction between two servers, improvement of metabolite annotation in metabolic networks and automatic loading of a mapping in genome scale metabolic network analysis tool MetExplore. It is important to note that this mapping can also be performed on a single or a selection of organisms of interest and is thus not limited to large facilities. PMID:26909353

  19. Library+

    ERIC Educational Resources Information Center

    Merrill, Alex

    2011-01-01

    This article discusses possible future directions for academic libraries in the post Web/Library 2.0 world. These possible directions include areas such as data literacy, linked data sets, and opportunities for libraries in support of digital humanities. The author provides a brief sketch of the background information regarding the topics and…

  20. Genome-Wide Sequence Comparison of Centromeric Regions and BAC-Landing on Chromosomes Provide New Insights into Centromere Evolution Among Wheat, Brachypodium, and Rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an emerging model system, the nearly finished sequence of Brachypodium distachyon will provide new insights into comparative and functional genomics of grass species. However, centromeres of B. distachyon are unlikely to be sequenced and assembled precisely similar to many other sequenced organis...

  1. Genome resource for the Indonesian coelacanth, Latimeria menadoensis.

    PubMed

    Danke, Joshua; Miyake, Tsutomu; Powers, Thomas; Schein, Jacqueline; Shin, Heesun; Bosdet, Ian; Erdmann, Mark; Caldwell, Roy; Amemiya, Chris T

    2004-03-01

    We have generated a BAC library from the Indonesian coelacanth, Latimeria menadoensis. This library was generated using genomic DNA of nuclei isolated from heart tissue, and has an average insert size of 171 kb. There are a total of 288 384-well microtiter dishes in the library (110,592 clones) and its genomic representation is estimated to encompass > or = 7X coverage based on the amount of DNA presumably cloned in the library as well as via hybridization with probes to a small set of single copy genes. This genomic resource has been made available to the public and should prove useful to the scientific community for many applications, including comparative genomics, molecular evolution and conservation genetics.

  2. A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish

    PubMed Central

    Fuentes, Fernando; Reynolds, Eric; Lewellis, Stephen W.; Venkiteswaran, Gayatri; Knaut, Holger

    2016-01-01

    Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels. PMID:26818072

  3. A genome-wide library of CB4856/N2 introgression lines of Caenorhabditis elegans

    PubMed Central

    Doroszuk, Agnieszka; Snoek, L. Basten; Fradin, Emilie; Riksen, Joost; Kammenga, Jan

    2009-01-01

    Recombinant inbred lines (RILs) derived from Caenorhabditis elegans wild-type N2 and CB4856 are increasingly being used for mapping genes underlying complex traits. To speed up mapping and gene discovery, introgression lines (ILs) offer a powerful tool for more efficient QTL identification. We constructed a library of 90 ILs, each carrying a single homozygous CB4856 genomic segment introgressed into the genetic background of N2. The ILs were genotyped by 123 single-nucleotide polymorphism (SNP) markers. The proportion of the CB4856 segments in most lines does not exceed 3%, and together the introgressions cover 96% of the CB4856 genome. The value of the IL library was demonstrated by identifying novel loci underlying natural variation in two ageing-related traits, i.e. lifespan and pharyngeal pumping rate. Bin mapping of lifespan resulted in six QTLs, which all have a lifespan-shortening effect on the CB4856 allele. We found five QTLs for the decrease in pumping rate, of which four colocated with QTLs found for average lifespan. This suggests pleiotropic or closely linked QTL associated with lifespan and pumping rate. Overall, the presented IL library provides a versatile resource toward easier and efficient fine mapping and functional analyses of loci and genes underlying complex traits in C. elegans. PMID:19542186

  4. Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

    PubMed

    Koo, Byoung-Mo; Kritikos, George; Farelli, Jeremiah D; Todor, Horia; Tong, Kenneth; Kimsey, Harvey; Wapinski, Ilan; Galardini, Marco; Cabal, Angelo; Peters, Jason M; Hachmann, Anna-Barbara; Rudner, David Z; Allen, Karen N; Typas, Athanasios; Gross, Carol A

    2017-03-22

    A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.

  5. A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

    PubMed Central

    Bassett, Andrew R.; Kong, Lesheng; Liu, Ji-Long

    2015-01-01

    The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies. PMID:26165496

  6. A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells.

    PubMed

    Bassett, Andrew R; Kong, Lesheng; Liu, Ji-Long

    2015-06-20

    The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines, enabling screening for cellular phenotypes resulting from genetic aberrations. Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi. This is in part due to the lower degree of redundancy between genes in this organism, whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes. The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques, but allows analysis over longer periods of time which can be critical for certain phenotypes. In this study, we have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies.

  7. Functional Selection of Vaccine Candidate Peptides from Staphylococcus aureus Whole-Genome Expression Libraries In Vitro

    PubMed Central

    Weichhart, Thomas; Horky, Markus; Söllner, Johannes; Gangl, Susanne; Henics, Tamàs; Nagy, Eszter; Meinke, Andreas; von Gabain, Alexander; Fraser, Claire M.; Gill, Steve R.; Hafner, Martin; von Ahsen, Uwe

    2003-01-01

    An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen Staphylococcus aureus. A library built up from genomic fragments of the virulent S. aureus COL strain (methicillin-resistant S. aureus) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a high antibody titer and opsonic activity against S. aureus were used, since these antibodies indicate the in vivo expression and immunoreactivity of the corresponding proteins. Identified clones cluster in distinct regions of 75 genes, most of them classifiable as secreted or surface-localized proteins, including previously identified virulence factors. In addition, 14 putative novel short open reading frames were identified and their immunoreactivity and in vivo mRNA expression were confirmed, underscoring the annotation-independent, true genomic nature of our approach. Evidence is provided that a large fraction of the identified peptides cannot be expressed in an in vivo-based surface display system. Thus, in vitro protein selection, not biased by the context of living entities, allows screening of genomic expression libraries with a large number of different ligands simultaneously. It is a powerful approach for fingerprinting the repertoire of immune reactive proteins serving as target candidates for active and passive vaccination against pathogens. PMID:12874343

  8. Construction and analysis of Pst I DNA library for RFLP mapping of the rye genome

    SciTech Connect

    Korzun, V.N.; Kartel, N.A.; Boerner, A.

    1995-06-01

    Pst I, a methylation-sensitive restriction enzyme, was used for producing a library of rye genome DNA rich in low-copy sequences, and intended as probes for genetic mapping. Dot-hybridization and Southern blot analysis showed that 43.6% of the library is represented by low-copy DNA sequences. To locate these sequences on chromosomes and determine the degree of their repetitiveness, 11 clones were hybridized with DNA of nulli-tetrasomic lines of Chinese Spring wheat, wheat-rye addition lines, and barley cleaved by Hind III, EcoR I, EcoR V, Dra I, and BamH I restriction enzymes. Each of the rye DNA clones studied hybridized with wheat and barley DNA, suggesting that low-copy Pst I clones of rye correspond to the evolutionary conservative DNA fraction in cereals. 21 refs., 3 figs., 2 tabs.

  9. Removal of Inserted BAC after linearizatiON (RIBON)-a novel strategy to excise the mini-F sequences from viral BAC vectors.

    PubMed

    Ishihara, Yukari; Esaki, Motoyuki; Yasuda, Atsushi

    2016-08-01

    The bacterial artificial chromosome (BAC) technology has been a mainstay approach for generating recombinant viruses, and several methods for excision of the mini-F sequences from the viral BAC vectors have been developed. However, these strategies either require complicated procedures or leave scars of inserted sequences. To overcome these problems, a new method to excise the mini-F sequences from viral BAC vectors based on the Removal of Inserted BAC after linearizatiON (RIBON) strategy was developed in this study for herpesvirus of turkeys (HVT). Enhanced green fluorescent protein (eGFP) DNA and the mini-F sequences were inserted into the gene encoding HVT thymidine kinase (TK) by homologous recombination in chicken embryo fibroblasts (CEFs), and the constructed HVT-BAC vector was used to transform Escherichia coli (pHVT-BAC). To remove the inserted eGFP and mini-F sequences, pHVT-BAC was linearized using a homing endonuclease I-SceI and used to cotransfect CEFs together with a plasmid containing the TK gene of HVT. The obtained viruses (44%) did not express eGFP, and DNA sequencing of isolated clones revealed that they were completely free of the inserted BAC sequences. Moreover, growth kinetics and plaque morphology of reconstituted viruses were comparable with those of the parental HVT. The results of this study demonstrate that the novel RIBON approach to remove mini-F sequences from the viral genome is simple and effective.

  10. A High-throughput AFLP-based Method for Constructing Integrated Genetic and Physical Maps: Progress Toward a Sorghum Genome Map

    PubMed Central

    Klein, Patricia E.; Klein, Robert R.; Cartinhour, Samuel W.; Ulanch, Paul E.; Dong, Jianmin; Obert, Jacque A.; Morishige, Daryl T.; Schlueter, Shannon D.; Childs, Kevin L.; Ale, Melissa; Mullet, John E.

    2000-01-01

    Sorghum is an important target for plant genomic mapping because of its adaptation to harsh environments, diverse germplasm collection, and value for comparing the genomes of grass species such as corn and rice. The construction of an integrated genetic and physical map of the sorghum genome (750 Mbp) is a primary goal of our sorghum genome project. To help accomplish this task, we have developed a new high-throughput PCR-based method for building BAC contigs and locating BAC clones on the sorghum genetic map. This task involved pooling 24,576 sorghum BAC clones (∼4× genome equivalents) in six different matrices to create 184 pools of BAC DNA. DNA fragments from each pool were amplified using amplified fragment length polymorphism (AFLP) technology, resolved on a LI-COR dual-dye DNA sequencing system, and analyzed using Bionumerics software. On average, each set of AFLP primers amplified 28 single-copy DNA markers that were useful for identifying overlapping BAC clones. Data from 32 different AFLP primer combinations identified ∼2400 BACs and ordered ∼700 BAC contigs. Analysis of a sorghum RIL mapping population using the same primer pairs located ∼200 of the BAC contigs on the sorghum genetic map. Restriction endonuclease fingerprinting of the entire collection of sorghum BAC clones was applied to test and extend the contigs constructed using this PCR-based methodology. Analysis of the fingerprint data allowed for the identification of 3366 contigs each containing an average of 5 BACs. BACs in ∼65% of the contigs aligned by AFLP analysis had sufficient overlap to be confirmed by DNA fingerprint analysis. In addition, 30% of the overlapping BACs aligned by AFLP analysis provided information for merging contigs and singletons that could not be joined using fingerprint data alone. Thus, the combination of fingerprinting and AFLP-based contig assembly and mapping provides a reliable, high-throughput method for building an integrated genetic and physical map

  11. Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences.

    PubMed

    Vardi, Oriya; Shamir, Inbal; Javasky, Elisheva; Goren, Alon; Simon, Itamar

    2017-01-01

    Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5' end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation.

  12. Biases in the SMART-DNA library preparation method associated with genomic poly dA/dT sequences

    PubMed Central

    Shamir, Inbal; Javasky, Elisheva; Goren, Alon; Simon, Itamar

    2017-01-01

    Avoiding biases in next generation sequencing (NGS) library preparation is crucial for obtaining reliable sequencing data. Recently, a new library preparation method has been introduced which has eliminated the need for the ligation step. This method, termed SMART (switching mechanism at the 5′ end of the RNA transcript), is based on template switching reverse transcription. To date, there has been no systematic analysis of the additional biases introduced by this method. We analysed the genomic distribution of sequenced reads prepared from genomic DNA using the SMART methodology and found a strong bias toward long (≥12bp) poly dA/dT containing genomic loci. This bias is unique to the SMART-based library preparation and does not appear when libraries are prepared with conventional ligation based methods. Although this bias is obvious only when performing paired end sequencing, it affects single end sequenced samples as well. Our analysis demonstrates that sequenced reads originating from SMART-DNA libraries are heavily skewed toward genomic poly dA/dT tracts. This bias needs to be considered when deciding to use SMART based technology for library preparation. PMID:28235101

  13. Pulling out the 1%: whole-genome capture for the targeted enrichment of ancient DNA sequencing libraries.

    PubMed

    Carpenter, Meredith L; Buenrostro, Jason D; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M Thomas P; Willerslev, Eske; Greenleaf, William J; Bustamante, Carlos D

    2013-11-07

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062-147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217-73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples.

  14. Pulling out the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries

    PubMed Central

    Carpenter, Meredith L.; Buenrostro, Jason D.; Valdiosera, Cristina; Schroeder, Hannes; Allentoft, Morten E.; Sikora, Martin; Rasmussen, Morten; Gravel, Simon; Guillén, Sonia; Nekhrizov, Georgi; Leshtakov, Krasimir; Dimitrova, Diana; Theodossiev, Nikola; Pettener, Davide; Luiselli, Donata; Sandoval, Karla; Moreno-Estrada, Andrés; Li, Yingrui; Wang, Jun; Gilbert, M. Thomas P.; Willerslev, Eske; Greenleaf, William J.; Bustamante, Carlos D.

    2013-01-01

    Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain <1% endogenous DNA, with the majority of sequencing capacity taken up by environmental DNA. Here we present a capture-based method for enriching the endogenous component of aDNA sequencing libraries. By using biotinylated RNA baits transcribed from genomic DNA libraries, we are able to capture DNA fragments from across the human genome. We demonstrate this method on libraries created from four Iron Age and Bronze Age human teeth from Bulgaria, as well as bone samples from seven Peruvian mummies and a Bronze Age hair sample from Denmark. Prior to capture, shotgun sequencing of these libraries yielded an average of 1.2% of reads mapping to the human genome (including duplicates). After capture, this fraction increased substantially, with up to 59% of reads mapped to human and enrichment ranging from 6- to 159-fold. Furthermore, we maintained coverage of the majority of regions sequenced in the precapture library. Intersection with the 1000 Genomes Project reference panel yielded an average of 50,723 SNPs (range 3,062–147,243) for the postcapture libraries sequenced with 1 million reads, compared with 13,280 SNPs (range 217–73,266) for the precapture libraries, increasing resolution in population genetic analyses. Our whole-genome capture approach makes it less costly to sequence aDNA from specimens containing very low levels of endogenous DNA, enabling the analysis of larger numbers of samples. PMID:24568772

  15. Rapid editing and evolution of bacterial genomes using libraries of synthetic DNA.

    PubMed

    Gallagher, Ryan R; Li, Zhe; Lewis, Aaron O; Isaacs, Farren J

    2014-10-01

    Multiplex automated genome engineering (MAGE) is a powerful technology for in vivo genome editing that uses synthetic single-stranded DNA (ssDNA) to introduce targeted modifications directly into the Escherichia coli chromosome. MAGE is a cyclical process that involves transformation of ssDNA (by electroporation) followed by outgrowth, during which bacteriophage homologous recombination proteins mediate annealing of ssDNAs to their genomic targets. By iteratively introducing libraries of mutagenic ssDNAs targeting multiple sites, MAGE can generate combinatorial genetic diversity in a cell population. Alternatively, MAGE can introduce precise mutant alleles at many loci for genome-wide editing or for recoding projects that are not possible with other methods. In recent technological advances, MAGE has been improved by strain modifications and selection techniques that enhance allelic replacement. This protocol describes the manual execution of MAGE wherein each cycle takes ≈ 2.5 h, which, if carried out by two people, allows ≈ 10 continuous cycles of MAGE-based mutagenesis per day.

  16. Analysis of genomic regions of Trichoderma harzianum IOC-3844 related to biomass degradation.

    PubMed

    Crucello, Aline; Sforça, Danilo Augusto; Horta, Maria Augusta Crivelente; dos Santos, Clelton Aparecido; Viana, Américo José Carvalho; Beloti, Lilian Luzia; de Toledo, Marcelo Augusto Szymanski; Vincentz, Michel; Kuroshu, Reginaldo Massanobu; de Souza, Anete Pereira

    2015-01-01

    Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes.

  17. Analysis of Genomic Regions of Trichoderma harzianum IOC-3844 Related to Biomass Degradation

    PubMed Central

    Crucello, Aline; Sforça, Danilo Augusto; Horta, Maria Augusta Crivelente; dos Santos, Clelton Aparecido; Viana, Américo José Carvalho; Beloti, Lilian Luzia; de Toledo, Marcelo Augusto Szymanski; Vincentz, Michel; Kuroshu, Reginaldo Massanobu; de Souza, Anete Pereira

    2015-01-01

    Trichoderma harzianum IOC-3844 secretes high levels of cellulolytic-active enzymes and is therefore a promising strain for use in biotechnological applications in second-generation bioethanol production. However, the T. harzianum biomass degradation mechanism has not been well explored at the genetic level. The present work investigates six genomic regions (~150 kbp each) in this fungus that are enriched with genes related to biomass conversion. A BAC library consisting of 5,760 clones was constructed, with an average insert length of 90 kbp. The assembled BAC sequences revealed 232 predicted genes, 31.5% of which were related to catabolic pathways, including those involved in biomass degradation. An expression profile analysis based on RNA-Seq data demonstrated that putative regulatory elements, such as membrane transport proteins and transcription factors, are located in the same genomic regions as genes related to carbohydrate metabolism and exhibit similar expression profiles. Thus, we demonstrate a rapid and efficient tool that focuses on specific genomic regions by combining a BAC library with transcriptomic data. This is the first BAC-based structural genomic study of the cellulolytic fungus T. harzianum, and its findings provide new perspectives regarding the use of this species in biomass degradation processes. PMID:25836973

  18. Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries.

    PubMed

    Goodman, Andrew L; Wu, Meng; Gordon, Jeffrey I

    2011-11-17

    Insertion sequencing (INSeq) is a method for determining the insertion site and relative abundance of large numbers of transposon mutants in a mixed population of isogenic mutants of a sequenced microbial species. INSeq is based on a modified mariner transposon containing MmeI sites at its ends, allowing cleavage at chromosomal sites 16-17 bp from the inserted transposon. Genomic regions adjacent to the transposons are amplified by linear PCR with a biotinylated primer. Products are bound to magnetic beads, digested with MmeI and barcoded with sample-specific linkers appended to each restriction fragment. After limited PCR amplification, fragments are sequenced using a high-throughput instrument. The sequence of each read can be used to map the location of a transposon in the genome. Read count measures the relative abundance of that mutant in the population. Solid-phase library preparation makes this protocol rapid (18 h), easy to scale up, amenable to automation and useful for a variety of samples. A protocol for characterizing libraries of transposon mutant strains clonally arrayed in a multiwell format is provided.

  19. Genomic Libraries and a Host Strain Designed for Highly Efficient Two-Hybrid Selection in Yeast

    PubMed Central

    James, P.; Halladay, J.; Craig, E. A.

    1996-01-01

    The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections. PMID:8978031

  20. High-throughput screening of a Corynebacterium glutamicum mutant library on genomic and metabolic level.

    PubMed

    Reimer, Lorenz C; Spura, Jana; Schmidt-Hohagen, Kerstin; Schomburg, Dietmar

    2014-01-01

    Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known

  1. A kingdom-specific protein domain HMM library for improved annotation of fungal genomes

    PubMed Central

    Alam, Intikhab; Hubbard, Simon J; Oliver, Stephen G; Rattray, Magnus

    2007-01-01

    Background Pfam is a general-purpose database of protein domain alignments and profile Hidden Markov Models (HMMs), which is very popular for the annotation of sequence data produced by genome sequencing projects. Pfam provides models that are often very general in terms of the taxa that they cover and it has previously been suggested that such general models may lack some of the specificity or selectivity that would be provided by kingdom-specific models. Results Here we present a general approach to create domain libraries of HMMs for sub-taxa of a kingdom. Taking fungal species as an example, we construct a domain library of HMMs (called Fungal Pfam or FPfam) using sequences from 30 genomes, consisting of 24 species from the ascomycetes group and two basidiomycetes, Ustilago maydis, a fungal pathogen of maize, and the white rot fungus Phanerochaete chrysosporium. In addition, we include the Microsporidion Encephalitozoon cuniculi, an obligate intracellular parasite, and two non-fungal species, the oomycetes Phytophthora sojae and Phytophthora ramorum, both plant pathogens. We evaluate the performance in terms of coverage against the original 30 genomes used in training FPfam and against five more recently sequenced fungal genomes that can be considered as an independent test set. We show that kingdom-specific models such as FPfam can find instances of both novel and well characterized domains, increases overall coverage and detects more domains per sequence with typically higher bitscores than Pfam for the same domain families. An evaluation of the effect of changing E-values on the coverage shows that the performance of FPfam is consistent over the range of E-values applied. Conclusion Kingdom-specific models are shown to provide improved coverage. However, as the models become more specific, some sequences found by Pfam may be missed by the models in FPfam and some of the families represented in the test set are not present in FPfam. Therefore, we recommend

  2. Ligation bias in illumina next-generation DNA libraries: implications for sequencing ancient genomes.

    PubMed

    Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel; Stagegaard, Julia; Alberdi, Maria T; Prado, José Luis; Prieto, Alfredo; Willerslev, Eske; Orlando, Ludovic

    2013-01-01

    Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries.

  3. Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

    PubMed Central

    Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel; Stagegaard, Julia; Alberdi, Maria T.; Prado, José Luis; Prieto, Alfredo; Willerslev, Eske; Orlando, Ludovic

    2013-01-01

    Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries. PMID:24205269

  4. Characterization of Uncultured Genome Fragment from Soil Metagenomic Library Exposed Rare Mismatch of Internal Tetranucleotide Frequency

    PubMed Central

    Liu, Yunpeng; Yang, Dongqing; Zhang, Nan; Chen, Lin; Cui, Zhongli; Shen, Qirong; Zhang, Ruifu

    2016-01-01

    Exploring the genomic information of a specific uncultured soil bacterium is vital to understand its function in the ecosystem but is still a challenge due to the lack of culture techniques. To examine the genomes of uncultured bacteria, a metagenomic bacterial artificial chromosome library derived from a soil sample was screened for 16S rDNA-containing clones. Five clones (4C6, 5E7, 5G4, 5G12, and 5H7) containing uncultured soil bacteria genome fragment (with low 16S rDNA similarity to isolated bacteria) were selected for sequencing. Clone 5E7 and 5G4 showed only 82 and 83% of 16S rDNA identity to known sequences. Phylogenetic analysis of 16S rDNA indicated that 5E7 and 5G4 were potentially from new class of Chloroflexi. Only one-third of the 5G4 open reading frames have significant hits against HMMER. Internal tetranucleotide frequency analysis indicated that the unknown region of 5G4 was poorly correlated with other parts of the clone, indicating that this section might be obtained through lateral transfer. It was suggested that this region rich for unknown genes is under fast evolution. PMID:28066395

  5. Effects of Blood-Alcohol Concentration (BAC) Feedback on BAC Estimates Over Time

    ERIC Educational Resources Information Center

    Bullers, Susan; Ennis, Melissa

    2006-01-01

    This study examines the effects of self-tested blood alcohol concentration (BAC) feedback, from personal hand-held breathalyzers, on the accuracy of BAC estimation. Using an e-mail prompted web-based questionnaire, 19 participants were asked to report both BAC estimates and subsequently measured BAC levels over the course of 27 days. Results from…

  6. Construction of DNA libraries from flow sorted human chromosomes

    SciTech Connect

    Deaven, L.L.; McCormick, M.K.; Grady, D.L.

    1994-09-01

    We have constructed a series of DNA libraries from flow-sorted chromosomes. Small insert, complete digest libraries cloned into the EcoRI insertion site of Charon 21A are available from the American Type Culture Collection, Rockville, MD. Partial digest libraries cloned into cosmid (sCos1) or phage (Charon 40) vectors have been constructed for chromosomes 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, X and Y. Purity estimates by in situ analysis of sorted chromosomes, flow karyotype analysis, and plaque or colony hybridization indicate that most of these libraries are 90-95% pure. Additional cosmid library constructions, 5-10X arrays of libraries into microtiter plates, and high density membrane arrays of libraries are in progress. Recently, we have completed YAC libraries for chromosomes 5, 9, 16, and 21. These libraries are made from complete DNA digests using the rare cutters Clal, SacII, EagI, or NotI/NheI. The average insert size is {similar_to}200 kb, and chimera frequencies are low (1-10%). Libraries have also been constructed using M13 or bluescript vectors (chromosomes 5, 7, 17) to generate STS markers for the selection of chromosome-specific inserts from total genomic AC libraries. Because of the advantages of insert size and stability associated with BAC and PAC cloning systems, we are currently attempting to adapt pBAC108L and pCYPAC1 vectors for use with flow-sorted chromosomal DNA.

  7. A low-copy-number plasmid for retrieval of toxic genes from BACs and generation of conditional targeting constructs.

    PubMed

    Na, Giyoun; Wolfe, Andrew; Ko, Chemyong; Youn, Hyesook; Lee, Young-Min; Byun, Sung June; Jeon, Iksoo; Koo, Yongbum

    2013-06-01

    Bacterial Artificial Chromosome (BAC) clones are widely used for retrieving genomic DNA sequences for gene targeting. In this study, low-copy-number plasmids pBAC-FB, pBAC-FC, and pBAC-DE, which carry the F plasmid replicon, were generated from pBACe3.6. pBAC-FB was successfully used to retrieve a sequence of a BAC that was resistant to retrieval by a high-copy-number plasmid via λ Red-mediated recombineering (gap-repair cloning). This plasmid was also used to retrieve two other genes from BAC, indicating its general usability retrieving genes from BAC. The retrieved genes were manipulated in generating targeting vectors for gene knockouts by recombineering. The functionality of the targeting vector was further validated in a targeting experiment with C57BL/6 embryonic stem cells. The low-copy-number plasmid pBAC-FB is a plasmid of choice to retrieve toxic DNA sequences from BACs and to manipulate them to generate gene-targeting constructs by recombineering.

  8. SiRNA sequence model: redesign algorithm based on available genome-wide libraries.

    PubMed

    Kozak, Karol

    2013-12-01

    The evolution of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in cells. Design tools have been developed based on experimental data to increase the knockdown efficiency of siRNAs. Not all siRNAs that are developed to a given target mRNA are equally effective. Currently available design algorithms take an accession, identify conserved regions among their transcript space, find accessible regions within the mRNA, design all possible siRNAs for these regions, filter them based on multi-scores thresholds, and then perform off-target filtration. These different criteria are used by commercial suppliers to produce siRNA genome-wide libraries for different organisms. In this article, we analyze existing siRNA design algorithms and evaluate weight of design parameters for libraries produced in the last decade. We proved that not all essential parameters are currently applied by siRNA vendors. Based on our evaluation results, we were able to suggest an siRNA sequence pattern. The findings in our study can be useful for commercial vendors improving the design of RNAi constructs, by addressing both the issue of potency and the issue of specificity.

  9. Development of SSR markers derived from SSR-enriched genomic library of eggplant (Solanum melongena L.).

    PubMed

    Nunome, Tsukasa; Negoro, Satomi; Kono, Izumi; Kanamori, Hiroyuki; Miyatake, Koji; Yamaguchi, Hirotaka; Ohyama, Akio; Fukuoka, Hiroyuki

    2009-10-01

    Eggplant (Solanum melongena L.), also known as aubergine or brinjal, is an important vegetable in many countries. Few useful molecular markers have been reported for eggplant. We constructed simple sequence repeat (SSR)-enriched genomic libraries in order to develop SSR markers, and sequenced more than 14,000 clones. From these sequences, we designed 2,265 primer pairs to flank SSR motifs. We identified 1,054 SSR markers from amplification of 1,399 randomly selected primer pairs. The markers have an average polymorphic information content of 0.27 among eight lines of S. melongena. Of the 1,054 SSR markers, 214 segregated in an intraspecific mapping population. We constructed cDNA libraries from several eggplant tissues and obtained 6,144 expressed sequence tag (EST) sequences. From these sequences, we designed 209 primer pairs, 7 of which segregated in the mapping population. On the basis of the segregation data, we constructed a linkage map, and mapped the 236 segregating markers to 14 linkage groups. The linkage map spans a total length of 959.1 cM, with an average marker distance of 4.3 cM. The markers should be a useful resource for qualitative and quantitative trait mapping and for marker-assisted selection in eggplant breeding.

  10. Recovery of a soybean urease genomic clone by sequential library screening with two synthetic oligodeoxynucleotides.

    PubMed

    Krueger, R W; Holland, M A; Chisholm, D; Polacco, J C

    1987-01-01

    We report the first isolation of a low-copy-number gene from a complex higher plant (soybean) genome by direct screening with synthetic oligodeoxynucleotide (oligo) probes. A synthetic, mixed, 21-nucleotide (nt) oligo (21-1) based on a seven amino acid (aa) sequence from soybean seed urease, was used to screen genomic libraries of soybean (Glycine max [L.] Merr.) in the lambda Charon 4 vector. Twenty homologous clones were recovered from a screen of 500,000 plaques. These were counterscreened with embryo-specific cDNA (15-2 cDNA) made by priming with a second, mixed 15-nt oligo (15-2), based on a Jack bean (Canavalia ensiformis) urease peptide [Takishima et al., J. Natl. Def. Med. Coll. 5 (1980) 19-23]. Five out of 20 clones were homologous to 15-2 cDNA and proved to be identical. Nucleotide sequence analysis of representative clone E15 confirmed that it contained urease sequences. Subclones of E15 homologous to the oligo probes contain a deduced amino acid sequence which matches 108 of 130 aa residues of an amino acid run in a recently published [Mamiya et al., Proc. Jap. Acad. 61B (1985) 359-398] complete protein sequence for Jack-bean seed urease. Using clone E15 as a probe of soybean embryonic mRNA revealed a homologous 3.8-kb species that is the size of the urease messenger. This species is absent from mRNA of embryos of a soybean seed urease-null mutant. However, both urease-positive and urease-null genomes contain the 11-kb DNA fragment bearing urease sequences.

  11. Advances in plant chromosome genomics.

    PubMed

    Doležel, Jaroslav; Vrána, Jan; Cápal, Petr; Kubaláková, Marie; Burešová, Veronika; Simková, Hana

    2014-01-01

    Next generation sequencing (NGS) is revolutionizing genomics and is providing novel insights into genome organization, evolution and function. The number of plant genomes targeted for sequencing is rising. For the moment, however, the acquisition of full genome sequences in large genome species remains difficult, largely because the short reads produced by NGS platforms are inadequate to cope with repeat-rich DNA, which forms a large part of these genomes. The problem of sequence redundancy is compounded in polyploids, which dominate the plant kingdom. An approach to overcoming some of these difficulties is to reduce the full nuclear genome to its individual chromosomes using flow-sorting. The DNA acquired in this way has proven to be suitable for many applications, including PCR-based physical mapping, in situ hybridization, forming DNA arrays, the development of DNA markers, the construction of BAC libraries and positional cloning. Coupling chromosome sorting with NGS offers opportunities for the study of genome organization at the single chromosomal level, for comparative analyses between related species and for the validation of whole genome assemblies. Apart from the primary aim of reducing the complexity of the template, taking a chromosome-based approach enables independent teams to work in parallel, each tasked with the analysis of a different chromosome(s). Given that the number of plant species tractable for chromosome sorting is increasing, the likelihood is that chromosome genomics - the marriage of cytology and genomics - will make a significant contribution to the field of plant genetics.

  12. A first generation integrated map of the rainbow trout genome

    PubMed Central

    2011-01-01

    Background Rainbow trout (Oncorhynchus mykiss) are the most-widely cultivated cold freshwater fish in the world and an important model species for many research areas. Coupling great interest in this species as a research model with the need for genetic improvement of aquaculture production efficiency traits justifies the continued development of genomics research resources. Many quantitative trait loci (QTL) have been identified for production and life-history traits in rainbow trout. An integrated physical and genetic map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS) programs for improving rainbow trout aquaculture production. Results The first generation integrated map of the rainbow trout genome is composed of 238 BAC contigs anchored to chromosomes of the genetic map. It covers more than 10% of the genome across segments from all 29 chromosomes. Anchoring of 203 contigs to chromosomes of the National Center for Cool and Cold Water Aquaculture (NCCCWA) genetic map was achieved through mapping of 288 genetic markers derived from BAC end sequences (BES), screening of the BAC library with previously mapped markers and matching of SNPs with BES reads. In addition, 35 contigs were anchored to linkage groups of the INRA (French National Institute of Agricultural Research) genetic map through markers that were not informative for linkage analysis in the NCCCWA mapping panel. The ratio of physical to genetic linkage distances varied substantially among chromosomes and BAC contigs with an average of 3,033 Kb/cM. Conclusions The integrated map described here provides a framework for a robust composite genome map for rainbow trout. This resource is needed for genomic analyses in this research model and economically important species and will facilitate comparative genome mapping with other salmonids and with model fish species. This resource will also facilitate efforts to

  13. Swine Genome Sequencing Consortium (SGSC): A Strategic Roadmap for Sequencing The Pig Genome

    PubMed Central

    Schook, Lawrence B.; Beever, Jonathan E.; Rogers, Jane; Humphray, Sean; Archibald, Alan; Chardon, Patrick; Milan, Denis; Rohrer, Gary; Eversole, Kellye

    2005-01-01

    The Swine Genome Sequencing Consortium (SGSC) was formed in September 2003 by academic, government and industry representatives to provide international coordination for sequencing the pig genome. The SGSC’s mission is to advance biomedical research for animal production and health by the development of DNAbased tools and products resulting from the sequencing of the swine genome. During the past 2 years, the SGSC has met bi-annually to develop a strategic roadmap for creating the required scientific resources, to integrate existing physical maps, and to create a sequencing strategy that captured international participation and a broad funding base. During the past year, SGSC members have integrated their respective physical mapping data with the goal of creating a minimal tiling path (MTP) that will be used as the sequencing template. During the recent Plant and Animal Genome meeting (January 16, 2005 San Diego, CA), presentations demonstrated that a human–pig comparative map has been completed, BAC fingerprint contigs (FPC) for each of the autosomes and X chromosome have been constructed and that BAC end-sequencing has permitted, through BLAST analysis and RH-mapping, anchoring of the contigs. Thus, significant progress has been made towards the creation of a MTP. In addition, whole-genome (WG) shotgun libraries have been constructed and are currently being sequenced in various laboratories around the globe. Thus, a hybrid sequencing approach in which 3x coverage of BACs comprising the MTP and 3x of the WG-shotgun libraries will be used to develop a draft 6x coverage of the pig genome. PMID:18629187

  14. Rapid Virulence Annotation (RVA): identification of virulence factors using a bacterial genome library and multiple invertebrate hosts.

