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Sample records for genomic bac library

  1. Construction and characterization of a bovine BAC library with four genome-equivalent coverage.

    PubMed

    Eggen, A; Gautier, M; Billaut, A; Petit, E; Hayes, H; Laurent, P; Urban, C; Pfister-Genskow, M; Eilertsen, K; Bishop, M D

    2001-01-01

    A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents. PMID:11712974

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

    PubMed

    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

  3. 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

  4. 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. PMID:26438131

  5. Selection of chromosome 22-specific clones from human genomic BAC library using a chromosome-specific cosmid library pool

    SciTech Connect

    Kim, U.J.; Shizuya, H.; Birren, B.

    1994-07-15

    A new approach to rapidly identify chromosome-specific subsets of clones from a total human genomic library is described. The authors report here the results of screening a human bacterial artificial chromosome (BAC) library using the total pool of clones from a chromosome 22-specific cosmid library as a composite probe. The human BAC library was gridded on filters at high density and hybridized with DNA from the pooled chromosome 22-specific Lawrist library under suppressive conditions. In a single hybridization, they picked 280 candidates from the BAC library representing over 30,000 clones (or 1.2 x coverage of human genome). This subset contained more than 60% of the chromosome 22-specific BAC clones that were previously found to be present in the original BAC library. In principle, this approach can be applied to select a subset of clones from other global libraries with relatively large inserts using a pool from a regional library as a composite probe. It is important to note that the target and probe libraries must be based on vectors that share no homology with each other. 8 refs., 2 figs., 2 tabs.

  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. Enhancing genome investigations in the mosquito Culex quinquefasciatus via BAC library construction and characterization

    PubMed Central

    2011-01-01

    Background Culex quinquefasciatus (Say) is a major species in the Culex pipiens complex and an important vector for several human pathogens including West Nile virus and parasitic filarial nematodes causing lymphatic filariasis. It is common throughout tropical and subtropical regions and is among the most geographically widespread mosquito species. Although the complete genome sequence is now available, additional genomic tools are needed to improve the sequence assembly. Findings We constructed a bacterial artificial chromosome (BAC) library using the pIndigoBAC536 vector and HindIII partially digested DNA isolated from Cx. quinquefasciatus pupae, Johannesburg strain (NDJ). Insert size was estimated by NotI digestion and pulsed-field gel electrophoresis of 82 randomly selected clones. To estimate genome coverage, each 384-well plate was pooled for screening with 29 simple sequence repeat (SSR) and five gene markers. The NDJ library consists of 55,296 clones arrayed in 144 384-well microplates. Fragment insert size ranged from 50 to 190 kb in length (mean = 106 kb). Based on a mean insert size of 106 kb and a genome size of 579 Mbp, the BAC library provides ~10.1-fold coverage of the Cx. quinquefasciatus genome. PCR screening of BAC DNA plate pools for SSR loci from the genetic linkage map and for four genes associated with reproductive diapause in Culex pipiens resulted in a mean of 9.0 positive plate pools per locus. Conclusion The NDJ library represents an excellent resource for genome assembly enhancement and characterization in Culex pipiens complex mosquitoes. PMID:21914202

  8. Construction and characterization of a Lipotes vexillifer genomic DNA BAC library.

    PubMed

    Du, Bo; Zhang, Xian-Feng; Fang, Sheng-Guo; Wang, Ding

    2007-04-01

    We constructed a genomic DNA library for Lipotes vexillifer (L. vexillifer), the Baiji or Yangtze River dolphin, one of the most endangered mammals in the world. The library consists of 149,000 BAC clones, with an average insert size of 83 kb, representing approximately 3.4 haploid genome equivalents. PCR amplification of four known L. vexillifer genes yielded two to four positive clones each. To demonstrate the utility of this library, we isolated and sequenced the L. vexillifer alpha lactalbumin gene, which is a gene specific to mammals and one which has been widely used as molecular tool in phylogenetic analysis. We also end-sequenced 20 randomly selected clones, resulting in the identification of at least five new L. vexillifer genes, five SSR loci, and one SINE locus. These results suggest that this library is a valuable resource for candidate gene cloning, physical mapping, and genome sequencing of this important and threatened species. PMID:17867838

  9. 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

  10. 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

  11. Genome Enablement of the Notothenioidei: Genome Size Estimates from 11 Species and BAC Libraries from 2 Representative Taxa

    PubMed Central

    DETRICH, H. WILLIAM; STUART, ANDREW; SCHOENBORN, MICHAEL; PARKER, SANDRA K.; METHÉ, BARBARA A.; AMEMIYA, CHRIS T.

    2013-01-01

    The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species flock in the cold Southern Ocean surrounding Antarctica. To enable genome-level studies of these psychrophilic fishes, we estimated the sizes of the genomes of 11 Antarctic species and generated high-quality BAC libraries for 2, the notothen Notothenia coriiceps and the icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families, [e.g., the icefishes (Channichthyidae)], was accompanied by genome expansion. Species (n = 6) of the basal family Nototheniidae had C values that ranged between 0.98 and 1.20 pg, whereas those of the icefishes, the notothenioid crown group, were 1.66–1.83 pg (n = 4 species). The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprised 12X and 10X coverage of the respective genomes and had average insert sizes of 138 and 168 kb. Greater than 60% of paired BAC ends sampled from each library (~0.1% of each genome) contained repetitive sequences, and the repetitive element landscapes of the 2 genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The representation and depth of coverage of the libraries were verified by identification of multiple Hox gene contigs: six discrete Hox clusters were found in N. coriiceps and at least five Hox clusters were found in C. aceratus. Given the unusual anatomical and physiological adaptations of the notothenioids, the availability of these BAC libraries sets the stage for expanded analysis of the psychrophilic mode of life. PMID:20235119

  12. 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...

  13. 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. PMID:27511172

  14. Development of BAC libraries and integrated physical mapping of human chromosome 22 using BACs. Annual report, July 1994--June 1995

    SciTech Connect

    Kim, U.J.; Shizuya, Hiroaki; Simon, M.I.

    1995-12-31

    BACs and fosmids are stable, nonchimeric, and highly representative cloning systems. BACs maintain large-fragment genomic inserts (100 to 300 kb) that are easily prepared for most types of experiments, including DNA sequencing. The authors have improved the methods for generating BACs and developed extensive BAC libraries. They have constructed human BAC libraries with more than 175,000 clones from male fibroblast and sperm, and a mouse BAC library with more than 200,000 clones. The authors are currently expanding human library with the aim of achieving total 50X coverage human genomic library using sperm samples from anonymous donors.

  15. Adventures in the enormous: a 1.8 million clone BAC library for the 21.7 Gb genome of loblolly pine.

    PubMed

    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 and characterization of a BAC library for the molecular dissection of a single wild beet centromere and sugar beet (Beta vulgaris) genome analysis.

    PubMed

    Gindullis, F; Dechyeva, D; Schmidt, T

    2001-10-01

    We have constructed a sugar beet bacterial artificial chromosome (BAC) library of the chromosome mutant PRO1. This Beta vulgaris mutant carries a single chromosome fragment of 6-9 Mbp that is derived from the wild beet Beta procumbens and is transmitted efficiently in meiosis and mitosis. The library consists of 50,304 clones, with an average insert size of 125 kb. Filter hybridizations revealed that approximately 3.1% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents eight genome equivalents. Thus, there is a greater than 99.96% probability that any sequence of the PROI genome can be found in the library. Approximately 0.2% of the clones hybridized with centromeric sequences of the PRO1 minichromosome. Using the identified BAC clones in fluorescence in situ hybridization experiments with PRO1 and B. procumbens chromosome spreads, their wild-beet origin and centromeric localization were demonstrated. Comparative Southern hybridization of pulsed-field separated PROI DNA and BAC inserts indicate that the centromeric region of the minichromosome is represented by overlapping clones in the library. Therefore, the PRO1 BAC library provides a useful tool for the characterization of a single plant centromere and is a valuable resource for sugar beet genome analysis. PMID:11681609

  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. CHARACTERIZATION AND PHYSICAL MAPPING OF MAIZE BAC LIBRARIES USING HIGH DENSITY BAC FILTER HYBRIDIZATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A HindIII and an EcoRI maize BAC library have been constructed from maize inbred line B73. Use of both libraries to make a physical map should minimize the under representation of certain genomic regions caused by the use of a particular restriction enzyme. High-density filter sets from the two libr...

  20. 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. PMID:24243195

  1. Construction and characterization of a deep-coverage carrot (Daucus carota L.) BAC library

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The first carrot (Daucus carota L.) BAC library was constructed using imbred line B8503, which is nematode-resistant and accumulates carotenes in its roots. The BAC library consists of 92,160 clones comprising 22.4 haploid genome equivalents based on a genome size of 473 Mb/1C. Upon the analysis of ...

  2. SEQUENCING THE PIG GENOME USING A 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 localized to the genome through integration with the UIVC RH map as well BAC end sequence alignments to the human genome. Over 265k HindIII restriction digest fingerprints totaling 16.2...

  3. Repetitive Genomic Elements in a European Corn Borer, Ostrinia nubilalis, BAC Library were Indicated by BAC End Sequencing and Development of Sequence Tag Site Markers: Implications for Lepidopteran Genomic Research

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia, and a model system for insect olfaction and speciation. A bacterial artificial chromosome (BAC) library constructed for O. nubilalis contains 36,864 clones with estim...

  4. 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.

  5. 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...

  6. 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...

  7. 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.

  8. BAC Libraries from Wheat Chromosome 7D – Efficient Tool for Positional Cloning of Aphid Resistance Genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Positional cloning in bread wheat is a tedious task due to its huge genome size (~17 Gbp) and polyploid character. BAC libraries represent an essential tool for positional cloning. However, wheat BAC libraries comprise more than million clones, which make their screening very laborious. Here we pres...

  9. BAC-Pool Sequencing and Assembly of 19 Mb of the Complex Sugarcane Genome.

    PubMed

    Okura, Vagner Katsumi; de Souza, Rafael S C; de Siqueira Tada, Susely F; Arruda, Paulo

    2016-01-01

    Sequencing plant genomes are often challenging because of their complex architecture and high content of repetitive sequences. Sugarcane has one of the most complex genomes. It is highly polyploid, preserves intact homeologous chromosomes from its parental species and contains >55% repetitive sequences. Although bacterial artificial chromosome (BAC) libraries have emerged as an alternative for accessing the sugarcane genome, sequencing individual clones is laborious and expensive. Here, we present a strategy for sequencing and assembly reads produced from the DNA of pooled BAC clones. A set of 178 BAC clones, randomly sampled from the SP80-3280 sugarcane BAC library, was pooled and sequenced using the Illumina HiSeq2000 and PacBio platforms. A hybrid assembly strategy was used to generate 2,451 scaffolds comprising 19.2 MB of assembled genome sequence. Scaffolds of ≥20 Kb corresponded to 80% of the assembled sequences, and the full sequences of forty BACs were recovered in one or two contigs. Alignment of the BAC scaffolds with the chromosome sequences of sorghum showed a high degree of collinearity and gene order. The alignment of the BAC scaffolds to the 10 sorghum chromosomes suggests that the genome of the SP80-3280 sugarcane variety is ∼19% contracted in relation to the sorghum genome. In conclusion, our data show that sequencing pools composed of high numbers of BAC clones may help to construct a reference scaffold map of the sugarcane genome. PMID:27047520

  10. BAC-Pool Sequencing and Assembly of 19 Mb of the Complex Sugarcane Genome

    PubMed Central

    Okura, Vagner Katsumi; de Souza, Rafael S. C.; de Siqueira Tada, Susely F.; Arruda, Paulo

    2016-01-01

    Sequencing plant genomes are often challenging because of their complex architecture and high content of repetitive sequences. Sugarcane has one of the most complex genomes. It is highly polyploid, preserves intact homeologous chromosomes from its parental species and contains >55% repetitive sequences. Although bacterial artificial chromosome (BAC) libraries have emerged as an alternative for accessing the sugarcane genome, sequencing individual clones is laborious and expensive. Here, we present a strategy for sequencing and assembly reads produced from the DNA of pooled BAC clones. A set of 178 BAC clones, randomly sampled from the SP80-3280 sugarcane BAC library, was pooled and sequenced using the Illumina HiSeq2000 and PacBio platforms. A hybrid assembly strategy was used to generate 2,451 scaffolds comprising 19.2 MB of assembled genome sequence. Scaffolds of ≥20 Kb corresponded to 80% of the assembled sequences, and the full sequences of forty BACs were recovered in one or two contigs. Alignment of the BAC scaffolds with the chromosome sequences of sorghum showed a high degree of collinearity and gene order. The alignment of the BAC scaffolds to the 10 sorghum chromosomes suggests that the genome of the SP80-3280 sugarcane variety is ∼19% contracted in relation to the sorghum genome. In conclusion, our data show that sequencing pools composed of high numbers of BAC clones may help to construct a reference scaffold map of the sugarcane genome. PMID:27047520

  11. BACTERIAL ARTIFICIAL CHROMOSOME(BAC)LIBRARIES CONSTRUCTED FROM THE GENETIC STANDARD OF UPLAND COTTON

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two BAC libraries and one plant transformation-competent BIBAC library were developed from the Gossypium hirsutum acc. TM-1 for the development of an integrative cotton physical and genetic map and other genomic applications. TM-1 is the most desirable choice for the physical map of Upland cotton be...