    PubMed

    Waterfield, Nicholas R; Sanchez-Contreras, Maria; Eleftherianos, Ioannis; Dowling, Andrea; Yang, Guowei; Wilkinson, Paul; Parkhill, Julian; Thomson, Nicholas; Reynolds, Stuart E; Bode, Helge B; Dorus, Steven; Ffrench-Constant, Richard H

    2008-10-14

    Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.

  15. Cytogenetic Analysis of Populus trichocarpa - Ribosomal DNA, Telomere Repeat Sequence, and Marker-selected BACs

    SciTech Connect

    Tuskan, Gerald A; Gunter, Lee E; DiFazio, Stephen P

    2009-01-01

    The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis -type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequence assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.

  16. Cytogenetic analysis of Populus trichocarpa--ribosomal DNA, telomere repeat sequence, and marker-selected BACs.

    PubMed

    Islam-Faridi, M N; Nelson, C D; DiFazio, S P; Gunter, L E; Tuskan, G A

    2009-01-01

    The 18S-28S rDNA and 5S rDNA loci in Populus trichocarpa were localized using fluorescent in situ hybridization (FISH). Two 18S-28S rDNA sites and one 5S rDNA site were identified and located at the ends of 3 different chromosomes. FISH signals from the Arabidopsis-type telomere repeat sequence were observed at the distal ends of each chromosome. Six BAC clones selected from 2 linkage groups based on genome sequence assembly (LG-I and LG-VI) were localized on 2 chromosomes, as expected. BACs from LG-I hybridized to the longest chromosome in the complement. All BAC positions were found to be concordant with sequence assembly positions. BAC-FISH will be useful for delineating each of the Populus trichocarpa chromosomes and improving the sequence assembly of this model angiosperm tree species.

  17. Towards a Library of Standard Operating Procedures (SOPs) for (meta)genomic annotation

    SciTech Connect

    Kyrpides, Nikos; Angiuoli, Samuel V.; Cochrane, Guy; Field, Dawn; Garrity, George; Gussman, Aaron; Kodira, Chinnappa D.; Klimke, William; Kyrpides, Nikos; Madupu, Ramana; Markowitz, Victor; Tatusova, Tatiana; Thomson, Nick; White, Owen

    2008-04-01

    Genome annotations describe the features of genomes and accompany sequences in genome databases. The methodologies used to generate genome annotation are diverse and typically vary amongst groups. Descriptions of the annotation procedure are helpful in interpreting genome annotation data. Standard Operating Procedures (SOPs) for genome annotation describe the processes that generate genome annotations. Some groups are currently documenting procedures but standards are lacking for structure and content of annotation SOPs. In addition, there is no central repository to store and disseminate procedures and protocols for genome annotation. We highlight the importance of SOPs for genome annotation and endorse a central online repository of SOPs.

  18. Construction of a genomic DNA library with a TA vector and its application in cloning of the phytoene synthase gene from the cyanobacterium Spirulina platensis M-135

    NASA Astrophysics Data System (ADS)

    Yoshikazu, Kawata; Shin-Ichi, Yano; Hiroyuki, Kojima

    1998-03-01

    An efficient and simple method for constructing a genomic DNA library using a TA cloning vector is presented. It is based on the sonicative cleavage of genomic DNA and modification of fragment ends with Taq DNA polymerase, followed by ligation using a TA vector. This method was applied for cloning of the phytoene synthase gene crt B from Spirulina platensis. This method is useful when genomic DNA cannot be efficiently digested with restriction enzymes, a problem often encountered during the construction of a genomic DNA library of cyanobacteria.

  19. Comparative sequence and genetic analyses of asparagus BACs reveal no microsynteny with onion or rice.

    PubMed

    Jakse, Jernej; Telgmann, Alexa; Jung, Christian; Khar, Anil; Melgar, Sergio; Cheung, Foo; Town, Christopher D; Havey, Michael J

    2006-12-01

    The Poales (includes the grasses) and Asparagales [includes onion (Allium cepa L.) and asparagus (Asparagus officinalis L.)] are the two most economically important monocot orders. The Poales are a member of the commelinoid monocots, a group of orders sister to the Asparagales. Comparative genomic analyses have revealed a high degree of synteny among the grasses; however, it is not known if this synteny extends to other major monocot groups such as the Asparagales. Although we previously reported no evidence for synteny at the recombinational level between onion and rice, microsynteny may exist across shorter genomic regions in the grasses and Asparagales. We sequenced nine asparagus BACs to reveal physically linked genic-like sequences and determined their most similar positions in the onion and rice genomes. Four of the asparagus BACs were selected using molecular markers tightly linked to the sex-determining M locus on chromosome 5 of asparagus. These BACs possessed only two putative coding regions and had long tracts of degenerated retroviral elements and transposons. Five asparagus BACs were selected after hybridization of three onion cDNAs that mapped to three different onion chromosomes. Genic-like sequences that were physically linked on the cDNA-selected BACs or genetically linked on the M-linked BACs showed significant similarities (e < -20) to expressed sequences on different rice chromosomes, revealing no evidence for microsynteny between asparagus and rice across these regions. Genic-like sequences that were linked in asparagus were used to identify highly similar (e < -20) expressed sequence tags (ESTs) of onion. These onion ESTs mapped to different onion chromosomes and no relationship was observed between physical or genetic linkages in asparagus and genetic linkages in onion. These results further indicate that synteny among grass genomes does not extend to a sister order in the monocots and that asparagus may not be an appropriate smaller genome

  20. Genomic Library Screens for Genes Involved in n-Butanol Tolerance in Escherichia coli

    PubMed Central

    Reyes, Luis H.; Almario, Maria P.; Kao, Katy C.

    2011-01-01

    Background n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance. Methodology/Principal Findings Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%. Conclusions/Significance We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the

  1. Development of one set of chromosome-specific microsatellite-containing BACs and their physical mapping in Gossypium hirsutum L.

    PubMed

    Wang, Kai; Guo, Wangzhen; Zhang, Tianzhen

    2007-09-01

    Fluorescence in situ hybridization (FISH), using bacterial artificial chromosome (BAC) clone as probe, is a reliable cytological technique for chromosome identification. It has been used in many plants, especially in those containing numerous small chromosomes. We previously developed eight chromosome-specific BAC clones from tetraploid cotton, which were used as excellent cytological markers for chromosomes identification. Here, we isolated the other chromosome-specific BAC clones to make a complete set for the identification of all 26 chromosome-pairs by this technology in tetraploid cotton (Gossypium hirsutum L.). This set of BAC markers was demonstrated to be useful to assign each chromosome to a genetic linkage group unambiguously. In addition, these BAC clones also served as convenient and reliable landmarks for establishing physical linkage with unknown targeted sequences. Moreover, one BAC containing an EST, with high sequence similarity to a G. hirsutum ethylene-responsive element-binding factor was located physically on the long arm of chromosome A7 with the help of a chromosome-A7-specific BAC FISH marker. Comparative analysis of physical marker positions in the chromosomes by BAC-FISH and genetic linkage maps demonstrated that most of the 26 BAC clones were localized close to or at the ends of their respective chromosomes, and indicated that the recombination active regions of cotton chromosomes are primarily located in the distal regions. This technology also enables us to make associations between chromosomes and their genetic linkage groups and re-assign each chromosome according to the corresponding genetic linkage group. This BAC clones and BAC-FISH technology will be useful for us to evaluate grossly the degree to which a linkage map provides adequate coverage for developing a saturated genetic map, and provides a powerful resource for cotton genomic researches.

  2. A Marker-Dense Physical Map of the Bradyrhizobium japonicum Genome

    PubMed Central

    Tomkins, Jeffrey P.; Wood, Todd C.; Stacey, Minviluz G.; Loh, John T.; Judd, Adam; Goicoechea, Jose L.; Stacey, Gary; Sadowsky, Michael J.; Wing, Rod A.

    2001-01-01

    Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes the development of a physical framework for the genome of Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybean. A BAC library for B. japonicum was constructed that provides a 77-fold genome coverage based on an estimated genome size of 8.7 Mb. The library contains 4608 clones with an average insert size of 146 kb. To generate a physical map, the entire library was fingerprinted with HindIII, and the fingerprinted clones were assembled into contigs using the Fingerprint Contig software (FPC; Sanger Centre, UK). The FPC analysis placed 3410 clones in six large contigs. The ends of 1152 BAC inserts were sequenced to generate a sequence-tagged connector (STC) framework. To join and orient the contigs, high-density BAC colony filters were probed with 41 known gene probes and 17 end sequences from contig boundaries. STC sequences were searched against the public databases using FASTA and BLASTX algorithms. Query results allowed the identification of 113 high probability matches with putative functional identities that were placed on the physical map. Combined with the hybridization data, a high-resolution physical map with 194 positioned markers represented in two large contigs was developed, providing a marker every 45 kb. Of these markers, 177 are known or putative B. japonicum genes. Additionally, 1338 significant BLASTX results (E < 10−4) were manually sorted by function to produce a functionally categorized database of relevant B. japonicum STC sequences that can also be traced to specific locations in the physical map. PMID:11483585

  3. Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms.

    PubMed

    Coorey, Namal V C; Matthews, James H; Bellows, David S; Atkinson, Paul H

    2015-11-01

    Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA). However, the biological activities of many xenobiotics are masked by the pleiotropic drug resistance (PDR) network which effluxes xenobiotics that are PDR substrates. The PDR network in S. cerevisiae is almost entirely under the control of two functionally homologous transcription factors Pdr1p and Pdr3p. Herein we report the construction of a PDR-attenuated haploid non-essential DMA (PA-DMA), lacking PDR1 and PDR3, which permits the MoA elucidation of xenobiotics that are PDR substrates at low concentrations. The functionality of four key cellular processes commonly activated in response to xenobiotic stress: oxidative stress response, general stress response, unfolded stress response and calcium signalling pathways were assessed in the absence of PDR1 and PDR3 genes and were found to unaltered, therefore, these key chemogenomic signatures are not lost when using the PA-DMA. Efficacy of the PA-DMA was demonstrated using cycloheximide and latrunculin A at low nanomolar concentrations to attain chemical genetic profiles that were more specific to their known main mechanisms. We also found a two-fold increase in the number of compounds that are bioactive in the pdr1Δpdr3Δ compared to the wild type strain in screening the commercially available LOPAC(1280) library. The PA-DMA should be particularly applicable to mechanism determination of xenobiotics that have limited availability, such as natural products.

  4. DNA sequences and composition from 12 BAC clones-derived MUSB SSR markers mapped to cotton (Gossypium Hirsutum L. x G. Barbadense L.)chromosomes 11 and 21

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To discover resistance (R) and/or pathogen-induced (PR) genes involved in disease response, 12 bacterial artificial chromosome (BAC) clones from cv. Acala Maxxa (G. hirsutum) were sequenced at the Clemson University, Genomics Institute, Clemson, SC. These BACs derived MUSB single sequence repeat (SS...

  5. Hybrid Nonviral/Viral Vector Systems for Improved piggyBac DNA Transposon In Vivo Delivery

    PubMed Central

    Cooney, Ashley L; Singh, Brajesh K; Sinn, Patrick L

    2015-01-01

    The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR. PMID:25557623

  6. Estimating P-coverage of biosynthetic pathways in DNA libraries and screening by genetic selection: biotin biosynthesis in the marine microorganism Chromohalobacter.

    PubMed

    Kim, Eun Jin; Angell, Scott; Janes, Jeff; Watanabe, Coran M H

    2008-06-01

    Traditional approaches to natural product discovery involve cell-based screening of natural product extracts followed by compound isolation and characterization. Their importance notwithstanding, continued mining leads to depletion of natural resources and the reisolation of previously identified metabolites. Metagenomic strategies aimed at localizing the biosynthetic cluster genes and expressing them in surrogate hosts offers one possible alternative. A fundamental question that naturally arises when pursuing such a strategy is, how large must the genomic library be to effectively represent the genome of an organism(s) and the biosynthetic gene clusters they harbor? Such an issue is certainly augmented in the absence of expensive robotics to expedite colony picking and/or screening of clones. We have developed an algorism, named BPC (biosynthetic pathway coverage), supported by molecular simulations to deduce the number of BAC clones required to achieve proper coverage of the genome and their respective biosynthetic pathways. The strategy has been applied to the construction of a large-insert BAC library from a marine microorganism, Hon6 (isolated from Honokohau, Maui) thought to represent a new species. The genomic library is constructed with a BAC yeast shuttle vector pClasper lacZ paving the way for the culturing of libraries in both prokaryotic and eukaryotic hosts. Flow cytometric methods are utilized to estimate the genome size of the organism and BPC implemented to assess P-coverage or percent coverage. A genetic selection strategy is illustrated, applications of which could expedite screening efforts in the identification and localization of biosynthetic pathways from marine microbial consortia, offering a powerful complement to genome sequencing and degenerate probe strategies. Implementing this approach, we report on the biotin biosynthetic pathway from the marine microorganism Hon6.

  7. Availability of birth defects and genetic disease information in public libraries -- implications for the Human Genome Project

    SciTech Connect

    Sell, S.; Gettig, E.; Mulvihill, J.J.

    1994-09-01

    In order to better educate the public about birth defects and genetic diseases/testing, access to information is critical. The public library system of the United States is extensive and serves as an invaluable resource to citizens. We surveyed reference librarians at each of 87 public libraries in Allegheny and Westmoreland Counties, Pennsylvania. The study design included a questionnaire to ascertain the genetic knowledge of reference librarians and cataloged current resources in print and via telecommunications available to the public. A high compliance rate was achieved due to the incentive of providing copies of the Alliance of Genetic Support Group Directory to those who responded to the survey along with complete sets of the forty-three March of Dimes Information Sheets currently available. Analysis of demographic data related to the age, gender, and educational background, in addition to the occurrence of personal experiences with genetic disease was ascertained. Reference librarians were chosen as the study group due to the common experience of families seeking further information from the public library after or prior to a genetic consultation. As the Human Genome Project identifies new genes for conditions, people will seek public information more frequently. The study shows that public libraries are an appropriate point of education to and for the public.

  8. Markerless modification of trinucleotide repeat loci in BACs.

    PubMed

    Benzow, Kellie A; Koob, Michael D

    2013-01-01

    Transcription and splicing of human genes are regulated by nucleotide sequences encoded across large segments of our genome, and trinucleotide repeat expansion mutations can have both profound and subtle effects on these processes. In the course of our work to understand the impact of the Spinocerebellar Ataxia type 8 (SCA8) CTG repeat expansion on the transcription and splicing of the RNAs encoded near the SCA8 locus, we have developed a set of reagents and protocols for modifying large genomic BAC clones of this region. We describe the two-step procedure that allows us to precisely replace unexpanded trinucleotide repeats with expanded variants of these repeat sequences without leaving any exogenous sequences in the final constructs, and we discuss how this approach can be adapted to make other desired sequence changes to these genomic clones.

  9. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.

    PubMed

    Zhou, Yuexin; Zhu, Shiyou; Cai, Changzu; Yuan, Pengfei; Li, Chunmei; Huang, Yanyi; Wei, Wensheng

    2014-05-22

    Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.

  10. Intra- and interchromosomal rearrangements between cowpea [Vigna unguiculata (L.) Walp.] and common bean (Phaseolus vulgaris L.) revealed by BAC-FISH.

    PubMed

    Vasconcelos, Emanuelle Varão; de Andrade Fonsêca, Artur Fellipe; Pedrosa-Harand, Andrea; de Andrade Bortoleti, Kyria Cilene; Benko-Iseppon, Ana Maria; da Costa, Antônio Félix; Brasileiro-Vidal, Ana Christina

    2015-06-01

    Cowpea (Vigna unguiculata) is an annual legume grown in tropical and subtropical regions, which is economically relevant due to high protein content in dried beans, green pods, and leaves. In this work, a comparative cytogenetic study between V. unguiculata and Phaseolus vulgaris (common bean) was conducted using BAC-FISH. Sequences previously mapped in P. vulgaris chromosomes (Pv) were used as probes in V. unguiculata chromosomes (Vu), contributing to the analysis of macrosynteny between both legumes. Thirty-seven clones from P. vulgaris 'BAT93' BAC library, corresponding to its 11 linkage groups, were hybridized in situ. Several chromosomal rearrangements were identified, such as translocations (between BACs from Pv1 and Pv8; Pv2 and Pv3; as well as Pv2 and Pv11), duplications (BAC from Pv3), as well as paracentric and pericentric inversions (BACs from Pv3, and Pv4, respectively). Two BACs (from Pv2 and Pv7), which hybridized at terminal regions in almost all P. vulgaris chromosomes, showed single-copy signal in Vu. Additionally, 17 BACs showed no signal in V. unguiculata chromosomes. The present results demonstrate the feasibility of using BAC libraries in comparative chromosomal mapping and karyotype evolution studies between Phaseolus and Vigna species, and revealed several macrosynteny and collinearity breaks among both legumes.

  11. Rhipicephalus (Boophilus) microplus strain Deutsch, 5 BAC clone sequencing, including two encoding Cytochrome P450s and one encoding CzEst9 carboxylesterase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. BAC clones give insight into the genome struct...

  12. Inference of subgenomic origin of BACs in an interspecific hybrid sugarcane cultivar by overlapping oligonucleotide hybridizations.

    PubMed

    Kim, Changsoo; Robertson, Jon S; Paterson, Andrew H

    2011-09-01

    Sugarcane (Saccharum spp.) breeders in the early 20th century made remarkable progress in increasing yield and disease resistance by crossing Saccharum spontaneum L., a wild relative, to Saccharum officinarum L., a traditional cultivar. Modern sugarcane cultivars have approximately 71%-83% of their chromosomes originating from S. officinarum, approximately 10%-21% from S. spontaneum, and approximately 2%-13% recombinant or translocated chromosomes. In the present work, C(0)t-based cloning and sequencing (CBCS) was implemented to further explore highly repetitive DNA and to seek species-specific repeated DNA in both S. officinarum and S. spontaneum. For putatively species-specific sequences, overlappping oligonucleotide probes (overgos) were designed and hybridized to BAC filters from the interspecific hybrid sugarcane cultivar 'R570' to try to deduce parental origins of BAC clones. We inferred that 12 967 BACs putatively originated from S. officinarum and 5117 BACs from S. spontaneum. Another 1103 BACs were hybridized by both species-specific overgos, too many to account for by conventional recombination, thus suggesting ectopic recombination and (or) translocation of DNA elements. Constructing a low C(0)t library is useful to collect highly repeated DNA sequences and to search for potentially species-specific molecular markers, especially among recently diverged species. Even in the absence of repeat families that are species-specific in their entirety, the identification of localized variations within consensus sequences, coupled with the site specificity of short synthetic overgos, permits researchers to monitor species-specific or species-enriched variants.

  13. BacS: an abundant bacteroid protein in Rhizobium etli whose expression ex planta requires nifA.

    PubMed

    Jahn, Olivia J; Davila, Guillermo; Romero, David; Noel, K Dale

    2003-01-01

    Rhizobium etli CFN42 bacteroids from bean nodules possessed an abundant 16-kDa protein (BacS) that was found in the membrane pellet after cell disruption. This protein was not detected in bacteria cultured in tryptone-yeast extract. In minimal media, it was produced at low oxygen concentration but not in a mutant whose nifA was disrupted. N-terminal sequencing of the protein led to isolation of a bacS DNA fragment. DNA hybridization and nucleotide sequencing revealed three copies of the bacS gene, all residing on the main symbiotic plasmid of strain CFN42. A stretch of 304 nucleotides, exactly conserved upstream of all three bacS open reading frames, had very close matches with the NifA and sigma 54 consensus binding sequences. The only bacS homology in the genetic sequence databases was to three hypothetical proteins of unknown function, all from rhizobial species. Mutation and genetic complementation indicated that each of the bacS genes gives rise to a BacS polypeptide. Mutants disrupted or deleted in all three genes did not produce the BacS polypeptide but were Nod+ and Fix+ on Phaseolus vulgaris.

  14. Local assemblies of paired-end reduced representation libraries sequenced with the illumina genome analyzer in maize.

    PubMed

    Deschamps, Stéphane; Nannapaneni, Kishore; Zhang, Yun; Hayes, Kevin

    2012-01-01

    The use of next-generation DNA sequencing technologies has greatly facilitated reference-guided variant detection in complex plant genomes. However, complications may arise when regions adjacent to a read of interest are used for marker assay development, or when reference sequences are incomplete, as short reads alone may not be long enough to ascertain their uniqueness. Here, the possibility of generating longer sequences in discrete regions of the large and complex genome of maize is demonstrated, using a modified version of a paired-end RAD library construction strategy. Reads are generated from DNA fragments first digested with a methylation-sensitive restriction endonuclease, sheared, enriched with biotin and a selective PCR amplification step, and then sequenced at both ends. Sequences are locally assembled into contigs by subgrouping pairs based on the identity of the read anchored by the restriction site. This strategy applied to two maize inbred lines (B14 and B73) generated 183,609 and 129,018 contigs, respectively, out of which at least 76% were >200 bps in length. A subset of putative single nucleotide polymorphisms from contigs aligning to the B73 reference genome with at least one mismatch was resequenced, and 90% of those in B14 were confirmed, indicating that this method is a potent approach for variant detection and marker development in species with complex genomes or lacking extensive reference sequences.

  15. Genomics of compositae weeds: EST libraries, microarrays, and evidence of introgression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    • Premise of Study: Weeds cause considerable environmental and economic damage. However, genomic characterization of weeds has lagged behind that of model plants and crop species. Here we report on the development of genomic tools and resources for 11 weeds from the Compositae family that can serve ...

  16. Identifying Distal cis-acting Gene-Regulatory Sequences by Expressing BACs Functionalized with loxP-Tn10 Transposons in Zebrafish.

    PubMed

    Chatterjee, Pradeep K; Shakes, Leighcraft A; Wolf, Hope M; Mujalled, Mohammad A; Zhou, Constance; Hatcher, Charles; Norford, Derek C

    2013-06-21

    Bacterial Artificial Chromosomes (BACs) are large pieces of DNA from the chromosomes of organisms propagated faithfully in bacteria as large extra-chromosomal plasmids. Expression of genes contained in BACs can be monitored after functionalizing the BAC DNA with reporter genes and other sequences that allow stable maintenance and propagation of the DNA in the new host organism. The DNA in BACs can be altered within its bacterial host in several ways. Here we discuss one such approach, using Tn10 mini-transposons, to introduce exogenous sequences into BACs for a variety of purposes. The largely random insertions of Tn10 transposons carrying lox sites have been used to position mammalian cell-selectable antibiotic resistance genes, enhancer-traps and inverted repeat ends of the vertebrate transposon Tol2 precisely at the ends of the genomic DNA insert in BACs. These modified BACs are suitable for expression in zebrafish or mouse, and have been used to functionally identify important long-range gene regulatory sequences in both species. Enhancer-trapping using BACs should prove uniquely useful in analyzing multiple discontinuous DNA domains that act in concert to regulate expression of a gene, and is not limited by genome accessibility issues of traditional enhancer-trapping methods.

  17. Remnants of the Legume Ancestral Genome Preserved in Gene-Rich Regions: Insights from Lupinus angustifolius Physical, Genetic, and Comparative Mapping.

    PubMed

    Książkiewicz, Michał; Zielezinski, Andrzej; Wyrwa, Katarzyna; Szczepaniak, Anna; Rychel, Sandra; Karlowski, Wojciech; Wolko, Bogdan; Naganowska, Barbara

    The narrow-leafed lupin (Lupinus angustifolius) was recently considered as a legume reference species. Genetic resources have been developed, including a draft genome sequence, linkage maps, nuclear DNA libraries, and cytogenetic chromosome-specific landmarks. Here, we used a complex approach, involving DNA fingerprinting, sequencing, genetic mapping, and molecular cytogenetics, to localize and analyze L. angustifolius gene-rich regions (GRRs). A L. angustifolius genomic bacterial artificial chromosome (BAC) library was screened with short sequence repeat (SSR)-based probes. Selected BACs were fingerprinted and assembled into contigs. BAC-end sequence (BES) annotation allowed us to choose clones for sequencing, targeting GRRs. Additionally, BESs were aligned to the scaffolds of the genome sequence. The genetic map was supplemented with 35 BES-derived markers, distributed in 14 linkage groups and tagging 37 scaffolds. The identified GRRs had an average gene density of 19.6 genes/100 kb and physical-to-genetic distance ratios of 11 to 109 kb/cM. Physical and genetic mapping was supported by multi-BAC-fluorescence in situ hybridization (FISH), and five new linkage groups were assigned to the chromosomes. Syntenic links to the genome sequences of five legume species (Medicago truncatula, Glycine max, Lotus japonicus, Phaseolus vulgaris, and Cajanus cajan) were identified. The comparative mapping of the two largest lupin GRRs provides novel evidence for ancient duplications in all of the studied species. These regions are conserved among representatives of the main clades of Papilionoideae. Furthermore, despite the complex evolution of legumes, some segments of the nuclear genome were not substantially modified and retained their quasi-ancestral structures. Cytogenetic markers anchored in these regions constitute a platform for heterologous mapping of legume genomes.

  18. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML

    PubMed Central

    Kurata, Morito; Rathe, Susan K.; Bailey, Natashay J.; Aumann, Natalie K.; Jones, Justine M.; Veldhuijzen, G. Willemijn; Moriarity, Branden S.; Largaespada, David A.

    2016-01-01

    Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML. PMID:27808171

  19. Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.

    PubMed

    Kurata, Morito; Rathe, Susan K; Bailey, Natashay J; Aumann, Natalie K; Jones, Justine M; Veldhuijzen, G Willemijn; Moriarity, Branden S; Largaespada, David A

    2016-11-03

    Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.

  20. Large Gap Size Paired-end Library Construction for Second Generation Sequencing

    SciTech Connect

    Peng, Ze; Hamilton, Matthew; Froula, Jeff; Ewing, Aren; Foster, Brian; Cheng, Jan-Fang

    2010-05-28

    Fosmid or BAC end sequencing plays an important role in de novo assembly of large genomes like fungi and plants. However construction and Sanger sequencing of fosmid or BAC libraries are laborious and costly. The current 454 Paired-End (PE) Library and Illumina Jumping Library construction protocols are limited with the gap sizes of approximately 20 kb and 8 kb, respectively. In the attempt to understand the limitations of constructing PE libraries with greater than 30Kb gaps, we have purified 18, 28, 45, and 65Kb sheared DNA fragments from yeast and circularized the ends using the Cre-loxP approach described in the 454 PE Library protocol. With the increasing fragment sizes, we found a general trend of decreasing library quality in several areas. First, redundant reads and reads containing multiple loxP linkers increase when the average fragment size increases. Second, the contamination of short distance pairs (<10Kb) increases as the fragment size increases. Third, chimeric rate increases with the increasing fragment sizes. We have modified several steps to improve the quality of the long span PE libraries. The modification includes (1) the use of special PFGE program to reduce small fragment contamination; (2) the increase of DNA samples in the circularization step and prior to the PCR to reduce redundant reads; and (3) the decrease of fragment size in the double SPRI size selection to get a higher frequency of LoxP linker containing reads. With these modifications we have generated large gap size PE libraries with a much better quality.

  1. Fluorescence in situ hybridization with Bacterial Artificial Chromosomes (BACs) to mitotic heterochromatin of Drosophila.

    PubMed

    Accardo, Maria Carmela; Dimitri, Patrizio

    2010-01-01

    The organization of eukaryotic chromosomes into euchromatin and heterochromatin represents an enigmatic aspect of genome evolution. Constitutive heterochromatin is a basic, yet still poorly understood component of eukaryotic genomes and its molecular characterization by means of standard genomic approaches is intrinsically difficult. Drosophila melanogaster polytene chromosomes do not seem to be particularly useful to map heterochromatin sequences because the typical features of heterochromatin, organized as it is into a chromocenter, limit cytogenetic analysis. In contrast, constitutive heterochromatin has been well-defined at the cytological level in mitotic chromosomes of neuroblasts and has been subdivided into several bands with differential staining properties. Fluorescence in situ hybridization (FISH) using Bacterial Artificial Chromosomes (BAC) probes that carry large genomic portions defined by sequence annotation has yielded a "revolution" in the field of cytogenetics because it has allowed the mapping of multiple genes at once, thus rendering constitutive heterochromatin amenable to easy and fast cytogenetics analyses. Indeed, BAC-based FISH approaches on Drosophila mitotic chromosomes have made it possible to correlate genomic sequences to their cytogenetic location, aiming to build an integrated map of the pericentric heterochromatin. This chapter presents our standard protocols for BAC-based FISH, aimed at mapping large chromosomal regions of mitotic heterochromatin in Drosophila melanogaster.

  2. Mitogenome assembly from genomic multiplex libraries: comparison of strategies and novel mitogenomes for five species of frogs.

    PubMed

    Machado, D J; Lyra, M L; Grant, T

    2016-05-01

    Next-generation sequencing continues to revolutionize biodiversity studies by generating unprecedented amounts of DNA sequence data for comparative genomic analysis. However, these data are produced as millions or billions of short reads of variable quality that cannot be directly applied in comparative analyses, creating a demand for methods to facilitate assembly. We optimized an in silico strategy to efficiently reconstruct high-quality mitochondrial genomes directly from genomic reads. We tested this strategy using sequences from five species of frogs: Hylodes meridionalis (Hylodidae), Hyloxalus yasuni (Dendrobatidae), Pristimantis fenestratus (Craugastoridae), and Melanophryniscus simplex and Rhinella sp. (Bufonidae). These are the first mitogenomes published for these species, the genera Hylodes, Hyloxalus, Pristimantis, Melanophryniscus and Rhinella, and the families Craugastoridae and Hylodidae. Sequences were generated using only half of one lane of a standard Illumina HiqSeq 2000 flow cell, resulting in fewer than eight million reads. We analysed the reads of Hylodes meridionalis using three different assembly strategies: (1) reference-based (using bowtie2); (2) de novo (using abyss, soapdenovo2 and velvet); and (3) baiting and iterative mapping (using mira and mitobim). Mitogenomes were assembled exclusively with strategy 3, which we employed to assemble the remaining mitogenomes. Annotations were performed with mitos and confirmed by comparison with published amphibian mitochondria. In most cases, we recovered all 13 coding genes, 22 tRNAs, and two ribosomal subunit genes, with minor gene rearrangements. Our results show that few raw reads can be sufficient to generate high-quality scaffolds, making any Illumina machine run using genomic multiplex libraries a potential source of data for organelle assemblies as by-catch.

  3. Integrated physical, genetic and genome map of chickpea (Cicer arietinum L.).

    PubMed

    Varshney, Rajeev K; Mir, Reyazul Rouf; Bhatia, Sabhyata; Thudi, Mahendar; Hu, Yuqin; Azam, Sarwar; Zhang, Yong; Jaganathan, Deepa; You, Frank M; Gao, Jinliang; Riera-Lizarazu, Oscar; Luo, Ming-Cheng

    2014-03-01

    Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance "QTL-hotspot" region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.

  4. Genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shRNA library.

    PubMed

    Yoshino, Seiko; Hara, Toshiro; Weng, Jane S; Takahashi, Yuka; Seiki, Motoharu; Sakamoto, Takeharu

    2012-01-01

    Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.

  5. Final report. Human artificial episomal chromosome (HAEC) for building large genomic libraries

    SciTech Connect

    Jean-Michael H. Vos

    1999-12-09

    Collections of human DNA fragments are maintained for research purposes as clones in bacterial host cells. However for unknown reasons, some regions of the human genome appear to be unclonable or unstable in bacteria. Their team has developed a system using episomes (extrachromosomal, autonomously replication DNA) that maintains large DNA fragments in human cells. This human artificial episomal chromosomal (HAEC) system may prove useful for coverage of these especially difficult regions. In the broader biomedical community, the HAEC system also shows promise for use in functional genomics and gene therapy. Recent improvements to the HAEC system and its application to mapping, sequencing, and functionally studying human and mouse DNA are summarized. Mapping and sequencing the human genome and model organisms are only the first steps in determining the function of various genetic units critical for gene regulation, DNA replication, chromatin packaging, chromosomal stability, and chromatid segregation. Such studies will require the ability to transfer and manipulate entire functional units into mammalian cells.

  6. As Blood Alcohol Content (BAC) Increases, So Does Impairment

    MedlinePlus

    ... turn JavaScript on. Feature: Rethinking Drinking As Blood Alcohol Content (BAC) Increases, So Does Impairment Past Issues / ... of Contents For purposes of law enforcement, blood alcohol content (BAC) is used to define intoxication and ...