  12. BAC libraries of Triticum urartu, Aegilops speltoides and Ae. tauschii, the diploid ancestors of polyploid wheat.

    PubMed

    Akhunov, E D; Akhunova, A R; Dvorák, J

    2005-11-01

    Triticum urartu, Aegilops speltoides and Ae. tauschii are respectively the immediate diploid sources, or their closest relatives, of the A, B and D genomes of polyploid wheats. Here we report the construction and characterization of arrayed large-insert libraries in a bacterial artificial chromosome (BAC) vector, one for each of these diploid species. The libraries are equivalent to 3.7, 5.4 and 4.1 of the T. urartu, Ae. speltoides, Ae. tauschii genomes, respectively. The predicted levels of genome coverage were confirmed by library hybridization with single-copy genes. The libraries were used to estimate the proportion of known repeated nucleotide sequences and gene content in each genome by BAC-end sequencing. Repeated sequence families previously detected in Triticeae accounted for 57, 61 and 57% of the T. urartu, Ae. speltoides and Ae. tauschii genomes, and coding regions accounted for 5.8, 4.5 and 4.8%, respectively. PMID:16177898

  13. 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.

  14. CHARACTERIZATION OF THREE MAIZE BAC LIBRARIES AND ANCHORING OF THE PHYSICAL MAP TO THE GENETIC MAP USING HIGH-DENSITY BAC FILTER HYBRIDIZATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three maize (Zea mays L.) bacterial artificial chromosome (BAC) libraries, HindIII, EcoRI and MboI, were constructed from inbred line B73 to minimize under-representation of certain genomic regions caused by the use of a single restriction enzyme library. High-density filter sets from all three lib...

  15. 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.

  16. Construction and characterization of a BAC library from the Coffea arabica genotype Timor Hybrid CIFC 832/2.

    PubMed

    Cação, S M B; Silva, N V; Domingues, D S; Vieira, L G E; Diniz, L E C; Vinecky, F; Alves, G S C; Andrade, A C; Carpentieri-Pipolo, V; Pereira, L F P

    2013-06-01

    Most of the world's coffee production originates from Coffea arabica, an allotetraploid species with low genetic diversity and for which few genomic resources are available. Genomic libraries with large DNA fragment inserts are useful tools for the study of plant genomes, including the production of physical maps, integration studies of physical and genetic maps, genome structure analysis and gene isolation by positional cloning. Here, we report the construction and characterization of a Bacterial Artificial Chromosome (BAC) library from C. arabica Timor Hybrid CIFC 832/2, a parental genotype for several modern coffee cultivars. The BAC library consists of 56,832 clones with an average insert size of 118 kb, which represents a dihaploid genome coverage of five to sixfold. The content of organellar DNA was estimated at 1.04 and 0.5 % for chloroplast and mitochondrial DNA, respectively. The BAC library was screened for the NADPH-dependent mannose-6-phosphate reductase gene (CaM6PR) with markers positioned on four linkage groups of a partial C. arabica genetic map. A mixed approach using PCR and membrane hybridization of BAC pools allowed for the discovery of nine BAC clones with the CaM6PR gene and 53 BAC clones that were anchored to the genetic map with simple sequence repeat markers. This library will be a useful tool for future studies on comparative genomics and the identification of genes and regulatory elements controlling major traits in this economically important crop species. PMID:23677718

  17. Distribution of Genes and Repetitive Elements in the Diabrotica virgifera virgifera Genome Estimated Using BAC Sequencing

    PubMed Central

    Coates, Brad S.; Alves, Analiza P.; Wang, Haichuan; Walden, Kimberly K. O.; French, B. Wade; Miller, Nicholas J.; Abel, Craig A.; Robertson, Hugh M.; Sappington, Thomas W.; Siegfried, Blair D.

    2012-01-01

    Feeding damage caused by the western corn rootworm, Diabrotica virgifera virgifera, is destructive to corn plants in North America and Europe where control remains challenging due to evolution of resistance to chemical and transgenic toxins. A BAC library, DvvBAC1, containing 109,486 clones with 104 ± 34.5 kb inserts was created, which has an ~4.56X genome coverage based upon a 2.58 Gb (2.80 pg) flow cytometry-estimated haploid genome size. Paired end sequencing of 1037 BAC inserts produced 1.17 Mb of data (~0.05% genome coverage) and indicated ~9.4 and 16.0% of reads encode, respectively, endogenous genes and transposable elements (TEs). Sequencing genes within BAC full inserts demonstrated that TE densities are high within intergenic and intron regions and contribute to the increased gene size. Comparison of homologous genome regions cloned within different BAC clones indicated that TE movement may cause haplotype variation within the inbred strain. The data presented here indicate that the D. virgifera virgifera genome is large in size and contains a high proportion of repetitive sequence. These BAC sequencing methods that are applicable for characterization of genomes prior to sequencing may likely be valuable resources for genome annotation as well as scaffolding. PMID:22919272

  18. Distribution of genes and repetitive elements in the Diabrotica virgifera virgifera genome estimated using BAC sequencing.

    PubMed

    Coates, Brad S; Alves, Analiza P; Wang, Haichuan; Walden, Kimberly K O; French, B Wade; Miller, Nicholas J; Abel, Craig A; Robertson, Hugh M; Sappington, Thomas W; Siegfried, Blair D

    2012-01-01

    Feeding damage caused by the western corn rootworm, Diabrotica virgifera virgifera, is destructive to corn plants in North America and Europe where control remains challenging due to evolution of resistance to chemical and transgenic toxins. A BAC library, DvvBAC1, containing 109,486 clones with 104 ± 34.5 kb inserts was created, which has an ~4.56X genome coverage based upon a 2.58 Gb (2.80 pg) flow cytometry-estimated haploid genome size. Paired end sequencing of 1037 BAC inserts produced 1.17 Mb of data (~0.05% genome coverage) and indicated ~9.4 and 16.0% of reads encode, respectively, endogenous genes and transposable elements (TEs). Sequencing genes within BAC full inserts demonstrated that TE densities are high within intergenic and intron regions and contribute to the increased gene size. Comparison of homologous genome regions cloned within different BAC clones indicated that TE movement may cause haplotype variation within the inbred strain. The data presented here indicate that the D. virgifera virgifera genome is large in size and contains a high proportion of repetitive sequence. These BAC sequencing methods that are applicable for characterization of genomes prior to sequencing may likely be valuable resources for genome annotation as well as scaffolding. PMID:22919272

  19. 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.

  20. Construction and characterization of a human chromosome 2-specific BAC library

    SciTech Connect

    Wang, M.; Shouse, S.; Manson, J.

    1994-12-01

    We have constructed a human chromosome 2-specific bacterial artificial chromosome (BAC) library using DNA from the somatic cell hybrid GM10826. The average size of the clones is about 63 kb. The coverage and distribution of the library were estimated by screening with known polymorphic genetic markers and fluorescence in situ hybridization (FISH). Twenty-one markers tested positive when DNA pools prepared from approximately one-sixth of the library were screened with 33 known markers. This is consistent with the theoretical calculation of 63% coverage at one genomic equivalent. This suggested that the coverage of the library is approximately 5-6X. FISH analysis with 54 BACs revealed single site hybridization to chromosome 2, and the clones were distributed randomly on the chromosome. We have also performed direct sequencing of the BAC insert ends to generate sequence-tagged sites suitable for mapping and chromosome walking. This is the first reported human chromosome 2-specific BAC library and should provide a resource for physical mapping and disease searching for this chromosome. 30 refs., 5 figs.

  1. 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...

  2. 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.

  3. A First Generation BAC Physical Map of the Rainbow Trout Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The physical map was constructed using the high-information content fingerprinting (HICF) method of Luo et al. (2003; Genomics, 82, 378-389). All the clones from the Swanson YY doubled haploid male BAC library (10X coverage; 184,704 clones) were fingerprinted and edited using FPMiner software. App...

  4. 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 ...

  5. Characterization of a deep-coverage carrot (Daucus carota L.) BAC library and initial analysis of BAC-end sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 17.3-fold redundant bacterial artificial chromosome (BAC) library has been synthesized for carrot, the most-economically important member of the family Apiaceae. The library consists of 92,160 clones with an average insert size of 121 kb and ~ 2 % organellar DNA content. To provide an overview of ...

  6. 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. PMID:16586115

  7. Construction of an expressible BAC library of the unculturable insect microorganism, stink bug Plautia stali symbiont, for the search of biologically active and useful symbiont products.

    PubMed

    Kobayashi, Hideaki; Fujii-Muramatsu, Rika; Noda, Hiroaki; Takeishi, Keiichi

    2014-01-01

    While gene products and metabolites of insect symbiotic bacteria may act as useful resources for insect-microbe studies and medicinal use, it is usually difficult to obtain the insect symbionts to some extent in quantity because most of them are unculturable. In this study, the possibility of using bacterial artificial chromosome (BAC) libraries as a heterologous gene expression tool for the discovery of novel symbiont metabolites was evaluated. A BAC library was constructed from the symbiont purified from the posterior midgut cecum of the stink bug Plautia stali. The BAC library, which consisted of 513 clones with an average insert size of 41 kb, represented greater than five-fold coverage of the genome. The ability of the BAC clones to express plural genes from large-sized insert DNA in Escherichia coli was examined by the growth of BAC-transformed leu operon-deficient DH10B cells on M9 minimal medium supplemented with glucose. Two BAC clones complemented leucine deficiency in DH10B cells; the clones contained the leu operon of the symbiont chromosome. The P. stali symbiont genes introduced into the BAC vector are functional in E. coli, and these genes are expressed in an operon unit. BAC libraries can be used to generate gene product- and metabolite-libraries, facilitating to characterize potential metabolites of the P. stali symbiont. PMID:24694601

  8. Sequencing of 6.7 Mb of the melon genome using a BAC pooling strategy

    PubMed Central

    2010-01-01

    Background Cucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has a high intra-specific genetic variation, morphologic diversity and a small genome size (454 Mb), which make it suitable for a great variety of molecular and genetic studies. A number of genetic and genomic resources have already been developed, such as several genetic maps, BAC genomic libraries, a BAC-based physical map and EST collections. Sequence information would be invaluable to complete the picture of the melon genomic landscape, furthering our understanding of this species' evolution from its relatives and providing an important genetic tool. However, to this day there is little sequence data available, only a few melon genes and genomic regions are deposited in public databases. The development of massively parallel sequencing methods allows envisaging new strategies to obtain long fragments of genomic sequence at higher speed and lower cost than previous Sanger-based methods. Results In order to gain insight into the structure of a significant portion of the melon genome we set out to perform massive sequencing of pools of BAC clones. For this, a set of 57 BAC clones from a double haploid line was sequenced in two pools with the 454 system using both shotgun and paired-end approaches. The final assembly consists of an estimated 95% of the actual size of the melon BAC clones, with most likely complete sequences for 50 of the BACs, and a total sequence coverage of 39x. The accuracy of the assembly was assessed by comparing the previously available Sanger sequence of one of the BACs against its 454 sequence, and the polymorphisms found involved only 1.7 differences every 10,000 bp that were localized in 15 homopolymeric regions and two dinucleotide tandem repeats. Overall, the study provides approximately 6.7 Mb or 1.5% of the melon genome. The analysis of this new data has allowed us to gain further

  9. CHARACTERIZATION AND PHYSICAL MAPPING OF MAIZE BAC LIBRARIES USING HIGH DENSITY BAC FILTER HYBRIDIZATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-density filter sets from two maize B73 libraries containing 6X (HindIII) and 7X (EcoRI) haploid genome equivalents, respectively, were evaluated with a set of complex probes. The complex probes will provide information on chromosome architecture and organellar DNA content. A second set of pro...