  7. Wheat Genomics: Present Status and Future Prospects

    PubMed Central

    Gupta, P. K.; Mir, R. R.; Mohan, A.; Kumar, J.

    2008-01-01

    Wheat (Triticum aestivum L.), with a large genome (16000 Mb) and high proportion (∼80%) of repetitive sequences, has been a difficult crop for genomics research. However, the availability of extensive cytogenetics stocks has been an asset, which facilitated significant progress in wheat genomic research in recent years. For instance, fairly dense molecular maps (both genetic and physical maps) and a large set of ESTs allowed genome-wide identification of gene-rich and gene-poor regions as well as QTL including eQTL. The availability of markers associated with major economic traits also allowed development of major programs on marker-assisted selection (MAS) in some countries, and facilitated map-based cloning of a number of genes/QTL. Resources for functional genomics including TILLING and RNA interference (RNAi) along with some new approaches like epigenetics and association mapping are also being successfully used for wheat genomics research. BAC/BIBAC libraries for the subgenome D and some individual chromosomes have also been prepared to facilitate sequencing of gene space. In this brief review, we discuss all these advances in some detail, and also describe briefly the available resources, which can be used for future genomics research in this important crop. PMID:18528518

  8. Genome Clone Libraries and Data from the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium

    DOE Data Explorer

    The I.M.A.G.E. Consortium was initiated in 1993 by four academic groups on a collaborative basis after informal discussions led to a common vision of how to achieve an important goal in the study of the human genome: the Integrated Molecular Analysis of Genomes and their Expression Consortium's primary goal is to create arrayed cDNA libraries and associated bioinformatics tools, and make them publicly available to the research community. The primary organisms of interest include intensively studied mammalian species, including human, mouse, rat and non-human primate species. The Consortium has also focused on several commonly studied model organisms; as part of this effort it has arrayed cDNAs from zebrafish, and Fugu (pufferfish) as well as Xenopus laevis and X. tropicalis (frog). Utilizing high speed robotics, over nine million individual cDNA clones have been arrayed into 384-well microtiter plates, and sufficient replicas have been created to distribute copies both to sequencing centers and to a network of five distributors located worldwide. The I.M.A.G.E. Consortium represents the world's largest public cDNA collection, and works closely with the National Institutes of Health's Mammalian Gene Collection(MGC) to help it achieve its goal of creating a full-length cDNA clone for every human and mouse gene. I.M.A.G.E. is also a member of the ORFeome Collaboration, working to generate a complete set of expression-ready open reading frame clones representing each human gene. Custom informatics tools have been developed in support of these projects to better allow the research community to select clones of interest and track and collect all data deposited into public databases about those clones and their related sequences. I.M.A.G.E. clones are publicly available, free of any royalties, and may be used by anyone agreeing with the Consortium's guidelines.

  9. Further evidence of microcolinearity between barley and rice genomes at two orthologous regions.

    PubMed

    Park, Yong-Jin; Dixit, Anupam; Yoo, Jae-Woong; Bennetzen, Jeff

    2004-06-30

    Two genetic markers, BCD135 and RZ567 were used to select clones from genomic BAC libraries of barley and rice for sequencing and subsequent sequence comparisons. A set of two orthologous BACs each from barley and rice was selected by hybridization with BCD135 and RZ567 cDNA probes. A total of 556-kb stretch including two barley BACs (773K135 and 745C13) and two orthologous rice BACs (24K23 and 49D11) was completely sequenced. Comparative sequence analysis between orthologous BACs from the two species revealed presence of two conserved genes at BCD135 region and only one gene at the RZ567 regions. The two conserved genes were in the same order and orientation in both the species however, separated by significantly larger distance in barley. The larger distance between two barley genes was mainly due to presence of different retrotransposable elements and their derivatives (78.9% of the intergenic region) that expanded the barley BCD135 region at the rate of 9.1X. An additional gene of unknown function was also inserted along with several retrotransposable elements between two conserved genes at barley BCD135 region. More genome expansion rate (10X) around barley RZ567 locus was estimated by extremely high proportion (> 70%) of retrotransposons. Among different retrotransposons, the Sabrina elements rather than BARE were more prevalent in both the regions. Contrary to it, the BCD135 region of rice was composed of only 17.1% retrotransposable elements and no significant retrotransposons except 14 miniature inverted transposable elements (MITEs) were observed in its RZ567 region. The sequence comparison between orthologous regions of rice and barley genomes was useful for gene identification and determination of individual gene structure indicating the possibility of effective utilization of rice genome sequences in understanding the large genome of barley. (The sequence data described in this paper have been submitted to the GenBank data library under the accession

  10. A Sequence-Ready BAC Clone Contig of a 2.2-Mb Segment of Human Chromosome 1q24

    PubMed Central

    Vollrath, Douglas; Jaramillo-Babb, Virna L.

    1999-01-01

    Human chromosomal region 1q24 encodes two cloned disease genes and lies within large genetic inclusion intervals for several disease genes that have yet to be identified. We have constructed a single bacterial artificial chromosome (BAC) clone contig that spans over 2 Mb of 1q24 and consists of 78 clones connected by 100 STSs. The average density of mapped STSs is one of the highest described for a multimegabase region of the human genome. The contig was efficiently constructed by generating STSs from clone ends, followed by library walking. Distance information was added by determining the insert sizes of all clones, and expressed sequence tags (ESTs) and genes were incorporated to create a partial transcript map of the region, providing candidate genes for local disease loci. The gene order and content of the region provide insight into ancient duplication events that have occurred on proximal 1q. The stage is now set for further elucidation of this interesting region through large-scale sequencing. [The sequence data described in this paper have been submitted to GenBank under accession nos. G42259–G42312 and G42330–G42335.] PMID:10022979

  11. Microsatellite Discovery from BAC End Sequences and Genetic Mapping to Anchor the Soybean Physical and Genetic Maps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Physical maps can be an invaluable resource for improving and assessing the quality of a whole-genome sequence assembly. Here we report the identification and screening of 3,290 microsatellites (SSRs) identified from BAC end sequences of clones comprising the physical map of the cultivar Williams 8...

  12. Evolution of sex chromosomes ZW of Schistosoma mansoni inferred from chromosome paint and BAC mapping analyses.

    PubMed

    Hirai, Hirohisa; Hirai, Yuriko; LoVerde, Philip T

    2012-12-01

    Chromosomes of schistosome parasites among digenetic flukes have a unique evolution because they exhibit the sex chromosomes ZW, which are not found in the other groups of flukes that are hermaphrodites. We conducted molecular cytogenetic analyses for investigating the sex chromosome evolution using chromosome paint analysis and BAC clones mapping. To carry this out, we developed a technique for making paint probes of genomic DNA from a single scraped chromosome segment using a chromosome microdissection system, and a FISH mapping technique for BAC clones. Paint probes clearly identified each of the 8 pairs of chromosomes by a different fluorochrome color. Combination analysis of chromosome paint analysis with Z/W probes and chromosome mapping with 93 BAC clones revealed that the W chromosome of Schistosoma mansoni has evolved by at least four inversion events and heterochromatinization. Nine of 93 BAC clones hybridized with both the Z and W chromosomes, but the locations were different between Z and W chromosomes. The homologous regions were estimated to have moved from the original Z chromosome to the differentiated W chromosome by three inversions events that occurred before W heterohcromatinization. An inversion that was observed in the heterochromatic region of the W chromosome likely occurred after W heterochromatinization. These inversions and heterochromatinization are hypothesized to be the key factors that promoted the evolution of the W chromosome of S. mansoni.

  13. Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon

    PubMed Central

    Katayama, Hokahiro; Yasuchika, Kentaro; Miyauchi, Yuya; Kojima, Hidenobu; Yamaoka, Ryoya; Kawai, Takayuki; Yukie Yoshitoshi, Elena; Ogiso, Satoshi; Kita, Sadahiko; Yasuda, Katsutaro; Sasaki, Naoya; Fukumitsu, Ken; Komori, Junji; Ishii, Takamichi; Uemoto, Shinji

    2017-01-01

    Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy. PMID:28295042

  14. Comparison of homoeolocus organisation in paired BAC clones from white clover (Trifolium repens L.) and microcolinearity with model legume species

    PubMed Central

    2010-01-01

    Background White clover (Trifolium repens L.) is an outbreeding allotetraploid species and an important forage legume in temperate grassland agriculture. Comparison of sub-genome architecture and study of nucleotide sequence diversity within allopolyploids provides insight into evolutionary divergence mechanisms, and is also necessary for the development of whole-genome sequencing strategies. This study aimed to evaluate the degree of divergence between the O and P' sub-genomes of white clover through sequencing of BAC clones containing paired homoeoloci. The microsyntenic relationships between the genomes of white clover and the model legumes Lotus japonicus and Medicago truncatula as well as Arabidopsis thaliana were also characterised. Results A total of four paired homoeologous BACs were selected and sequenced to generate 173 kb of overlapping sequence between the O and P' sub-genomes. Equivalent gene content was generally observed, apart from small-scale deletions, in contrast to conservation of intergenic sequences, which varied between the four selected regions. Measurement of the number of synonymous substitutions between homoeologous genes led to estimation of a 4.2 million year divergence time between the two sub-genomes. Microsynteny was observed between the genomes of white clover and L. japonicus for all four targeted regions, but corresponding M. truncatula genomic regions were only identified for two BAC pairs. Conclusions This study describes the first analysis of sub-genome structural conservation across selected genomic regions in white clover. Although the high levels of sequence conservation between the O and P' sub-genomes would complicate efforts for whole genome sequence assembly, the conserved microsynteny with model legume genomes, especially that of L. japonicus, will be highly valuable for the future of white clover genomics and molecular breeding. PMID:20492736

  15. Extraction of high-molecular-weight genomic DNA for long-read sequencing of single molecules.

    PubMed

    Mayjonade, Baptiste; Gouzy, Jérôme; Donnadieu, Cécile; Pouilly, Nicolas; Marande, William; Callot, Caroline; Langlade, Nicolas; Muños, Stéphane

    2016-10-01

    De novo sequencing of complex genomes is one of the main challenges for researchers seeking high-quality reference sequences. Many de novo assemblies are based on short reads, producing fragmented genome sequences. Third-generation sequencing, with read lengths >10 kb, will improve the assembly of complex genomes, but these techniques require high-molecular-weight genomic DNA (gDNA), and gDNA extraction protocols used for obtaining smaller fragments for short-read sequencing are not suitable for this purpose. Methods of preparing gDNA for bacterial artificial chromosome (BAC) libraries could be adapted, but these approaches are time-consuming, and commercial kits for these methods are expensive. Here, we present a protocol for rapid, inexpensive extraction of high-molecular-weight gDNA from bacteria, plants, and animals. Our technique was validated using sunflower leaf samples, producing a mean read length of 12.6 kb and a maximum read length of 80 kb.

  16. Estimation of long terminal repeat element content in the Helicoverpa zea genome from high-throughput sequencing of bacterial artificial chromosome pools.

    PubMed

    Coates, Brad S; Abel, Craig A; Perera, Omaththage P

    2017-04-01

    The lepidopteran pest insect Helicoverpa zea feeds on cultivated corn and cotton across the Americas where control remains challenging owing to the evolution of resistance to chemical and transgenic insecticidal toxins, yet genomic resources remain scarce for this species. A bacterial artificial chromosome (BAC) library having a mean genomic insert size of 145 ± 20 kbp was created from a laboratory strain of H. zea, which provides ∼12.9-fold coverage of a 362.8 ± 8.8 Mbp (0.37 ± 0.09 pg) flow cytometry estimated haploid genome size. Assembly of Illumina HiSeq 2000 reads generated from 14 pools that encompassed all BAC clones resulted in 165 485 genomic contigs (N50 = 3262 bp; 324.6 Mbp total). Long terminal repeat (LTR) protein coding regions annotated from 181 contigs included 30 Ty1/copia, 78 Ty3/gypsy, and 73 BEL/Pao elements, of which 60 (33.1%) encoded all five functional polyprotein (pol) domains. Approximately 14% of LTR elements are distributed non-randomly across pools of BAC clones.

  17. Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2).

    PubMed

    Rodrigues, F; Zeeman, A M; Alves, C; Sousa, M J; Steensma, H Y; Côrte-Real, M; Leão, C

    2001-04-01

    A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.

  18. BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in upland cotton.

    PubMed

    Buyyarapu, Ramesh; Kantety, Ramesh V; Yu, John Z; Xu, Zhanyou; Kohel, Russell J; Percy, Richard G; Macmil, Simone; Wiley, Graham B; Roe, Bruce A; Sharma, Govind C

    2013-01-01

    Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes.

  19. Reevaluation of the Coding Potential and Proteomic Analysis of the BAC Derived Rhesus Cytomegalovirus Strain 68-1

    SciTech Connect

    Malouli, Daniel; Nakayasu, Ernesto S.; Viswanathan, Kasinath; Camp, David G.; Chang, W. L.; Barry, Peter A.; Smith, Richard D.; Fruh, Klaus

    2012-09-01

    Cytomegaloviruses are highly host restricted resulting in co-speciation with their hosts. As a natural pathogen of rhesus macaques (RM), Rhesus Cytomegalovirus (RhCMV) has therefore emerged as a highly relevant experimental model for pathogenesis and vaccine development due to its close evolutionary relationship to human CMV (HCMV). To date, most in vivo experiments performed with RhCMV employed strain 68-1 cloned as bacterial artificial chromosome (BAC). However, the complete genome sequence of the 68-1 BAC has not been determined. Furthermore, the gene content of the RhCMV genome is unknown and previous open reading frame (ORF) predictions relied solely on uninterrupted ORFs with an arbitrary cutoff of 300bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV we re-evaluated the RhCMV 68-1 BAC-genome by whole genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By additionally comparing the RhCMV genome to that of several closely related Old World Monkey (OWM) CMVs we were able to filter out many unlikely ORFs and obtain a simplified map of the RhCMV genome. This comparative genomics analysis eliminated many genes previously characterized as RhCMV-specific while consolidating a high conservation of ORFs among OWM-CMVs and between RhCMV and HCMV. Moreover, virion proteomics independently validated the revised ORF predictions since only proteins encoded by predicted ORFs could be detected. Taken together these data suggest a much higher conservation of genome and virion structure between CMVs of humans, apes and OWMs than previously assumed. Remarkably, BAC-derived RhCMV is able to establish and maintain persistent infection despite the lack of multiple genes homologous to HCMV genes involved in tissue tropism.

  20. Characterization of rubber tree microRNA in phytohormone response using large genomic DNA libraries, promoter sequence and gene expression analysis.

    PubMed

    Kanjanawattanawong, Supanath; Tangphatsornruang, Sithichoke; Triwitayakorn, Kanokporn; Ruang-areerate, Panthita; Sangsrakru, Duangjai; Poopear, Supannee; Somyong, Suthasinee; Narangajavana, Jarunya

    2014-10-01

    The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs.

  1. Fungicidal mechanisms of the antimicrobial peptide Bac8c.

    PubMed

    Lee, Wonyoung; Lee, Dong Gun

    2015-02-01

    Bac8c (RIWVIWRR-NH2) is an analogue peptide derived through complete substitution analysis of the linear bovine host defense peptide variant Bac2A. In the present study, the antifungal mechanism of Bac8c against pathogenic fungi was investigated, with a particular focus on the effects of Bac8c on the cytoplasmic membrane. We used bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] staining and 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] assays to show that Bac8c induced disturbances in the membrane potential of Candida albicans. An increase in membrane permeability and suppression of cell wall regeneration were also observed in Bac8c-treated C. albicans. We studied the effects of Bac8c treatment on model membranes to elucidate its antifungal mechanism. Using calcein and FITC-labeled dextran leakage assays from Bac8c-treated large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs), we found that Bac8c has a pore-forming action on fungal membranes, with an estimated pore radius of between 2.3 and 3.3 nm. A membrane-targeted mechanism of action was also supported by the observation of potassium release from the cytosol of Bac8c-treated C. albicans. These results indicate that Bac8c is considered as a potential candidate to develop a novel antimicrobial agent because of its low-cost production characteristics and high antimicrobial activity via its ability to induce membrane perturbations in fungi.

  2. Size matters: use of YACs, BACs and PACs in transgenic animals.

    PubMed

    Giraldo, P; Montoliu, L

    2001-04-01

    In 1993, several groups, working independently, reported the successful generation of transgenic mice with yeast artificial chromosomes (YACs) using standard techniques. The transfer of these large fragments of cloned genomic DNA correlated with optimal expression levels of the transgenes, irrespective of their location in the host genome. Thereafter, other groups confirmed the advantages of YAC transgenesis and position-independent and copy number-dependent transgene expression were demonstrated in most cases. The transfer of YACs to the germ line of mice has become popular in many transgenic facilities to guarantee faithful expression of transgenes. This technique was rapidly exported to livestock and soon transgenic rabbits, pigs and other mammals were produced with YACs. Transgenic animals were also produced with bacterial or P1-derived artificial chromosomes (BACs/PACs) with similar success. The use of YACs, BACs and PACs in transgenesis has allowed the discovery of new genes by complementation of mutations, the identification of key regulatory sequences within genomic loci that are crucial for the proper expression of genes and the design of improved animal models of human genetic diseases. Transgenesis with artificial chromosomes has proven useful in a variety of biological, medical and biotechnological applications and is considered a major breakthrough in the generation of transgenic animals. In this report, we will review the recent history of YAC/BAC/PAC-transgenic animals indicating their benefits and the potential problems associated with them. In this new era of genomics, the generation and analysis of transgenic animals carrying artificial chromosome-type transgenes will be fundamental to functionally identify and understand the role of new genes, included within large pieces of genomes, by direct complementation of mutations or by observation of their phenotypic consequences.

  3. Generation of a Genome Scale Lentiviral Vector Library for EF1α Promoter-Driven Expression of Human ORFs and Identification of Human Genes Affecting Viral Titer

    PubMed Central

    Škalamera, Dubravka; Dahmer, Mareike; Purdon, Amy S.; Wilson, Benjamin M.; Ranall, Max V.; Blumenthal, Antje; Gabrielli, Brian; Gonda, Thomas J.

    2012-01-01

    The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors. PMID:23251614

  4. Modification of bacterial artificial chromosomes (BACs) and preparation of intact BAC DNA for generation of transgenic mice.

    PubMed

    Gong, Shiaoching; Yang, X William

    2005-05-01

    BAC transgenesis is a powerful tool for the study of gene expression and gene function in the mouse in vivo. In this unit, detailed protocols are provided for modification (i.e., marker gene insertion, deletion, or point mutation) of BACs by homologous recombination in E. coli. This method utilizes a shuttle vector that allows transient expression of the E. coli RecA gene to support homologous recombination in the BAC host bacteria. In addition, two protocols are provided for purification of BAC DNA for microinjection to generate transgenic mice. Since BAC DNA is prone to degradation, which may introduce positional effects in transgenic mice, two methods are given for purification of intact BAC DNA for subsequent microinjection.

  5. Observing copepods through a genomic lens

    PubMed Central

    2011-01-01

    Background Copepods outnumber every other multicellular animal group. They are critical components of the world's freshwater and marine ecosystems, sensitive indicators of local and global climate change, key ecosystem service providers, parasites and predators of economically important aquatic animals and potential vectors of waterborne disease. Copepods sustain the world fisheries that nourish and support human populations. Although genomic tools have transformed many areas of biological and biomedical research, their power to elucidate aspects of the biology, behavior and ecology of copepods has only recently begun to be exploited. Discussion The extraordinary biological and ecological diversity of the subclass Copepoda provides both unique advantages for addressing key problems in aquatic systems and formidable challenges for developing a focused genomics strategy. This article provides an overview of genomic studies of copepods and discusses strategies for using genomics tools to address key questions at levels extending from individuals to ecosystems. Genomics can, for instance, help to decipher patterns of genome evolution such as those that occur during transitions from free living to symbiotic and parasitic lifestyles and can assist in the identification of genetic mechanisms and accompanying physiological changes associated with adaptation to new or physiologically challenging environments. The adaptive significance of the diversity in genome size and unique mechanisms of genome reorganization during development could similarly be explored. Genome-wide and EST studies of parasitic copepods of salmon and large EST studies of selected free-living copepods have demonstrated the potential utility of modern genomics approaches for the study of copepods and have generated resources such as EST libraries, shotgun genome sequences, BAC libraries, genome maps and inbred lines that will be invaluable in assisting further efforts to provide genomics tools for

  6. Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

    PubMed

    Al-Jarbou, Ahmed Nasser

    2012-01-01

    Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

  7. [Construction of a recombinant BAC-HSV-1 strain HF with a GFP reporter gene and characterization of its infectious progeny virus].

    PubMed

    Liu, Xin-Jing; Song, Bo; Lu, Jia-Meng; Wang, Qing-Zhi; Han, Zhi-Qiang; Xu, Yu-Ming

    2011-05-01

    To construct the plasmid of BAC-HSV-1 with GFP reporter gene and research the biological property of its infectious progeny virus. We constructed the plasmid C223-UL43-left-arms-UL47-right-arms which carried the homologous sequences of HSV-1. Liposome embedding method was used to transfect HSV-1 genome and the plasmid C223-UL43-left-arms-UL47-right-arms linearized by Mlu I digestion into Vero cells. After the successful homologous recombination in the eukaryotic cells, the recombinant BAC-HSV-1 with GFP reporter gene was generated. Then, the positive CPE were taken by plaque purification and by hirt extraction during the moment of the circularization of HSV-1 DNA, and the plasmid of BAC -HSV-1 was acquired. Electroporation was used to transfect the BAC -HSV-1 into DH10B, and then the single colonies of interest were confirmed both by MluI digestion and PCR. Experimental group and the control group cells were given BAC-HSV-1 plasmid and HSV-1 genomic DNA respectively to produce the BAC-HSV-1 and HSV-1 progeny virions. Vero cells were inoculated with the progeny virions at MOI = 0.1 and then a TCID50 assay was performed to determine the titers of virons in the two groups at 48 hours post inoculation. The plasmid BAC-HSV-1 was successfully constructed by the restriction enzyme analysis and the PCR. The titers of progeny virions were calculated by the TCID50 assay. No significant difference in the titers of virions between two groups was observed (P > 0.05). The infectious BAC-HSV-1 shuttle virus/plasmid between eukaryotic and prokaryotic cells was successfully constructed.

  8. Generation of BAC transgenic epithelial organoids.

    PubMed

    Schwank, Gerald; Andersson-Rolf, Amanda; Koo, Bon-Kyoung; Sasaki, Nobuo; Clevers, Hans

    2013-01-01

    Under previously developed culture conditions, mouse and human intestinal epithelia can be cultured and expanded over long periods. These so-called organoids recapitulate the three-dimensional architecture of the gut epithelium, and consist of all major intestinal cell types. One key advantage of these ex vivo cultures is their accessibility to live imaging. So far the establishment of transgenic fluorescent reporter organoids has required the generation of transgenic mice, a laborious and time-consuming process, which cannot be extended to human cultures. Here we present a transfection protocol that enables the generation of recombinant mouse and human reporter organoids using BAC (bacterial artificial chromosome) technology.

  9. Partial complementation of Sinorhizobium meliloti bacA mutant phenotypes by the Mycobacterium tuberculosis BacA protein.

    PubMed

    Arnold, M F F; Haag, A F; Capewell, S; Boshoff, H I; James, E K; McDonald, R; Mair, I; Mitchell, A M; Kerscher, B; Mitchell, T J; Mergaert, P; Barry, C E; Scocchi, M; Zanda, M; Campopiano, D J; Ferguson, G P

    2013-01-01

    The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human β-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac7(1-16). This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections.

  10. Cas-Database: web-based genome-wide guide RNA library design for gene knockout screens using CRISPR-Cas9

    PubMed Central

    Park, Jeongbin; Kim, Jin-Soo; Bae, Sangsu

    2016-01-01

    Motivation: CRISPR-derived RNA guided endonucleases (RGENs) have been widely used for both gene knockout and knock-in at the level of single or multiple genes. RGENs are now available for forward genetic screens at genome scale, but single guide RNA (sgRNA) selection at this scale is difficult. Results: We develop an online tool, Cas-Database, a genome-wide gRNA library design tool for Cas9 nucleases from Streptococcus pyogenes (SpCas9). With an easy-to-use web interface, Cas-Database allows users to select optimal target sequences simply by changing the filtering conditions. Furthermore, it provides a powerful way to select multiple optimal target sequences from thousands of genes at once for the creation of a genome-wide library. Cas-Database also provides a web application programming interface (web API) for advanced bioinformatics users. Availability and implementation: Free access at http://www.rgenome.net/cas-database/. Contact: sangsubae@hanyang.ac.kr or jskim01@snu.ac.kr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153724

  11. Rapid genomic DNA changes in allotetraploid fish hybrids.

    PubMed

    Wang, J; Ye, L H; Liu, Q Z; Peng, L Y; Liu, W; Yi, X G; Wang, Y D; Xiao, J; Xu, K; Hu, F Z; Ren, L; Tao, M; Zhang, C; Liu, Y; Hong, Y H; Liu, S J

    2015-06-01

    Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F20) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine-cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F1 hybrids (2nF1) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF1 and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF1. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F20 hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.

  12. Sequence-level comparative analysis of the Brassica napus genome around two stearoyl-ACP desaturase loci.

    PubMed

    Cho, Kwangsoo; O'Neill, Carmel M; Kwon, Soo-Jin; Yang, Tae-Jin; Smooker, Andrew M; Fraser, Fiona; Bancroft, Ian

    2010-02-01

    We conducted a sequence-level comparative analyses, at the scale of complete bacterial artificial chromosome (BAC) clones, between the genome of the most economically important Brassica species, Brassica napus (oilseed rape), and those of Brassica rapa, the genome of which is currently being sequenced, and Arabidopsis thaliana. We constructed a new B. napus BAC library and identified and sequenced clones that contain homoeologous regions of the genome including stearoyl-ACP desaturase-encoding genes. We sequenced the orthologous region of the genome of B. rapa and conducted comparative analyses between the Brassica sequences and those of the orthologous region of the genome of A. thaliana. The proportion of genes conserved (approximately 56%) is lower than has been reported previously between A. thaliana and Brassica (approximately 66%). The gene models for sets of conserved genes were used to determine the extent of nucleotide conservation of coding regions. This was found to be 84.2 +/- 3.9% and 85.8 +/- 3.7% between the B. napus A and C genomes, respectively, and that of A. thaliana, which is consistent with previous results for other Brassica species, and 97.5 +/- 3.1% between the B. napus A genome and B. rapa, and 93.1 +/- 4.9% between the B. napus C genome and B. rapa. The divergence of the B. napus genes from the A genome and the B. rapa genes was greater than anticipated and indicates that the A genome ancestor of the B. napus cultivar studied was relatively distantly related to the cultivar of B. rapa selected for genome sequencing.

  13. Generation of an inducible and optimized piggyBac transposon system†

    PubMed Central

    Cadiñanos, Juan; Bradley, Allan

    2007-01-01

    Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5′-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation. PMID:17576687

  14. Palaeohexaploid ancestry for Caryophyllales inferred from extensive gene-based physical and genetic mapping of the sugar beet genome (Beta vulgaris).

    PubMed

    Dohm, Juliane C; Lange, Cornelia; Holtgräwe, Daniela; Sörensen, Thomas Rosleff; Borchardt, Dietrich; Schulz, Britta; Lehrach, Hans; Weisshaar, Bernd; Himmelbauer, Heinz

    2012-05-01

    Sugar beet (Beta vulgaris) is an important crop plant that accounts for 30% of the world's sugar production annually. The genus Beta is a distant relative of currently sequenced taxa within the core eudicotyledons; the genomic characterization of sugar beet is essential to make its genome accessible to molecular dissection. Here, we present comprehensive genomic information in genetic and physical maps that cover all nine chromosomes. Based on this information we identified the proposed ancestral linkage groups of rosids and asterids within the sugar beet genome. We generated an extended genetic map that comprises 1127 single nucleotide polymorphism markers prepared from expressed sequence tags and bacterial artificial chromosome (BAC) end sequences. To construct a genome-wide physical map, we hybridized gene-derived oligomer probes against two BAC libraries with 9.5-fold cumulative coverage of the 758 Mbp genome. More than 2500 probes and clones were integrated both in genetic maps and the physical data. The final physical map encompasses 535 chromosomally anchored contigs that contains 8361 probes and 22 815 BAC clones. By using the gene order established with the physical map, we detected regions of synteny between sugar beet (order Caryophyllales) and rosid species that involves 1400-2700 genes in the sequenced genomes of Arabidopsis, poplar, grapevine, and cacao. The data suggest that Caryophyllales share the palaeohexaploid ancestor proposed for rosids and asterids. Taken together, we here provide extensive molecular resources for sugar beet and enable future high-resolution trait mapping, gene identification, and cross-referencing to regions sequenced in other plant species.

  15. Chromosome region-specific libraries for human genome analysis. Progress report, September 1, 1991--August 31, 1992

    SciTech Connect

    Kao, Fa-Ten

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  16. Whole Genome Mapping with Feature Sets from High-Throughput Sequencing Data

    PubMed Central

    Pan, Yonglong; Wang, Xiaoming; Liu, Lin; Wang, Hao; Luo, Meizhong

    2016-01-01

    A good physical map is essential to guide sequence assembly in de novo whole genome sequencing, especially when sequences are produced by high-throughput sequencing such as next-generation-sequencing (NGS) technology. We here present a novel method, Feature sets-based Genome Mapping (FGM). With FGM, physical map and draft whole genome sequences can be generated, anchored and integrated using the same data set of NGS sequences, independent of restriction digestion. Method model was created and parameters were inspected by simulations using the Arabidopsis genome sequence. In the simulations, when ~4.8X genome BAC library including 4,096 clones was used to sequence the whole genome, ~90% of clones were successfully connected to physical contigs, and 91.58% of genome sequences were mapped and connected to chromosomes. This method was experimentally verified using the existing physical map and genome sequence of rice. Of 4,064 clones covering 115 Mb sequence selected from ~3 tiles of 3 chromosomes of a rice draft physical map, 3,364 clones were reconstructed into physical contigs and 98 Mb sequences were integrated into the 3 chromosomes. The physical map-integrated draft genome sequences can provide permanent frameworks for eventually obtaining high-quality reference sequences by targeted sequencing, gap filling and combining other sequences. PMID:27611682

  17. A reliable and efficient method for deleting operational sequences in PACs and BACs.

    PubMed

    Nistala, Ravi; Sigmund, Curt D

    2002-05-15

    P1-derived artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) have become very useful as tools to study gene expression and regulation in cells and in transgenic mice. They carry large fragments of genomic DNA (> or =100 kb) and therefore may contain all of the cis-regulatory elements required for expression of a gene. Because of this, even when inserted randomly in the genome, they can emulate the native environment of a gene resulting in a tightly regulated pattern of expression. Because these large genomic clones often contain DNA sequences which can manipulate chromatin at the local level, they become immune to position effects which affect expression of smaller transgenes, and thus their expression is proportional to copy number. Transgenic mice containing large BACs and PACs have become excellent models to examine the regulation of gene expression. Their usefulness would certainly be increased if easy and efficient methods are developed to manipulate them. We describe herein a method to make deletion mutations reliably and efficiently using a novel modification of the Chi-stimulated homologous recombination method. Specifically, we generated and employed a Lox511 'floxed' CAM resistance marker that first affords selection for homologous recombination in Escherichia coli, and then can be easily deleted leaving only a single Lox511 site as the footprint.

  18. [Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121].

    PubMed

    Liu, H; Dai, B; Jing, B; Wu, W; Li, S; Fang, Z; Zhao, H; Ye, D; Yan, R; Liu, J; Song, S; Yang, Y; Zhang, Y; Liu, F; Tu, Y; Yang, H; Huang, Z; Liang, L; Hu, L; Zhao, M

    1997-03-01

    A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017.

  19. Genomic Resources for Water Yam (Dioscorea alata L.): Analyses of EST-Sequences, De Novo Sequencing and GBS Libraries.

    PubMed

    Saski, Christopher A; Bhattacharjee, Ranjana; Scheffler, Brian E; Asiedu, Robert

    2015-01-01

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources in several model and non-model plant species. Yam (Dioscorea spp.) is a major food and cash crop in many countries but research efforts have been limited to understand the genetics and generate genomic information for the crop. The availability of a large number of genomic resources including genome-wide molecular markers will accelerate the breeding efforts and application of genomic selection in yams. In the present study, several methods including expressed sequence tags (EST)-sequencing, de novo sequencing, and genotyping-by-sequencing (GBS) profiles on two yam (Dioscorea alata L.) genotypes (TDa 95/00328 and TDa 95-310) was performed to generate genomic resources for use in its improvement programs. This includes a comprehensive set of EST-SSRs, genomic SSRs, whole genome SNPs, and reduced representation SNPs. A total of 1,152 EST-SSRs were developed from >40,000 EST-sequences generated from the two genotypes. A set of 388 EST-SSRs were validated as polymorphic showing a polymorphism rate of 34% when tested on two diverse parents targeted for anthracnose disease. In addition, approximately 40X de novo whole genome sequence coverage was generated for each of the two genotypes, and a total of 18,584 and 15,952 genomic SSRs were identified for TDa 95/00328 and TDa 95-310, respectively. A custom made pipeline resulted in the selection of 573 genomic SSRs common across the two genotypes, of which only eight failed, 478 being polymorphic and 62 monomorphic indicating a polymorphic rate of 83.5%. Additionally, 288,505 high quality SNPs were also identified between these two genotypes. Genotyping by sequencing reads on these two genotypes also revealed 36,790 overlapping SNP positions that are distributed throughout the genome. Our efforts in using different approaches

  20. Integration of draft sequence and physical map as framework for genomic research in soybean (Glycine max)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three independent BAC libraries, consisting of 223,640 clones, combined with genetic and gene-based markers were used to construct a minimal tiling path (MTP) of BAC clones. Out of the 134,182 fingerprinted clones, 107,214 clones were assembled into contigs and 1,355 FPC contigs were aligned to buil...

  1. In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries.

    PubMed Central

    Jacobs, W R; Barrett, J F; Clark-Curtiss, J E; Curtiss, R

    1986-01-01

    Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts. Images PMID:2937735

  2. Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum.

    PubMed

    Grativol, Clícia; Regulski, Michael; Bertalan, Marcelo; McCombie, W Richard; da Silva, Felipe Rodrigues; Zerlotini Neto, Adhemar; Vicentini, Renato; Farinelli, Laurent; Hemerly, Adriana Silva; Martienssen, Robert A; Ferreira, Paulo Cavalcanti Gomes

    2014-07-01

    Many economically important crops have large and complex genomes that hamper their sequencing by standard methods such as whole genome shotgun (WGS). Large tracts of methylated repeats occur in plant genomes that are interspersed by hypomethylated gene-rich regions. Gene-enrichment strategies based on methylation profiles offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration with McrBC endonuclease digestion to enrich for euchromatic regions in the sugarcane genome. To verify the efficiency of methylation filtration and the assembly quality of sequences submitted to gene-enrichment strategy, we have compared assemblies using methyl-filtered (MF) and unfiltered (UF) libraries. The use of methy filtration allowed a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5× more scaffolds and 1.7× more assembled Mb in length compared with unfiltered dataset. The coverage of sorghum coding sequences (CDS) by MF scaffolds was at least 36% higher than by the use of UF scaffolds. Using MF technology, we increased by 134× the coverage of gene regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds that covered all genes of the sugarcane bacterial artificial chromosomes (BACs), 97.2% of sugarcane expressed sequence tags (ESTs), 92.7% of sugarcane RNA-seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds from encoded enzymes of the sucrose/starch pathway discovered 291 single-nucleotide polymorphisms (SNPs) in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes was also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and for improvement of sugarcane as a biofuel crop.

  3. Sugarcane genome sequencing by methylation filtration provides tools for genomic research in the genus Saccharum

    PubMed Central

    Grativol, Clícia; Regulski, Michael; Bertalan, Marcelo; McCombie, W. Richard; da Silva, Felipe Rodrigues; Neto, Adhemar Zerlotini; Vicentini, Renato; Farinelli, Laurent; Hemerly, Adriana Silva; Martienssen, Robert A.; Ferreira, Paulo Cavalcanti Gomes

    2015-01-01

    SUMMARY Many economically important crops have large and complex genomes, which hampers sequencing of their genome by standard methods such as WGS. Large tracts of methylated repeats occur at plant genomes interspersed by hypomethylated gene-rich regions. Gene enrichment strategies based on methylation profile offer an alternative to sequencing repetitive genomes. Here, we have applied methyl filtration (MF) with McrBC digestion to enrich for euchromatic regions of sugarcane genome. To verify the efficiency of MF and the assembly quality of sequences submitted to gene-enrichment strategy, we have compared assemblies using MF and unfiltered (UF) libraries. The MF allowed the achievement of a better assembly by filtering out 35% of the sugarcane genome and by producing 1.5 times more scaffolds and 1.7 times more assembled Mb compared to unfiltered scaffolds. The coverage of sorghum CDS by MF scaffolds was at least 36% higher than by UF scaffolds. Using MF technology, we increased by 134X the coverage of genic regions of the monoploid sugarcane genome. The MF reads assembled into scaffolds covering all genes at sugarcane BACs, 97.2% of sugarcane ESTs, 92.7% of sugarcane RNA-seq reads and 98.4% of sorghum protein sequences. Analysis of MF scaffolds encoding enzymes of the sucrose/starch pathway discovered 291 SNPs in the wild sugarcane species, S. spontaneum and S. officinarum. A large number of microRNA genes were also identified in the MF scaffolds. The information achieved by the MF dataset provides a valuable tool for genomic research in the genus Saccharum and improvement of sugarcane as a biofuel crop. PMID:24773339

  4. Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

    PubMed

    Owens, Jesse B; Mauro, Damiano; Stoytchev, Ilko; Bhakta, Mital S; Kim, Moon-Soo; Segal, David J; Moisyadi, Stefan

    2013-10-01

    Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

  5. Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system.

    PubMed

    Lin, Bo; Liu, Kun; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Zhu, Mingyue; Li, Mengsen

    2017-02-28

    α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72-96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP.

  6. Expression and bioactivity of human α-fetoprotein in a Bac-to-Bac system

    PubMed Central

    Lin, Bo; Liu, Kun; Wang, Wenting; Li, Wei; Dong, Xu; Chen, Yi; Lu, Yan; Guo, Junli; Li, Mengsen

    2016-01-01

    α-fetoprotein (AFP) is an early serum growth factor in foetal embryonic development and hepatic oncogenesis. A growing number of investigations of AFP as a tumour-specific biomarker have concluded that AFP is an important target for cancer treatment. AFP also plays an immunomodulatory role in the treatment of several autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, myasthenia gravis and thyroiditis. In an effort to support biochemical screening and drug design and discovery, we attempted to express and purify human AFP in a Bac-to-Bac system. Two key factors affecting the expression of recombinant human AFP (R-AFP), namely the infectious baculovirus inoculum volume and the culturing time post-infection, were optimized to maximize the yield. We achieved a high yield of approximately 1.5 mg/l of harvested medium with a 72–96 h incubation period after infection and an inoculum volume ratio of 1:100. We also assessed the role of R-AFP in the proliferation of the human liver cancer cell line Bel 7402, and the results indicated that R-AFP promoted the growth of hepatoma cells. We concluded that this method can produce high yields of R-AFP, which can be used for studies related to AFP. PMID:27913752

  7. Efficient expression of acetylcholine-binding protein from Aplysia californica in Bac-to-Bac system.

    PubMed

    Lin, Bo; Meng, Hailing; Bing, Hui; Zhangsun, Dongting; Luo, Sulan

    2014-01-01

    The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 10(6) cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

  8. A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles | Office of Cancer Genomics

    Cancer.gov

    Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells.

  9. Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection.

    PubMed

    Murata, Natsuko; Masuda, Kiyoshi; Nishiyama, Ryutaro; Nomura, Koji

    2005-06-01

    In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.

  10. Copy-Number Variations Observed in a Japanese Population by BAC Array CGH: Summary of Relatively Rare CNVs

    PubMed Central

    Satoh, Yasunari; Sasaki, Keiko; Shimoichi, Yuko; Sugita, Keiko; Katayama, Hiroaki; Takahashi, Norio

    2012-01-01

    Copy-number variations (CNVs) may contribute to genetic variation in humans. Reports regarding existence and characteristics of CNVs in a large apparently healthy Japanese cohort are quite limited. We report the data from a screening of 213 unrelated Japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray (BAC aCGH). In a previous paper, we summarized the data by focusing on highly polymorphic CNVs (in ≥5.0 % of the individuals). However, rare variations have recently received attention from scientists who espouse a hypothesis called “common disease and rare variants.” Here, we report CNVs identified in fewer than 10 individuals in our study population. We found a total of 126 CNVs at 52 different BAC regions in the genome. The CNVs observed at 27 of the 52 BAC-regions were found in only one unrelated individual. The majority of CNVs found in this study were not identified in the Japanese who were examined in the other studies. Family studies were conducted, and the results demonstrated that the CNVs were inherited from one parent in the families. PMID:22315515

  11. Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer.

    PubMed

    Zeng, Fang; Li, Zicong; Cai, Gengyuan; Gao, Wenchao; Jiang, Gelong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2016-10-01

    Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.

  12. Comparative analysis of a BAC contig of the porcine RN region and the human transcript map: implications for the cloning of trait loci.

    PubMed

    Jeon, J T; Amarger, V; Rogel-Gaillard, C; Robic, A; Bongcam-Rudloff, E; Paul, S; Looft, C; Milan, D; Chardon, P; Andersson, L

    2001-03-15

    The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals.

  13. Genomic resources for water yam (Dioscorea alata L.): analyses of EST-Sequences, De Novo sequencing and GBS libraries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The reducing cost and rapid progress in next-generation sequencing techniques coupled with high performance computational approaches have resulted in large-scale discovery of advanced genomic resources such as SSRs, SNPs and InDels in several model and non-model plant species. Yam (Dioscorea spp.) i...

  14. Losing Libraries, Saving Libraries

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2010-01-01

    This summer, as public libraries continued to get budget hit after budget hit across the country, several readers asked for a comprehensive picture of the ravages of the recession on library service. In partnership with 2010 Movers & Shakers Laura Solomon and Mandy Knapp, Ohio librarians who bought the Losing Libraries domain name,…

  15. Construction and Characterization of a Repetitive DNA Library in Parodontidae (Actinopterygii: Characiformes): A Genomic and Evolutionary Approach to the Degeneration of the W Sex Chromosome

    PubMed Central

    Oliveira, Jordana Inácio Nascimento; Nogaroto, Viviane; Almeida, Mara Cristina; Artoni, Roberto Ferreira; Cestari, Marta Margarete; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo

    2014-01-01

    Abstract Repetitive DNA sequences, including tandem and dispersed repeats, comprise a large portion of eukaryotic genomes and are important for gene regulation, sex chromosome differentiation, and karyotype evolution. In Parodontidae, only the repetitive DNAs WAp and pPh2004 and rDNAs were previously studied using fluorescence in situ hybridization. This study aimed to build a library of repetitive DNA in Parodontidae. We isolated 40 clones using Cot-1; 17 of these clones exhibited similarity to repetitive DNA sequences, including satellites, minisatellites, microsatellites, and class I and class II transposable elements (TEs), from Danio rerio and other organisms. The physical mapping of the clones to chromosomes revealed the presence of a satellite DNA, a Helitron element, and degenerate short interspersed element (SINE), long interspersed element (LINE), and tc1-mariner elements on the sex chromosomes. Some clones exhibited dispersed signals; other sequences were not detected. The 5S rDNA was detected on an autosomal pair. These elements likely function in the molecular degeneration of the W chromosome in Parodontidae. Thus, the location of these elements on the chromosomes is important for understanding the function of these repetitive DNAs and for integrative studies with genome sequencing. The presented data demonstrate that an intensive invasion of TEs occurred during W sex chromosome differentiation in the Parodontidae. PMID:25122415

  16. The genome of the sea urchin Strongylocentrotus purpuratus.

    PubMed

    Sodergren, Erica; Weinstock, George M; Davidson, Eric H; Cameron, R Andrew; Gibbs, Richard A; Angerer, Robert C; Angerer, Lynne M; Arnone, Maria Ina; Burgess, David R; Burke, Robert D; Coffman, James A; Dean, Michael; Elphick, Maurice R; Ettensohn, Charles A; Foltz, Kathy R; Hamdoun, Amro; Hynes, Richard O; Klein, William H; Marzluff, William; McClay, David R; Morris, Robert L; Mushegian, Arcady; Rast, Jonathan P; Smith, L Courtney; Thorndyke, Michael C; Vacquier, Victor D; Wessel, Gary M; Wray, Greg; Zhang, Lan; Elsik, Christine G; Ermolaeva, Olga; Hlavina, Wratko; Hofmann, Gretchen; Kitts, Paul; Landrum, Melissa J; Mackey, Aaron J; Maglott, Donna; Panopoulou, Georgia; Poustka, Albert J; Pruitt, Kim; Sapojnikov, Victor; Song, Xingzhi; Souvorov, Alexandre; Solovyev, Victor; Wei, Zheng; Whittaker, Charles A; Worley, Kim; Durbin, K James; Shen, Yufeng; Fedrigo, Olivier; Garfield, David; Haygood, Ralph; Primus, Alexander; Satija, Rahul; Severson, Tonya; Gonzalez-Garay, Manuel L; Jackson, Andrew R; Milosavljevic, Aleksandar; Tong, Mark; Killian, Christopher E; Livingston, Brian T; Wilt, Fred H; Adams, Nikki; Bellé, Robert; Carbonneau, Seth; Cheung, Rocky; Cormier, Patrick; Cosson, Bertrand; Croce, Jenifer; Fernandez-Guerra, Antonio; Genevière, Anne-Marie; Goel, Manisha; Kelkar, Hemant; Morales, Julia; Mulner-Lorillon, Odile; Robertson, Anthony J; Goldstone, Jared V; Cole, Bryan; Epel, David; Gold, Bert; Hahn, Mark E; Howard-Ashby, Meredith; Scally, Mark; Stegeman, John J; Allgood, Erin L; Cool, Jonah; Judkins, Kyle M; McCafferty, Shawn S; Musante, Ashlan M; Obar, Robert A; Rawson, Amanda P; Rossetti, Blair J; Gibbons, Ian R; Hoffman, Matthew P; Leone, Andrew; Istrail, Sorin; Materna, Stefan C; Samanta, Manoj P; Stolc, Viktor; Tongprasit, Waraporn; Tu, Qiang; Bergeron, Karl-Frederik; Brandhorst, Bruce P; Whittle, James; Berney, Kevin; Bottjer, David J; Calestani, Cristina; Peterson, Kevin; Chow, Elly; Yuan, Qiu Autumn; Elhaik, Eran; Graur, Dan; Reese, Justin T; Bosdet, Ian; Heesun, Shin; Marra, Marco A; Schein, Jacqueline; Anderson, Michele K; Brockton, Virginia; Buckley, Katherine M; Cohen, Avis H; Fugmann, Sebastian D; Hibino, Taku; Loza-Coll, Mariano; Majeske, Audrey J; Messier, Cynthia; Nair, Sham V; Pancer, Zeev; Terwilliger, David P; Agca, Cavit; Arboleda, Enrique; Chen, Nansheng; Churcher, Allison M; Hallböök, F; Humphrey, Glen W; Idris, Mohammed M; Kiyama, Takae; Liang, Shuguang; Mellott, Dan; Mu, Xiuqian; Murray, Greg; Olinski, Robert P; Raible, Florian; Rowe, Matthew; Taylor, John S; Tessmar-Raible, Kristin; Wang, D; Wilson, Karen H; Yaguchi, Shunsuke; Gaasterland, Terry; Galindo, Blanca E; Gunaratne, Herath J; Juliano, Celina; Kinukawa, Masashi; Moy, Gary W; Neill, Anna T; Nomura, Mamoru; Raisch, Michael; Reade, Anna; Roux, Michelle M; Song, Jia L; Su, Yi-Hsien; Townley, Ian K; Voronina, Ekaterina; Wong, Julian L; Amore, Gabriele; Branno, Margherita; Brown, Euan R; Cavalieri, Vincenzo; Duboc, Véronique; Duloquin, Louise; Flytzanis, Constantin; Gache, Christian; Lapraz, François; Lepage, Thierry; Locascio, Annamaria; Martinez, Pedro; Matassi, Giorgio; Matranga, Valeria; Range, Ryan; Rizzo, Francesca; Röttinger, Eric; Beane, Wendy; Bradham, Cynthia; Byrum, Christine; Glenn, Tom; Hussain, Sofia; Manning, Gerard; Miranda, Esther; Thomason, Rebecca; Walton, Katherine; Wikramanayke, Athula; Wu, Shu-Yu; Xu, Ronghui; Brown, C Titus; Chen, Lili; Gray, Rachel F; Lee, Pei Yun; Nam, Jongmin; Oliveri, Paola; Smith, Joel; Muzny, Donna; Bell, Stephanie; Chacko, Joseph; Cree, Andrew; Curry, Stacey; Davis, Clay; Dinh, Huyen; Dugan-Rocha, Shannon; Fowler, Jerry; Gill, Rachel; Hamilton, Cerrissa; Hernandez, Judith; Hines, Sandra; Hume, Jennifer; Jackson, Laronda; Jolivet, Angela; Kovar, Christie; Lee, Sandra; Lewis, Lora; Miner, George; Morgan, Margaret; Nazareth, Lynne V; Okwuonu, Geoffrey; Parker, David; Pu, Ling-Ling; Thorn, Rachel; Wright, Rita

    2006-11-10

    We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.

  17. Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

    PubMed Central

    Hurst, Sheldon; Rowedder, Holli; Michaels, Brandye; Bullock, Hannah; Jackobeck, Ryan; Abebe-Akele, Feseha; Durakovic, Umjia; Gately, Jon; Janicki, Erik

    2015-01-01

    ABSTRACT The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont. PMID

  18. Whole genome linkage disequilibrium maps in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine whole genome linkage disequilibrium maps were constructed for eight breeds of cattle. These data provide fundamental information concerning bovine genome organization which will allow the design of studies to associate genetic variation with economically important traits and also provides bac...

  19. Novel SSR Markers from BAC-End Sequences, DArT Arrays and a Comprehensive Genetic Map with 1,291 Marker Loci for Chickpea (Cicer arietinum L.)

    PubMed Central

    Nayak, Spurthi N.; Varghese, Nicy; Shah, Trushar M.; Penmetsa, R. Varma; Thirunavukkarasu, Nepolean; Gudipati, Srivani; Gaur, Pooran M.; Kulwal, Pawan L.; Upadhyaya, Hari D.; KaviKishor, Polavarapu B.; Winter, Peter; Kahl, Günter; Town, Christopher D.; Kilian, Andrzej; Cook, Douglas R.; Varshney, Rajeev K.

    2011-01-01

    Chickpea (Cicer arietinum L.) is the third most important cool season food legume, cultivated in arid and semi-arid regions of the world. The goal of this study was to develop novel molecular markers such as microsatellite or simple sequence repeat (SSR) markers from bacterial artificial chromosome (BAC)-end sequences (BESs) and diversity arrays technology (DArT) markers, and to construct a high-density genetic map based on recombinant inbred line (RIL) population ICC 4958 (C. arietinum)×PI 489777 (C. reticulatum). A BAC-library comprising 55,680 clones was constructed and 46,270 BESs were generated. Mining of these BESs provided 6,845 SSRs, and primer pairs were designed for 1,344 SSRs. In parallel, DArT arrays with ca. 15,000 clones were developed, and 5,397 clones were found polymorphic among 94 genotypes tested. Screening of newly developed BES-SSR markers and DArT arrays on the parental genotypes of the RIL mapping population showed polymorphism with 253 BES-SSR markers and 675 DArT markers. Segregation data obtained for these polymorphic markers and 494 markers data compiled from published reports or collaborators were used for constructing the genetic map. As a result, a comprehensive genetic map comprising 1,291 markers on eight linkage groups (LGs) spanning a total of 845.56 cM distance was developed (http://cmap.icrisat.ac.in/cmap/sm/cp/thudi/). The number of markers per linkage group ranged from 68 (LG 8) to 218 (LG 3) with an average inter-marker distance of 0.65 cM. While the developed resource of molecular markers will be useful for genetic diversity, genetic mapping and molecular breeding applications, the comprehensive genetic map with integrated BES-SSR markers will facilitate its anchoring to the physical map (under construction) to accelerate map-based cloning of genes in chickpea and comparative genome evolution studies in legumes. PMID:22102885

  20. BacHbpred: Support Vector Machine Methods for the Prediction of Bacterial Hemoglobin-Like Proteins

    PubMed Central

    Selvaraj, MuthuKrishnan; Puri, Munish; Dikshit, Kanak L.; Lefevre, Christophe

    2016-01-01

    The recent upsurge in microbial genome data has revealed that hemoglobin-like (HbL) proteins may be widely distributed among bacteria and that some organisms may carry more than one HbL encoding gene. However, the discovery of HbL proteins has been limited to a small number of bacteria only. This study describes the prediction of HbL proteins and their domain classification using a machine learning approach. Support vector machine (SVM) models were developed for predicting HbL proteins based upon amino acid composition (AC), dipeptide composition (DC), hybrid method (AC + DC), and position specific scoring matrix (PSSM). In addition, we introduce for the first time a new prediction method based on max to min amino acid residue (MM) profiles. The average accuracy, standard deviation (SD), false positive rate (FPR), confusion matrix, and receiver operating characteristic (ROC) were analyzed. We also compared the performance of our proposed models in homology detection databases. The performance of the different approaches was estimated using fivefold cross-validation techniques. Prediction accuracy was further investigated through confusion matrix and ROC curve analysis. All experimental results indicate that the proposed BacHbpred can be a perspective predictor for determination of HbL related proteins. BacHbpred, a web tool, has been developed for HbL prediction. PMID:27034664

  1. Genomic Instability of the Sex-Determining Locus in Atlantic Salmon (Salmo salar).

    PubMed

    Lubieniecki, Krzysztof P; Lin, Song; Cabana, Emily I; Li, Jieying; Lai, Yvonne Y Y; Davidson, William S

    2015-09-22

    Atlantic salmon and rainbow trout, like other members of the subfamily Salmoninae, are gonochoristic with male heterogamety. The finding that sex-linked genetic markers varied between species suggested that the sex-determining gene differs among salmonid species, or that there is one sex-determining gene that has the capacity to move around the genome. The discovery of sdY, the sex-determining gene in rainbow trout, and its presence in many male salmonids gave support to the latter. Additional evidence for a salmonid-specific, sex-determining jumping gene came from the mapping of the sex-determining locus to three different chromosomes in Tasmanian male Atlantic salmon lineages. To characterize the sex-determining region, we isolated three sdY containing BACs from an Atlantic salmon male library. Sequencing of these BACs yielded two contigs, one of which contained the sdY gene. Sequence analysis of the borders of male-specific and female/male common regions revealed highly repetitive sequences associated with mobile elements, which may allow an sdY cassette to jump around the genome. FISH analysis using a BAC or a plasmid containing the sdY gene showed that the sdY gene did indeed localize to the chromosomes where SEX had been mapped in different Tasmanian Atlantic salmon families. Moreover, the plasmid sdY gene probe hybridized primarily to one of the sex chromosomes as would be expected of a male-specific gene. Our results suggest that a common salmonid sex-determining gene (sdY) can move between three specific loci on chromosomes 2, 3, and 6, giving the impression that there are multiple SEX loci both within and between salmonid species.

  2. Characterizing a novel strain of Bacillus amyloliquefaciens BAC03 for potential biological control application

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: Identify and characterize a bacterial strain from suppressive soil, BAC03, evaluate its antimicrobial activity against Streptomyces scabies and other microorganisms, and characterize an antimicrobial substance produced by this strain. Methods and Results: Bacterial strain BAC03 (isolated from ...

  3. A whole-genome, radiation hybrid map of wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Generating a reference sequence of bread wheat (Triticum aestivum L.) is a challenging task because of its large, highly repetitive and allopolyploid genome. Ordering of BAC- and NGS-based contigs in ongoing wheat genome-sequencing projects primarily uses recombination and comparative genomics-base...

  4. Progress in the characterization of a human genomic YAC library selected on the basis of homology to T{sub 2}AG{sub 3}

    SciTech Connect

    Vocero-Akbani, A.; Sanjurjo, H.; Fair, K.

    1994-09-01

    Using a combination of physical and genetic mapping methods we have characterized more than 190 YAC clones originally isolated on the basis of hybridization to the human telomere regions by FISH (using Alu-PCR products or YAC subclones individually or pooled as probes). Thirty-seven of the YACs mapped to single telomeres while 16 mapped to more than one telomere, or to interstitial regions, including centromeres. Subclone libraries were constructed for a subset of YACs, genetic markers developed, and the loci incorporated into genetic maps for chromosomes 2, 6, 7, 8, 10, 12, 13, 14 and 20. Altogether 28 different telomeres are now defined by chromosomally mapped STSs which were derived from YACs that were FISH mapped to the termini of 1p, 2p{sup *}, 2q{sup +}, 3p, 3q, 4q, 5q, 6q{sup *}, 7p, 7q{sup *+}, 8p{sup +}, 9q, 10p{sup *}, 10q, 11q, 12p{sup *}, 13q{sup *+}, 14q{sup *+}, 16p, 16q, 17p, 17q, 18p, 18q, 20p, 21q, and 22q ({sup *} microsatellite marker, {sup +}RFLP). Development of microsatellite genetic markers for the five additional telomeres is currently in progress [7p (50 b), 10q (275 kb). 17p (100 kb), 17q (175 kb), and 18p (225 kb)]. For YACs that have been localized to telomeres by FISH and to chromosomes by STS mapping to a rodent/human somatic cell hybrid chromosome panel, five genome equivalent bacteriophage lamda subclone libraries have been constructed and screened for the presence of human DNA and CA{sub n} dinucleotide repeats by plaque filter hybridization. A number of CA positive clones have been sequenced revealing simple repeats of 12 or more CAs per clone. STS development and testing for polymorphism using the CEPH pedigree resource is in progress.

  5. Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures.

    PubMed

    Kim, Wonyong; Park, Hee Kuk; Thanh, Hien Dang; Lee, Bo-Young; Shin, Jong Wook; Shin, Hyoung-Shik

    2011-12-01

    Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene-targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.

  6. A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila.

    PubMed Central

    Bonin, Christopher P; Mann, Richard S

    2004-01-01

    P-element-based gene and enhancer trap strategies have provided a wealth of information on the expression and function of genes in Drosophila melanogaster. Here we present a new vector that utilizes the simple insertion requirements of the piggyBac transposon, coupled to a splice acceptor (SA) site fused to the sequence encoding enhanced green fluorescent protein (EGFP) and a transcriptional terminator. Mobilization of the piggyBac splice site gene trap vector (PBss) was accomplished by heat-shock-induced expression of piggyBac transposase (PBase). We show that insertion of PBss into genes leads to fusions between the gene's mRNA and the PBss-encoded EGFP transcripts. As heterozygotes, these fusions report the normal pattern of expression of the trapped gene. As homozygotes, these fusions can inactivate the gene and lead to lethality. Molecular characterization of PBss insertion events shows that they are single copy, that they always occur at TTAA sequences, and that splicing utilizes the engineered splice site in PBss. In those instances where protein-EGFP fusions are predicted to occur, the subcellular localization of the wild-type protein can be inferred from the localization of the EGFP fusion protein. These experiments highlight the utility of the PBss system for expanding the functional genomics tools that are available in Drosophila. PMID:15342518

  7. Recombinase-mediated cassette exchange (RMCE) and BAC engineering via VCre/VloxP and SCre/SloxP systems.

    PubMed

    Minorikawa, Sachiko; Nakayama, Manabu

    2011-04-01

    Site-specific recombination is a powerful biotechnological tool for genome engineering. We previously reported two novel site-specific recombination systems, VCre/VloxP and SCre/SloxP, that do not cross-react with Cre/loxP and Flp/FRT in culture cells and mouse embryonic stem (ES) cells. In this study, a site-specific recombination assay in Escherichia coli was used to examine the activity of mutant VCre (H314L and Y349F) and mutant SCre (H317L and Y352F), in which both mutated residues lie within the active center of Cre recombination. The site-specific recombination activity of both mutants was significantly decreased. Recombinase-mediated cassette exchange (RMCE) using VloxP and the Vlox2272 mutant site was performed in E. coli by introducing a cassette bearing VloxP and Vlox2272 into a recipient plasmid bearing the same sites. RMCE using SloxP and Slox2272 was also performed by SCre recombinase. Moreover, BAC engineering via Red recombination and VCre/VloxP were demonstrated. First, the DNA cassette for modification was introduced into a BAC clone via Red recombination; second, the antibiotics resistance gene flanked by VloxP was removed from the BAC clone by induction of VCre recombinase. Such site-specific recombination systems may effectively be used in combination with other site-specific recombination systems or engineering tools (e.g., Red recombination).

  8. Simple and efficient vectors for retrofitting BACs and PACs with mammalian neoR and EGFP marker genes.

    PubMed

    Kaname, T; Huxley, C

    2001-03-21

    Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) are widely used to investigate the functions of genes and genomes in mammalian cells in vitro and in vivo. We have developed a series of vectors which can simply and efficiently be retrofitted onto BACs or PACs. These vectors carry a neoR gene for selection in cells in tissue culture, including ES cells, and also an EGFP gene driven by the strong CAG promoter for quick detection of the DNA in cells. All the plasmids are retrofitted using the loxP site and Cre recombinase and some carry the gamma origin of plasmid R6K which does not function in commonly used bacteria such as DH10B. Retrofitting of PACs and BACs carrying alphoid DNA was very efficient with almost no rearrangement of the highly repetitive alphoid DNA. Following transfer into HT1080 cells and mouse oocytes in tissue culture the DNA could easily be monitored by the EGFP fluorescence.

  9. Suicidal autointegration of sleeping beauty and piggyBac transposons in eukaryotic cells.

    PubMed

    Wang, Yongming; Wang, Jichang; Devaraj, Anatharam; Singh, Manvendra; Jimenez Orgaz, Ana; Chen, Jia-Xuan; Selbach, Matthias; Ivics, Zoltán; Izsvák, Zsuzsanna

    2014-03-01

    Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.

  10. Genome Improvement at JGI-HAGSC

    SciTech Connect

    Grimwood, Jane; Schmutz, Jeremy J.; Myers, Richard M.

    2012-03-03

    Since the completion of the sequencing of the human genome, the Joint Genome Institute (JGI) has rapidly expanded its scientific goals in several DOE mission-relevant areas. At the JGI-HAGSC, we have kept pace with this rapid expansion of projects with our focus on assessing, assembling, improving and finishing eukaryotic whole genome shotgun (WGS) projects for which the shotgun sequence is generated at the Production Genomic Facility (JGI-PGF). We follow this by combining the draft WGS with genomic resources generated at JGI-HAGSC or in collaborator laboratories (including BAC end sequences, genetic maps and FLcDNA sequences) to produce an improved draft sequence. For eukaryotic genomes important to the DOE mission, we then add further information from directed experiments to produce reference genomic sequences that are publicly available for any scientific researcher. Also, we have continued our program for producing BAC-based finished sequence, both for adding information to JGI genome projects and for small BAC-based sequencing projects proposed through any of the JGI sequencing programs. We have now built our computational expertise in WGS assembly and analysis and have moved eukaryotic genome assembly from the JGI-PGF to JGI-HAGSC. We have concentrated our assembly development work on large plant genomes and complex fungal and algal genomes.

  11. Generation of a transgenic cashmere goat using the piggyBac transposition system.

    PubMed

    Bai, Ding-Ping; Yang, Ming-Ming; Qu, Lei; Chen, Yu-Lin

    2017-04-15

    The development of transgenic technologies in the Cashmere goat (Capra hircus) has the potential to improve the quality of the meat and wool. The piggyBac (PB) transposon system is highly efficient and can be used to transpose specific target genes into the genome. Here, we developed a PB transposon system to produce transgenic Cashmere goat fetal fibroblasts (GFFs) with the enhanced green fluorescent protein (EGFP). We then used the genetically modified GFFs as nuclear donors to generate transgenic embryos by somatic cell nuclear transfer (SCNT). The embryos (n = 40) were implanted into female goats (n = 20). One transgenic kid that expressed EGFP throughout the surface features of its body was born. This result demonstrated the usefulness of PB transposon system in generating transgenic Cashmere goats.

  12. Bacteriocin protein BacL1 of Enterococcus faecalis is a peptidoglycan D-isoglutamyl-L-lysine endopeptidase.

    PubMed

    Kurushima, Jun; Hayashi, Ikue; Sugai, Motoyuki; Tomita, Haruyoshi

    2013-12-27

    Enterococcus faecalis strains are commensal bacteria in humans and other animals, and they are also the causative agent of opportunistic infectious diseases. Bacteriocin 41 (Bac41) is produced by certain E. faecalis clinical isolates, and it is active against other E. faecalis strains. Our genetic analyses demonstrated that the extracellular products of the bacL1 and bacA genes, which are encoded in the Bac41 operon, coordinately express the bacteriocin activity against E. faecalis. In this study, we investigated the molecular functions of the BacL1 and BacA proteins. Immunoblotting and N-terminal amino acid sequence analysis revealed that BacL1 and BacA are secreted without any processing. The coincidental treatment with the recombinant BacL1 and BacA showed complete bacteriocin activity against E. faecalis, but neither BacL1 nor BacA protein alone showed the bacteriocin activity. Interestingly, BacL1 alone demonstrated substantial degrading activity against the cell wall fraction of E. faecalis in the absence of BacA. Furthermore, MALDI-TOF MS analysis revealed that BacL1 has a peptidoglycan D-isoglutamyl-L-lysine endopeptidase activity via a NlpC/P60 homology domain. These results collectively suggest that BacL1 serves as a peptidoglycan hydrolase and, when BacA is present, results in the lysis of viable E. faecalis cells.

  13. Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis.

    PubMed

    Zhao, Zi-Xia; Xu, Peng; Cao, Ding-Chen; Kuang, You-Yi; Deng, Hai-Xia; Zhang, Yan; Xu, Li-Ming; Li, Jiong-Tang; Xu, Jian; Sun, Xiao-Wen

    2014-09-15

    Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is a hypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein analysis has shown functional specialization of the two mb transcripts. In this work, analysis for mb-containing bacterial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1) and myoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed that mb-1 and mb-2 are located on separate chromosomes. In both of the mb-1 and mb-2 containing BAC clones, gene synteny was well conserved with the homologous region on zebrafish chromosome 1, supporting that the common carp specific mb-2 gene originated from the recent whole genome duplication event in cyprinid lineage. Transcription factor binding sites search indicated that both common carp mb genes lacked specificity Protein 1 (Sp1) and myocyte enhancer factor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the well-studied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region of common carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted the hypoxia-inducible, non-muscle expression pattern of mb-1. In the case of mb-2, a 10 bp insertion to the binding site of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might cause the non-response to hypoxia treatment of mb-2. The case of common carp mb gene duplication and subsequent differentiation in expression pattern and protein function provided an example for adaptive evolution toward aquatic hypoxia tolerance.