  10. Physical mapping in large genomes: accelerating anchoring of BAC contigs to genetic maps through in silico analysis.

    PubMed

    Paux, Etienne; Legeai, Fabrice; Guilhot, Nicolas; Adam-Blondon, Anne-Françoise; Alaux, Michaël; Salse, Jérôme; Sourdille, Pierre; Leroy, Philippe; Feuillet, Catherine

    2008-02-01

    Anchored physical maps represent essential frameworks for map-based cloning, comparative genomics studies, and genome sequencing projects. High throughput anchoring can be achieved by polymerase chain reaction (PCR) screening of bacterial artificial chromosome (BAC) library pools with molecular markers. However, for large genomes such as wheat, the development of high dimension pools and the number of reactions that need to be performed can be extremely large making the screening laborious and costly. To improve the cost efficiency of anchoring in such large genomes, we have developed a new software named Elephant (electronic physical map anchoring tool) that combines BAC contig information generated by FingerPrinted Contig with results of BAC library pools screening to identify BAC addresses with a minimal amount of PCR reactions. Elephant was evaluated during the construction of a physical map of chromosome 3B of hexaploid wheat. Results show that a one dimensional pool screening can be sufficient to anchor a BAC contig while reducing the number of PCR by 384-fold thereby demonstrating that Elephant is an efficient and cost-effective tool to support physical mapping in large genomes. PMID:18038165

  11. Libraries for genomic SELEX.

    PubMed Central

    Singer, B S; Shtatland, T; Brown, D; Gold, L

    1997-01-01

    An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo. A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism. As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids. The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism. We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase. We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX. PMID:9016629

  12. 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. PMID:22403630

  13. 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

  14. 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.

  15. Construction of a BAC library and a physical map of a major QTL for CBB resistance of common bean (Phaseolus vulgaris L.).

    PubMed

    Liu, S Y; Yu, K; Huffner, M; Park, S J; Banik, M; Pauls, K P; Crosby, W

    2010-07-01

    A major quantitative trait loci (QTL) conditioning common bacterial blight (CBB) resistance in common bean (Phaseolus vulgaris L.) lines HR45 and HR67 was derived from XAN159, a resistant line obtained from an interspecific cross between common bean lines and the tepary bean (P. acutifolius L.) line PI319443. This source of CBB resistance is widely used in bean breeding. Several other CBB resistance QTL have been identified but none of them have been physically mapped. Four molecular markers tightly linked to this QTL have been identified suitable for marker assisted selection and physical mapping of the resistance gene. A bacterial artificial chromosome (BAC) library was constructed from high molecular weight DNA of HR45 and is composed of 33,024 clones. The size of individual BAC clone inserts ranges from 30 kb to 280 kb with an average size of 107 kb. The library is estimated to represent approximately sixfold genome coverage. The BAC library was screened as BAC pools using four PCR-based molecular markers. Two to seven BAC clones were identified by each marker. Two clones were found to have both markers PV-tttc001 and STS183. One preliminary contig was assembled based on DNA finger printing of those positive BAC clones. The minimum tiling path of the contig contains 6 BAC clones spanning an estimated size of 750 kb covering the QTL region. PMID:20419470

  16. A BAC library of Beta vulgaris L. for the targeted isolation of centromeric DNA and molecular cytogenetics of Beta species.

    PubMed

    Jacobs, Gunnar; Dechyeva, Daryna; Wenke, Torsten; Weber, Beatrice; Schmidt, Thomas

    2009-03-01

    We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis. PMID:18386131

  17. 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.

  18. Amniote phylogenomics: testing evolutionary hypotheses with BAC library scanning and targeted clone analysis of large-scale DNA sequences from reptiles.

    PubMed

    Shedlock, Andrew M; Janes, Daniel E; Edwards, Scott V

    2008-01-01

    Phylogenomics research integrating established principles of systematic biology and taking advantage of the wealth of DNA sequences being generated by genome science holds promise for answering long-standing evolutionary questions with orders of magnitude more primary data than in the past. Although it is unrealistic to expect whole-genome initiatives to proceed rapidly for commercially unimportant species such as reptiles, practical approaches utilizing genomic libraries of large-insert clones pave the way for a phylogenomics of species that are nevertheless essential for testing evolutionary hypotheses within a phylogenetic framework. This chapter reviews the case for adopting genome-enabled approaches to evolutionary studies and outlines a program for using bacterial artificial chromosome (BAC) libraries or plasmid libraries as a basis for completing "genome scans" of reptiles. We have used BACs to close a critical gap in the genome database for Reptilia, the sister group of mammals, and present the methodological approaches taken to achieve this as a guideline for designing similar comparative studies. In addition, we provide a detailed step-by-step protocol for BAC-library screening and shotgun sequencing of specific clones containing target genes of evolutionary interest. Taken together, the genome scanning and shotgun sequencing techniques offer complementary diagnostic potential and can substantially increase the scale and power of analyses aimed at testing evolutionary hypotheses for nonmodel species. PMID:18629663

  19. An overview of the Phalaenopsis orchid genome through BAC end sequence analysis

    PubMed Central

    2011-01-01

    Background Phalaenopsis orchids are popular floral crops, and development of new cultivars is economically important to floricultural industries worldwide. Analysis of orchid genes could facilitate orchid improvement. Bacterial artificial chromosome (BAC) end sequences (BESs) can provide the first glimpses into the sequence composition of a novel genome and can yield molecular markers for use in genetic mapping and breeding. Results We used two BAC libraries (constructed using the BamHI and HindIII restriction enzymes) of Phalaenopsis equestris to generate pair-end sequences from 2,920 BAC clones (71.4% and 28.6% from the BamHI and HindIII libraries, respectively), at a success rate of 95.7%. A total of 5,535 BESs were generated, representing 4.5 Mb, or about 0.3% of the Phalaenopsis genome. The trimmed sequences ranged from 123 to 1,397 base pairs (bp) in size, with an average edited read length of 821 bp. When these BESs were subjected to sequence homology searches, it was found that 641 (11.6%) were predicted to represent protein-encoding regions, whereas 1,272 (23.0%) contained repetitive DNA. Most of the repetitive DNA sequences were gypsy- and copia-like retrotransposons (41.9% and 12.8%, respectively), whereas only 10.8% were DNA transposons. Further, 950 potential simple sequence repeats (SSRs) were discovered. Dinucleotides were the most abundant repeat motifs; AT/TA dimer repeats were the most frequent SSRs, representing 253 (26.6%) of all identified SSRs. Microsynteny analysis revealed that more BESs mapped to the whole-genome sequences of poplar than to those of grape or Arabidopsis, and even fewer mapped to the rice genome. This work will facilitate analysis of the Phalaenopsis genome, and will help clarify similarities and differences in genome composition between orchids and other plant species. Conclusion Using BES analysis, we obtained an overview of the Phalaenopsis genome in terms of gene abundance, the presence of repetitive DNA and SSR markers

  20. 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. PMID:16404584

  1. Construction of two BAC libraries from the wild Mexican diploid potato, Solanum pinnatisectum, and the identification of clones near the late blight and Colorado potato beetle resistance loci.

    PubMed

    Chen, Q; Sun, S; Ye, Q; McCuine, S; Huff, E; Zhang, H-B

    2004-04-01

    To facilitate isolation and characterization of disease and insect resistance genes important to potato, two bacterial artificial chromosome (BAC) libraries were constructed from genomic DNA of the Mexican wild diploid species, Solanum pinnatisectum, which carries high levels of resistance to the most important potato pathogen and pest, the late blight and the Colorado potato beetle (CPB). One of the libraries was constructed from the DNA, partially digested with BamHI, and it consists of 40328 clones with an average insert size of 125 kb. The other library was constructed from the DNA partially digested with EcoRI, and it consists of 17280 clones with an average insert size of 135 kb. The two libraries, together, represent approximately six equivalents of the wild potato haploid genome. Both libraries were evaluated for contamination with organellar DNA sequences and were shown to have a very low percentage (0.65-0.91%) of clones derived from the chloroplast genome. High-density filters, prepared from the two libraries, were screened with ten restriction fragment length polymorphism (RFLP) markers linked to the resistance genes for late blight, CPB, Verticillium wilt and potato cyst nematodes, and the gene Sr1 for the self-incompatibility S-locus. Thirty nine positive clones were identified and at least two positive BAC clones were detected for each RFLP marker. Four markers that are linked to the late blight resistance gene Rpi1 hybridized to 14 BAC clones. Fifteen BAC clones were shown to harbor the PPO (polyphenol oxidase) locus for the CPB resistance by three RFLP probes. Two RFLP markers detected five BAC clones that were linked to the Sr1 gene for self-incompatibility. These results agree with the library's predicted extent of coverage of the potato genome, and indicated that the libraries are useful resources for the molecular isolation of disease and insect resistance genes, as well as other economically important genes in the wild potato species. The

  2. 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.

  3. 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

    2014-01-01

    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. PMID:24608928

  4. Construction and Analysis of Siberian Tiger Bacterial Artificial Chromosome Library with Approximately 6.5-Fold Genome Equivalent Coverage

    PubMed Central

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

    2014-01-01

    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. PMID:24608928

  5. Genome-wide end-sequenced BAC resources for the NOD/MrkTac☆ and NOD/ShiLtJ☆☆ mouse genomes

    PubMed Central

    Steward, Charles A.; Humphray, Sean; Plumb, Bob; Jones, Matthew C.; Quail, Michael A.; Rice, Stephen; Cox, Tony; Davies, Rob; Bonfield, James; Keane, Thomas M.; Nefedov, Michael; de Jong, Pieter J.; Lyons, Paul; Wicker, Linda; Todd, John; Hayashizaki, Yoshihide; Gulban, Omid; Danska, Jayne; Harrow, Jen; Hubbard, Tim; Rogers, Jane; Adams, David J.

    2010-01-01

    Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes (T1D) due to the progressive loss of insulin-secreting β-cells by an autoimmune driven process. NOD mice represent a valuable tool for studying the genetics of T1D and for evaluating therapeutic interventions. Here we describe the development and characterization by end-sequencing of bacterial artificial chromosome (BAC) libraries derived from NOD/MrkTac (DIL NOD) and NOD/ShiLtJ (CHORI-29), two commonly used NOD substrains. The DIL NOD library is composed of 196,032 BACs and the CHORI-29 library is composed of 110,976 BACs. The average depth of genome coverage of the DIL NOD library, estimated from mapping the BAC end-sequences to the reference mouse genome sequence, was 7.1-fold across the autosomes and 6.6-fold across the X chromosome. Clones from this library have an average insert size of 150 kb and map to over 95.6% of the reference mouse genome assembly (NCBIm37), covering 98.8% of Ensembl mouse genes. By the same metric, the CHORI-29 library has an average depth over the autosomes of 5.0-fold and 2.8-fold coverage of the X chromosome, the reduced X chromosome coverage being due to the use of a male donor for this library. Clones from this library have an average insert size of 205 kb and map to 93.9% of the reference mouse genome assembly, covering 95.7% of Ensembl genes. We have identified and validated 191,841 single nucleotide polymorphisms (SNPs) for DIL NOD and 114,380 SNPs for CHORI-29. In total we generated 229,736,133 bp of sequence for the DIL NOD and 121,963,211 bp for the CHORI-29. These BAC libraries represent a powerful resource for functional studies, such as gene targeting in NOD embryonic stem (ES) cell lines, and for sequencing and mapping experiments. PMID:19909804

  6. 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...

  7. BAC to the future: functional genomics in mammals

    PubMed Central

    Roguev, Assen; Krogan, Nevan J

    2012-01-01

    A high-throughput pipeline to engineer bacterial artificial chromosomes (BACs) expressing tagged genes of higher eukaryotes allows large-scale protein localization and interaction studies. PMID:18446155

  8. An integrated BAC/BIBAC-based physical and genetic map of the cotton genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Integrated genome-wide genetic and physical maps are crucial to many aspects of cotton genome research. We report a genome-wide BAC/BIBAC-based physical and genetic map of the upland cotton genome using a high-resolution and high-throughput capillary-based fingerprinting method. The map was constr...

  9. 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. PMID:24671676

  10. Insights into the Loblolly Pine Genome: Characterization of BAC and Fosmid Sequences

    PubMed Central

    Dougherty, William M.; Martínez-García, Pedro J.; Koriabine, Maxim; Holtz-Morris, Ann; deJong, Pieter; Crepeau, Marc; Langley, Charles H.; Puiu, Daniela; Salzberg, Steven L.; Neale, David B.; Stevens, Kristian A.