  14. Creating Library Spaces: Libraries 2040.

    ERIC Educational Resources Information Center

    Bruijnzeels, Rob

    This paper suggests that by 2004, the traditional public libraries will have ceased to exist and new, attractive future libraries will have taken their place. The Libraries 2040 project of the Netherlands is initiating seven different libraries of the future. The Brabant library is the "ultimate library of the future" for the Dutch…

  15. Characterization of the porcine sperm adhesion molecule gene SPAM1- expression analysis, genomic structure, and chromosomal mapping.

    PubMed

    Day, A E; Quilter, C R; Sargent, C A; Mileham, A J

    2002-06-01

    Sequence analysis of cDNA products, derived from adult porcine testis mRNA, gave overlapping nucleotide sequence correlating to 1952 bp of the sperm adhesion molecule 1 (SPAM1) gene. This sequence was shown to be homologous to SPAM1 genes known in other mammalian species and contained an open reading frame encoding a 493-amino acid protein. Fluorescence in situ hybridization (FISH), using a bacterial artificial chromosome (BAC) clone from the PigE BAC library, was used to map SPAM1 to chromosome 18 of the pig. This finding is consistent with comparative mapping experiments performed between pig and human chromosomes. Polymerase chain reaction (PCR) analysis of genomic DNA has shown that the 1952 bp of cDNA sequence spans approximately 9 kb of genomic DNA and comprises of at least four exons, with its size and structure being relatively conserved between mouse, human and pig. Reverse transcriptase (RT)-PCR analysis of mRNA from nine porcine tissues has also suggested that expression of SPAM1 is limited to the testis.

  16. The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.

    PubMed

    Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo; Zhu, Shilin; Shi, Daihu; McDill, Joshua; Yang, Linfeng; Hawkins, Simon; Neutelings, Godfrey; Datla, Raju; Lambert, Georgina; Galbraith, David W; Grassa, Christopher J; Geraldes, Armando; Cronk, Quentin C; Cullis, Christopher; Dash, Prasanta K; Kumar, Polumetla A; Cloutier, Sylvie; Sharpe, Andrew G; Wong, Gane K-S; Wang, Jun; Deyholos, Michael K

    2012-11-01

    Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.

  17. Effects of enhanced sanctions for high-BAC DWI offenders on case dispositions and rates of recidivism.

    PubMed

    McCartt, Anne T; Northrup, Veronika Shabanova

    2004-09-01

    Implemented January 1, 1998, Minnesota's high-BAC law mandates more severe administrative pre-conviction penalties and more severe post-conviction penalties for offenses with BACs > or = 0.20%. Most notably, the law provides for the administrative impoundment of the license plate of first-time DWI offenders with BACs > or = 0.20. During the three years after the law took effect, a large majority of first-time and repeat offenders with BACs > or = 0.20% did, in fact, receive high-BAC administrative dispositions and/or high-BAC court convictions, which carried more severe penalties. For example, in 1998 85.6% of first offenders with BACs > or = 0.20% received a high-BAC administrative disposition and/or a high-BAC court conviction; 65.0% received both high-BAC administrative and high-BAC court dispositions. The proportion of high-BAC first-time offenders who received the statutory high-BAC dispositions declined from 1998 to 1999 and 2000. Based on survival analysis, the one-year recidivism rate among first offenders arrested in 1998 with BACs > or = 0.20% was significantly lower than for offenders with BACs 0.17-0.19% (who also had relatively high BACs but were not subject to enhanced sanctions), after controlling for age and gender. There were similar, but not significant, results for first offenders arrested in 1999.

  18. Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica

    PubMed Central

    Liu, Xianjun; Lu, Ying; Yan, Mingli; Sun, Donghong; Hu, Xuefang; Liu, Shuyan; Chen, Sheyuan; Guan, Chunyun; Liu, Zhongsong

    2016-01-01

    Proanthocyanidins (PA) is a type of prominent flavonoid compound deposited in seed coats which controls the pigmentation in all Brassica species. Annotation of Brassica juncea genome survey sequences showed 72 PA genes; however, a functional description of these genes, especially how their interactions regulate seed pigmentation, remains elusive. In the present study, we designed 19 primer pairs to screen a bacterial artificial chromosome (BAC) library of B. juncea. A total of 284 BAC clones were identified and sequenced. Alignment of the sequences confirmed that 55 genes were cloned, with every Arabidopsis PA gene having 2–7 homologs in B. juncea. BLAST analysis using the recently released B. rapa or B. napus genome database identified 31 and 58 homologous genes, respectively. Mapping and phylogenetic analysis indicated that 30 B. juncea PA genes are located in the A-genome chromosomes except A04, whereas the remaining 25 genes are mapped to the B-genome chromosomes except B05 and B07. RNA-seq data and Fragments Per Kilobase of a transcript per Million mapped reads (FPKM) analysis showed that most of the PA genes were expressed in the seed coat of B. juncea and B. napus, and that BjuTT3, BjuTT18, BjuANR, BjuTT4-2, BjuTT4-3, BjuTT19-1, and BjuTT19-3 are transcriptionally regulated, and not expressed or downregulated in yellow-seeded testa. Importantly, our study facilitates in better understanding of the molecular mechanism underlying Brassica PA profiles and accumulation, as well as in further characterization of PA genes. PMID:28018375

  19. Genome-Wide Identification, Localization, and Expression Analysis of Proanthocyanidin-Associated Genes in Brassica.

    PubMed

    Liu, Xianjun; Lu, Ying; Yan, Mingli; Sun, Donghong; Hu, Xuefang; Liu, Shuyan; Chen, Sheyuan; Guan, Chunyun; Liu, Zhongsong

    2016-01-01

    Proanthocyanidins (PA) is a type of prominent flavonoid compound deposited in seed coats which controls the pigmentation in all Brassica species. Annotation of Brassica juncea genome survey sequences showed 72 PA genes; however, a functional description of these genes, especially how their interactions regulate seed pigmentation, remains elusive. In the present study, we designed 19 primer pairs to screen a bacterial artificial chromosome (BAC) library of B. juncea. A total of 284 BAC clones were identified and sequenced. Alignment of the sequences confirmed that 55 genes were cloned, with every Arabidopsis PA gene having 2-7 homologs in B. juncea. BLAST analysis using the recently released B. rapa or B. napus genome database identified 31 and 58 homologous genes, respectively. Mapping and phylogenetic analysis indicated that 30 B. juncea PA genes are located in the A-genome chromosomes except A04, whereas the remaining 25 genes are mapped to the B-genome chromosomes except B05 and B07. RNA-seq data and Fragments Per Kilobase of a transcript per Million mapped reads (FPKM) analysis showed that most of the PA genes were expressed in the seed coat of B. juncea and B. napus, and that BjuTT3, BjuTT18, BjuANR, BjuTT4-2, BjuTT4-3, BjuTT19-1, and BjuTT19-3 are transcriptionally regulated, and not expressed or downregulated in yellow-seeded testa. Importantly, our study facilitates in better understanding of the molecular mechanism underlying Brassica PA profiles and accumulation, as well as in further characterization of PA genes.

  20. PiggyBac as a novel vector in cancer gene therapy: current perspective.

    PubMed

    Mirzaei, H; Sahebkar, A; Jaafari, M R; Hadjati, J; Javanmard, S H; Mirzaei, H R; Salehi, R

    2016-01-01

    Selection of suitable delivery system is one of the crucial aspects in gene therapy that determines the efficiency of gene therapy. The past two decades have witnessed extensive efforts for finding safe and efficient vectors to overcome the limitations of viral vectors. The utilization of DNA transposon-based vectors for gene therapy has emerged as a promising non-viral alternative. DNA 'cut-and-paste' is one of the main mechanisms of genome engineering by transposon elements. However, the lack of an efficient transposition system has limited the utilization of transposon vectors in mice and mammalian systems. PiggyBac (PB) is known as a highly efficient DNA transposon originally isolated from Trichoplusia ni as an alternative to Sleeping Beauty (SB). It has been shown that PB can be functional in various species including mammalian systems. This vector could overcome some limitations of other vectors in cancer gene therapy. Some advantages of PB include the capacity for integration into the genome and providing a stable expression, capacity to harbor 10 and 9.1 kb of foreign DNA into the host genome, without a significant reduction in their transposition activity and display non-overlapping targeting preferences. However, to advance PB to clinical applications, some obstacles still require to be overcome to improve its safety and efficiency. Hence, it seems that this vector could open new horizons in gene and cancer therapy.

  1. Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur.

    PubMed

    Brosch, R; Gordon, S V; Buchrieser, C; Pym, A S; Garnier, T; Cole, S T

    2000-06-30

    On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine.

  2. [Developing a physical map of human chromosome 22 using Pace electrophoresis and large fragment cloning]. Annual report, October 1, 1991--July 1, 1994

    SciTech Connect

    Simon, M.I.

    1994-12-31

    In the past two years, the authors have made a great deal of progress in establishing Fosmid and BAC libraries and in using large BAC libraries for gene mapping. In addition, they initiated work on the application of BAC clones to long range genome sequencing. They continue to increase the ability to rapidly generate large BAC libraries and to efficiently apply these libraries to genome mapping. The BACs provide a very effective means of developing physical maps. The current work suggests that BAC contigs will be extremely useful as source material for genome sequencing.

  3. Clinical comparison of the Bactec Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN blood culture vials for the detection of candidemia.

    PubMed

    Ericson, Eva-Lena; Klingspor, Lena; Ullberg, Måns; Ozenci, Volkan

    2012-06-01

    The present study analyzed the performance of Bactec Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN vials in detection and time to detection (TTD) of Candida spp. in 179 simultaneous blood cultures. The Mycosis IC/F, BacT/Alert FA, and BacT/Alert FN vials could detect Candida spp. in 144 (80.45%) of 179, 149 (83.24%) of 179, and 8 (4.47%) of 179 samples, respectively. With the presence of antifungal therapy, the numbers of positive vials were higher in BacT/Alert FA compared to Mycosis IC/F, 87/99 versus 73/99, respectively (P < 0.05). TTD (SD) for C. albicans was shorter in Mycosis IC/F than in BacT/Alert FA vials without antifungal therapy, 20.89 (9.33) versus 28.26 (9.77), respectively (P < 0.01). The detection of Candida spp., with concomitant bacteremia, was higher in Mycosis IC/F than in BacT/Alert FA vials, 28/30 and 19/30, respectively (P = 0.01). The present data show that the use of Bactec Mycosis IC/F together with BacT/Alert FA vials might improve the detection of Candida spp.

  4. The BepiColombo Archive Core System (BACS)

    NASA Astrophysics Data System (ADS)

    Macfarlane, A. J.; Osuna, P.; Pérez-López, F.; Vallejo, J. C.; Martinez, S.; Arviset, C.; Casale, M.

    2015-09-01

    BepiColombo is an interdisciplinary ESA mission to explore the planet Mercury in cooperation with JAXA. The mission consists of two separate orbiters: ESA's Mercury Planetary Orbiter (MPO) and JAXA's Mercury Magnetospheric Orbiter (MMO), which are dedicated to the detailed study of the planet and its magnetosphere. The MPO scientific payload comprises 11 instruments covering different scientific disciplines developed by several European teams. The MPO science operations will be prepared by the MPO Science Ground Segment (SGS) located at the European Space Astronomy Centre (ESAC) in Madrid. The BepiColombo Archive Core System (BACS) will be the central archive in which all mission operational data will be stored and is being developed by the Science Archives and Virtual Observatory Team (SAT) also at ESAC. The BACS will act as one of the modular subsystems within the BepiColombo Science Operations Control System (BSCS), (Vallejo 2014; Pérez-López 2014) which is under the responsibility of the SGS, with the purpose of facilitating the information exchange of data and metadata between the other subsystems of the BSCS as well as with the MPO Instrument Teams. This paper gives an overview of the concept and design of the BACS and how it integrates into the science ground segment workflow.

  5. Partial Diversity Generates Effector Immunity Specificity of the Bac41-Like Bacteriocins of Enterococcus faecalis Clinical Strains

    PubMed Central

    Kurushima, Jun; Ike, Yasuyoshi

    2016-01-01

    ABSTRACT Bacteriocin 41 (Bac41) is the plasmid-encoded bacteriocin produced by the opportunistic pathogen Enterococcus faecalis. Its genetic determinant consists of bacL1 (effector), bacL2 (regulator), bacA (effector), and bacI (immunity). The secreted effectors BacL1 and BacA coordinate to induce the lytic cell death of E. faecalis. Meanwhile, the immunity factor BacI provides self-resistance to the Bac41 producer, E. faecalis, against the action of BacL1 and BacA. In this study, we demonstrated that more than half of the 327 clinical strains of E. faecalis screened had functional Bac41 genes. Analysis of the genetic structure of the Bac41 genes in the DNA sequences of the E. faecalis strains revealed that the Bac41-like genes consist of a relatively conserved region and a variable region located downstream from bacA. Based on similarities in the variable region, the Bac41-like genes could be classified into type I, type IIa, and type IIb. Interestingly, the distinct Bac41 types had specific immunity factors for self-resistance, BacI1 or BacI2, and did not show cross-immunity to the other type of effector. We also demonstrated experimentally that the specificity of the immunity was determined by the combination of the C-terminal region of BacA and the presence of the unique BacI1 or BacI2 factor. These observations suggested that Bac41-like bacteriocin genes are extensively disseminated among E. faecalis strains in the clinical environment and can be grouped into at least three types. It was also indicated that the partial diversity results in specificity of self-resistance which may offer these strains a competitive advantage. IMPORTANCE Bacteriocins are antibacterial effectors produced by bacteria. In general, a bacteriocin-coding gene is accompanied by a cognate immunity gene that confers self-resistance on the bacteriocin-producing bacterium itself. We demonstrated that one of the bacteriocins, Bac41, is disseminated among E. faecalis clinical strains and the

  6. Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA.

    PubMed

    Gould, Matthew K; Bachmaier, Sabine; Ali, Juma A M; Alsford, Sam; Tagoe, Daniel N A; Munday, Jane C; Schnaufer, Achim C; Horn, David; Boshart, Michael; de Koning, Harry P

    2013-10-01

    One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development.

  7. The value of avian genomics to the conservation of wildlife

    PubMed Central

    2009-01-01

    Background Genomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution. Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care). In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes. Results Genotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current population's genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology. Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one. Conclusion Mining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the

  8. Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons.

    PubMed

    Kim, Shin-Il; Oceguera-Yanez, Fabian; Sakurai, Chiho; Nakagawa, Masato; Yamanaka, Shinya; Woltjen, Knut

    2016-01-01

    Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.

  9. Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae.

    PubMed

    Collinson, S K; Clouthier, S C; Doran, J L; Banser, P A; Kay, W W

    1996-02-01

    Salmonella enteritidis produces thin, aggregative fimbriae, named SEF17, which are composed of polymerized AgfA fimbrin proteins. DNA sequence analysis of a 2-kb region of S. enteritidis DNA revealed three contiguous genes, agfBAC. The 453-bp agfA gene encodes the AgfA fimbrin, which was predicted to be 74% identical and 86% similar in primary sequence to the Escherichia coli curli structural protein, CsgA. pHAG, a pUC18 derivative containing a 3.0-kb HindIII fragment encoding agfBAC, directed the in vitro expression of the major AgfA fimbrin, with an M(r) of 17,000, and a minor AgfB protein, with an M(r) of 16,000, encoded by the 453-bp agfB gene. AgfA was not expressed from pDAG, a pUC18 derivative containing a 3.1-kb DraI DNA fragment encoding agfA but not agfB. Primer extension analysis identified two adjacent transcription start sites located immediately upstream of agfB in positions analogous to those of the E. coli curlin csgBA operon. No transcription start sites were located immediately upstream of agfA or agfC. Northern (RNA) blot analysis confirmed that transcription of agfA was initiated from the agfB promoter region. Secondary-structure analysis of the putative mRNA transcript for agfBAC predicted the formation of a stem-loop structure (delta Gzero, -22 kcal/mol [-91 kJ/mol]) in the intercistronic region between agfA and agfC, which may be involved in stabilization of the agfBA portion of the agfBAC transcript. agfBAC and flanking regions had a high degree of sequence similarity with those counterparts of the E. coli curlin csgBA region for which sequence data are available. These data are demonstrative of the high degree of similarity between S. enteritidis SEF17 fimbriae and E. coli curli with respect to fimbrin amino acid sequence and genetic organization and, therefore, are indicative of a common and relatively recent ancestry.

  10. Radiation hybrid maps of D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high-resolution genome maps saturated with ordered markers to assist in anchoring and orienting BAC contigs/ sequence scaffolds for whole genome sequence assembly. Radiation hybrid (RH) mapping has proven to be an e...

  11. Expression and isolation of antimicrobial small molecules from soil DNA libraries.

    PubMed

    MacNeil, I A; Tiong, C L; Minor, C; August, P R; Grossman, T H; Loiacono, K A; Lynch, B A; Phillips, T; Narula, S; Sundaramoorthi, R; Tyler, A; Aldredge, T; Long, H; Gilman, M; Holt, D; Osburne, M S

    2001-04-01

    Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

  12. Library 2000.

    ERIC Educational Resources Information Center

    Drake, Miriam A.

    In fall 1984, the Georgia Institute of Technology administration and library staff began planning for Library 2000, a project aimed at creating a showcase library to demonstrate the application of the latest information technology in an academic and research environment. The purposes of Library 2000 include: increasing awareness of students,…

  13. Library Computing.

    ERIC Educational Resources Information Center

    Dayall, Susan A.; And Others

    1987-01-01

    Six articles on computers in libraries discuss training librarians and staff to use new software; appropriate technology; system upgrades of the Research Libraries Group's information system; pre-IBM PC microcomputers; multiuser systems for small to medium-sized libraries; and a library user's view of the traditional card catalog. (EM)

  14. Library Buildings.

    ERIC Educational Resources Information Center

    Manley, Will; And Others

    1989-01-01

    The innovative designs of three libraries are described: the Tempe (Arizona) Public Library, which emphasizes services for children and students; an underground library at Park College, Missouri; and a public library located in the Vancouver (Washington) Mall. The fourth article describes the work going on to restore the Los Angeles (California)…

  15. Suicidal Autointegration of Sleeping Beauty and piggyBac Transposons in Eukaryotic Cells

    PubMed Central

    Devaraj, Anatharam; Singh, Manvendra; Jimenez Orgaz, Ana; Chen, Jia-Xuan; Selbach, Matthias; Ivics, Zoltán; Izsvák, Zsuzsanna

    2014-01-01

    Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB. PMID:24625543

  16. A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

    PubMed Central

    2011-01-01

    Background Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome. Results Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella. Conclusions When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution. PMID:21619600

  17. Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System.

    PubMed

    Fornwald, James A; Lu, Quinn; Boyce, Frederick M; Ames, Robert S

    2016-01-01

    BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. BacMams have become an integral part of the recombinant mammalian gene expression toolbox in research labs worldwide. Construction of transfer vectors is straightforward using basic molecular biology protocols. Virus generation is based on common methods used with the baculovirus insect cell expression system. BacMam transduction of mammalian cells requires minimal modifications to familiar cell culture methods. This chapter highlights the BacMam transfer vector pHTBV.

  18. FPGA based implementation of hardware diagnostic layer for local trigger of BAC calorimeter for ZEUS detector

    NASA Astrophysics Data System (ADS)

    Pozniak, Krzysztof T.

    2004-07-01

    The paper describes design and construction of hardware diagnostics layer dedicated to the local trigger of the Backing Calorimeter (BAC). The BAC is a part of the ZEUS experiment in DESY, Hamburg. A general characteristic of the hardware of BAC trigger was presented. The design of hardware diagnostic and calibration sub-systems for BAC trigger bases on the continuous monitoring of consecutive electronic and photonic blocks. The monitoring process is performed via the specialized tests. The standardized diagnostic components were realized in the algorithmic and parameterized description in AHDL. There were presented the implementation results in ALTERA ACEX chips.

  19. RiBAC: Role Interaction Based Access Control Model for Community Computing

    NASA Astrophysics Data System (ADS)

    Jung, Youna; Masoumzadeh, Amirreza; Joshi, James B. D.; Kim, Minkoo

    Community computing is an agent-based development paradigm for ubiquitous computing systems. In a community computing system, ubiquitous services are provided by cooperation among agents. While agents cooperate, they interact with each other continuously to access data of other agents and/or to execute other agent’s actions. However, in cases of security-critical ubiquitous services such as medical or military services, an access control mechanism is necessary to prevent unauthorized access to critical data or action. In this paper, we propose a family of Role interaction Based Access Control (RiBAC) models for Community Computing, by extending the existing RBAC model to consider role interactions. As a basic model, we propose the core RiBAC model. For the convenience of management and to provide more fine-grained access control, we propose Hierarchical RiBAC (H-RiBAC), Constrained RiBAC (C-RiBAC), and Constrained Hierarchical RiBAC (CH-RiBAC) models. Finally, we extend the existing community computing framework to accommodate the specification and enforcement of RiBAC policies.

  20. A comprehensive whole-genome integrated cytogenetic map for the alpaca (Lama pacos).

    PubMed

    Avila, Felipe; Baily, Malorie P; Perelman, Polina; Das, Pranab J; Pontius, Joan; Chowdhary, Renuka; Owens, Elaine; Johnson, Warren E; Merriwether, David A; Raudsepp, Terje

    2014-01-01

    Genome analysis of the alpaca (Lama pacos, LPA) has progressed slowly compared to other domestic species. Here, we report the development of the first comprehensive whole-genome integrated cytogenetic map for the alpaca using fluorescence in situ hybridization (FISH) and CHORI-246 BAC library clones. The map is comprised of 230 linearly ordered markers distributed among all 36 alpaca autosomes and the sex chromosomes. For the first time, markers were assigned to LPA14, 21, 22, 28, and 36. Additionally, 86 genes from 15 alpaca chromosomes were mapped in the dromedary camel (Camelus dromedarius, CDR), demonstrating exceptional synteny and linkage conservation between the 2 camelid genomes. Cytogenetic mapping of 191 protein-coding genes improved and refined the known Zoo-FISH homologies between camelids and humans: we discovered new homologous synteny blocks (HSBs) corresponding to HSA1-LPA/CDR11, HSA4-LPA/CDR31 and HSA7-LPA/CDR36, and revised the location of breakpoints for others. Overall, gene mapping was in good agreement with the Zoo-FISH and revealed remarkable evolutionary conservation of gene order within many human-camelid HSBs. Most importantly, 91 FISH-mapped markers effectively integrated the alpaca whole-genome sequence and the radiation hybrid maps with physical chromosomes, thus facilitating the improvement of the sequence assembly and the discovery of genes of biological importance.

  1. Production of Green Fluorescent Protein Transgenic Embryonic Stem Cells Using the GENSAT Bacterial Artificial Chromosome Library

    PubMed Central

    Tomishima, Mark J.; Hadjantonakis, Anna-Katerina; Gong, Shiaoching; Studer, Lorenz

    2010-01-01

    Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology. PMID:16990587

  2. Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library.

    PubMed

    Tomishima, Mark J; Hadjantonakis, Anna-Katerina; Gong, Shiaoching; Studer, Lorenz

    2007-01-01

    Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology.

  3. Sex Chromosome Evolution in Amniotes: Applications for Bacterial Artificial Chromosome Libraries

    PubMed Central

    Janes, Daniel E.; Valenzuela, Nicole; Ezaz, Tariq; Amemiya, Chris; Edwards, Scott V.

    2011-01-01

    Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently. These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200 kb of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced. With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries. PMID:20981143

  4. Legume Genome Initiative at the University of Oklahoma

    SciTech Connect

    Bruce A. Roe

    2004-02-27

    Consolidated Appropriations Resolution, 2003 Conference Report for the Department of Energy's Biological and Environmental Research (BER) program provided $481,000 for the Legume Genome Initiative at the University of Oklahoma. These funds were used to support our research that is aimed at determining the entire sequence of the gene rich regions of the genome of the legume, Medicago truncatula, by allowing us to obtain a greater degree of finished BAC sequences from the draft sequences we have already obtained through research funded by the Noble Foundation. During the funding period we increased the number of Medicago truncatula BACs with finished (Bermuda standard) sequences from 109 to 359, and the total number of BACs for which we collected sequence data from 584 to 842, 359 of which reached phase 2 (ordered and oriented contigs). We also sequenced a series of pooled BAC clones that cover additional euchromatic (gene rich) genomic regions. This work resulted in 6 refereed publications, see below. Genes whose sequence was determined during this study included multiple members of the plant disease resistance (R-gene) family as well as several genes involved in flavinoid biosynthesis, nitrogen fixation and plant-microbial symbosis. This work also served as a prelude to obtaining NSF funding for the international collaborative effort to complete the entire sequence of the Medicago truncatula genomic euchromatic regions using a BAC based approach.

  5. PBmice: an integrated database system of piggyBac (PB) insertional mutations and their characterizations in mice

    PubMed Central

    Sun, Ling V.; Jin, Ke; Liu, Yiming; Yang, Wenwei; Xie, Xing; Ye, Lin; Wang, Li; Zhu, Lin; Ding, Sheng; Su, Yi; Zhou, Jie; Han, Min; Zhuang, Yuan; Xu, Tian; Wu, Xiaohui; Gu, Ning; Zhong, Yang

    2008-01-01

    DNA transposon piggyBac (PB) is a newly established mutagen for large-scale mutagenesis in mice. We have designed and implemented an integrated database system called PBmice (PB Mutagenesis Information CEnter) for storing, retrieving and displaying the information derived from PB insertions (INSERTs) in the mouse genome. This system is centered on INSERTs with information including their genomic locations and flanking genomic sequences, the expression levels of the hit genes, and the expression patterns of the trapped genes if a trapping vector was used. It also archives mouse phenotyping data linked to INSERTs, and allows users to conduct quick and advanced searches for genotypic and phenotypic information relevant to a particular or a set of INSERT(s). Sequence-based information can be cross-referenced with other genomic databases such as Ensembl, BLAST and GBrowse tools used in PBmice offer enhanced search and display for additional information relevant to INSERTs. The total number and genomic distribution of PB INSERTs, as well as the availability of each PB insertional LINE can also be viewed with user-friendly interfaces. PBmice is freely available at http://www.idmshanghai.cn/PBmice or http://www.scbit.org/PBmice/. PMID:17932058

  6. Facile Recovery of Individual High-Molecular-Weight, Low-Copy-Number Natural Plasmids for Genomic Sequencing

    SciTech Connect

    Williams, L.E.; Detter, C,; Barrie, K.; Lapidus, A.; Summers, A.O.

    2006-06-01

    Sequencing of the large (>50 kb), low-copy-number (<5 per cell) plasmids that mediate horizontal gene transfer has been hindered by the difficulty and expense of isolating DNA from individual plasmids of this class. We report here that a kit method previously devised for purification of bacterial artificial chromosomes (BACs) can be adapted for effective preparation of individual plasmids up to 220 kb from wild gram-negative and gram-positive bacteria. Individual plasmid DNA recovered from less than 10 ml of Escherichia coli, Staphylococcus, and Corynebacterium cultures was of sufficient quantity and quality for construction of highcoverage libraries, as shown by sequencing five native plasmids ranging in size from 30 kb to 94 kb. We also report recommendations for vector screening to optimize plasmid sequence assembly, preliminary annotation of novel plasmid genomes, and insights on mobile genetic element biology derived from these sequences. Adaptation of this BAC method for large plasmid isolation removes one major technical hurdle to expanding our knowledge of the natural plasmid gene pool.

  7. BACs-on-Beads technology: a reliable test for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.

    PubMed

    García-Herrero, Sandra; Campos-Galindo, Inmaculada; Martínez-Conejero, José Antonio; Serra, Vicente; Olmo, Inés; Lara, Coral; Simón, Carlos; Rubio, Carmen

    2014-01-01

    The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples.

  8. BACs-on-Beads Technology: A Reliable Test for Rapid Detection of Aneuploidies and Microdeletions in Prenatal Diagnosis

    PubMed Central

    Martínez-Conejero, José Antonio; Serra, Vicente; Olmo, Inés; Lara, Coral; Simón, Carlos

    2014-01-01

    The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples. PMID:24795887

  9. Sperm-mediated transgenesis in chicken using a PiggyBac transposon system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Towards development of transgenic chickens without the use of viral vectors, factors affecting sperm mediated gene transfer (SMGT) using a piggyBac vector are being studied. The piggyBac pPBCAG-LacZ contains 13bp terminal inverted repeats flanking a LacZ gene driven by the CAG promoter. A helper pla...

  10. Excision of the piggyBac transposable element in maize cells is a precise event

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The piggyBac transposable element (TE) from the moth Trichoplusia ni encodes a ‘cut and paste’ DNA transposase that has been used to transform a number of insects, as well as planaria, mammalian cells, and mice. The wild type and a mutated piggyBac TE excised from a DNA vector in transient assays u...

  11. A non-autonomous insect piggyBac trasposable element is mobile in tobacco

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid onl...

  12. Validation of the German version of the brief assessment of cognition in Schizophrenia (BACS) - preliminary results.

    PubMed

    Sachs, G; Winklbaur, B; Jagsch, R; Keefe, R S E

    2011-03-01

    The German version of the BACS showed high test-retest reliability. Sensitivity and specificity scores demonstrated good ability to differentiate between patients and controls. The study suggests that the German Version of the BACS is a useful scale to evaluate cognitive functioning.

  13. Special Libraries

    ERIC Educational Resources Information Center

    Lavendel, Giuliana

    1977-01-01

    Discusses problems involved in maintaining special scientific or engineering libraries, including budget problems, remote storage locations, rental computer retrieval systems, protecting trade secrets, and establishing a magnetic tape library. (MLH)

  14. Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies.

    PubMed

    Plomion, Christophe; Aury, Jean-Marc; Amselem, Joëlle; Alaeitabar, Tina; Barbe, Valérie; Belser, Caroline; Bergès, Hélène; Bodénès, Catherine; Boudet, Nathalie; Boury, Christophe; Canaguier, Aurélie; Couloux, Arnaud; Da Silva, Corinne; Duplessis, Sébastien; Ehrenmann, François; Estrada-Mairey, Barbara; Fouteau, Stéphanie; Francillonne, Nicolas; Gaspin, Christine; Guichard, Cécile; Klopp, Christophe; Labadie, Karine; Lalanne, Céline; Le Clainche, Isabelle; Leplé, Jean-Charles; Le Provost, Grégoire; Leroy, Thibault; Lesur, Isabelle; Martin, Francis; Mercier, Jonathan; Michotey, Célia; Murat, Florent; Salin, Franck; Steinbach, Delphine; Faivre-Rampant, Patricia; Wincker, Patrick; Salse, Jérôme; Quesneville, Hadi; Kremer, Antoine

    2016-01-01

    The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

  15. Library Skills.

    ERIC Educational Resources Information Center

    Paul, Karin; Kuhlthau, Carol C.; Branch, Jennifer L.; Solowan, Diane Galloway; Case, Roland; Abilock, Debbie; Eisenberg, Michael B.; Koechlin, Carol; Zwaan, Sandi; Hughes, Sandra; Low, Ann; Litch, Margaret; Lowry, Cindy; Irvine, Linda; Stimson, Margaret; Schlarb, Irene; Wilson, Janet; Warriner, Emily; Parsons, Les; Luongo-Orlando, Katherine; Hamilton, Donald

    2003-01-01

    Includes 19 articles that address issues related to library skills and Canadian school libraries. Topics include information literacy; inquiry learning; critical thinking and electronic research; collaborative inquiry; information skills and the Big 6 approach to problem solving; student use of online databases; library skills; Internet accuracy;…

  16. Validation of the tablet-administered Brief Assessment of Cognition (BAC App).

    PubMed

    Atkins, Alexandra S; Tseng, Tina; Vaughan, Adam; Twamley, Elizabeth W; Harvey, Philip; Patterson, Thomas; Narasimhan, Meera; Keefe, Richard S E

    2016-10-19

    Computerized tests benefit from automated scoring procedures and standardized administration instructions. These methods can reduce the potential for rater error. However, especially in patients with severe mental illnesses, the equivalency of traditional and tablet-based tests cannot be assumed. The Brief Assessment of Cognition in Schizophrenia (BACS) is a pen-and-paper cognitive assessment tool that has been used in hundreds of research studies and clinical trials, and has normative data available for generating age- and gender-corrected standardized scores. A tablet-based version of the BACS called the BAC App has been developed. This study compared performance on the BACS and the BAC App in patients with schizophrenia and healthy controls. Test equivalency was assessed, and the applicability of paper-based normative data was evaluated. Results demonstrated the distributions of standardized composite scores for the tablet-based BAC App and the pen-and-paper BACS were indistinguishable, and the between-methods mean differences were not statistically significant. The discrimination between patients and controls was similarly robust. The between-methods correlations for individual measures in patients were r>0.70 for most subtests. When data from the Token Motor Test was omitted, the between-methods correlation of composite scores was r=0.88 (df=48; p<0.001) in healthy controls and r=0.89 (df=46; p<0.001) in patients, consistent with the test-retest reliability of each measure. Taken together, results indicate that the tablet-based BAC App generates results consistent with the traditional pen-and-paper BACS, and support the notion that the BAC App is appropriate for use in clinical trials and clinical practice.

  17. Genome-wide annotation of mutations in a phenotyped mutant library provides an efficient platform for discovery of casual gene mutations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. Conventionally, these mutations are identified by techniques that can detect single-nucleotide mismatches in heteroduplexes of individual PCR amplicons. We applied whole-genome sequencing to 256-phenotyped mutant l...

  18. Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon

    PubMed Central

    Li, Tianda; Shuai, Ling; Mao, Junjie; Wang, Xuepeng; Wang, Mei; Zhang, Xinxin; Wang, Leyun; Li, Yanni; Li, Wei; Zhou, Qi

    2016-01-01

    Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids. We showed that the transgenic rats expressed the RFP or eGFP gene in many organs and had the capability to transmit the marker gene to the next generation through germline integration. In addition, rat embryonic stem cells (ESCs) carrying an RFP reporter gene can be derived from the blastocysts of the transgenic rats. Moreover, the RFP gene can be detected in chimeras derived from RFP ESCs via blastocyst injection. This work suggests that PB-mediated transgenesis is a powerful tool to generate transgenic rats expressing fluorescent proteins with high efficiency, and this technique can be used to derive rat ESCs expressing a reporter protein. PMID:27624004

  19. FISH analysis of Drosophila melanogaster heterochromatin using BACs and P elements.

    PubMed

    Corradini, Nicoletta; Rossi, Fabrizio; Vernì, Fiammetta; Dimitri, Patrizio

    2003-07-01

    The heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster contains about 30 essential genes defined by genetic analysis. In the last decade only a few of these genes have been molecularly characterized and found to correspond to protein-coding genes involved in important cellular functions. Moreover, several predicted genes have been identified by annotation of genomic sequence that are associated with polytene chromosome divisions 40, 41 and 80 but their locations on the cytogenetic map of the heterochromatin are still uncertain. To expand our current knowledge of the genetic functions located in heterochromatin, we have performed fluorescence in situ hybridization (FISH) mapping to mitotic chromosomes of nine bacterial artificial chromosomes (BACs) carrying several predicted genes and of 13 P element insertions assigned to the proximal regions of 2R and 3L. We found that 22 predicted genes map to the h46 region of 2R and eight map to the h47 regions of 3L. This amounts to at least 30 predicted genes located in these heterochromatic regions, whereas previous studies detected only seven vital genes. Finally, another 58 genes localize either in the euchromatin-heterochromatin transition regions or in the proximal euchromatin of 2R and 3L.

  20. Comparative analysis of the pig BAC sequence involved in the regulation of myostatin gene.

    PubMed

    Yu, Zhengquan; Li, Yan; Meng, Qingyong; Yuan, Jing; Zhao, Zhihui; Li, Wei; Hu, Xiaoxiang; Yan, Bingxue; Fan, Baoliang; Yu, Shuyang; Li, Ning

    2005-04-01

    Myostatin (GDF8, MSTN) is a member of the transforming growth factor beta superfamily that is essential for proper regulation of skeletal muscle mass. In order to study its expression and regulatory mechanism deeply, we have presented a comparative analysis of about 170-kb pig BAC sequence containing the myostatin gene among pig, human and mouse. The genomic region is characterized by high interspersed repeats and low G+C content. As for the myostatin gene, a higher sequence similarity is found between human and pig than between these species and the mouse. One striking feature is that the structure of two TATA-boxes in the nearby downstream of CCAAT-box is identified in the promoter. Further analysis reveals that the TATA-box1 is responsible for the transcription in pig and human, but the TATA-box2 acts on the transcription in mouse. The other interesting feature is that two polyadenylation signal sequences (AATAAA) exist in 3'UTR of the pig myostatin gene. Moreover, a large number of potential transcription factor-binding sites are also identified in evolutionary conserved regions (ECRs), which may be associated with the regulation of myostatin. Many putative transcription factors play an important role in the muscle development, and the complex interaction between myostatin and these factors may be required for proper muscle development.

  1. Genome Mapping and Molecular Breeding of Tomato

    PubMed Central

    Foolad, Majid R.

    2007-01-01

    The cultivated tomato, Lycopersicon esculentum, is the second most consumed vegetable worldwide and a well-studied crop species in terms of genetics, genomics, and breeding. It is one of the earliest crop plants for which a genetic linkage map was constructed, and currently there are several molecular maps based on crosses between the cultivated and various wild species of tomato. The high-density molecular map, developed based on an L. esculentum × L. pennellii cross, includes more than 2200 markers with an average marker distance of less than 1 cM and an average of 750 kbp per cM. Different types of molecular markers such as RFLPs, AFLPs, SSRs, CAPS, RGAs, ESTs, and COSs have been developed and mapped onto the 12 tomato chromosomes. Markers have been used extensively for identification and mapping of genes and QTLs for many biologically and agriculturally important traits and occasionally for germplasm screening, fingerprinting, and marker-assisted breeding. The utility of MAS in tomato breeding has been restricted largely due to limited marker polymorphism within the cultivated species and economical reasons. Also, when used, MAS has been employed mainly for improving simply-inherited traits and not much for improving complex traits. The latter has been due to unavailability of reliable PCR-based markers and problems with linkage drag. Efforts are being made to develop high-throughput markers with greater resolution, including SNPs. The expanding tomato EST database, which currently includes ∼214 000 sequences, the new microarray DNA chips, and the ongoing sequencing project are expected to aid development of more practical markers. Several BAC libraries have been developed that facilitate map-based cloning of genes and QTLs. Sequencing of the euchromatic portions of the tomato genome is paving the way for comparative and functional analysis of important genes and QTLs. PMID:18364989

  2. Rhipicephalus microplus strain Deutsch, 10 BAC clone sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and difficult to assemble and analyze. We used labeled DNA probes from the coding reg...

  3. Superconductivity in the Graphite Intercalation Compound BaC(6).

    PubMed

    Heguri, Satoshi; Kawade, Naoya; Fujisawa, Takumi; Yamaguchi, Akira; Sumiyama, Akihiko; Tanigaki, Katsumi; Kobayashi, Mototada

    2015-06-19

    Among many two-dimensional (2D) high T(C) superconductors, graphite intercalation compounds (GICs) are the most famous intercalation family, which are classified as typical electron-phonon mediated superconductors. We show unambiguous experimental facts that BaC(6), the superconductivity of which has been missing for many years so far among various alkaline earth metal (Ca, Sr, and Ba) intercalted GICs, exhibits superconductivity at T(C)=65  mK. By adding this finding as the additional experimental point, a complete figure displaying the relationship between T(C) and interlayer distance (d) for GICs is now provided, and their possible superconducting mechanisms raised so far are revisited. The present study settles a long-running debate between theories and experiments on the superconductivity in the first stage GICs.

  4. Superconductivity in the Graphite Intercalation Compound BaC 6

    NASA Astrophysics Data System (ADS)

    Heguri, Satoshi; Kawade, Naoya; Fujisawa, Takumi; Yamaguchi, Akira; Sumiyama, Akihiko; Tanigaki, Katsumi; Kobayashi, Mototada

    2015-06-01

    Among many two-dimensional (2D) high TC superconductors, graphite intercalation compounds (GICs) are the most famous intercalation family, which are classified as typical electron-phonon mediated superconductors. We show unambiguous experimental facts that BaC 6 , the superconductivity of which has been missing for many years so far among various alkaline earth metal (Ca, Sr, and Ba) intercalted GICs, exhibits superconductivity at TC=65 mK . By adding this finding as the additional experimental point, a complete figure displaying the relationship between TC and interlayer distance (d ) for GICs is now provided, and their possible superconducting mechanisms raised so far are revisited. The present study settles a long-running debate between theories and experiments on the superconductivity in the first stage GICs.

  5. Exploiting BAC-end sequences for the mining, characterization and utility of new short sequences repeat (SSR) markers in Citrus.

    PubMed

    Biswas, Manosh Kumar; Chai, Lijun; Mayer, Christoph; Xu, Qiang; Guo, Wenwu; Deng, Xiuxin

    2012-05-01

    The aim of this study was to develop a large set of microsatellite markers based on publicly available BAC-end sequences (BESs), and to evaluate their transferability, discriminating capacity of genotypes and mapping ability in Citrus. A set of 1,281 simple sequence repeat (SSR) markers were developed from the 46,339 Citrus clementina BAC-end sequences (BES), of them 20.67% contained SSR longer than 20 bp, corresponding to roughly one perfect SSR per 2.04 kb. The most abundant motifs were di-nucleotide (16.82%) repeats. Among all repeat motifs (TA/AT)n is the most abundant (8.38%), followed by (AG/CT)n (4.51%). Most of the BES-SSR are located in the non-coding region, but 1.3% of BES-SSRs were found to be associated with transposable element (TE). A total of 400 novel SSR primer pairs were synthesized and their transferability and polymorphism tested on a set of 16 Citrus and Citrus relative's species. Among these 333 (83.25%) were successfully amplified and 260 (65.00%) showed cross-species transferability with Poncirus trifoliata and Fortunella sp. These cross-species transferable markers could be useful for cultivar identification, for genomic study of Citrus, Poncirus and Fortunella sp. Utility of the developed SSR marker was demonstrated by identifying a set of 118 markers each for construction of linkage map of Citrus reticulata and Poncirus trifoliata. Genetic diversity and phylogenetic relationship among 40 Citrus and its related species were conducted with the aid of 25 randomly selected SSR primer pairs and results revealed that citrus genomic SSRs are superior to genic SSR for genetic diversity and germplasm characterization of Citrus spp.

  6. OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA

    PubMed Central

    Mietzsch, Mario; Casteleyn, Vincent; Weger, Stefan; Zolotukhin, Sergei; Heilbronn, Regine

    2015-01-01

    Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application. PMID:26134901

  7. OneBac 2.0: Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA.

    PubMed

    Mietzsch, Mario; Casteleyn, Vincent; Weger, Stefan; Zolotukhin, Sergei; Heilbronn, Regine

    2015-10-01

    Scalable production of recombinant adeno-associated virus vectors (rAAV) in baculovirus-infected Sf9 cells yields high burst sizes but variable infectivity rates per packaged AAV vector genome depending on the chosen serotype. Infectivity rates are particularly low for rAAV5 vectors, based on the genetically most divergent AAV serotype. In this study we describe key improvements of the OneBac system for the generation of rAAV5 vectors, whose manufacturing has been unsatisfactory in all current insect cell-based production systems. The Sf9 cell-based expression strategy for AAV5 capsid proteins was modified to enhance relative AAV5 VP1 levels. This resulted in a 100-fold boost of infectivity per genomic AAV5 particle with undiminished burst sizes per producer cell. Furthermore, the issue of collateral packaging of helper DNA into AAV capsids was approached. By modifications of the AAV rep and cap expression constructs used for the generation of stable Sf9 cell lines, collateral packaging of helper DNA sequences during rAAV vector production was dramatically reduced down to 0.001% of packaged rAAV genomes, while AAV5 burst sizes and infectivity rates were maintained. OneBac 2.0 represents the first insect cell-based scalable production system for high per-particle AAV5 infectivity rates combined with minimal collateral packaging of helper DNA, allowing the manufacturing of safe AAV5-based gene therapies for clinical application.

  8. The Genome Sequence of the Fungal Pathogen Fusarium virguliforme That Causes Sudden Death Syndrome in Soybean

    PubMed Central

    Srivastava, Subodh K.; Huang, Xiaoqiu; Brar, Hargeet K.; Fakhoury, Ahmad M.; Bluhm, Burton H.; Bhattacharyya, Madan K.

    2014-01-01

    Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease. Methodology/Principal Findings We have generated the draft genome sequence of F. virguliforme by conducting whole-genome shotgun sequencing on a 454 GS-FLX Titanium sequencer. Initially, single-end reads of a 400-bp shotgun library were assembled using the PCAP program. Paired end sequences from 3 and 20 Kb DNA fragments and approximately 100 Kb inserts of 1,400 BAC clones were used to generate the assembled genome. The assembled genome sequence was 51 Mb. The N50 scaffold number was 11 with an N50 Scaffold length of 1,263 Kb. The AUGUSTUS gene prediction program predicted 14,845 putative genes, which were annotated with Pfam and GO databases. Gene distributions were uniform in all but one of the major scaffolds. Phylogenic analyses revealed that F. virguliforme was closely related to the pea pathogen, Nectria haematococca. Of the 14,845 F. virguliforme genes, 11,043 were conserved among five Fusarium species: F. virguliforme, F. graminearum, F. verticillioides, F. oxysporum and N. haematococca; and 1,332 F. virguliforme-specific genes, which may include pathogenicity genes. Additionally, searches for candidate F. virguliforme pathogenicity genes using gene sequences of the pathogen-host interaction database identified 358 genes. Conclusions The F. virguliforme genome sequence and putative pathogenicity genes presented here will facilitate identification of pathogenicity mechanisms involved in SDS development. Together, these resources will expedite our efforts towards discovering

  9. BAC: A computer program for calculating shielding in buildings against initial radiation

    NASA Astrophysics Data System (ADS)

    Danielson, G.

    1980-10-01

    Calculation methodology and transmission data for BAC in the event of a nuclear explosion are considered. The shielding factor is the rate between the radiation dose at one point in the building and the dose in open air. It is separately calculated for neutrons, gamma rays from fission products, and secondary gamma rays. For this calculation, BAC uses data for radiation transmission in concrete. This program is utilized for fallout shelters and other buildings where walls and floors/roofs are mostly made of concrete and bricks. Instructions for the program are given, and BAC results and values are in certain cases compared with those obtained with the Monte Carlo method.

  10. Libraries program

    USGS Publications Warehouse

    2011-01-01

    The U.S. Congress authorized a library for the U.S. Geological Survey (USGS) in 1879. The library was formally established in 1882 with the naming of the first librarian and began with a staff of three and a collection of 1,400 books. Today, the USGS Libraries Program is one of the world's largest Earth and natural science repositories and a resource of national significance used by researchers and the public worldwide.

  11. America's Star Libraries: Top-Rated Libraries

    ERIC Educational Resources Information Center

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

    "Library Journal"'s national rating of public libraries, the "LJ" Index of Public Library Service 2009, Round 2, identifies 258 "star" libraries. Created by Keith Curry Lance and Ray Lyons and based on 2007 data from the IMLS, it rates 7,268 public libraries. The top libraries in each group get five, four, or three stars. All included libraries,…

  12. Preparation of PAC libraries. Final technical report

    SciTech Connect

    Pieter J. de Jong

    1997-12-31

    The goals of this project were to create P1 Artificial Chromosome (PAC) cloning vectors and use these vectors to generate, characterize, and distribute both human and mouse genomic PAC libraries to the scientific community.

  13. [Integration of plasmid pPL 7065 into chromosome of Bac. pumilis].

    PubMed

    Lysenko, A M; Koz'mipa, L M; Abromova, M A; Lukin, A A

    1980-01-01

    Hybridization of tritium-labelled plasmid 7065 with total DNA of several Bac. pumilis strains differing in the degree of spore-formation showed that strain 7065-k contains the plasmid in an integral state.

  14. Genetically-directed Sparse Neuronal Labeling in BAC Transgenic Mice through Mononucleotide Repeat Frameshift

    PubMed Central

    Lu, Xiao-Hong; Yang, X. William

    2017-01-01

    Mosaicism with Repeat Frameshift (MORF) allows a single Bacterial Artificial Chromosome (BAC) transgene to direct sparse labeling of genetically-defined neuronal populations in mice. The BAC transgene drives cell-type-specific transcription of an out-of-frame mononucleotide repeat that is placed between a translational start codon and a membrane-bound fluorescent protein lacking its start codon. The stochastic frameshift of the unstable repeat DNA in a subset of BAC-expressing neurons results in the in-frame translation of the reporter protein hence the sparse neuronal labeling. As a proof-of-concept, we generated D1-dopamine receptor (D1) BAC MORF mice that label about 1% striatal D1-expressing medium spiny neurons and allow visualization of their dendrites. These mice enable the study of D1-MSN dendrite development in wildtype mice, and its degeneration in a mouse model of Huntington’s disease. PMID:28272512

  15. Library Advocacy

    ERIC Educational Resources Information Center

    Plunkett, Kate

    2010-01-01

    This paper is about the issue of advocacy. Standing at the vanguard of literacy, library media specialists have a unique role. However, it is time for media specialists to advocate their services in a proactive way. If library media specialists cannot, both individually and collectively, put advocacy at the forefront, then students will suffer the…

  16. Privatizing Libraries

    ERIC Educational Resources Information Center

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

    This timely special report from ALA Editions provides a succinct but comprehensive overview of the "privatization" of public libraries. It provides a history of the trend of local and state governments privatizing public services and assets, and then examines the history of public library privatization right up to the California…

  17. Library Research.

    ERIC Educational Resources Information Center

    Wright, Nancy Kirkpatrick

    This workbook, designed for a Library Research course at Yavapai College, provides 15 lessons in advanced library reference skills. Each lesson provides explanatory text and reinforcement exercises. After Lesson I introduces specialized dictionaries and encyclopedias (e.g., for foreign languages, medicine, music, economics, social sciences, and…

  18. 23 CFR 1225.4 - Adoption of 0.08 BAC per se law.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 23 Highways 1 2010-04-01 2010-04-01 false Adoption of 0.08 BAC per se law. 1225.4 Section 1225.4... TRANSPORTATION GUIDELINES OPERATION OF MOTOR VEHICLES BY INTOXICATED PERSONS § 1225.4 Adoption of 0.08 BAC per se... is enforcing a law that provides that any person with a blood or breath alcohol concentration...

  19. Status and opportunities for genomics research with rainbow trout

    USGS Publications Warehouse

    Thorgaard, G.H.; Bailey, G.S.; Williams, D.; Buhler, D.R.; Kaattari, S.L.; Ristow, S.S.; Hansen, J.D.; Winton, J.R.; Bartholomew, J.L.; Nagler, J.J.; Walsh, P.J.; Vijayan, M.M.; Devlin, R.H.; Hardy, R.W.; Overturf, K.E.; Young, W.P.; Robison, B.D.; Rexroad, C.; Palti, Y.

    2002-01-01

    The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes. ?? 2002 Elsevier Science Inc. All rights reserved.

  20. Exploration of BAC versus plasmid expression vectors in recombinant CHO cells.

    PubMed

    Mader, Alexander; Prewein, Bernhard; Zboray, Katalin; Casanova, Emilio; Kunert, Renate

    2013-05-01

    Vector engineering approaches are commonly used to increase recombinant protein production in mammalian cells, and among various concepts, bacterial artificial chromosomes (BAC) have been proposed to serve as open chromatin regions to omit chromosome positional effects. For proof of concept, we developed stable recombinant Chinese hamster ovary (CHO) cell lines using different expression vector systems: the plasmid vectors contained the identical expression cassette as the BAC constructs. Two anti-HIV1 antibody derivates served as model proteins (3D6scFc and 2F5scFc) for generation of four stable recombinant CHO cell lines. The BAC-derived clones showed three to four times higher specific productivity, and therefore, gene copy numbers and transcript level were quantified. The active chromatin region provided with the BAC environment significantly improved transcription evidenced with both model proteins. Specific transcription was approximately six times higher from BAC-based vectors compared to the corresponding plasmid vectors for both single-chain fragment crystallizable (scFc) proteins. Our accurate investigations elucidated also differences between translational activities related to the protein of choice. 3D6scFc expressed specifically three to four times more product than 2F5scFc indicating that the product by itself also contributes to enhanced productivity. This study indicated comparable increase of transcription level for both scFc proteins when using the BAC system, but translation, maturation, and secretion of individual proteins seem to be protein specific.

  1. The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

    PubMed Central

    Troyanovsky, Boris; Bitko, Vira; Pastukh, Viktor; Fouty, Brian; Solodushko, Victor

    2016-01-01

    Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo. PMID:27701401

  2. The medicago genome provides insight into evolution of rhizobial symbiosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Medicago truncatula is an excellent model for the study of legume-specific biology, especially endosymbiotic interactions with bacteria and fungi. This paper describes the sequence of the euchromatic portion of the M. truncatula genome based on a recently completed BAC-based assembly supplemented by...

  3. Constructing a Cytogenetic Map of the Maize Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are developing a pachytene cytogenetic FISH (Fluorescence in situ Hybridization) map of the maize (Zea mays L.) genome using maize marker-selected sorghum BACs (Bacterial Artificial Chromosome) as described by Koumbaris and Bass (2003, Plant J. 35:647). The two main projects are the production of...

  4. A complete mitochondrial genome of wheat (Triticum aestivum cv. Chinese Yumai), and fast evolving mitochondrial genes in higher plants.

    PubMed

    Cui, Peng; Liu, Huitao; Lin, Qiang; Ding, Feng; Zhuo, Guoyin; Hu, Songnian; Liu, Dongcheng; Yang, Wenlong; Zhan, Kehui; Zhang, Aimin; Yu, Jun

    2009-12-01

    Plant mitochondrial genomes, encoding necessary proteins involved in the system of energy production, play an important role in the development and reproduction of the plant. They occupy a specific evolutionary pattern relative to their nuclear counterparts. Here, we determined the winter wheat (Triticum aestivum cv. Chinese Yumai) mitochondrial genome in a length of 452 and 526 bp by shotgun sequencing its BAC library. It contains 202 genes, including 35 known protein-coding genes, three rRNA and 17 tRNA genes, as well as 149 open reading frames (ORFs; greater than 300 bp in length). The sequence is almost identical to the previously reported sequence of the spring wheat (T. aestivum cv. Chinese Spring); we only identified seven SNPs (three transitions and four transversions) and 10 indels (insertions and deletions) between the two independently acquired sequences, and all variations were found in non-coding regions. This result confirmed the accuracy of the previously reported mitochondrial sequence of the Chinese Spring wheat. The nucleotide frequency and codon usage of wheat are common among the lineage of higher plant with a high AT-content of 58%. Molecular evolutionary analysis demonstrated that plant mitochondrial genomes evolved at different rates, which may correlate with substantial variations in metabolic rate and generation time among plant lineages. In addition, through the estimation of the ratio of non-synonymous to synonymous substitution rates between orthologous mitochondrion-encoded genes of higher plants, we found an accelerated evolutionary rate that seems to be the result of relaxed selection.

  5. Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes

    PubMed Central

    2013-01-01

    Background Small supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA). To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms. Results By FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a

  6. OneBac: Platform for Scalable and High-Titer Production of Adeno-Associated Virus Serotype 1–12 Vectors for Gene Therapy

    PubMed Central

    Mietzsch, Mario; Grasse, Sabrina; Zurawski, Catherine; Weger, Stefan; Bennett, Antonette; Agbandje-McKenna, Mavis; Muzyczka, Nicholas; Zolotukhin, Sergei

    2014-01-01

    Abstract Scalable and genetically stable recombinant adeno-associated virus (rAAV) production systems combined with facile adaptability for an extended repertoire of AAV serotypes are required to keep pace with the rapidly increasing clinical demand. For scalable high-titer production of the full range of rAAV serotypes 1–12, we developed OneBac, consisting of stable insect Sf9 cell lines harboring silent copies of AAV1–12 rep and cap genes induced upon infection with a single baculovirus that also carries the rAAV genome. rAAV burst sizes reach up to 5×105 benzonase-resistant, highly infectious genomic particles per cell, exceeding typical yields of current rAAV production systems. In contrast to recombinant rep/cap baculovirus strains currently employed for large-scale rAAV production, the Sf9rep/cap cell lines are genetically stable, leading to undiminished rAAV burst sizes over serial passages. Thus, OneBac combines full AAV serotype options with the capacity for stable scale-up production, the current bottleneck for the transition of AAV from gene therapy trials to routine clinical treatment. PMID:24299301

  7. Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

    PubMed

    Kamboj, Aman; Saini, Mohini; Rajan, Lekshmi S; Patel, Chhabi Lal; Chaturvedi, V K; Gupta, Praveen K

    2015-12-15

    To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

  8. Making Your Own Gene Library.

    ERIC Educational Resources Information Center

    Perez-Ortin, Jose E.; Li Del Olmo, Marcel; Matallana, Emilia; Tordera, Vicente

    1997-01-01

    Presents an experiment aimed at constructing a genomic library that can be carried out over a week. Helps students learn concepts such as donor and vector DNAs, construction of recombinant DNA, host strain, and experiments in gene cloning more clearly. (PVD)

  9. Ataxin-2 Regulates RGS8 Translation in a New BAC-SCA2 Transgenic Mouse Model

    PubMed Central

    Figueroa, Karla P.; Rinehart, Marc D.; Wiest, Shaina; Pflieger, Lance T.; Scoles, Daniel R.; Pulst, Stefan M.

    2015-01-01

    Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling. PMID:25902068

  10. The phytophthora genome initiative database: informatics and analysis for distributed pathogenomic research.

    PubMed

    Waugh, M; Hraber, P; Weller, J; Wu, Y; Chen, G; Inman, J; Kiphart, D; Sobral, B

    2000-01-01

    The Phytophthora Genome Initiative (PGI) is a distributed collaboration to study the genome and evolution of a particularly destructive group of plant pathogenic oomycete, with the goal of understanding the mechanisms of infection and resistance. NCGR provides informatics support for the collaboration as well as a centralized data repository. In the pilot phase of the project, several investigators prepared Phytophthora infestans and Phytophthora sojae EST and Phytophthora sojae BAC libraries and sent them to another laboratory for sequencing. Data from sequencing reactions were transferred to NCGR for analysis and curation. An analysis pipeline transforms raw data by performing simple analyses (i.e., vector removal and similarity searching) that are stored and can be retrieved by investigators using a web browser. Here we describe the database and access tools, provide an overview of the data therein and outline future plans. This resource has provided a unique opportunity for the distributed, collaborative study of a genus from which relatively little sequence data are available. Results may lead to insight into how better to control these pathogens. The homepage of PGI can be accessed at http:www.ncgr.org/pgi, with database access through the database access hyperlink.

  11. Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report the stable genetic transformation of the Queensland fruit fly Bactrocera tryoni using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP.A transformation frequency of 5–10% was obtained.Inheritance of the transgenes has remained stable over eight generations despite...

  12. Callpath Library

    SciTech Connect

    Gamblin, T.

    2013-11-09

    The "Callpath Library" is a software abstraction layer over a number of stack tracing utilities. It allows tool develoopers to conveniently represent and mNipulate call paths gathered fro U. Wisconsin's Stackwalker API and GNU Backtrace.

  13. Digital Libraries.

    ERIC Educational Resources Information Center

    Fox, Edward A.; Urs, Shalini R.

    2002-01-01

    Provides an overview of digital libraries research, practice, and literature. Highlights include new technologies; redefining roles; historical background; trends; creating digital content, including conversion; metadata; organizing digital resources; services; access; information retrieval; searching; natural language processing; visualization;…

  14. Academic Libraries

    ERIC Educational Resources Information Center

    Library Journal, 1970

    1970-01-01

    Building data is given for the following academic libraries: (1) Rosary College, River Forest, Illinois; (2) Abilene Christian College, Abilene, Texas; (3) University of California, San Diego, La Jolla, California. (MF)

  15. Minibactenecins ChBac7.Nα and ChBac7. Nβ - Antimicrobial Peptides from Leukocytes of the Goat Capra hircus.

    PubMed Central

    Shamova, O.V.; Orlov, D.S.; Zharkova, M.S.; Balandin, S.V.; Yamschikova, E.V.; Knappe, D.; Hoffmann, R.; Kokryakov, V.N.; Ovchinnikova, T.V.

    2016-01-01

    Antimicrobial peptides (AMPs) of neutrophils play an important role in the animal and human host defenses. We have isolated two AMPs (average molecular masses of 2895.5 and 2739.3 Da), with potent antimicrobial activity from neutrophils of the domestic goat (Capra hircus). A structural analysis of the obtained peptides revealed that they encompass N-terminal fragments (1–21 and 1–22) of the proline-rich peptide bactenecin 7.5. The primary structure of caprine bactenecin 7.5 had been previously deduced from the nucleotide sequence, but the corresponding protein had not been isolated from leukocytes until now. The obtained caprine AMPs were designated as mini-batenecins (mini-ChBac7.5Nα and mini-ChBac7.5Nβ), analogously to the reported C-terminal fragment of the ovine bactenecin 7.5 named Bac7.5mini [Anderson, Yu, 2003]. Caprine mini-ChBac7.5Nα and mini-ChBac7.5Nβ exhibit significant antimicrobial activity against Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii at a range of concentrations of 0.5–4 μM, as well as against some species of Gram-positive bacteria (Listeria monocytogenes EGD, Micrococcus luteus). The peptides demonstrate lipopolysaccharide-binding activity. Similarly to most proline-rich AMPs, caprine peptides inactivate bacteria without appreciable damage of their membranes. Mini-ChBac7.5Nα and mini-ChBac7.5Nβ have no hemolytic effect on human red blood cells and are nontoxic to various cultured human cells. Therefore, they might be considered as promising templates for the development of novel antibiotic pharmaceuticals. Isolation of highly active fragments of the antimicrobial peptide from goat neutrophils supports the hypothesis that fragmentation of cathelicidin-related AMPs is an important process that results in the generation of potent effector molecules, which are in some cases more active than full-size AMPs. These truncated AMPs may play a crucial role in

  16. Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening.

    PubMed

    Xu, Xiaodong; Chen, Yuanrong; Zhao, Yu; Liu, Xiaofen; Dong, Beitao; Jones, Ian M; Chen, Hongying

    2013-01-01

    In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.

  17. The effectiveness of a 0.05 blood alcohol concentration (BAC) limit for driving in the United States.

    PubMed

    Fell, James C; Voas, Robert B

    2014-06-01

    The National Transportation Safety Board recently recommended that states establish a per se blood alcohol concentration (BAC) limit of 0.05 or lower for all drivers who are not already required to adhere to lower BAC limits in a national effort to reduce alcohol-impaired driving. There is strong evidence for adopting this recommendation. A comprehensive review of the literature on BAC limits was conducted. The research indicates that virtually all drivers are impaired regarding at least some driving performance measures at a 0.05 BAC. The risk of being involved in a crash increases significantly at 0.05 BAC and above. The relative risk of being killed in a single-vehicle crash with BACs of 0.05-0.079 is 7-21 times higher than for drivers at 0.00 BAC. Lowering the BAC limit from 0.08 to 0.05 has been a proven effective countermeasure in numerous countries around the world. Most Americans do not believe a person should drive after having two or three drinks in 2 hours. It takes at least four drinks for the average 170-pound male to exceed 0.05 BAC in 2 hours (three drinks for the 137-pound female). Most industrialized nations have established a 0.05 BAC limit or lower for driving. Progress in reducing the proportion of drivers in fatal crashes with illegal BACs has stalled over the past 15 years. Lowering the BAC limit for driving from the current 0.08 to 0.05 has substantial potential to reduce the number of people who drink and drive in the United States and get involved in fatal crashes.

  18. Stereochemical Outcome at Four Stereogenic Centers During Conversion of Prephenate to Tetrahydrotyrosine by BacABGF in the Bacilysin Pathway†

    PubMed Central

    Parker, Jared B.; Walsh, Christopher T.

    2012-01-01

    The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H4Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-Δ4,Δ8-7R-dihydrohydroxyphenylpyruvate (en-H2HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclic-Δ3,Δ5-dihydrohydroxyphenylpyruvate (ex-H2HPP). The NADH-utilizing BacG then catalyzes a conjugate reduction, adding a pro-S hydride equivalent to C4 to yield tetrahydrohydroxyphenylpyruvate (H4HPP), a transamination away (via BacF) from 2S-H4Tyr. Incubations of the pathway enzymes in D2O yield deuterium incorporation at C8 from BacA and then C9 from BacB action. By 1H-NMR analysis on samples of H4Tyr, the stereochemistry at C4, C8 and C9 can be assigned. BacG (followed by BacF) converts 3E-ex-H2HPP to 2S, 4R, 7R-H4Tyr. The 3Z isomer is instead reduced and transaminated to the opposite diastereomer at C4: 2S, 4S, 7R-H4Tyr. Given that bacilysin has the 2S, 4S stereochemistry in its anticapsin moiety, it is likely that the 2S, 4S-H4Tyr is the diastereomer “on pathway”. NMR determination of the stereochemistry of the CHD samples at C8 and C9 allow assignment of all stereogenic centers (except C3) in this unusual tetrahydro- aromatic amino acid building block, giving insights and constraints on the BacA, BacB, and BacG mechanisms. PMID:22765234

  19. Chromatin states shape insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus

    PubMed Central

    Yoshida, Junko; Akagi, Keiko; Misawa, Ryo; Kokubu, Chikara; Takeda, Junji; Horie, Kyoji

    2017-01-01

    DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter–enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications. PMID:28252665

  20. Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids1

    PubMed Central

    Li, Zicong; Zeng, Fang; Meng, Fanming; Xu, Zhiqian; Zhang, Xianwei; Huang, Xiaoling; Tang, Fei; Gao, Wenchao; Shi, Junsong; He, Xiaoyan; Liu, Dewu; Wang, Chong; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2014-01-01

    ABSTRACT The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis. PMID:24671876

  1. Chromatin states shape insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus.

    PubMed

    Yoshida, Junko; Akagi, Keiko; Misawa, Ryo; Kokubu, Chikara; Takeda, Junji; Horie, Kyoji

    2017-03-02

    DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter-enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications.