    2013-01-01

    Despite their prevalence and importance, the genome sequences of loblolly pine, Norway spruce, and white spruce, three ecologically and economically important conifer species, are just becoming available to the research community. Following the completion of these large assemblies, annotation efforts will be undertaken to characterize the reference sequences. Accurate annotation of these ancient genomes would be aided by a comprehensive repeat library; however, few studies have generated enough sequence to fully evaluate and catalog their non-genic content. In this paper, two sets of loblolly pine genomic sequence, 103 previously assembled BACs and 90,954 newly sequenced and assembled fosmid scaffolds, were analyzed. Together, this sequence represents 280 Mbp (roughly 1% of the loblolly pine genome) and one of the most comprehensive studies of repetitive elements and genes in a gymnosperm species. A combination of homology and de novo methodologies were applied to identify both conserved and novel repeats. Similarity analysis estimated a repetitive content of 27% that included both full and partial elements. When combined with the de novo investigation, the estimate increased to almost 86%. Over 60% of the repetitive sequence consists of full or partial LTR (long terminal repeat) retrotransposons. Through de novo approaches, 6,270 novel, full-length transposable element families and 9,415 sub-families were identified. Among those 6,270 families, 82% were annotated as single-copy. Several of the novel, high-copy families are described here, with the largest, PtPiedmont, comprising 133 full-length copies. In addition to repeats, analysis of the coding region reported 23 full-length eukaryotic orthologous proteins (KOGS) and another 29 novel or orthologous genes. These discoveries, along with other genomic resources, will be used to annotate conifer genomes and address long-standing questions about gymnosperm evolution. PMID:24023741

  11. Insights into the loblolly pine genome: characterization of BAC and fosmid sequences.

    PubMed

    Wegrzyn, Jill L; Lin, Brian Y; Zieve, Jacob J; Dougherty, William M; Martínez-García, Pedro J; Koriabine, Maxim; Holtz-Morris, Ann; deJong, Pieter; Crepeau, Marc; Langley, Charles H; Puiu, Daniela; Salzberg, Steven L; Neale, David B; Stevens, Kristian A

    2013-01-01

    Despite their prevalence and importance, the genome sequences of loblolly pine, Norway spruce, and white spruce, three ecologically and economically important conifer species, are just becoming available to the research community. Following the completion of these large assemblies, annotation efforts will be undertaken to characterize the reference sequences. Accurate annotation of these ancient genomes would be aided by a comprehensive repeat library; however, few studies have generated enough sequence to fully evaluate and catalog their non-genic content. In this paper, two sets of loblolly pine genomic sequence, 103 previously assembled BACs and 90,954 newly sequenced and assembled fosmid scaffolds, were analyzed. Together, this sequence represents 280 Mbp (roughly 1% of the loblolly pine genome) and one of the most comprehensive studies of repetitive elements and genes in a gymnosperm species. A combination of homology and de novo methodologies were applied to identify both conserved and novel repeats. Similarity analysis estimated a repetitive content of 27% that included both full and partial elements. When combined with the de novo investigation, the estimate increased to almost 86%. Over 60% of the repetitive sequence consists of full or partial LTR (long terminal repeat) retrotransposons. Through de novo approaches, 6,270 novel, full-length transposable element families and 9,415 sub-families were identified. Among those 6,270 families, 82% were annotated as single-copy. Several of the novel, high-copy families are described here, with the largest, PtPiedmont, comprising 133 full-length copies. In addition to repeats, analysis of the coding region reported 23 full-length eukaryotic orthologous proteins (KOGS) and another 29 novel or orthologous genes. These discoveries, along with other genomic resources, will be used to annotate conifer genomes and address long-standing questions about gymnosperm evolution. PMID:24023741

  12. BAC-end sequences analysis provides first insights into coffee (Coffea canephora P.) genome composition and evolution.

    PubMed

    Dereeper, Alexis; Guyot, Romain; Tranchant-Dubreuil, Christine; Anthony, François; Argout, Xavier; de Bellis, Fabien; Combes, Marie-Christine; Gavory, Frederick; de Kochko, Alexandre; Kudrna, Dave; Leroy, Thierry; Poulain, Julie; Rondeau, Myriam; Song, Xiang; Wing, Rod; Lashermes, Philippe

    2013-10-01

    Coffee is one of the world's most important agricultural commodities. Coffee belongs to the Rubiaceae family in the euasterid I clade of dicotyledonous plants, to which the Solanaceae family also belongs. Two bacterial artificial chromosome (BAC) libraries of a homozygous doubled haploid plant of Coffea canephora were constructed using two enzymes, HindIII and BstYI. A total of 134,827 high quality BAC-end sequences (BESs) were generated from the 73,728 clones of the two libraries, and 131,412 BESs were conserved for further analysis after elimination of chloroplast and mitochondrial sequences. This corresponded to almost 13 % of the estimated size of the C. canephora genome. 6.7 % of BESs contained simple sequence repeats, the most abundant (47.8 %) being mononucleotide motifs. These sequences allow the development of numerous useful marker sites. Potential transposable elements (TEs) represented 11.9 % of the full length BESs. A difference was observed between the BstYI and HindIII libraries (14.9 vs. 8.8 %). Analysis of BESs against known coding sequences of TEs indicated that 11.9 % of the genome corresponded to known repeat sequences, like for other flowering plants. The number of genes in the coffee genome was estimated at 41,973 which is probably overestimated. Comparative genome mapping revealed that microsynteny was higher between coffee and grapevine than between coffee and tomato or Arabidopsis. BESs constitute valuable resources for the first genome wide survey of coffee and provide new insights into the composition and evolution of the coffee genome. PMID:23708951

  13. 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...

  14. 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...

  15. Analysis Of Papaya BAC End Sequences: Insights Into The Organization Of A Tree Fruit Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Papaya (Carica papaya L.) is a major tree fruit crop of tropical and subtropical regions with an estimated genome size of 372 Mbp. We present the analysis of 4.7% of the papaya genome based on BAC end sequences (BESs) representing 17 million high-quality bases. Microsatellites discovered in 5,452 BE...

  16. Assembly and sorting of homologous BAC contigs in allotetraploid cotton genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Upland cotton (G. hirsutum) is a diploidized allopolyploid species containing At and Dt sub-genomes that have partial homology. Assembly and sorting of homologous BAC contigs into their subgenomes and further to individual chromosomes are of both great interest and great challenge for genome-wide i...

  17. Genome size evolution in pufferfish: an insight from BAC clone-based Diodon holocanthus genome sequencing

    PubMed Central

    2010-01-01

    Background Variations in genome size within and between species have been observed since the 1950 s in diverse taxonomic groups. Serving as model organisms, smooth pufferfish possess the smallest vertebrate genomes. Interestingly, spiny pufferfish from its sister family have genome twice as large as smooth pufferfish. Therefore, comparative genomic analysis between smooth pufferfish and spiny pufferfish is useful for our understanding of genome size evolution in pufferfish. Results Ten BAC clones of a spiny pufferfish Diodon holocanthus were randomly selected and shotgun sequenced. In total, 776 kb of non-redundant sequences without gap representing 0.1% of the D. holocanthus genome were identified, and 77 distinct genes were predicted. In the sequenced D. holocanthus genome, 364 kb is homologous with 265 kb of the Takifugu rubripes genome, and 223 kb is homologous with 148 kb of the Tetraodon nigroviridis genome. The repetitive DNA accounts for 8% of the sequenced D. holocanthus genome, which is higher than that in the T. rubripes genome (6.89%) and that in the Te. nigroviridis genome (4.66%). In the repetitive DNA, 76% is retroelements which account for 6% of the sequenced D. holocanthus genome and belong to known families of transposable elements. More than half of retroelements were distributed within genes. In the non-homologous regions, repeat element proportion in D. holocanthus genome increased to 10.6% compared with T. rubripes and increased to 9.19% compared with Te. nigroviridis. A comparison of 10 well-defined orthologous genes showed that the average intron size (566 bp) in D. holocanthus genome is significantly longer than that in the smooth pufferfish genome (435 bp). Conclusion Compared with the smooth pufferfish, D. holocanthus has a low gene density and repeat elements rich genome. Genome size variation between D. holocanthus and the smooth pufferfish exhibits as length variation between homologous region and different accumulation of non

  18. Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project.

    PubMed

    Park, Tae-Ho; Park, Beom-Seok; Kim, Jin-A; Hong, Joon Ki; Jin, Mina; Seol, Young-Joo; Mun, Jeong-Hwan

    2011-01-01

    As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa. PMID:21338952

  19. High-resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization.

    PubMed

    Jönsson, Göran; Staaf, Johan; Olsson, Eleonor; Heidenblad, Markus; Vallon-Christersson, Johan; Osoegawa, Kazutoyo; de Jong, Pieter; Oredsson, Stina; Ringnér, Markus; Höglund, Mattias; Borg, Ake

    2007-06-01

    A BAC-array platform for comparative genomic hybridization was constructed from a library of 32,433 clones providing complete genome coverage, and evaluated by screening for DNA copy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK-BR-3, L56Br-C1, ZR-75-1, JIMT1, MDA-MB-231, MDA-MB-361, and HCC2218) and one cell line derived from fibrocystic disease of the breast (MCF10A). These were also characterized by gene expression analysis and found to represent all five recently described breast cancer subtypes using the "intrinsic gene set" and centroid correlation. Three cell lines, HCC1937 and L56BrC1 derived from BRCA1 mutation carriers and MDA-MB-231, were of basal-like subtype and characterized by a high frequency of low-level gains and losses of typical pattern, including limited deletions on 5q. Four estrogen receptor positive cell lines were of luminal A subtype and characterized by a different pattern of aberrations and high-level amplifications, including ERBB2 and other 17q amplicons in BT474 and MDA-MB-361. SK-BR-3 cells, characterized by a complex genome including ERBB2 amplification, massive high-level amplifications on 8q and a homozygous deletion of CDH1 at 16q22, had an expression signature closest to luminal B subtype. The effects of gene amplifications were verified by gene expression analysis to distinguish targeted genes from silent amplicon passengers. JIMT1, derived from an ERBB2 amplified trastuzumab resistant tumor, was of the ERBB2 subtype. Homozygous deletions included other known targets such as PTEN (HCC1937) and CDKN2A (MDA-MB-231, MCF10A), but also new candidate suppressor genes such as FUSSEL18 (HCC1937) and WDR11 (L56Br-C1) as well as regions without known genes. The tiling BAC-arrays constitute a powerful tool for high-resolution genomic profiling suitable for cancer research and clinical diagnostics. PMID:17334996

  20. Mobilization of giant piggyBac transposons in the mouse genome

    PubMed Central

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

    2011-01-01

    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. PMID:21948799

  1. 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. PMID:25239744

  2. 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

  3. Biomedical applications and studies of molecular evolution: a proposal for a primate genomic library resource.

    PubMed

    Eichler, Evan E; DeJong, Pieter J

    2002-05-01

    The anticipated completion of two of the most biomedically relevant genomes, mouse and human, within the next three years provides an unparalleled opportunity for the large-scale exploration of genome evolution. Targeted sequencing of genomic regions in a panel of primate species and comparison to reference genomes will provide critical insight into the nature of single-base pair variation, mechanisms of chromosomal rearrangement, patterns of selection, and species adaptation. Although not recognized as model "genetic organisms" because of their longevity and low fecundity, 30 of the approximately 300 primate species are targets of biomedical research. The existence of a human reference sequence and genomic primate BAC libraries greatly facilitates the recovery of genes/genomic regions of high biological interest because of an estimated maximum neutral nucleotide sequence divergence of 25%. Primate species, therefore, may be regarded as the ideal model "genomic organisms". Based on existing BAC library resources, we propose the construction of a panel of primate BAC libraries from phylogenetic anchor species for the purpose of comparative medicine as well as studies of genome evolution. PMID:11997334

  4. 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...

  5. 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. PMID:26252423

  6. Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library.

    PubMed

    Li, R; Mignot, E; Faraco, J; Kadotani, H; Cantanese, J; Zhao, B; Lin, X; Hinton, L; Ostrander, E A; Patterson, D F; de Jong, P J

    1999-05-15

    A large insert canine genomic bacterial artificial chromosome (BAC) library was built from a Doberman pinscher. Approximately 166,000 clones were gridded on nine high-density hybridization filters. Insert analysis of randomly selected clones indicated a mean insert size of 155 kb and predicted 8.1 coverage of the canine genome. Two percent of the clones were nonrecombinant. Chromosomal fluorescence in situ hybridization studies of 60 BAC clones indicated no chimerism. The library was hybridized with dog PCR products representing eight genes (ADA, TNFA, GCA, MYB, HOXA, GUSB, THY1, and TOP1). The resulting positive clones were characterized and shown to be compatible with an eightfold redundant library. PMID:10331940

  7. 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...

  8. 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).

  9. DNA shuttling between plasmid vectors and a genome vector: systematic conversion and preservation of DNA libraries using the Bacillus subtilis genome (BGM) vector.