  2. The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs

    PubMed Central

    Sato, Masahiro; Maeda, Kosuke; Koriyama, Miyu; Inada, Emi; Saitoh, Issei; Miura, Hiromi; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi; Miyoshi, Kazuchika

    2016-01-01

    The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes. PMID:27589724

  3. Personal Virtual Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2004-01-01

    Virtual libraries are becoming more and more common. Most states have a virtual library. A growing number of public libraries have a virtual presence on the Web. Virtual libraries are a growing addition to school library media collections. The next logical step would be personal virtual libraries. A personal virtual library (PVL) is a collection…

  4. America's Star Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  5. Mouse and human BAC transgenes recapitulate tissue-specific expression of the vitamin D receptor in mice and rescue the VDR-null phenotype.

    PubMed

    Lee, Seong Min; Bishop, Kathleen A; Goellner, Joseph J; O'Brien, Charles A; Pike, J Wesley

    2014-06-01

    The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

  6. Application of the BacT/ALERTR 3D system for sterility testing of injectable products.

    PubMed

    Bugno, Adriana; Lira, Rodolfo Santos; Oliveira, Wesley Anderson; Almodovar, Adriana Aparecida Buzzo; Saes, Deborah Pita Sanches; Pinto, Terezinha de Jesus Andreoli

    2015-01-01

    Sterility testing as described in the pharmacopoeia compendia requires a 14-day incubation period to obtain an analytical result. Alternative methods that could be applied to evaluating product sterility are especially interesting due to the possibility of reducing this incubation period and thus the associated costs. The aims of this study were to evaluate the performance of the BacT/ALERT(R) 3D system in detecting microorganisms in large-volume parenteral solutions that were intentionally contaminated and to compare this system to pharmacopoeia sterility testing using the membrane filtration method. The results indicated that there were no significant differences between the methods regarding the ability to detect microbial contamination; however, detection with the BacT/ALERT(R) 3D system was faster compared to the pharmacopoeia method. Therefore, the BacT/ALERT(R) 3D system is a viable alternative for assessing the sterility of injectable products.

  7. Application of the BacT/ALERTR 3D system for sterility testing of injectable products

    PubMed Central

    Bugno, Adriana; Lira, Rodolfo Santos; Oliveira, Wesley Anderson; Almodovar, Adriana Aparecida Buzzo; Saes, Deborah Pita Sanches; de Jesus Andreoli Pinto, Terezinha

    2015-01-01

    Sterility testing as described in the pharmacopoeia compendia requires a 14-day incubation period to obtain an analytical result. Alternative methods that could be applied to evaluating product sterility are especially interesting due to the possibility of reducing this incubation period and thus the associated costs. The aims of this study were to evaluate the performance of the BacT/ALERTR 3D system in detecting microorganisms in large-volume parenteral solutions that were intentionally contaminated and to compare this system to pharmacopoeia sterility testing using the membrane filtration method. The results indicated that there were no significant differences between the methods regarding the ability to detect microbial contamination; however, detection with the BacT/ALERTR 3D system was faster compared to the pharmacopoeia method. Therefore, the BacT/ALERTR 3D system is a viable alternative for assessing the sterility of injectable products. PMID:26413055

  8. Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool.

    PubMed

    Kim, Adele; Pyykko, Ilmari

    2011-08-01

    Transposons have been promising elements for gene integration, and the Sleeping Beauty (SB) system has been the major one for many years, although there have been several other transposon systems available, for example, Tol2. However, recently another system known as PiggyBac (PB) has been introduced and developed for fulfilling the same purposes, for example, mutagenesis, transgenesis and gene therapy and in some cases with improved transposition efficiency and advantages over the Sleeping Beauty transposon system, although improved hyperactive transposase has highly increased the transposition efficacy for SB. The PB systems have been used in many different scientific research fields; therefore, the purpose of this review is to describe some of these versatile uses of the PiggyBac system to give readers an overview on the usage of PiggyBac system.

  9. A Tie2-driven BAC-TRAP transgenic line for in vivo endothelial gene profiling

    PubMed Central

    Santhosh, Devi; Huang, Zhen

    2016-01-01

    Recent technological innovations including bacterial artificial chromosome-based translating ribosome affinity purification (BAC-TRAP) have greatly facilitated analysis of cell type-specific gene expression in vivo, especially in the nervous system. To better study endothelial gene expression in vivo, we have generated a BAC-TRAP transgenic mouse line where the L10a ribosomal subunit is tagged with EGFP and placed under the control of the endothelium-specific Tie2 (Tek) promoter. We show that transgene expression in this line is widely, but specifically, detected in endothelial cells in several brain regions throughout pre- and postnatal development, as well as in other organs. We also show that this line results in highly significant enrichment of endothelium-specific mRNAs from brain tissues at different stages. This BAC-TRAP line therefore provides a useful genetic tool for in vivo endothelial gene profiling under various developmental, physiological, and pathological conditions. PMID:26817747

  10. Genome-wide analysis of transposable elements in the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae): description of novel families.

    PubMed

    Hernandez-Hernandez, Eric M; Fernández-Medina, Rita Daniela; Navarro-Escalante, Lucio; Nuñez, Jonathan; Benavides-Machado, Pablo; Carareto, Claudia M A

    2017-02-15

    The coffee berry borer (CBB) Hypothenemus hampei is the most limiting pest of coffee production worldwide. The CBB genome has been recently sequenced; however, information regarding the presence and characteristics of transposable elements (TEs) was not provided. Using systematic searching strategies based on both de novo and homology-based approaches, we present a library of TEs from the draft genome of CBB sequenced by the Colombian Coffee Growers Federation. The library consists of 880 sequences classified as 66% Class I (LTRs: 46%, non-LTRs: 20%) and 34% Class II (DNA transposons: 8%, Helitrons: 16% and MITEs: 10%) elements, including families of the three main LTR (Gypsy, Bel-Pao and Copia) and non-LTR (CR1, Daphne, I/Nimb, Jockey, Kiri, R1, R2 and R4) clades and DNA superfamilies (Tc1-mariner, hAT, Merlin, P, PIF-Harbinger, PiggyBac and Helitron). We propose the existence of novel families: Hypo, belonging to the LTR Gypsy superfamily; Hamp, belonging to non-LTRs; and rosa, belonging to Class II or DNA transposons. Although the rosa clade has been previously described, it was considered to be a basal subfamily of the mariner family. Based on our phylogenetic analysis, including Tc1, mariner, pogo, rosa and Lsra elements from other insects, we propose that rosa and Lsra elements are subfamilies of an independent family of Class II elements termed rosa. The annotations obtained indicate that a low percentage of the assembled CBB genome (approximately 8.2%) consists of TEs. Although these TEs display high diversity, most sequences are degenerate, with few full-length copies of LTR and DNA transposons and several complete and putatively active copies of non-LTR elements. MITEs constitute approximately 50% of the total TEs content, with a high proportion associated with DNA transposons in the Tc1-mariner superfamily.

  11. A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map

    SciTech Connect

    Kelleher, Colin; CHIU, Dr. R.; Shin, Dr. H.; Krywinski, Martin; Fjell, Chris; Wilkin, Jennifer; Yin, Tongming; Difazio, Stephen P.

    2007-01-01

    As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence assembly to be 485 {+-} 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of these haplotype sequence differences.

  12. Estimating Driver Risk Using Alcohol Biomarkers, Interlock BAC Tests and Psychometric Assessments: Initial Descriptives

    PubMed Central

    Marques, Paul; Tippetts, Scott; Allen, John; Javors, Martin; Alling, Christer; Yegles, Michel; Pragst, Fritz; Wurst, Friedrich

    2009-01-01

    Aim To identify alcohol biomarker and psychometric measures that relate to drivers’ blood alcohol concentration (BAC) patterns from ignition interlock devices (IIDs). Design, Setting, Participants, Measurements In Alberta, Canada, 534 drivers, convicted of driving under the influence of alcohol (DUI), installed IIDs and agreed to participate in a research study. IID BAC tests are an established proxy for predicting future DUI convictions. Three risk groups were defined by rates of failed BAC tests. Program entry and followup blood samples (n=302, 171) were used to measure phosphatidyl ethanol (PETH), carbohydrate deficient transferrin (%CDT), gamma glutamyltransferase (GGT) and other biomarkers. Program entry urine (n=130) was analyzed for ethyl glucuronide (ETG) and ethyl sulfate (ETS). Entry hair samples were tested for fatty acid ethyl esters (FAEE) (n=92) and ETG (n=146). Psychometric measures included the DSM-4 Diagnostic Interview Schedule Alcohol Module, Alcohol Use Disorders Identification Test (AUDIT), the Timeline Followback (TLFB), the Drinker Inventory of Consequences (DRINC), and the Temptation and Restraint Inventory (TRI). Findings Except for FAEE, all alcohol biomarkers were significantly related to the interlock BAC test profiles; higher marker levels predicted higher rates of interlock BAC test failures. PETH, the strongest with an overall ANOVA F ratio of 35.5, had significant correlations with all nine of the other alcohol biomarkers and with 16 of 19 psychometric variables. Urine ETG and ETS were strongly correlated with the IID BAC tests. Conclusions The findings suggest several alcohol biomarkers and assessments could play an important role in the prediction and control of driver alcohol risk when relicensing. PMID:19922520

  13. Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools.

    PubMed

    Rajendra, Yashas; Balasubramanian, Sowmya; Peery, Robert B; Swartling, James R; McCracken, Neil A; Norris, Dawn L; Frye, Christopher C; Barnard, Gavin C

    2017-02-11

    Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017.

  14. Development of novel simple sequence repeat markers in bitter gourd (Momordica charantia L.) through enriched genomic libraries and their utilization in analysis of genetic diversity and cross-species transferability.

    PubMed

    Saxena, Swati; Singh, Archana; Archak, Sunil; Behera, Tushar K; John, Joseph K; Meshram, Sudhir U; Gaikwad, Ambika B

    2015-01-01

    Microsatellite or simple sequence repeat (SSR) markers are the preferred markers for genetic analyses of crop plants. The availability of a limited number of such markers in bitter gourd (Momordica charantia L.) necessitates the development and characterization of more SSR markers. These were developed from genomic libraries enriched for three dinucleotide, five trinucleotide, and two tetranucleotide core repeat motifs. Employing the strategy of polymerase chain reaction-based screening, the number of clones to be sequenced was reduced by 81 % and 93.7 % of the sequenced clones contained in microsatellite repeats. Unique primer-pairs were designed for 160 microsatellite loci, and amplicons of expected length were obtained for 151 loci (94.4 %). Evaluation of diversity in 54 bitter gourd accessions at 51 loci indicated that 20 % of the loci were polymorphic with the polymorphic information content values ranging from 0.13 to 0.77. Fifteen Indian varieties were clearly distinguished indicative of the usefulness of the developed markers. Markers at 40 loci (78.4 %) were transferable to six species, viz. Momordica cymbalaria, Momordica subangulata subsp. renigera, Momordica balsamina, Momordica dioca, Momordica cochinchinesis, and Momordica sahyadrica. The microsatellite markers reported will be useful in various genetic and molecular genetic studies in bitter gourd, a cucurbit of immense nutritive, medicinal, and economic importance.

  15. Comparison of the chromosome maps around a resistance hot spot on chromosome 5 of potato and tomato using BAC-FISH painting.

    PubMed

    Achenbach, Ute C; Tang, Xiaomin; Ballvora, Agim; de Jong, Hans; Gebhardt, Christiane

    2010-02-01

    Potato chromosome 5 harbours numerous genes for important qualitative and quantitative traits, such as resistance to the root cyst nematode Globodera pallida and the late blight fungus, Phytophthora infestans. The genes make up part of a "hot spot" for resistances to various pathogens covering a genetic map length of 3 cM between markers GP21 and GP179. We established the physical size and position of this region on chromosome 5 in potato and tomato using fluorescence in situ hybridization (FISH) on pachytene chromosomes. Five potato bacterial artificial chromosome (BAC) clones with the genetically anchored markers GP21, R1-contig (proximal end), CosA, GP179, and StPto were selected, labeled with different fluorophores, and hybridized in a five-colour FISH experiment. Our results showed the location of the BAC clones in the middle of the long arm of chromosome 5 in both potato and tomato. Based on chromosome measurements, we estimate the physical size of the GP21-GP179 interval at 0.85 Mb and 1.2 Mb in potato and tomato, respectively. The GP21-GP179 interval is part of a genome segment known to have inverted map positions between potato and tomato.

  16. Underground Libraries.

    ERIC Educational Resources Information Center

    Fuhlrott, Rolf

    1986-01-01

    Discussion of underground buildings constructed primarily during last two decades for various reasons (energy conservation, density of environment, preservation of landscape and historic buildings) notes advantages, disadvantages, and psychological and design considerations. Examples of underground libraries, built mainly in United States, are…

  17. Comparison of biological activated carbon (BAC) and membrane bioreactor (MBR) for pollutants removal in drinking water treatment.

    PubMed

    Tian, J Y; Chen, Z L; Liang, H; Li, X; Wang, Z Z; Li, G B

    2009-01-01

    Biological activated carbon (BAC) and membrane bioreactor (MBR) were systematically compared for the drinking water treatment from slightly polluted raw water under the same hydraulic retention time (HRT) of 0.5 h. MBR exhibited excellent turbidity removal capacity due to the separation of the membrane; while only 60% of influent turbidity was intercepted by BAC. Perfect nitrification was achieved by MBR with the 89% reduction in ammonia; by contrast, BAC only eliminated a moderate amount of influent ammonia (by 54.5%). However, BAC was able to remove more dissolved organic matter (DOM, especially for organic molecules of 3,000 approximately 500 Daltons) and corresponding disinfection by-product formation potential (DBPFP) in raw water than MBR. Unfortunately, particulate organic matter (POM) was detected in the BAC effluent. On the other hand, BAC and MBR displayed essentially the same capacity for biodegradable organic matter (BOM) removal. Fractionation of DOM showed that the removal efficiencies of hydrophobic neutrals, hydrophobic acids, weakly hydrophobic acids and hydrophilic organic matter through BAC treatment were 11.7%, 8.8%, 13.9% and 4.8% higher than that through MBR; while MBR achieved 13.8% higher hydrophobic bases removal as compared with BAC.

  18. Minnesota Zoological Garden Library.

    ERIC Educational Resources Information Center

    Norell, Angela

    1988-01-01

    Describes the history and functions of the Minnesota Zoological Garden library. Topics covered include the library collections; library services, including online search capabilities; and the various groups of users served by the library. (three references) (CLB)

  19. Pharmacological Screening Using an FXN-EGFP Cellular Genomic Reporter Assay for the Therapy of Friedreich Ataxia

    PubMed Central

    Li, Lingli; Voullaire, Lucille; Sandi, Chiranjeevi; Pook, Mark A.

    2013-01-01

    Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol. PMID:23418481

  20. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

    PubMed Central

    Freeman, Jennifer L; Adeniyi, Adeola; Banerjee, Ruby; Dallaire, Stephanie; Maguire, Sean F; Chi, Jianxiang; Ng, Bee Ling; Zepeda, Cinthya; Scott, Carol E; Humphray, Sean; Rogers, Jane; Zhou, Yi; Zon, Leonard I; Carter, Nigel P; Yang, Fengtang; Lee, Charles

    2007-01-01

    Background The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data. Results Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG) chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC) clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH) and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed. Conclusion The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models. PMID:17597531

  1. Turkish Libraries: Historical Context.

    ERIC Educational Resources Information Center

    Cakin, Irfan

    1984-01-01

    Summary of the development of libraries in Turkey highlights the existence of libraries in the ninth century, the Shamssaddin Altunaba Medrese library in Konya, libraries established during the Ottoman era, reports to reform libraries (1869-70, 1909), and reports and library developments attributed to the Republican Era beginning in 1923. (EJS)

  2. Library Research and Statistics.

    ERIC Educational Resources Information Center

    Lynch, Mary Jo; St. Lifer, Evan; Halstead, Kent; Fox, Bette-Lee; Miller, Marilyn L.; Shontz, Marilyn L.

    2001-01-01

    These nine articles discuss research and statistics on libraries and librarianship, including libraries in the United States, Canada, and Mexico; acquisition expenditures in public, academic, special, and government libraries; price indexes; state rankings of public library data; library buildings; expenditures in school library media centers; and…

  3. Methods for combinatorial and parallel library design.

    PubMed

    Schnur, Dora M; Beno, Brett R; Tebben, Andrew J; Cavallaro, Cullen

    2011-01-01

    Diversity has historically played a critical role in design of combinatorial libraries, screening sets and corporate collections for lead discovery. Large library design dominated the field in the 1990s with methods ranging anywhere from purely arbitrary through property based reagent selection to product based approaches. In recent years, however, there has been a downward trend in library size. This was due to increased information about the desirable targets gleaned from the genomics revolution and to the ever growing availability of target protein structures from crystallography and homology modeling. Creation of libraries directed toward families of receptors such as GPCRs, kinases, nuclear hormone receptors, proteases, etc., replaced the generation of libraries based primarily on diversity while single target focused library design has remained an important objective. Concurrently, computing grids and cpu clusters have facilitated the development of structure based tools that screen hundreds of thousands of molecules. Smaller "smarter" combinatorial and focused parallel libraries replaced those early un-focused large libraries in the twenty-first century drug design paradigm. While diversity still plays a role in lead discovery, the focus of current library design methods has shifted to receptor based methods, scaffold hopping/bio-isostere searching, and a much needed emphasis on synthetic feasibility. Methods such as "privileged substructures based design" and pharmacophore based design still are important methods for parallel and small combinatorial library design. This chapter discusses some of the possible design methods and presents examples where they are available.

  4. A Hybrid Adenoviral Vector System Achieves Efficient Long-Term Gene Expression in the Liver via piggyBac Transposition

    PubMed Central

    Smith, Ryan P.; Riordan, Jesse D.; Feddersen, Charlotte R.

    2015-01-01

    Abstract Much research has gone into the development of hybrid gene delivery systems that combine the broad tropism and efficient transduction of adenoviral vectors with the ability to achieve stable expression of cargo genes. In addition to gene therapy applications, such a system has considerable advantages for studies of gene function in vivo, permitting fine-tuned genetic manipulation with higher throughput than can be achieved using standard transgenic and DNA targeting techniques. Existing strategies are limited, however, by low integration efficiencies, small cargo capacity, and/or a dependence on target cell division. The utility of this approach could be enhanced by a system that provides all of the following: (1) efficient delivery, (2) stable expression in a high percentage of target cells (whether mitotic or not), (3) large cargo capacity, (4) flexibility to use with a wide range of additional experimental conditions, and (5) simple experimental technique. Here we report the initial characterization of a hybrid system that meets these criteria by utilizing piggyBac (PB) transposition to achieve genomic integration from adenoviral vectors. We demonstrate stable expression of an adenovirus (Ad)-PB-delivered reporter gene in ∼20–40% of hepatocytes following standard tail vein injection. Its high efficiency and flexibility relative to existing hybrid adenoviral gene delivery approaches indicate a considerable potential utility of the Ad-PB system for therapeutic gene delivery and in vivo studies of gene function. PMID:25808258

  5. Measuring brain activity cycling (BAC) in long term EEG monitoring of preterm babies.

    PubMed

    Stevenson, Nathan J; Palmu, Kirsi; Wikström, Sverre; Hellström-Westas, Lena; Vanhatalo, Sampsa

    2014-07-01

    Measuring fluctuation of vigilance states in early preterm infants undergoing long term intensive care holds promise for monitoring their neurological well-being. There is currently, however, neither objective nor quantitative methods available for this purpose in a research or clinical environment. The aim of this proof-of-concept study was, therefore, to develop quantitative measures of the fluctuation in vigilance states or brain activity cycling (BAC) in early preterm infants. The proposed measures of BAC were summary statistics computed on a frequency domain representation of the proportional duration of spontaneous activity transients (SAT%) calculated from electroencephalograph (EEG) recordings. Eighteen combinations of three statistics and six frequency domain representations were compared to a visual interpretation of cycling in the SAT% signal. Three high performing measures (band energy/periodogram: R = 0.809, relative band energy/nonstationary frequency marginal: R = 0.711, g-statistic/nonstationary frequency marginal: R = 0.638) were then compared to a grading of sleep wake cycling based on the visual interpretation of the amplitude-integrated EEG trend. These measures of BAC are conceptually straightforward, correlate well with the visual scores of BAC and sleep wake cycling, are robust enough to cope with the technically compromised monitoring data available in intensive care units, and are recommended for further validation in prospective studies.

  6. BAC and Beer: Operationalizing Drunk Driving Laws in a Research Methods Course Exercise.

    ERIC Educational Resources Information Center

    Taylor, Ralph B.; McConnell, Patrick

    2001-01-01

    Focuses on an exercise utilized in a research methods class and based on social problems that invites student interest. Explains the exercise has students determine their blood alcohol level (BAC) by asking them to estimate the number of beers it would take to have them just reach driving under the influence (DUI) status. (CMK)

  7. Insect transformation with piggyBac: getting the number of injections just right

    PubMed Central

    Morrison, N. I.; Shimeld, S. M.

    2016-01-01

    Abstract The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision‐making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co‐opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov‐Chain Monte‐Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac. PMID:27027400

  8. Sperm-mediated transgenesis in chicken using a PiggyBac transposon system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sperm-mediated transgenesis in chicken using a PiggyBac transposon system Emmanuel Quansah1,2, Julie Long2, David Donovan2, Stephen Becker2, Bhanu Telugu2, Juli Frey2, Nigel Urwin1 1,Charles Sturt University, Graham Center of Agricultural Innovation, Wagga Wagga. Australia and 2Beltsville Agricultu...

  9. Cloning and characterization of piggyBac-like elements in lepidopteran insects.

    PubMed

    Wu, Min; Sun, Zhichan; Luo, Guanghua; Hu, Chunlin; Zhang, Wei; Han, Zhaojun

    2011-01-01

    PiggyBac-like elements (PLE) are widespread in variety of organisms, however, few of them are active or have an intact transposon structure. To further define the distribution PLEs in Lepidoptera, where the original active piggyBac IFP2 was discovered, and potentially isolate new functional elements, a survey for PLEs by PCR amplification and Southern dot blots was performed. Two new PLEs, AyPLE and AaPLE, were successfully isolated from the noctuid species, Agrotis ypsilon and Argyrogramma agnate, respectively. These elements were found to be closely related to each other by sequence similarity, and by sharing the same 16 bp inverted terminal repeat sequences. The AyPLE1.1 and AaPLE1.1 elements are structurally intact having characteristic TTAA target site duplications, inverted terminal repeats and intact open reading frames encoding putative transposases with the presumed piggyBac DDD domains, which are features consistent with autonomous functional transposons. Phylogenetic analysis revealed that AyPLE1.1 and AaPLE1.1 cluster with another noctuid species element, HaPLE1.1, suggesting a common ancestor for the three types of PLEs. This contributes to our understanding of the distribution and evolution of piggyBac in Lepidoptera.

  10. Paradoxical bronchospasm from benzalkonium chloride (BAC) preservative in albuterol nebulizer solution in a patient with acute severe asthma. A case report and literature review of airway effects of BAC.

    PubMed

    George, Mathew; Joshi, Saumya Vinod; Concepcion, Emily; Lee, Haesoon

    2017-01-01

    Nebulized bronchodilator solutions are available in the United States as both nonsterile and sterile-filled products. Sulfites, benzalkonium chloride (BAC), or chlorobutanol are added to nonsterile products to prevent bacterial growth. Bronchoconstriction from inhaled BAC is cumulative, prolonged, and correlates directly with basal airway responsiveness. The multi-dose dropper bottle of albuterol sulfate solution contains 50 μg BAC per/2.5 mg of albuterol, which may be below or at the lower limit of the threshold dose for bronchoconstriction. However, with repeated albuterol nebulization, the effect can be additive and cumulative, often exceeding the bronchoconstriction threshold. We report a case of a 17 years old patient, who received 32 mg of BAC via nebulization over a period of 3.5 days that probably caused persistent bronchospasm evidenced by failure to improve clinically and to increase peak expiratory flow rate (PEFR) from 125 L/min (27% of predicted value) to 300 L/min (68% of predicted value) within 2 hours of withdrawing BAC. The patient's respiratory status and PEFR improved dramatically once the nebulization solution was switched to BAC free lev-albuterol solution. The pediatric providers, particularly the emergency department physicians, intensivists and pulmonologists need to be aware of this rare albeit possible toxicity to the respiratory system caused by BAC used as a preservative in albuterol nebulizer solution.

  11. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Papers on art libraries, librarianship, and documentation presented at the 1982 International Federation of Library Associations (IFLA) conference include: (1) "The Tyranny of Distance: Art Libraries in Canada," a description by Mary F. Williamson of Canada's regional art libraries which serve both art students and the general public;…

  12. Strengthening State Library Administrative Agency (Territorial Library).

    ERIC Educational Resources Information Center

    Nieves M. Flores Memorial Library, Agana, Guam.

    This document describes the Basic State Plan Amendments for the Library Services and Construction Act in Guam and the regulations promulgated thereunder. The major projects described under the plan are: Strengthening State Library Administrative Agency; Staff Development; Library Collections, Extention Services, Institutional Libraries; and…

  13. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

    PubMed

    Manat, Guillaume; El Ghachi, Meriem; Auger, Rodolphe; Baouche, Karima; Olatunji, Samir; Kerff, Frédéric; Touzé, Thierry; Mengin-Lecreulx, Dominique; Bouhss, Ahmed

    2015-01-01

    Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane.

  14. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase

    PubMed Central

    Manat, Guillaume; El Ghachi, Meriem; Auger, Rodolphe; Baouche, Karima; Olatunji, Samir; Kerff, Frédéric; Touzé, Thierry; Mengin-Lecreulx, Dominique; Bouhss, Ahmed

    2015-01-01

    Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP) phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2) catalyze the dephosphorylation of C55-PP molecules on the same (outer) side of the plasma membrane. PMID:26560897

  15. Engineering large viral DNA genomes using the CRISPR-Cas9 system.

    PubMed

    Suenaga, Tadahiro; Kohyama, Masako; Hirayasu, Kouyuki; Arase, Hisashi

    2014-09-01

    Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus-infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time-consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat-Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene-ablated HSV but also gene knock-in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein-Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.

  16. The map-based genome sequence of Spirodela polyrhiza aligned with its chromosomes, a reference for karyotype evolution.

    PubMed

    Cao, Hieu Xuan; Vu, Giang Thi Ha; Wang, Wenqin; Appenroth, Klaus J; Messing, Joachim; Schubert, Ingo

    2016-01-01

    Duckweeds are aquatic monocotyledonous plants of potential economic interest with fast vegetative propagation, comprising 37 species with variable genome sizes (0.158-1.88 Gbp). The genomic sequence of Spirodela polyrhiza, the smallest and the most ancient duckweed genome, needs to be aligned to its chromosomes as a reference and prerequisite to study the genome and karyotype evolution of other duckweed species. We selected physically mapped bacterial artificial chromosomes (BACs) containing Spirodela DNA inserts with little or no repetitive elements as probes for multicolor fluorescence in situ hybridization (mcFISH), using an optimized BAC pooling strategy, to validate its physical map and correlate it with its chromosome complement. By consecutive mcFISH analyses, we assigned the originally assembled 32 pseudomolecules (supercontigs) of the genomic sequences to the 20 chromosomes of S. polyrhiza. A Spirodela cytogenetic map containing 96 BAC markers with an average distance of 0.89 Mbp was constructed. Using a cocktail of 41 BACs in three colors, all chromosome pairs could be individualized simultaneously. Seven ancestral blocks emerged from duplicated chromosome segments of 19 Spirodela chromosomes. The chromosomally integrated genome of S. polyrhiza and the established prerequisites for comparative chromosome painting enable future studies on the chromosome homoeology and karyotype evolution of duckweed species.

  17. BACs-on-Beads™ (BoBs™) assay for the genetic evaluation of prenatal samples and products of conception.

    PubMed

    Grati, Francesca Romana; Vialard, François; Gross, Susan

    2015-01-01

    BACs-on-Beads™ (BoBs™) is a new emerging technology, a modification of comparative genomic hybridization that can be used to detect DNA copy number gains and losses. Here, we describe the application of two different types of BoBs™ assays: (1) Prenatal BoBs (CE-IVD) to detect the most frequent syndromes associated with chromosome microdeletions, as well as the trisomy 13, 18 and 21, and (2) KaryoLite BoBs (RUO) which can detect aneuploidy in all chromosomes by quantifying proximal and terminal regions of each chromosomal arm. The interpretation of the results by BoBsoft™ software is also described. Although BoBs™ may not have the breadth and scope to replace chromosomal microarrays (array comparative genomic hybridization and single nucleotide polymorphism array) in the prenatal setting, particularly when a fetal anomaly has been detected, it is a well suited alternative for FISH or QF-PCR because BoBs™ is comparable, if not superior in terms of cost, turnaround time (TAT) and throughput and accuracy. BoBs™ also has the ability to detect significant fetal mosaicism (≥30% with Prenatal BoBs and ≥50% with KaryoLite BoBs). However, perhaps the greatest strength of this new technology is the fact that unlike FISH or QF-PCR, it has the ability to detect common microdeletion syndromes or additional aneuploidies, both of which may be easily missed despite excellent prenatal sonography. Thus, when BoBs™ is applied in the correct clinical setting and run and analyzed in appropriate laboratories this technique can improve and augment best practices with a personalization of prenatal care.

  18. Investigation of decolorization of textile wastewater in an anaerobic/aerobic biological activated carbon system (A/A BAC).

    PubMed

    Pasukphun, N; Vinitnantharat, S; Gheewala, S

    2010-04-01

    The aim of this study is to investigate the decolorization in anaerobic/aerobic biological activated carbon (A/A BAC) system. The experiment was divided into 2 stages; stage I is batch test for preliminary study of dye removal equilibrium time. The preliminary experiment (stage I) provided the optimal data for experimental design of A/A BAC system in SBR (stage II). Stage II is A/A BAC system imitated Sequencing Batch Reactor (SBR) which consist of 5 main periods; fill, react, settle, draw and idle. React period include anaerobic phase followed by aerobic phase. The BAC main media; Granular Activated Carbon (GAC), Mixed Cultures (MC) and Biological Activated Carbon (BAC) were used for dye and organic substances removal in three different solutions; Desizing Agent Solution (DAS), dye Solution (DS) and Synthetic Textile Wastewater (STW). Results indicate that GAC adsorption plays role in dye removal followed by BAC and MC activities, respectively. In the presence desizing agent, decolorization by MC was improved because desizing agent acts as co-substrates for microorganisms. It was found that 50% of dye removal efficiency was achieved in Fill period by MC. GC/MS analysis was used to identify dye intermediate from decolorization. Dye intermediate containing amine group was found in the solution and on BAC surfaces. The results demonstrated that combination of MC and BAC in the system promotes decolorization and dye intermediate removal. In order to improve dye removal efficiency in an A/A BAC system, replacement of virgin GAC, sufficient co-substrates supply and the appropriate anaerobic: aerobic period should be considered.

  19. Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.

    PubMed

    Beier, Sebastian; Himmelbach, Axel; Schmutzer, Thomas; Felder, Marius; Taudien, Stefan; Mayer, Klaus F X; Platzer, Matthias; Stein, Nils; Scholz, Uwe; Mascher, Martin

    2016-07-01

    Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.

  20. Structural characterization of Brachypodium genome and its syntenic relationship with rice and wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brachypodium distachyon (Brachypodium) has been recently recognized as an emerging model system for both comparative and functional genomics in grass species. In this study, 55,221 repeat masked Brachypodium BAC end sequences (BES) were used for comparative analysis against the 12 rice pseudomolecul...

  1. Graph Library

    SciTech Connect

    Schulz, Martin; Arnold, Dorian

    2007-06-12

    GraphLib is a support library used by other tools to create, manipulate, store, and export graphs. It provides a simple interface to specifS’ arbitrary directed and undirected graphs by adding nodes and edges. Each node and edge can be associated with a set of attributes describing size, color, and shape. Once created, graphs can be manipulated using a set of graph analysis algorithms, including merge, prune, and path coloring operations. GraphLib also has the ability to export graphs into various open formats such as DOT and GML.

  2. Cell Libraries

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA contract led to the development of faster and more energy efficient semiconductor materials for digital integrated circuits. Gallium arsenide (GaAs) conducts electrons 4-6 times faster than silicon and uses less power at frequencies above 100-150 megahertz. However, the material is expensive, brittle, fragile and has lacked computer automated engineering tools to solve this problem. Systems & Processes Engineering Corporation (SPEC) developed a series of GaAs cell libraries for cell layout, design rule checking, logic synthesis, placement and routing, simulation and chip assembly. The system is marketed by Compare Design Automation.

  3. Modification of the GS LT Paired-end Library Protocol for Constructing Longer Insert Size Libraries

    SciTech Connect

    Peng, Ze; Peng, Ze; Hamilton, Matthew; Ting, Sara; Tu, Hank; Goltsman, Eugene; Lapidus, Alla; Lucas, Susan; Cheng, Jan-Fang

    2008-05-22

    Paired-end library sequencing has been proven useful in scaffold construction during de novo assembly of genomic sequences. The ability of generating mate pairs with 8 Kb or greater insert sizes is especially important for genomes containing long repeats. While the current 454 GS LT Paired-end library preparation protocol can successfully construct libraries with 3 Kb insert size, it fails to generate longer insert sizes because the protocol is optimized to purify shorter fragments. We have made several changes in the protocol in order to increase the fragment length. These changes include the use of Promega column to increase the yield of large size DNA fragments, two gel purification steps to remove contaminated short fragments, and a large reaction volume in the circularization step to decrease the formation of chimeras. We have also made additional changes in the protocol to increase the overall quality of the libraries. The quality of the libraries are measured by a set of metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverage after the assemblies are completed. Our data indicated that all these changes have improved the quality of the longer insert size libraries.

  4. State Virtual Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2003-01-01

    Virtual library? Electronic library? Digital library? Online information network? These all apply to the growing number of Web-based resource collections managed by consortiums of state library entities. Some, like "INFOhio" and "KYVL" ("Kentucky Virtual Library"), have been available for a few years, but others are just starting. Searching for…

  5. Library Directions in 1988.

    ERIC Educational Resources Information Center

    DeCandido, GraceAnne A.

    1989-01-01

    Reviews major library issues and events of 1988, including: (1) the Federal Bureau of Investigation's Library Awareness Program; (2) cooperation with USSR libraries; (3) library finance; (4) preservation; and (5) special programing. News about a number of prominent library professionals is included in a sidebar. (MES)

  6. Genealogy and Libraries.

    ERIC Educational Resources Information Center

    Carothers, Diane Foxhill; And Others

    1983-01-01

    Ten articles describe genealogical collections of the National Archives and Library of Congress, state archives programs, academic research libraries, Allen County Public Library (Indiana), Newberry Library, Church of Jesus Christ of Latter-Day Saints genealogical library, and New England Historic Genealogical Society, and approaches to Black…

  7. Marketing the Virtual Library

    ERIC Educational Resources Information Center

    Fagan, Jody Condit

    2009-01-01

    Far more people are familiar with their local public or college library facility than their library's website and online resources. In fact, according to a recent survey, 96% of Americans said they had visited a library in person, but less than one-third have visited their online library. Since everyone agrees that online library resources are…

  8. Library Handbook for Faculty.

    ERIC Educational Resources Information Center

    Martinez, Angelina, Ed.

    Discussions of library resources, services and related activities as well as library materials selection and acquisition are provided for faculty to facilitate and enhance their use of the library. Included in the library resources section are books, periodicals, microforms, and special collections and archives. Instruction in library use,…

  9. Genome sequence of cultivated Upland cotton (Gossypium hirsutum TM-1) provides insights into genome evolution.

    PubMed

    Li, Fuguang; Fan, Guangyi; Lu, Cairui; Xiao, Guanghui; Zou, Changsong; Kohel, Russell J; Ma, Zhiying; Shang, Haihong; Ma, Xiongfeng; Wu, Jianyong; Liang, Xinming; Huang, Gai; Percy, Richard G; Liu, Kun; Yang, Weihua; Chen, Wenbin; Du, Xiongming; Shi, Chengcheng; Yuan, Youlu; Ye, Wuwei; Liu, Xin; Zhang, Xueyan; Liu, Weiqing; Wei, Hengling; Wei, Shoujun; Huang, Guodong; Zhang, Xianlong; Zhu, Shuijin; Zhang, He; Sun, Fengming; Wang, Xingfen; Liang, Jie; Wang, Jiahao; He, Qiang; Huang, Leihuan; Wang, Jun; Cui, Jinjie; Song, Guoli; Wang, Kunbo; Xu, Xun; Yu, John Z; Zhu, Yuxian; Yu, Shuxun

    2015-05-01

    Gossypium hirsutum has proven difficult to sequence owing to its complex allotetraploid (AtDt) genome. Here we produce a draft genome using 181-fold paired-end sequences assisted by fivefold BAC-to-BAC sequences and a high-resolution genetic map. In our assembly 88.5% of the 2,173-Mb scaffolds, which cover 89.6%∼96.7% of the AtDt genome, are anchored and oriented to 26 pseudochromosomes. Comparison of this G. hirsutum AtDt genome with the already sequenced diploid Gossypium arboreum (AA) and Gossypium raimondii (DD) genomes revealed conserved gene order. Repeated sequences account for 67.2% of the AtDt genome, and transposable elements (TEs) originating from Dt seem more active than from At. Reduction in the AtDt genome size occurred after allopolyploidization. The A or At genome may have undergone positive selection for fiber traits. Concerted evolution of different regulatory mechanisms for Cellulose synthase (CesA) and 1-Aminocyclopropane-1-carboxylic acid oxidase1 and 3 (ACO1,3) may be important for enhanced fiber production in G. hirsutum.

  10. Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries.

    PubMed

    Bouhajja, E; McGuire, M; Liles, M R; Bataille, G; Agathos, S N; George, I F

    2017-01-01

    The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

  11. Bacteriocin protein BacL1 of Enterococcus faecalis targets cell division loci and specifically recognizes L-Ala2-cross-bridged peptidoglycan.

    PubMed

    Kurushima, Jun; Nakane, Daisuke; Nishizaka, Takayuki; Tomita, Haruyoshi

    2015-01-01

    Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins, BacL1 and BacA. We previously reported that BacL1 protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan D-isoglutamyl-L-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, the target of BacL1 remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL1 specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL1 and BacA. The static growth state also abolished the binding and targeting of BacL1 to the cell division-associated site. Furthermore, the targeting of BacL1 was detectable among Gram-positive bacteria with an L-Ala-L-Ala-cross-bridging peptidoglycan, including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL1 specifically targets the L-Ala-L-Ala-cross-bridged peptidoglycan and potentially lyses the E. faecalis cells during cell division.

  12. Databases and information integration for the Medicago truncatula genome and transcriptome.

    PubMed

    Cannon, Steven B; Crow, John A; Heuer, Michael L; Wang, Xiaohong; Cannon, Ethalinda K S; Dwan, Christopher; Lamblin, Anne-Francoise; Vasdewani, Jayprakash; Mudge, Joann; Cook, Andrew; Gish, John; Cheung, Foo; Kenton, Steve; Kunau, Timothy M; Brown, Douglas; May, Gregory D; Kim, Dongjin; Cook, Douglas R; Roe, Bruce A; Town, Chris D; Young, Nevin D; Retzel, Ernest F

    2005-05-01

    An international consortium is sequencing the euchromatic genespace of Medicago truncatula. Extensive bioinformatic and database resources support the marker-anchored bacterial artificial chromosome (BAC) sequencing strategy. Existing physical and genetic maps and deep BAC-end sequencing help to guide the sequencing effort, while EST databases provide essential resources for genome annotation as well as transcriptome characterization and microarray design. Finished BAC sequences are joined into overlapping sequence assemblies and undergo an automated annotation process that integrates ab initio predictions with EST, protein, and other recognizable features. Because of the sequencing project's international and collaborative nature, data production, storage, and visualization tools are broadly distributed. This paper describes databases and Web resources for the project, which provide support for physical and genetic maps, genome sequence assembly, gene prediction, and integration of EST data. A central project Web site at medicago.org/genome provides access to genome viewers and other resources project-wide, including an Ensembl implementation at medicago.org, physical map and marker resources at mtgenome.ucdavis.edu, and genome viewers at the University of Oklahoma (www.genome.ou.edu), the Institute for Genomic Research (www.tigr.org), and Munich Information for Protein Sequences Center (mips.gsf.de).

  13. Transposase mediated construction of RNA-seq libraries.

    PubMed

    Gertz, Jason; Varley, Katherine E; Davis, Nicholas S; Baas, Bradley J; Goryshin, Igor Y; Vaidyanathan, Ramesh; Kuersten, Scott; Myers, Richard M

    2012-01-01

    RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions. We generated high-quality RNA-seq libraries from as little as 10 pg of mRNA (∼1 ng of total RNA) with this approach. We also present a strand-specific RNA-seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. The directional RNA-seq libraries maintain the same quality as the nondirectional libraries, while showing a high degree of strand specificity, such that 99.5% of reads map to the expected genomic strand. Each transposon-based library construction method performed well when compared with standard RNA-seq library construction methods with regard to complexity of the libraries, correlation between biological replicates, and the percentage of reads that align to the genome as well as exons. Our results show that high-quality RNA-seq libraries can be constructed efficiently and in an automatable fashion using transposition technology.

  14. Homecoming for Library Symbol.

    ERIC Educational Resources Information Center

    Egan, Bessie

    1987-01-01

    Discusses the significance and development of the library symbol and the history of its acceptance by the American Library Association (ALA) and the Canadian Library Association (CLA). Suggestions are made for its use. (CLB)

  15. Structural identification of DnaK binding sites within bovine and sheep bactenecin Bac7.

    PubMed

    Zahn, Michael; Kieslich, Bjorn; Berthold, Nicole; Knappe, Daniel; Hoffmann, Ralf; Strater, Norbert

    2014-04-01

    Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified.

  16. The BAC-process for treatment of waste water containing non-ionogenic synthetic surfactants.

    PubMed

    Sirotkin, A S; Koshkina LYu; Ippolitov, K G

    2001-09-01

    In this paper experimental results on the biological-activated carbon (BAC)-process for biological degradation and adsorption of non-ionogenic surfactants (NISS) in the waste water treatment are discussed. It is shown that the mechanism of the BAC-process is not the simple addition but the synergetic combination of biodegradation and carbon adsorption. The major aspects of such synergism are the biological regeneration (bioregeneration) of the adsorbent and the reduction of the toxic effect of waste water contaminants on microorganisms. It is shown that the basis of the bioregeneration process is the desorption of substances previously adsorbed on the activated carbon. The desorption from micropores takes place because of the reverse concentration gradient, due to the microbial degradation of waste water contaminants in the liquid phase. The desorption from mesopores is also supported by the activity of microorganisms exoenzymes. Thus, the process of bioregeneration is featured by two non-contradictory hypotheses.

  17. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer.

    PubMed

    Wu, Zhenfang; Xu, Zhiqian; Zou, Xian; Zeng, Fang; Shi, Junsong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Li, Zicong

    2013-12-01

    The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.

  18. Cloning and expression of manganese superoxide dismutase of the silkworm, Bombyx mori by Bac-to-Bac/BmNPV Baculovirus expression system.

    PubMed

    Yue, Wanfu; Miao, Yungen; Li, Xinghua; Wu, Xiaofeng; Zhao, Aichun; Nakagaki, Masao

    2006-11-01

    Superoxide dismutase (SODs) are metalloenzymes that catalyze the dismutation of the superoxide anion to molecular oxygen and hydrogen peroxide and, thus, form a crucial part of the cellular antioxidant defense mechanism. In this paper, we used the total fat body RNA of silkworm, Bombyx mori L. to clone and sequence a 648-bp Mn-SOD cDNA fragment through RT-PCR. Furthermore, a newly established Bac-to-Bac/BmNPV Baculovirus expression system was used to overexpress the recombinant Mn-SOD enzyme in silkworm larvae. The hemolymph was collected from the infected larvae 96 h post-infection and subjected to a 12 % SDS-PAGE and Western blotting. A 18.0-kDa protein was visualized after rBacmid/BmNPV/SOD infection. The SOD enzyme activity was determined with a tetrazolium salt for detection of superoxide radicals generated by xanthine and xanthine oxidase and its peak appeared in 96 h post-infection with 2.7 times of the control larvae. The availability of large quantities of SOD that the silkworm provides should greatly facilitate the future research and testing of this protein for potential application in medicine.

  19. Efficient production of canine interferon-alpha in silkworm Bombyx mori by use of a BmNPV/Bac-to-Bac expression system.

    PubMed

    Na, Zhao; Huipeng, Yao; Lipan, Lan; Cuiping, Cao; Umashankar, M L; Xingmeng, Lu; Xiaofeng, Wu; Bing, Wang; Weizheng, Cui; Cenis, J L

    2008-02-01

    We exploited the silkworm Bombyx mori for the production of recombinant canine interferon-alpha (CaIFN-alpha). The recombinant baculovirus harboring canine interferon gene was rapidly generated by the BmNPV/Bac-to-Bac system that was recently developed. In B. mori-derived cell lines, the expression of the recombinant protein reached maximal levels around 72-96 h post-infection. For the isolation of the expressed recombinant protein from B. mori larvae, the whole bodies of the infected larvae were homogenized, and the expressed protein was purified by affinity chromatography. Based on the fact that the recombinant CaIFN-alpha showed two bands on the sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern, the expressed protein was thought to be glycosylated. The rCaIFN-alpha yield was about 528 microg per larva, showing that the expression in silkworm was successful. Furthermore, the recombinant protein was proven to be able to inhibit the infection of Madin-Darby canine kidney cells by the vesicular stomatitis virus, indicating that it is biologically active in vitro. The method established in this study provides an efficient way to produce a large amount of CaIFN-alpha and paves the way for further utilization of this protein as a therapeutic agent or vaccine adjuvant in dogs.

  20. GENSAT BAC Cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits

    PubMed Central

    Gerfen, Charles R.; Paletzki, Ronald; Heintz, Nathaniel

    2013-01-01

    Summary Recent development of molecular genetic techniques are rapidly advancing understanding of the functional role of brain circuits in behavior. Critical to this approach is the ability to target specific neuron populations and circuits. The collection of over 250 BAC Cre-recombinase driver lines produced by the GENSAT project provides a resource for such studies. Here we provide characterization of GENSAT BAC-Cre driver lines with expression in specific neuroanatomical pathways within the cerebral cortex and basal ganglia. PMID:24360541

  1. Molecular Evolution of piggyBac Superfamily: From Selfishness to Domestication

    PubMed Central

    Bouallègue, Maryem; Rouault, Jacques-Deric; Hua-Van, Aurélie; Makni, Mohamed

    2017-01-01

    The piggyBac transposable element was originally isolated from the cabbage looper moth, Trichoplusia ni, in the 1980s. Despite its early discovery and specificity compared to the other Class II elements, the diversity and evolution of this superfamily have been only partially analyzed. Two main types of elements can be distinguished: the piggyBac-like elements (PBLE) with terminal inverted repeats, untranslated region, and an open reading frame encoding a transposase, and the piggyBac-derived sequences (PGBD), containing a sequence derived from a piggyBac transposase, and which correspond to domesticated elements. To define the distribution, their structural diversity and phylogenetic relationships, analyses were conducted using known PBLE and PGBD sequences to scan databases. From this data mining, numerous new sequences were characterized (50 for PBLE and 396 for PGBD). Structural analyses suggest that four groups of PBLE can be defined according to the presence/absence of sub-terminal repeats. The transposase is characterized by highly variable catalytic domain and C-terminal region. There is no relationship between the structural groups and the phylogeny of these PBLE elements. The PGBD are clearly structured into nine main groups. A new group of domesticated elements is suspected in Neopterygii and the remaining eight previously described elements have been investigated in more detail. In all cases, these sequences are no longer transposable elements, the catalytic domain of the ancestral transposase is not always conserved, but they are under strong purifying selection. The phylogeny of both PBLE and PGBD suggests multiple and independent domestication events of PGBD from different PBLE ancestors. PMID:28082605

  2. Efficacy of VectoBac (Bacillus thuringiensis variety israelensis) formulations for mosquito control in Australia.

    PubMed

    Russell, Tanya L; Brown, Michael D; Purdie, David M; Ryan, Peter A; Kay, Brian H

    2003-12-01

    Laboratory bioassays were conducted on the efficacy of a water-dispersible granule (WG) formulation of Bacillus thuringiensis variety israelensis (VectoBac WG; active ingredient [AI]: 3,000 Bti international toxic units [ITU]/mg) against third instars of six common Australian mosquito species, Aedes aegypti (L.), Ochlerotatus vigilax (Skuse), Ochlerotatus notoscriptus (Skuse), Culex sitiens Wiedemann, Culex annulirostris Skuse, and Culex quinquefasciatus Say. The normal model for log-linear mortality data was used to determine laboratory 48-h LC50 and LC95 values. The target mosquito species tested were extremely sensitive to the VectoBac WG formulation, with the most sensitive species (Cx. annulirostris and Cx. quinquefasciatus, LC95 value of 0.019 ppm) being twice as susceptible as the most tolerant (Oc. notoscriptus, LC95 value of 0.037 ppm). Cx. annulirostris was selected as a target species for a small-plot evaluation of VectoBac WG and VectoBac 12 aqueous solution (AS) ([AI]: 1,200 Bti ITU/mg) efficacy over time, in freshwater in southeastern Queensland, Australia. Replicated cohorts of caged third instars were exposed weekly to six concentrations of WG formulation (0.004-0.13 ppm) and three concentrations of the 12AS formulation (0.04-0.13 ppm). In water with high organic content, treatment concentrations of 0.008 ppm WG and 0.04 ppm 12AS and above produced significant larval control (> or = 96%) at 48 h posttreatment, with no residual control at week 1. Water quality was not affected by treatment with either formulation.

  3. BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus.

    PubMed

    Argilaguet, Jordi M; Pérez-Martín, Eva; López, Sergio; Goethe, Martin; Escribano, J M; Giesow, Katrin; Keil, Günther M; Rodríguez, Fernando

    2013-04-01

    Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17 days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

  4. Molecular evolution of piggyBac superfamily: from selfishness to domestication.

    PubMed

    Bouallègue, Maryem; Rouault, Jacques-Deric; Hua-Van, Aurélie; Makni, Mohamed; Capy, Pierre

    2017-01-12

    The piggyBac transposable element was originally isolated from the cabbage looper moth, Trichoplusia ni, in the 1980s. Despite its early discovery and specificity compared to the other Class II elements, the diversity and evolution of this superfamily have been only partially analyzed. Two main types of elements can be distinguished: the piggyBac-like elements (PBLE) with terminal inverted repeats (TIR), UTR and an ORF encoding a transposase, and the piggyBac-derived sequences (PGBD), containing a sequence derived from a piggyBac transposase, and which correspond to domesticated elements. To define the distribution, their structural diversity and phylogenetic relationships, analyses were conducted using known PBLE and PGBD sequences to scan databases. From this data mining, numerous new sequences were characterized (50 for PBLE and 396 for PGBD). Structural analyses suggest that four groups of PBLE can be defined according to the presence/absence of sub-terminal repeats. The transposase is characterized by highly variable catalytic domain and C-terminal region. There is no relationship between the structural groups and the phylogeny of these PBLE elements. The PGBD are clearly structured into nine main groups. A new group of domesticated elements is suspected in Neopterygii and the remaining eight previously described elements have been investigated in more detail. In all cases, these sequences are no longer transposable elements, the catalytic domain of the ancestral transposase is not always conserved, but they are under strong purifying selection. The phylogeny of both PBLE and PGBD suggests multiple and independent domestication events of PGBD from different PBLE ancestors.

  5. Generation of bioactive peptides by biological libraries.

    PubMed

    Mersich, Christa; Jungbauer, Alois

    2008-01-15

    Biological libraries are powerful tools for discovery of new ligands as well as for identification of cellular interaction partners. Since the first development of the first biological libraries in form of phage displays, numerous biological libraries have been developed. For the development of new ligands, the usage of synthetic oligonucleotides is the method of choice. Generation of random oligonucleotides has been refined and various strategies for random oligonucleotide design were developed. We trace the progress and design of new strategies for the generation of random oligonucleotides, and include a look at arising diversity biases. On the other hand, genomic libraries are widely employed for investigation of cellular protein-protein interactions and targeted search of proteomic binding partners. Expression of random peptides and proteins in a linear form or integrated in a scaffold can be facilitated both in vitro and in vivo. A typical in vitro system, ribosome display, provides the largest available library size. In vivo methods comprise smaller libraries, the size of which depends on their transformation efficiency. Libraries in different hosts such as phage, bacteria, yeast, insect cells, mammalian cells exhibit higher biosynthetic capabilities. The latest library systems are compared and their strengths and limitations are reviewed.

  6. Enforcement following 0.08% BAC law change: Sex-specific consequences of changing arrest practices?

    PubMed Central

    Schwartz, Jennifer; Davaran, Ardavan

    2013-01-01

    This research evaluated effects of stricter 0.08% BAC drunken driving law on changes in sex-specific DUI arrest rates, controlling for increased law enforcement resources and shifts in DUI-related behaviors. Another main purpose, the study assessed female/male differences in arrest increases due to broader enforcement standards and efforts. Panel data was assembled for 24 states over 1990–2007 on DUI arrests, alcohol policy, law enforcement resources, drinking and drunken driving prevalence. Two-way fixed-effects seemingly unrelated regression models predicted female versus male changes in DUI arrests following implementation of lower legal limits of intoxication, net controls. Findings suggest, first, a broader legal definition of drunken driving intending to officially sanction less serious offenders (0.08% vs. 0.10% BAC) was associated with increased DUI arrests for both sexes. Second, growth in specialized DUI-enforcement units also was related to increased arrests. Whereas male and female arrest trends were equally affected by the direct net-widening effects of 0.08% BAC alcohol-policy, specialized DUI-enforcement efforts to dig deeper into the offender-pool had stronger arrest-producing effects on females, particularly prior to law change. Specifying how changes in law and enforcement resources affect arrest outcomes is an important precursor to alcohol-policy analyses of effectiveness. A potential unintended consequence, effects of law and enforcement may differ across population segments. PMID:23773958

  7. A high-resolution PAC and BAC map of the SCA2 region.

    PubMed

    Nechiporuk, T; Nechiporuk, A; Sahba, S; Figueroa, K; Shibata, H; Chen, X N; Korenberg, J R; de Jong, P; Pulst, S M

    1997-09-15

    The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1. To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene. The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers. We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before. In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable. Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric. The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.

  8. Evaluating the biosafety of conventional and O3-BAC process and its relationship with NOM characteristics.

    PubMed

    Liao, Xiaobin; Zou, Rusen; Chen, Chao; Yuan, Baoling; Zhou, Zhenming; Zhang, Xiaojian

    2017-03-08

    It is the priority to guarantee biosafety for drinking water treatment. The objective of this study was to evaluate the impact of widely applied conventional and ozone-biological activated carbon (O3-BAC) advanced treatment technology on biosafety of drinking water. The items, including assimilable organic carbon (AOC), biodegradable dissolved organic carbon (BDOC), heterotrophic plate counts (HPCs) and the microorganism community structures, were used to evaluate the biosafety. Moreover, their relationships with molecular weights (MWs) and fluorescence intensity of dissolved organic matter were investigated. The results indicated that the technology provided a considerable gain in potable water quality by decreasing dissolved organic carbon (DOC, from 5.05 to 1.71 mg/L), AOC (from 298 to 131 μg/L), BDOC (from 1.39 to 0.24 mg/L) and HPCs (from 275 to 10 CFU/mL). Ozone brought an increase in DOC with low MW <1 kDa, which accompanies with an increase in AOC/BDOC concentration, which could be reduced effectively by subsequent BAC process. The formation of AOC/BDOC was closely related to DOC with low MWs and aromatic protein. Bacteria could be released from BAC filter, resulting in an increase in HPC and the presence of pathogenic bacteria in effluent, while the post sand filter could further guarantee the biosafety of finished water.

  9. [Study on the combination of preozonation and post-ozonation-BAC process for drinking water treatment].

    PubMed

    Guo, Zhao-hai; Yang, Min; Zhang, Yu; Pei, Yi-shan; Zhang, Jun-zhi; Hirotsuji, Juni

    2005-11-01

    This study was conducted to illustrate the effectiveness of preozonation or O3-BAC or combination of these two process in controlling DBPs for treating a dam source water in South China through SBR and continuous flow (200 L/h) experiments and the variables in the treatment train included the point of preozonation with respect to coagulation; the point of ozonation with respect to BAC, the ozone dosage required for preozonation and the Br- content of raw water. Results indicated the reduction in DBP formation potential closely paralleled the reduction in UV absorbance, and trihalomethane and haloacetic acid formation potential can be removed under a dose of preozone at 0.5 - 1.0 mg/L, at the same time, ozone by-products (bromate and formaldehyde) can be controlled in low level. The continuous flow experiment results show that combination of preozonation and O3-BAC process has obviously positive effects on the removals of particles with a size above 2 microm, COD(Mn), and TOC. However, DBPs formation can be controlled by the removal of organic matters.

  10. A Tie2-driven BAC-TRAP transgenic line for in vivo endothelial gene profiling.

    PubMed

    Santhosh, Devi; Huang, Zhen

    2016-03-01

    Recent technological innovations including bacterial artificial chromosome-based translating ribosome affinity purification (BAC-TRAP) have greatly facilitated analysis of cell type-specific gene expression in vivo, especially in the nervous system. To better study endothelial gene expression in vivo, we have generated a BAC-TRAP transgenic mouse line where the L10a ribosomal subunit is tagged with EGFP and placed under the control of the endothelium-specific Tie2 (Tek) promoter. We show that transgene expression in this line is widely, but specifically, detected in endothelial cells in several brain regions throughout pre- and postnatal development, as well as in other organs. We also show that this line results in highly significant enrichment of endothelium-specific mRNAs from brain tissues at different stages. This BAC-TRAP line therefore provides a useful genetic tool for in vivo endothelial gene profiling under various developmental, physiological, and pathological conditions. genesis 54:136-145, 2016. © 2016 Wiley Periodicals, Inc.

  11. Virtual Library on Genetics from Oak Ridge National Laboratory

    DOE Data Explorer

    The World Wide Web (WWW) Virtual Library is a collaborative effort to provide topic indices that break down into many subtopics guiding users to vast resources of information around the world. ORNL hosts the Virtual Library on Genetics as part of the WWWVL's Biosciences topic area. The VL on Genetics is also a collection of links to information resources that supported the DOE Human Genome Project. That project has now evolved into Genomics: GTL. GTL is DOE's next step in genomics--builds on data and resources from the Human Genome Project, the Microbial Genome Program, and systems biology. GTL will accelerate understanding of dynamic living systems for solutions to DOE mission challenges in energy and the environment. The section of the Virtual Library on Genetics that is titled Organisms guides users to genetic information resources and gene sequences for animals, insects, microbes, and plant life.

  12. Combined BAC and MIEX pre-treatment of secondary wastewater effluent to reduce fouling of nanofiltration membranes.

    PubMed

    Aryal, Ashok; Sathasivan, Arumugam; Heitz, Anna; Zheng, Gang; Nikraz, Hamid; Ginige, Maneesha P

    2015-03-01

    Biological activated carbon (BAC) and magnetic ion exchange resin (MIEX) were used to pre-treat secondary wastewater effluent (SWWE) and assessed for their capacity to reduce fouling of a nanofiltration membrane. BAC pre-treated water facilitated a lower but a steady flux while MIEX treated water resulted in a higher but a rapidly declining flux. Their combined use increased average flux from 58 to 89%. MIEX combined with BAC, in that order, was superior in reducing membrane fouling. Measurement of average Stokes radius (m) and apparent molecular weight distribution of dissolved organic matter (DOM), by nuclear magnetic resonance (NMR) and liquid chromatography organic carbon detection (LC-OCD), respectively, revealed that the microbial activity of BAC changed the nature of organic matter, probably by increasing the size of DOM molecules. BAC generally decreased the lower apparent molecular weight (LMW) fraction of dissolved organic carbon (DOC). Hence, the removal of LMW DOC and an increase of average Stokes radius (m) of DOM appeared to be important in facilitating a longer steady flux. Specifically, the combined MIEX/BAC pre-treatments appeared to target and reduce the foulants in SWWE that are largely responsible for the reduction of flux in nanofiltration membranes.

  13. Shearing DNA for genomic library construction.

    PubMed

    Hengen, P N

    1997-07-01

    Methods and reagents is a unique monthly column that highlights current discussion in the newsgroup bionet.molibio.methds-reagnts, available on the internet. This month's column discusses the pros and cons of various techniques used to shear DNA for shotgun cloning. For details on how to partake in the newsgroup, see the accompanying box.

  14. Construction of a Rye BAC Library for Utilization in Physical Mapping of an Aluminum Tolerance Gene Complex

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminium, the most abundant metal on earth, is highly toxic to plant growth and is found in about 2.5 billion hectares of acid soils, including more than 130 million hectares in the United States. Many of the world’s farmers are living on marginal soils that offer a stressful environment for plant ...

  15. Scaling up the 454 Titanium Library Construction and Pooling of Barcoded Libraries

    SciTech Connect

    Phung, Wilson; Hack, Christopher; Shapiro, Harris; Lucas, Susan; Cheng, Jan-Fang

    2009-03-23

    We have been developing a high throughput 454 library construction process at the Joint Genome Institute to meet the needs of de novo sequencing a large number of microbial and eukaryote genomes, EST, and metagenome projects. We have been focusing efforts in three areas: (1) modifying the current process to allow the construction of 454 standard libraries on a 96-well format; (2) developing a robotic platform to perform the 454 library construction; and (3) designing molecular barcodes to allow pooling and sorting of many different samples. In the development of a high throughput process to scale up the number of libraries by adapting the process to a 96-well plate format, the key process change involves the replacement of gel electrophoresis for size selection with Solid Phase Reversible Immobilization (SPRI) beads. Although the standard deviation of the insert sizes increases, the overall quality sequence and distribution of the reads in the genome has not changed. The manual process of constructing 454 shotgun libraries on 96-well plates is a time-consuming, labor-intensive, and ergonomically hazardous process; we have been experimenting to program a BioMek robot to perform the library construction. This will not only enable library construction to be completed in a single day, but will also minimize any ergonomic risk. In addition, we have implemented a set of molecular barcodes (AKA Multiple Identifiers or MID) and a pooling process that allows us to sequence many targets simultaneously. Here we will present the testing of pooling a set of selected fosmids derived from the endomycorrhizal fungus Glomus intraradices. By combining the robotic library construction process and the use of molecular barcodes, it is now possible to sequence hundreds of fosmids that represent a minimal tiling path of this genome. Here we present the progress and the challenges of developing these scaled-up processes.

  16. Validation of the French version of the BACS (the brief assessment of cognition in schizophrenia) among 50 French schizophrenic patients.

    PubMed

    Bralet, Marie-Cécile; Falissard, Bruno; Neveu, Xavier; Lucas-Ross, Margaret; Eskenazi, Anne-Marie; Keefe, Richard S E

    2007-09-01

    Schizophrenic patients demonstrate impairments in several key dimensions of cognition. These impairments are correlated with important aspects of functional outcome. While assessment of these cognition disorders is increasingly becoming a part of clinical and research practice in schizophrenia, there is no standard and easily administered test battery. The BACS (Brief Assessment of Cognition in Schizophrenia) has been validated in English language [Keefe RSE, Golberg TE, Harvey PD, Gold JM, Poe MP, Coughenour L. The Brief Assessment of Cognition in Schizophrenia: reliability, sensibility, and comparison with a standard neurocognitive battery. Schizophr. Res 2004;68:283-97], and was found to be as sensitive to cognitive dysfunction as a standard battery of tests, with the advantage of requiring less than 35 min to complete. We developed a French adaptation of the BACS and this study tested its ease of administration and concurrent validity. Correlation analyses between the BACS (version A) and a standard battery were performed. A sample of 50 stable schizophrenic patients received the French Version A of the BACS in a first session, and in a second session a standard battery. All the patients completed each of the subtests of the French BACS . The mean duration of completion for the BACS French version was 36 min (S.D.=5.56). A correlation analysis between the BACS (version A) global score and the standard battery global score showed a significant result (r=0.81, p<0.0001). The correlation analysis between the BACS (version A) sub-scores and the standard battery sub-scores showed significant results for verbal memory, working memory, verbal fluency, attention and speed of information processing and executive functions (p<0.001) and for motor speed (p<0.05). The French Version of the BACS is easier to use in French schizophrenic patients compared to a standard battery (administration shorter and completion rate better) and its good psychometric properties suggest

  17. DNA libraries for the construction of phage libraries: statistical and structural requirements and synthetic methods.

    PubMed

    Lindner, Thomas; Kolmar, Harald; Haberkorn, Uwe; Mier, Walter

    2011-02-15

    Peptide-based molecular probes identified by bacteriophage (phage) display technology expand the peptide repertoire for in vivo diagnosis and therapy of cancer. Numerous peptides that bind cancer-associated antigens have been discovered by panning phage libraries. However, until now only few of the peptides selected by phage display have entered clinical applications. The success of phage derived peptides essentially depends on the quality of the library screened. This review summarizes the methods to achieve highly homogenous libraries that cover a maximal sequence space. Biochemical and chemical strategies for the synthesis of DNA libraries and the techniques for their integration into the viral genome are discussed in detail. A focus is set on the methods that enable the exclusion of disturbing sequences. In addition, the parameters that define the variability, the minimal numbers of copies per library and the use of alternating panning cycles to avoid the loss of selected hits are evaluated.

  18. The New Library Professional

    ERIC Educational Resources Information Center

    Wilder, Stanley

    2007-01-01

    This article discusses what the growing generation gap among library employees mean for academic research libraries and for the profession. Viewed collectively, the members of the under-35 cohort are a harbinger of a new kind of academic library professional, one whose traits bear directly on the ability of libraries to thrive amid the continuing…

  19. Growing Competition for Libraries.

    ERIC Educational Resources Information Center

    Gibbons, Susan

    2001-01-01

    Describes the Questia subscription-based online academic digital books library. Highlights include weaknesses of the collection; what college students want from a library; importance of marketing; competition for traditional academic libraries that may help improve library services; and the ability of Questia to overcome barriers and…

  20. California: Library Information Technologies.

    ERIC Educational Resources Information Center

    Will, Barbara, Ed.

    1996-01-01

    Describes six information technology projects in California libraries, including Internet access in public libraries; digital library developments at the University of California, Berkeley; the World Wide Web home page for the state library; Pacific Bell's role in statewide connectivity; state government initiatives; and services of the state…