    PubMed

    Kaneko, Shinya; Akioka, Manami; Tsuge, Kenji; Itaya, Mitsuhiro

    2005-06-24

    The combined use of the contemporary vector systems, the bacterial artificial chromosome (BAC) vector and the Bacillus subtilis genome (BGM) vector, makes possible the handling of giant-length DNA (above 100 kb). Our newly constructed BGM vector efficiently integrated DNA prepared in the BAC vector. A BAC library comprised of 18 independent clones prepared from mitochondrial DNA (mtDNA) of Arabidopsis thaliana was converted to a parallel BGM library using the new BGM vector. The effectiveness of the combined use of the vector systems was confirmed by the stable recovery of all 18 DNAs as BAC clones from the respective BGM clones. We show that DNA in BGM was stably preserved at room temperature after spore formation of the host B.subtilis. Rapid and stable shuttling between Escherichiacoli and the B. subtilis host, combined with spore-mediated DNA storage, may facilitate the long-term and low-cost preservation and the transportation of DNA resources. PMID:15913652

  10. THE WHEAT D-GENOME HMW-GLUTENIN LOCUS:BAC SEQUENCING, GENE DISTRIBUTION, AND RETROTRANSPOSON CLUSTERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A bacterial-artificial-chromosome (BAC) clone from the genome of Triticum tauschii, the D-genome ancestor of hexaploid bread wheat, was sequenced and the presence of the two paralogous x- and y- type high-molecular-weight (HMW) glutenin genes of the Glu-D1 locus was confirmed. These two genes occur...

  11. Construction of a bacterial artificial chromosome library from the spikemoss Selaginella moellendorffii: a new resource for plant comparative genomics

    PubMed Central

    Wang, Wenming; Tanurdzic, Milos; Luo, Meizhong; Sisneros, Nicholas; Kim, Hye Ran; Weng, Jing-Ke; Kudrna, Dave; Mueller, Christopher; Arumuganathan, K; Carlson, John; Chapple, Clint; de Pamphilis, Claude; Mandoli, Dina; Tomkins, Jeff; Wing, Rod A; Banks, Jo Ann

    2005-01-01

    Background The lycophytes are an ancient lineage of vascular plants that diverged from the seed plant lineage about 400 Myr ago. Although the lycophytes occupy an important phylogenetic position for understanding the evolution of plants and their genomes, no genomic resources exist for this group of plants. Results Here we describe the construction of a large-insert bacterial artificial chromosome (BAC) library from the lycophyte Selaginella moellendorffii. Based on cell flow cytometry, this species has the smallest genome size among the different lycophytes tested, including Huperzia lucidula, Diphaiastrum digita, Isoetes engelmanii and S. kraussiana. The arrayed BAC library consists of 9126 clones; the average insert size is estimated to be 122 kb. Inserts of chloroplast origin account for 2.3% of the clones. The BAC library contains an estimated ten genome-equivalents based on DNA hybridizations using five single-copy and two duplicated S. moellendorffii genes as probes. Conclusion The S. moellenforffii BAC library, the first to be constructed from a lycophyte, will be useful to the scientific community as a resource for comparative plant genomics and evolution. PMID:15955246

  12. Screening and chromosome localization of two cotton BAC clones.

    PubMed

    Cui, Xinglei; Liu, Fang; Liu, Yuling; Zhou, Zhongli; Wang, Chunying; Yanyan Zhao; Meng, Fei; Wang, Xingxing; Cai, Xiaoyan; Wang, Yuhong; Peng, Renhai; Wang, Kunbo

    2016-01-01

    Two bacterial artificial chromosome (BAC) clones (350B21 and 299N22) of Pima 90-53 cotton [Gossypium barbadense Linnaeus, 1753 (2n=4x=52)] were screened from a BAC library using SSR markers. Strong hybridization signals were detected at terminal regions of all A genome (sub-genome) chromosomes, but were almost absent in D genome (sub-genome) chromosomes with BAC clone 350B21 as the probe. The results indicate that specific sequences, which only exist at the terminal parts of A genome (sub-genome) chromosomes with a huge repeat number, may be contained in BAC clone 350B21. When utilizing FISH with the BAC clone 299N22 as probe, a pair of obvious signals was detected on chromosome 13 of D genome (sub-genome), while strong dispersed signals were detected on all A genome (sub-genome) chromosomes. The results showed that peculiar repetitive sequence, which was distributed throughout all A genome (sub-genome) chromosomes, may exist in BAC clone 299N22. The absence of the repetitive sequences, which exist in the two BAC clones, in D genome may account for the genome-size variation between A and D genomes. In addition, the microcolinearity analysis of the clone 299N22 and its homologous region on Gossypium raimondii Ulbrich, 1932 chromosome 13 (D513) indicated that the clone 299N22 might come from A sub-genome of sea island cotton (Gossypium barbadense), and a huge number of small deletions, illegitimate recombination, translocation and rearrangements may have occurred during the genus evolution. The two BAC clones studied here can be used as cytological markers but will be also be helpful to research in cotton genome evolution and comparative genomics. PMID:27186333

  13. Screening and chromosome localization of two cotton BAC clones

    PubMed Central

    Cui, Xinglei; Liu, Fang; Liu, Yuling; Zhou, Zhongli; Wang, Chunying; Yanyan Zhao; Meng, Fei; Wang, Xingxing; Cai, Xiaoyan; Wang, Yuhong; Peng, Renhai; Wang, Kunbo

    2016-01-01

    Abstract Two bacterial artificial chromosome (BAC) clones (350B21 and 299N22) of Pima 90-53 cotton [Gossypium barbadense Linnaeus, 1753 (2n=4x=52)] were screened from a BAC library using SSR markers. Strong hybridization signals were detected at terminal regions of all A genome (sub-genome) chromosomes, but were almost absent in D genome (sub-genome) chromosomes with BAC clone 350B21 as the probe. The results indicate that specific sequences, which only exist at the terminal parts of A genome (sub-genome) chromosomes with a huge repeat number, may be contained in BAC clone 350B21. When utilizing FISH with the BAC clone 299N22 as probe, a pair of obvious signals was detected on chromosome 13 of D genome (sub-genome), while strong dispersed signals were detected on all A genome (sub-genome) chromosomes. The results showed that peculiar repetitive sequence, which was distributed throughout all A genome (sub-genome) chromosomes, may exist in BAC clone 299N22. The absence of the repetitive sequences, which exist in the two BAC clones, in D genome may account for the genome-size variation between A and D genomes. In addition, the microcolinearity analysis of the clone 299N22 and its homologous region on Gossypium raimondii Ulbrich, 1932 chromosome 13 (D513) indicated that the clone 299N22 might come from A sub-genome of sea island cotton (Gossypium barbadense), and a huge number of small deletions, illegitimate recombination, translocation and rearrangements may have occurred during the genus evolution. The two BAC clones studied here can be used as cytological markers but will be also be helpful to research in cotton genome evolution and comparative genomics. PMID:27186333

  14. A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R

    PubMed Central

    Bartoš, Jan; Paux, Etienne; Kofler, Robert; Havránková, Miroslava; Kopecký, David; Suchánková, Pavla; Šafář, Jan; Šimková, Hana; Town, Christopher D; Lelley, Tamas; Feuillet, Catherine; Doležel, Jaroslav

    2008-01-01

    Background Rye (Secale cereale L.) belongs to tribe Triticeae and is an important temperate cereal. It is one of the parents of man-made species Triticale and has been used as a source of agronomically important genes for wheat improvement. The short arm of rye chromosome 1 (1RS), in particular is rich in useful genes, and as it may increase yield, protein content and resistance to biotic and abiotic stress, it has been introgressed into wheat as the 1BL.1RS translocation. A better knowledge of the rye genome could facilitate rye improvement and increase the efficiency of utilizing rye genes in wheat breeding. Results Here, we report on BAC end sequencing of 1,536 clones from two 1RS-specific BAC libraries. We obtained 2,778 (90.4%) useful sequences with a cumulative length of 2,032,538 bp and an average read length of 732 bp. These sequences represent 0.5% of 1RS arm. The GC content of the sequenced fraction of 1RS is 45.9%, and at least 84% of the 1RS arm consists of repetitive DNA. We identified transposable element junctions in BESs and developed insertion site based polymorphism markers (ISBP). Out of the 64 primer pairs tested, 17 (26.6%) were specific for 1RS. We also identified BESs carrying microsatellites suitable for development of 1RS-specific SSR markers. Conclusion This work demonstrates the utility of chromosome arm-specific BAC libraries for targeted analysis of large Triticeae genomes and provides new sequence data from the rye genome and molecular markers for the short arm of rye chromosome 1. PMID:18803819

  15. [Integration sites and their characteristic analysis of piggyBac transposon in cattle genome].

    PubMed

    Du, Xin-Hua; Gao, Xue; Zhang, Lu-Pei; Gao, Hui-Jiang; Li, Jun-Ya; Xu, Shang-Zhong

    2013-06-01

    As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were detected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on chromosomes 1, 2, 11, and X chromosome. We found that PB transposon was inserted into the "TTAA" location and integrated into the intergenic non-regulatory sites between two genes. Analysis of the composition of the five bases, which was close to the side of the PB integration sites "TTAA", showed that PB 5' tended to be inserted into region rich in GC (62.5%). From the study, we got that transposition occurred in cattle genome by PB transposons and the integration site information acquired from the research will provide theoretical references for bovine study by PB transposon. PMID:23774022

  16. 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...

  17. Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision

    PubMed Central

    Nishizawa-Yokoi, Ayako; Endo, Masaki; Ohtsuki, Namie; Saika, Hiroaki; Toki, Seiichi

    2015-01-01

    Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants. PMID:25284193

  18. Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries

    SciTech Connect

    Liehr, T.; Weise, A.; Heller, A.; Starke, H.; Mrasek, K.; Kuechler, A.; Weier, H.-U.G.; Claussen, U.

    2003-06-23

    Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few mega base pairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of over lapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wave length intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific micro dissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region specific paints, but do not readily allow positioning of break points on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses.

  19. A first generation BAC-based physical map of the channel catfish genome

    PubMed Central

    Quiniou, Sylvie M-A; Waldbieser, Geoffrey C; Duke, Mary V

    2007-01-01

    Background Channel catfish, Ictalurus punctatus, is the leading species in North American aquaculture. Genetic improvement of catfish is performed through selective breeding, and genomic tools will help improve selection efficiency. A physical map is needed to integrate the genetic map with the karyotype and to support fine mapping of phenotypic trait alleles such as Quantitative Trait Loci (QTL) and the effective positional cloning of genes. Results A genome-wide physical map of the channel catfish was constructed by High-Information-Content Fingerprinting (HICF) of 46,548 Bacterial Artificial Chromosomes (BAC) clones using the SNaPshot technique. The clones were assembled into contigs with FPC software. The resulting assembly contained 1,782 contigs and covered an estimated physical length of 0.93 Gb. The validity of the assembly was demonstrated by 1) anchoring 19 of the largest contigs to the microsatellite linkage map 2) comparing the assembly of a multi-gene family to Restriction Fragment Length Polymorphism (RFLP) patterns seen in Southern blots, and 3) contig sequencing. Conclusion This is the first physical map for channel catfish. The HICF technique allowed the project to be finished with a limited amount of human resource in a high throughput manner. This physical map will greatly facilitate the detailed study of many different genomic regions in channel catfish, and the positional cloning of genes controlling economically important production traits. PMID:17284319

  20. Chimeric piggyBac transposases for genomic targeting in human cells.

    PubMed

    Owens, Jesse B; Urschitz, Johann; Stoytchev, Ilko; Dang, Nong C; Stoytcheva, Zoia; Belcaid, Mahdi; Maragathavally, Kommineni J; Coates, Craig J; Segal, David J; Moisyadi, Stefan

    2012-08-01

    Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy. PMID:22492708

  1. Vast potential for using the piggyBac transposon to engineer transgenic plants at specific genomic locations.

    PubMed

    Johnson, Eric T; Owens, Jesse B; Moisyadi, Stefan

    2016-01-01

    The acceptance of bioengineered plants by some nations is hampered by a number of factors, including the random insertion of a transgene into the host genome. Emerging technologies, such as site-specific nucleases, are enabling plant scientists to promote recombination or mutations at specific plant loci. Off target activity of these nucleases may limit widespread use. Insertion of transgenes by transposases engineered with a specific DNA binding domain has been accomplished in a number of organisms, but not in plants. The piggyBac transposon system, originally isolated from an insect, has been utilized to transform a variety of organisms. The piggyBac transposase is amendable to structural modifications, and was able to insert a transgene at a specific human locus through fusion of a DNA binding domain to its N-terminus. Recent developments demonstrating the activity of piggyBac transposase in plants is an important first step toward the potential use of engineered versions of piggyBac transposase for site-specific transgene insertion in plants. PMID:26930269

  2. A bacterial artificial chromosome library for the Australian saltwater crocodile (Crocodylus porosus) and its utilization in gene isolation and genome characterization

    PubMed Central

    2009-01-01

    Background Crocodilians (Order Crocodylia) are an ancient vertebrate group of tremendous ecological, social, and evolutionary importance. They are the only extant reptilian members of Archosauria, a monophyletic group that also includes birds, dinosaurs, and pterosaurs. Consequently, crocodilian genomes represent a gateway through which the molecular evolution of avian lineages can be explored. To facilitate comparative genomics within Crocodylia and between crocodilians and other archosaurs, we have constructed a bacterial artificial chromosome (BAC) library for the Australian saltwater crocodile, Crocodylus porosus. This is the first BAC library for a crocodile and only the second BAC resource for a crocodilian. Results The C. porosus BAC library consists of 101,760 individually archived clones stored in 384-well microtiter plates. NotI digestion of random clones indicates an average insert size of 102 kb. Based on a genome size estimate of 2778 Mb, the library affords 3.7 fold (3.7×) coverage of the C. porosus genome. To investigate the utility of the library in studying sequence distribution, probes derived from CR1a and CR1b, two crocodilian CR1-like retrotransposon subfamilies, were hybridized to C. porosus macroarrays. The results indicate that there are a minimum of 20,000 CR1a/b elements in C. porosus and that their distribution throughout the genome is decidedly non-random. To demonstrate the utility of the library in gene isolation, we probed the C. porosus macroarrays with an overgo designed from a C-mos (oocyte maturation factor) partial cDNA. A BAC containing C-mos was identified and the C-mos locus was sequenced. Nucleotide and amino acid sequence alignment of the C. porosus C-mos coding sequence with avian and reptilian C-mos orthologs reveals greater sequence similarity between C. porosus and birds (specifically chicken and zebra finch) than between C. porosus and squamates (green anole). Conclusion We have demonstrated the utility of the

  3. Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome

    PubMed Central

    2011-01-01

    Background Flax (Linum usitatissimum L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome. Results The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb). The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%), followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding. Conclusion The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable elements was low. The SSRs

  4. Characterizing the composition and evolution of homoeologous genomes in hexaploid wheat through BAC-end sequencing on chromosome 3B.

    PubMed

    Paux, Etienne; Roger, Delphine; Badaeva, Ekatherina; Gay, Georges; Bernard, Michel; Sourdille, Pierre; Feuillet, Catherine

    2006-11-01

    Bread wheat (Triticum aestivum) is one of the most important crops worldwide. However, because of its large, hexaploid, highly repetitive genome it is a challenge to develop efficient means for molecular analysis and genetic improvement in wheat. To better understand the composition and molecular evolution of the hexaploid wheat homoeologous genomes and to evaluate the potential of BAC-end sequences (BES) for marker development, we have followed a chromosome-specific strategy and generated 11 Mb of random BES from chromosome 3B, the largest chromosome of bread wheat. The sequence consisted of about 86% of repetitive elements, 1.2% of coding regions, and 13% remained unknown. With 1.2% of the sequence length corresponding to coding sequences, 6000 genes were estimated for chromosome 3B. New repetitive sequences were identified, including a Triticineae-specific tandem repeat (Fat) that represents 0.6% of the B-genome and has been differentially amplified in the homoeologous genomes before polyploidization. About 10% of the BES contained junctions between nested transposable elements that were used to develop chromosome-specific markers for physical and genetic mapping. Finally, sequence comparison with 2.9 Mb of random sequences from the D-genome of Aegilops tauschii suggested that the larger size of the B-genome is due to a higher content in repetitive elements. It also indicated which families of transposable elements are mostly responsible for differential expansion of the homoeologous wheat genomes during evolution. Our data demonstrate that BAC-end sequencing from flow-sorted chromosomes is a powerful tool for analysing the structure and evolution of polyploid and highly repetitive genomes. PMID:17010109

  5. 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...

  6. Construction and characterization of a human bacterial artificial chromosome library

    SciTech Connect

    Kim, Ung-Jin; Birren, B.W.; Slepak, T.

    1996-06-01

    We have constructed an arrayed human genomic BAC library with approximately 4X coverage that is represented by 96,000 BAC clones with average insert size of nearly 140 kb. A new BAC vector that allows color-based positive screening to identify transformants with inserts has increased BAC cloning efficiency. The library was gridded onto hybridization filters at high density for efficient identification of BAC clones by colony hybridization. The library was also formulated into characteristic DNA pools to allow for PCR screening of the library mainly by screening with more than 300 different landmarks that include cDNA, STSs, and cosmid clones. We describe methods for using BAC clones and discuss the implications for genome characterization, mapping, and sequencing. 25 refs., 5 figs., 1 tab.

  7. Construction of a BAC Library of the Leymus Cinereus X L. Triticoides Hybrid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leymus cinereus and L. triticoides are two wildrye grasses with many contrasting morphological and agronomic traits. The interspecific hybrid Leymus cinereus X L. triticoides and its progenies had been used to develop linkage maps of traits and molecular markers. To generate genomic DNA sequence i...

  8. Metagenomic analysis of Candidatus Liberibacter asiaticus in naturally populated psyllids (Diaphorina citri) using BAC libraries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Candidatus Liberibacter asiaticus (Las) is the most prevalent species of three species of Ca. Liberibacter causing citrus huanglongbing (HLB) in the world. The Las genome sequence published in 2009 was obtained from a single Las-infected psyllid using metagenomic approach. Studies of genetic divers...

  9. Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells.

    PubMed

    Saha, Sunandan; Woodard, Lauren E; Charron, Elizabeth M; Welch, Richard C; Rooney, Cliona M; Wilson, Matthew H

    2015-02-18

    Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5'TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects. PMID:25605795

  10. Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells

    PubMed Central

    Saha, Sunandan; Woodard, Lauren E.; Charron, Elizabeth M.; Welch, Richard C.; Rooney, Cliona M.; Wilson, Matthew H.

    2015-01-01

    Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5′TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects. PMID:25605795

  11. 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...

  12. 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

  13. Aquaculture Genomics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genomics chapter covers the basics of genome mapping and sequencing and the current status of several relevant species. The chapter briefly describes the development and use of (cDNA, BAC, etc.) libraries for mapping and obtaining specific sequence information. Other topics include comparative ...

  14. Generation of BAC reporter cell lines for drug discovery.

    PubMed

    Kao, Betty R; McColl, Bradley; Vadolas, Jim

    2015-01-01

    Bacterial artificial chromosome (BAC) reporter cell lines are generated through stable transfection of a BAC reporter construct wherein the gene of interest is tagged with a reporter gene such as eGFP. The large capacity of BACs (up to 350 kb of genomic sequence) enables the inclusion of all regulatory elements that ensure appropriate regulation of the gene of interest. Furthermore, the reporter gene allows the expression of the gene of interest to be readily detected by flow cytometry. Cell lines can also be easily cultured for extended periods with minimal cost. These features of BAC reporter cell lines make them highly amenable for use in high-throughput screening of large drug libraries for compounds that induce the expression of the gene of interest. This chapter describes a method for generation of BAC reporter cell lines that are suitable as cellular assay systems in high-throughput screening. Briefly, this method involves (A) generation of cell clones stably transfected with a BAC reporter construct, (B) selection of "candidate" cell clones based on the responsiveness to known inducers, (C) confirmation of the integrity of the BAC reporter construct integrated within the candidate clones, and (D) assessment of the developmental regulation of the BAC reporter construct. As an example, we describe the generation of a BAC reporter cell line containing the human β-globin locus modified to express γ-globin as eGFP for use as a cellular reporter assay for screening of drugs that can reactivate expression of developmentally silenced γ-globin for the treatment of β-hemoglobin disorders. PMID:25239756

  15. Comparison between BAC and oligo array platforms in detecting submicroscopic genomic rearrangements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Array-based comparative genomic hybridization (array CGH) has emerged as a powerful diagnostic technique for high resolution analysis of the human genome. It is a specific, sensitive, and rapid technique enabling detection of genomic arrangements and copy number changes. A variety of array CGH platf...

  16. Making BAC transgene constructs with lambda-red recombineering system for transgenic animals or cell lines.

    PubMed

    Holmes, Scott; Lyman, Suzanne; Hsu, Jen-Kang; Cheng, JrGang

    2015-01-01

    The genomic DNA libraries based on Bacteria Artificial Chromosomes (BAC) are the foundation of whole genomic mapping, sequencing, and annotation for many species like mice and humans. With their large insert size, BACs harbor the gene-of-interest and nearby transcriptional regulatory elements necessary to direct the expression of the gene-of-interest in a temporal and cell-type specific manner. When replacing a gene-of-interest with a transgene in vivo, the transgene can be expressed with the same patterns and machinery as that of the endogenous gene. This chapter describes in detail a method of using lambda-red recombineering to make BAC transgene constructs with the integration of a transgene into a designated location within a BAC. As the final BAC construct will be used for transfection in cell lines or making transgenic animals, specific considerations with BAC transgenes such as genotyping, BAC coverage and integrity as well as quality of BAC DNA will be addressed. Not only does this approach provide a practical and effective way to modify large DNA constructs, the same recombineering principles can apply to smaller high copy plasmids as well as to chromosome engineering. PMID:25239742

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

    PubMed Central

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

    2016-01-01

    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. PMID:27436955

  18. 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. PMID:27436955

  19. 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.

  20. BAC TransgeneOmics

    PubMed Central

    Poser, Ina; Sarov, Mihail; Hutchins, James R A; Hériché, Jean-Karim; Toyoda, Yusuke; Pozniakovsky, Andrei; Weigl, Daniela; Nitzsche, Anja; Hegemann, Björn; Bird, Alexander W; Pelletier, Laurence; Kittler, Ralf; Hua, Sujun; Naumann, Ronald; Augsburg, Martina; Sykora, Martina M; Hofemeister, Helmut; Zhang, Youming; Nasmyth, Kim; White, Kevin P; Dietzel, Steffen; Mechtler, Karl; Durbin, Richard; Stewart, A Francis; Peters, Jan-Michael; Buchholz, Frank; Hyman, Anthony A

    2009-01-01

    The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems. PMID:18391959

  1. PiggyBac transposon-based polyadenylation-signal trap for genome-wide mutagenesis in mice

    PubMed Central

    Li, Limei; Liu, Peng; Sun, Liangliang; Bin Zhou; Fei, Jian

    2016-01-01

    We designed a new type of polyadenylation-signal (PAS) trap vector system in living mice, the piggyBac (PB) (PAS-trapping (EGFP)) gene trapping vector, which takes advantage of the efficient transposition ability of PB and efficient gene trap and insertional mutagenesis of PAS-trapping. The reporter gene of PB(PAS-trapping (EGFP)) is an EGFP gene with its own promoter, but lacking a poly(A) signal. Transgenic mouse lines carrying PB(PAS-trapping (EGFP)) and protamine 1 (Prm1) promoter-driven PB transposase transgenes (Prm1-PBase) were generated by microinjection. Male mice doubly positive for PB(PAS-trapping (EGFP)) and Prm1-PBase were crossed with WT females, generating offspring with various insertion mutations. We found that 44.8% (26/58) of pups were transposon-positive progenies. New transposon integrations comprised 26.9% (7/26) of the transposon-positive progenies. We found that 100% (5/5) of the EGFP fluorescence-positive mice had new trap insertions mediated by a PB transposon in transcriptional units. The direction of the EGFP gene in the vector was consistent with the direction of the endogenous gene reading frame. Furthermore, mice that were EGFP-PCR positive, but EGFP fluorescent negative, did not show successful gene trapping. Thus, the novel PB(PAS-trapping (EGFP)) system is an efficient genome-wide gene-trap mutagenesis in mice. PMID:27292714

  2. 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...

  3. 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 (...

  4. Distribution of genes and repetitive elements in the Diabrotica virgifera virgifera genome estimated using BAC sequencing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Feeding damage caused by the corn rootworm, Diabrotica virgifera virgifera, is destructive to corn plants in North America and Europe where control remains challenging due to evolution of resistance traits that allow survival when exposed to chemical and transgenic toxins. Genome sequencing of an i...

  5. A First Generation Bac-Based Physical Map of the Channel Catfish Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Channel catfish, Ictalurus punctatus, is the leading species in North American aquaculture. Genetic improvement of catfish is performed through selective breeding, and genomic tools will help improve selection efficiency. A physical map is needed to integrate the genetic map with the kar...

  6. [An optimized method for construction of genomic library].

    PubMed

    Xu, Song; Zhang, Juan; Ma, Li-Xin

    2006-06-01

    Construction of genomic libraries is basic and important. Because of the laboriousness and high background of traditional methods for constructing genomic libraries, we improved them by overcoming these disadvantages. Two Ear I sites were chosen as the cloning sites, which can produce variable 3-base cohesive ends. Therefore the two overhangs could be devised to prevent a match and to avoid self-ligation of vector. Genomic DNA is cleaved partially with Sau3A I and subsequently incubated with dGTP and Klenow fragment of DNA polymerase Iso the self-ligation of fragments and ligation between them are blocked. In this study, the ARS probe vector (pHBM803/Trp) based on the improved method was constructed and then we constructed the Oryza sativa genomic library separately with the traditional method and improved method and compared them. The result of experiment indicated that the improved method could optimize the quality of library. PMID:16818436

  7. BAC sequencing using pooled methods.

    PubMed

    Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

    2015-01-01

    Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly. PMID:25239741

  8. TECHNIQUE FOR SCREENING AND MAINTAINING SMALLER GENOMIC LIBRARIES

    EPA Science Inventory

    A technique for screening and simultaneously maintaining individual clones of the gene library for long-term storage is described. his method is particularly useful for identification and cloning of genes from cosmid-based genomic libraries of prokaryotes that constitute a smalle...

  9. A 12-Mb complete coverage BAC contig map in human chromosome 16p13.1-p11.2.

    PubMed

    Cao, Y; Kang, H L; Xu, X; Wang, M; Dho, S H; Huh, J R; Lee, B J; Kalush, F; Bocskai, D; Ding, Y; Tesmer, J G; Lee, J; Moon, E; Jurecic, V; Baldini, A; Weier, H U; Doggett, N A; Simon, M I; Adams, M D; Kim, U J

    1999-08-01

    We have constructed a complete coverage BAC contig map that spans a 12-Mb genomic segment in the human chromosome 16p13.1-p11.2 region. The map consists of 68 previously mapped STSs and 289 BAC clones, 51 of which-corresponding to a total of 7.721 Mb of genomic DNA-have been sequenced, and provides a high resolution physical map of the region. Contigs were initially built based mainly on the analysis of STS contents and restriction fingerprint patterns of the clones. To close the gaps, probes derived from BAC clone ends were used to screen deeper BAC libraries. Clone end sequence data obtained from chromosome 16-specific BACs, as well as from public databases, were used for the identification of BACs that overlap with fully sequenced BACs by means of sequence match. This approach allowed precise alignment of clone overlaps in addition to restriction fingerprint comparison. A freehand contig drawing software tool was developed and used to manage the map data graphically and generate a real scale physical map. The map we present here is approximately 3.5 x deep and provides a minimal tiling path that covers the region in an array of contigous, overlapping BACs. PMID:10447511

  10. 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.

  11. Individual chromosome identification, chromosomal collinearity and genetic-physical integrated map in Gossypium darwinii and four D genome cotton species revealed by BAC-FISH.

    PubMed

    Gan, Yimei; Liu, Fang; Peng, Renhai; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Wang, Kunbo

    2012-01-01

    The study was conducted on the individual chromosome identification in Gossypium darwinii (A(d)D(d)), G. klotzschianum (D(3k)), G. davidsonii (D(3d)), G. armourianum (D(2-1)) and G. aridum (D(4)) using a multi-probe fluorescence of in situ hybridization (FISH) system. Comparative analysis on their genetic maps with that of physical maps was made as well. The FISH probes used contained two sets of bacterial artificial chromosome (BAC) clones [one is specific to 26 individual chromosomes from A and D subgenomes of G. hirsutum (A(h) and D(h)) while the other is a D genome centromere-specific BAC clone 150D24], 45S and 5S rDNA clones. The results showed that all A(d) chromosomes were marked with the 13 A(h) chromosome-specific BAC clones, whilst all D(d), D(3k), D(3d), D(2-1) and D(4) chromosomes and chromosomal arms were identified with the 13 D(h) chromosome-specific BAC clones and the D genome centromere-specific BAC. According to the homology within D subgenomes which are between A (D) genome and A (D) subgenome, the systematic nomenclature for individual chromosome in the five species was established. The chromosomes of A (D) subgenomes in G. darwinii were named as A(d)01-A(d)13 (D(d)01-D(d)13). The chromosomes in D(3k), D(3d), D(2-1) and D(4) were named as D(3k)01-D(3k)13, D(3d)01-D(3d)13, D(2-1)01-D(2-1)13 and D(4)01-D(4)13, respectively. Based on the successful identification for individual chromosomes, 45S and 5S rDNA were located to the special chromosomes and chromosomal arms for all five species. And there appeared chromosomal collinearity from the BAC clones among different species by comparing BACs positions, which suggested that the majority of chromosome segment homology may exist between D genomes and D subgenome. Moreover, as the genetic map and physical map were integrated, the orientations of genetic maps for D(d) and D genomes of diploid cotton were established. The orientations of some of chromosomes in genetic maps (D(d)03, D(d)04, D(d)06, D(d)09

  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. PMID:18931486

  13. Recombineering-Based Procedure for Creating BAC Transgene Constructs for Animals and Cell Lines

    PubMed Central

    Hollenback, Steven M.; Lyman, Suzanne; Cheng, JrGang

    2011-01-01

    The use of BAC/P1 as a vector for the generation of a transgene has gained popularity after the genomic annotation of many organisms was completed (often based on the respective BAC library). Large-scale generation of BAC transgenic mice has proven that BAC transgene approaches have less integration position effects and dosage artifacts when compared with traditional transgenic approaches. Also, a BAC can achieve the same tissue-specific expression as a Knock-in of the same gene with less effort and shorter time of establishment. The λ-RED recombinogenic system has been used to manipulate DNA constructs with site-directed mutagenesis, truncation, and tagging with an epitope tag or as a fusion protein by homologous recombination, as well as used here to modify many BACs with various transgenes. The recombineering plasmid, pKD46, is used to fabricate BAC transgenic constructs that can be used in generating transgenic organisms as well as used in mammalian cell culture. PMID:21732318

  14. Construction and characterization of a bacterial artificial chromosome library of Sorghum bicolor.

    PubMed Central

    Woo, S S; Jiang, J; Gill, B S; Paterson, A H; Wing, R A

    1994-01-01

    The construction of representative large insert DNA libraries is critical for the analysis of complex genomes. The predominant vector system for such work is the yeast artificial chromosome (YAC) system. Despite the success of YACs, many problems have been described including: chimerism, tedious steps in library construction and low yields of YAC insert DNA. Recently a new E.coli based system has been developed, the bacterial artificial chromosome (BAC) system, which offers many potential advantages over YACs. We tested the BAC system in plants by constructing an ordered 13,440 clone sorghum BAC library. The library has a combined average insert size, from single and double size selections, of 157 kb. Sorghum inserts of up to 315 kb were isolated and shown to be stable when grown for over 100 generations in liquid media. No chimeric clones were detected as determined by fluorescence in situ hybridization of ten BAC clones to metaphase and interphase S.bicolor nuclei. The library was screened with six sorghum probes and three maize probes and all but one sorghum probe hybridized to at least one BAC clone in the library. To facilitate chromosome walking with the BAC system, methods were developed to isolate the proximal ends of restriction fragments inserted into the BAC vector and used to isolate both the left and right ends of six randomly selected BAC clones. These results demonstrate that the S. bicolor BAC library will be useful for several physical mapping and map-based cloning applications not only in sorghum but other related cereal genomes, such as maize. Furthermore, we conclude that the BAC system is suitable for most large genome applications, is more 'user friendly' than the YAC system, and will likely lead to rapid progress in cloning biologically significant genes from plants. Images PMID:7800481

  15. 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

  16. Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones.

    PubMed

    Taboada, Xoana; Pansonato-Alves, Jose C; Foresti, Fausto; Martínez, Paulino; Viñas, Ana; Pardo, Belén G; Bouza, Carmen

    2014-06-01

    Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly. PMID:24473579

  17. Directing enhancer-traps and iTol2 end-sequences to deleted BAC ends with loxP- and lox511-Tn10 transposons.

    PubMed

    Chatterjee, Pradeep K

    2015-01-01

    A step-by-step detailed procedure is presented to progressively truncate genomic DNA inserts from either end in BACs. The bacterial transposon Tn10 carrying a loxP or a lox511 site is inserted at random into BAC DNA inside the bacterial cell. The cells are then infected with bacteriophage P1. The Cre protein expressed by phage P1 generates end-deletions by specifically recombining the inserted loxP (or lox511) with the loxP (or lox511) endogenous to and flanking insert DNA in BACs from the respective end. The Cre protein also helps phage P1 transduce the BAC DNA by packaging it in P1 heads. This packaging by P1 not only recovers the rare BAC clones containing Tn10 insertions efficiently but also selects end-truncated BACs from those containing inversions of portions of their DNA caused by transposition of Tn10 in the opposite orientation. The libraries of end-deleted BACs generated by this procedure are suitable for numerous mapping studies. Because DNA in front of the loxP (or lox511) arrowheads in the Tn10 transposon is retained at the newly created BAC end, exogenous DNA cassettes such as enhancer-traps and iTol2 ends can be efficiently introduced into BAC ends for germline expression in zebrafish or mice. The methodology should facilitate functional mapping studies of long-range cis-acting gene regulatory sequences in these organisms. PMID:25239743

  18. 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

  19. 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...

  20. Sequencing of the human IG light chain loci from a hydatidiform mole BAC library reveals locus-specific signatures of genetic diversity.

    PubMed

    Watson, C T; Steinberg, K M; Graves, T A; Warren, R L; Malig, M; Schein, J; Wilson, R K; Holt, R A; Eichler, E E; Breden, F

    2015-01-01

    Germline variation at immunoglobulin (IG) loci is critical for pathogen-mediated immunity, but establishing complete haplotype sequences in these regions has been problematic because of complex sequence architecture and diploid source DNA. We sequenced BAC clones from the effectively haploid human hydatidiform mole cell line, CHM1htert, across the light chain IG loci, kappa (IGK) and lambda (IGL), creating single haplotype representations of these regions. The IGL haplotype generated here is 1.25 Mb of contiguous sequence, including four novel IGLV alleles, one novel IGLC allele, and an 11.9-kb insertion. The CH17 IGK haplotype consists of two 644 kb proximal and 466 kb distal contigs separated by a large gap of unknown size; these assemblies added 49 kb of unique sequence extending into this gap. Our analysis also resulted in the characterization of seven novel IGKV alleles and a 16.7-kb region exhibiting signatures of interlocus sequence exchange between distal and proximal IGKV gene clusters. Genetic diversity in IGK/IGL was compared with that of the IG heavy chain (IGH) locus within the same haploid genome, revealing threefold (IGK) and sixfold (IGL) higher diversity in the IGH locus, potentially associated with increased levels of segmental duplication and the telomeric location of IGH. PMID:25338678

  1. A physical map of the bovine genome

    PubMed Central

    Snelling, Warren M; Chiu, Readman; Schein, Jacqueline E; Hobbs, Matthew; Abbey, Colette A; Adelson, David L; Aerts, Jan; Bennett, Gary L; Bosdet, Ian E; Boussaha, Mekki; Brauning, Rudiger; Caetano, Alexandre R; Costa, Marcos M; Crawford, Allan M; Dalrymple, Brian P; Eggen, André; Everts-van der Wind, Annelie; Floriot, Sandrine; Gautier, Mathieu; Gill, Clare A; Green, Ronnie D; Holt, Robert; Jann, Oliver; Jones, Steven JM; Kappes, Steven M; Keele, John W; de Jong, Pieter J; Larkin, Denis M; Lewin, Harris A; McEwan, John C; McKay, Stephanie; Marra, Marco A; Mathewson, Carrie A; Matukumalli, Lakshmi K; Moore, Stephen S; Murdoch, Brenda; Nicholas, Frank W; Osoegawa, Kazutoyo; Roy, Alice; Salih, Hanni; Schibler, Laurent; Schnabel, Robert D; Silveri, Licia; Skow, Loren C; Smith, Timothy PL; Sonstegard, Tad S; Taylor, Jeremy F; Tellam, Ross; Van Tassell, Curtis P; Williams, John L; Womack, James E; Wye, Natasja H; Yang, George; Zhao, Shaying

    2007-01-01

    Background Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The BAC libraries and clone maps are essential for the hybrid clone-by-clone/whole-genome shotgun sequencing approach taken by the bovine genome sequencing project. Results A bovine BAC map was constructed with HindIII restriction digest fragments of 290,797 BAC clones from animals of three different breeds. Comparative mapping of 422,522 BAC end sequences assisted with BAC map ordering and assembly. Genotypes and pedigree from two genetic maps and marker scores from three whole-genome RH panels were consolidated on a 17,254-marker composite map. Sequence similarity allowed integrating the BAC and composite maps with the bovine draft assembly (Btau3.1), establishing a comprehensive resource describing the bovine genome. Agreement between the marker and BAC maps and the draft assembly is high, although discrepancies exist. The composite and BAC maps are more similar than either is to the draft assembly. Conclusion Further refinement of the maps and greater integration into the genome assembly process may contribute to a high quality assembly. The maps provide resources to associate phenotypic variation with underlying genomic variation, and are crucial resources for understanding the biology underpinning this important ruminant species so closely associated with humans. PMID:17697342

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

    PubMed

    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

  3. Theobroma cacao: A genetically integrated physical map and genome-scale comparative synteny analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A comprehensive integrated genomic framework is considered a centerpiece of genomic research. In collaboration with the USDA-ARS (SHRS) and Mars Inc., the Clemson University Genomics Institute (CUGI) has developed a genetically anchored physical map of the T. cacao genome. Three BAC libraries contai...

  4. Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes

    PubMed Central

    Desai, Michael M.; Kishony, Roy

    2015-01-01

    Whole-genome sequencing has become an indispensible tool of modern biology. However, the cost of sample preparation relative to the cost of sequencing remains high, especially for small genomes where the former is dominant. Here we present a protocol for rapid and inexpensive preparation of hundreds of multiplexed genomic libraries for Illumina sequencing. By carrying out the Nextera tagmentation reaction in small volumes, replacing costly reagents with cheaper equivalents, and omitting unnecessary steps, we achieve a cost of library preparation of $8 per sample, approximately 6 times cheaper than the standard Nextera XT protocol. Furthermore, our procedure takes less than 5 hours for 96 samples. Several hundred samples can then be pooled on the same HiSeq lane via custom barcodes. Our method will be useful for re-sequencing of microbial or viral genomes, including those from evolution experiments, genetic screens, and environmental samples, as well as for other sequencing applications including large amplicon, open chromosome, artificial chromosomes, and RNA sequencing. PMID:26000737

  5. Herpesvirus BACs: Past, Present, and Future

    PubMed Central

    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. PMID:21048927

  6. 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. PMID:24090869

  7. 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.

  8. 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. PMID:25415307

  9. 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

  10. 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.

  11. Library construction for ancient genomics: single strand or double strand?

    PubMed

    Bennett, E Andrew; Massilani, Diyendo; Lizzo, Giulia; Daligault, Julien; Geigl, Eva-Maria; Grange, Thierry

    2014-06-01

    A novel method of library construction that takes advantage of a single-stranded DNA ligase has been recently described and used to generate high-resolution genomes from ancient DNA samples. While this method is effective and appears to recover a greater fraction of endogenous ancient material, there has been no direct comparison of results from different library construction methods on a diversity of ancient DNA samples. In addition, the single-stranded method is limited by high cost and lengthy preparation time and is restricted to the Illumina sequencing platform. Here we present in-depth comparisons of the different available library construction methods for DNA purified from 16 ancient and modern faunal and human remains, covering a range of different taphonomic and climatic conditions. We further present a DNA purification method for ancient samples that permits the concentration of a large volume of dissolved extract with minimal manipulation and methodological improvements to the single-stranded method to render it more economical and versatile, in particular to expand its use to both the Illumina and the Ion Torrent sequencing platforms. We show that the single-stranded library construction method improves the relative recovery of endogenous to exogenous DNA for most, but not all, of our ancient extracts. PMID:24924389

  12. 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

  13. 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....

  14. GenomeTools: a comprehensive software library for efficient processing of structured genome annotations.

    PubMed

    Gremme, Gordon; Steinbiss, Sascha; Kurtz, Stefan

    2013-01-01

    Genome annotations are often published as plain text files describing genomic features and their subcomponents by an implicit annotation graph. In this paper, we present the GenomeTools, a convenient and efficient software library and associated software tools for developing bioinformatics software intended to create, process or convert annotation graphs. The GenomeTools strictly follow the annotation graph approach, offering a unified graph-based representation. This gives the developer intuitive and immediate access to genomic features and tools for their manipulation. To process large annotation sets with low memory overhead, we have designed and implemented an efficient pull-based approach for sequential processing of annotations. This allows to handle even the largest annotation sets, such as a complete catalogue of human variations. Our object-oriented C-based software library enables a developer to conveniently implement their own functionality on annotation graphs and to integrate it into larger workflows, simultaneously accessing compressed sequence data if required. The careful C implementation of the GenomeTools does not only ensure a light-weight memory footprint while allowing full sequential as well as random access to the annotation graph, but also facilitates the creation of bindings to a variety of script programming languages (like Python and Ruby) sharing the same interface. PMID:24091398

  15. 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.

  16. 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...

  17. Genomic Resources for Gene Discovery, Functional Genome Annotation, and Evolutionary Studies of Maize and Its Close Relatives

    PubMed Central

    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

    2013-01-01

    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. PMID:24037269

  18. 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

  19. 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...

  20. 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...

  1. 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 users…

  2. 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.

  3. Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries

    PubMed Central

    Gaida, Stefan M.; Sandoval, Nicholas R.; Nicolaou, Sergios A.; Chen, Yili; Venkataramanan, Keerthi P.; Papoutsakis, Eleftherios T.

    2015-01-01

    A key limitation in using heterologous genomic or metagenomic libraries in functional genomics and genome engineering is the low expression of heterologous genes in screening hosts, such as Escherichia coli. To overcome this limitation, here we generate E. coli strains capable of recognizing heterologous promoters by expressing heterologous sigma factors. Among seven sigma factors tested, RpoD from Lactobacillus plantarum (Lpl) appears to be able of initiating transcription from all sources of DNA. Using the promoter GFP-trap concept, we successfully screen several heterologous and metagenomic DNA libraries, thus enlarging the genomic space that can be functionally sampled in E. coli. For an application, we show that screening fosmid-based Lpl genomic libraries in an E. coli strain with a chromosomally integrated Lpl rpoD enables the identification of Lpl genetic determinants imparting strong ethanol tolerance in E. coli. Transcriptome analysis confirms increased expression of heterologous genes in the engineered strain. PMID:25944046

  4. Genomic analysis of Ovis aries (Ovar)MHC Class IIa loci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Determining the genomic organization of the Ovis aries (Ovar) major histocompatibility complex class IIa region is essential for future functional studies related to antigen presentation. In this study, a bacterial artificial chromosome (BAC) library of genomic DNA from peripheral blood leukocytes ...

  5. Consequences of Normalizing Transcriptomic and Genomic Libraries of Plant Genomes Using a Duplex-Specific Nuclease and Tetramethylammonium Chloride

    PubMed Central

    Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

    2013-01-01

    Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce. PMID:23409088

  6. 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

  7. 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

  8. A new strategy for genome assembly using short sequence reads and reduced representation libraries.

    PubMed

    Young, Andrew L; Abaan, Hatice Ozel; Zerbino, Daniel; Mullikin, James C; Birney, Ewan; Margulies, Elliott H

    2010-02-01

    We have developed a novel approach for using massively parallel short-read sequencing to generate fast and inexpensive de novo genomic assemblies comparable to those generated by capillary-based methods. The ultrashort (<100 base) sequences generated by this technology pose specific biological and computational challenges for de novo assembly of large genomes. To account for this, we devised a method for experimentally partitioning the genome using reduced representation (RR) libraries prior to assembly. We use two restriction enzymes independently to create a series of overlapping fragment libraries, each containing a tractable subset of the genome. Together, these libraries allow us to reassemble the entire genome without the need of a reference sequence. As proof of concept, we applied this approach to sequence and assembled the majority of the 125-Mb Drosophila melanogaster genome. We subsequently demonstrate the accuracy of our assembly method with meaningful comparisons against the current available D. melanogaster reference genome (dm3). The ease of assembly and accuracy for comparative genomics suggest that our approach will scale to future mammalian genome-sequencing efforts, saving both time and money without sacrificing quality. PMID:20123915

  9. Separation of homologous BAC contigs in the tetraploid Upland cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Upland cotton has an allotetraploid genome. Separation of homologous BAC contigs to their sub-genomes and further to individual chromosomes is a great challenge for genome-wide integrated genetic and physical mapping. As a pilot experiment to test the feasibility of separating the contigs in sub-g...

  10. Construction and characterization of three wheat bacterial artificial chromosome libraries.

    PubMed

    Cao, Wenjin; Fu, Bisheng; Wu, Kun; Li, Na; Zhou, Yan; Gao, Zhongxia; Lin, Musen; Li, Guoqiang; Wu, Xinyi; Ma, Zhengqiang; Jia, Haiyan

    2014-01-01

    We have constructed three bacterial artificial chromosome (BAC) libraries of wheat cultivar Triticum aestivum Wangshuibai, germplasms T. monococcum TA2026 and TA2033. A total of 1,233,792,170,880 and 263,040 clones were picked and arrayed in 384-well plates. On the basis of genome sizes of 16.8 Gb for hexaploid wheat and 5.6 Gb for diploid wheat, the three libraries represented 9.05-, 2.60-, and 3.71-fold coverage of the haploid genomes, respectively. An improved descending pooling system for BAC libraries screening was established. This improved strategy can save 80% of the time and 68% of polymerase chain reaction (PCR) with the same successful rate as the universal 6D pooling strategy. PMID:25464379

  11. Construction and Characterization of Three Wheat Bacterial Artificial Chromosome Libraries

    PubMed Central

    Cao, Wenjin; Fu, Bisheng; Wu, Kun; Li, Na; Zhou, Yan; Gao, Zhongxia; Lin, Musen; Li, Guoqiang; Wu,  Xinyi; Ma, Zhengqiang; Jia, Haiyan

    2014-01-01

    We have constructed three bacterial artificial chromosome (BAC) libraries of wheat cultivar Triticum aestivum Wangshuibai, germplasms T. monococcum TA2026 and TA2033. A total of 1,233,792,170,880 and 263,040 clones were picked and arrayed in 384-well plates. On the basis of genome sizes of 16.8 Gb for hexaploid wheat and 5.6 Gb for diploid wheat, the three libraries represented 9.05-, 2.60-, and 3.71-fold coverage of the haploid genomes, respectively. An improved descending pooling system for BAC libraries screening was established. This improved strategy can save 80% of the time and 68% of polymerase chain reaction (PCR) with the same successful rate as the universal 6D pooling strategy. PMID:25464379

  12. Rapid construction of a Bacterial Artificial Chromosomal (BAC) expression vector using designer DNA fragments.

    PubMed

    Chen, Chao; Zhao, Xinqing; Jin, Yingyu; Zhao, Zongbao Kent; Suh, Joo-Won

    2014-11-01

    Bacterial artificial chromosomal (BAC) vectors are increasingly being used in cloning large DNA fragments containing complex biosynthetic pathways to facilitate heterologous production of microbial metabolites for drug development. To express inserted genes using Streptomyces species as the production hosts, an integration expression cassette is required to be inserted into the BAC vector, which includes genetic elements encoding a phage-specific attachment site, an integrase, an origin of transfer, a selection marker and a promoter. Due to the large sizes of DNA inserted into the BAC vectors, it is normally inefficient and time-consuming to assemble these fragments by routine PCR amplifications and restriction-ligations. Here we present a rapid method to insert fragments to construct BAC-based expression vectors. A DNA fragment of about 130 bp was designed, which contains upstream and downstream homologous sequences of both BAC vector and pIB139 plasmid carrying the whole integration expression cassette. In-Fusion cloning was performed using the designer DNA fragment to modify pIB139, followed by λ-RED-mediated recombination to obtain the BAC-based expression vector. We demonstrated the effectiveness of this method by rapid construction of a BAC-based expression vector with an insert of about 120 kb that contains the entire gene cluster for biosynthesis of immunosuppressant FK506. The empty BAC-based expression vector constructed in this study can be conveniently used for construction of BAC libraries using either microbial pure culture or environmental DNA, and the selected BAC clones can be directly used for heterologous expression. Alternatively, if a BAC library has already been constructed using a commercial BAC vector, the selected BAC vectors can be manipulated using the method described here to get the BAC-based expression vectors with desired gene clusters for heterologous expression. The rapid construction of a BAC-based expression vector facilitates

  13. Final progress report, Construction of a genome-wide highly characterized clone resource for genome sequencing

    SciTech Connect

    Nierman, William C.

    2000-02-14

    At TIGR, the human Bacterial Artificial Chromosome (BAC) end sequencing and trimming were with an overall sequencing success rate of 65%. CalTech human BAC libraries A, B, C and D as well as Roswell Park Cancer Institute's library RPCI-11 were used. To date, we have generated >300,000 end sequences from >186,000 human BAC clones with an average read length {approx}460 bp for a total of 141 Mb covering {approx}4.7% of the genome. Over sixty percent of the clones have BAC end sequences (BESs) from both ends representing over five-fold coverage of the genome by the paired-end clones. The average phred Q20 length is {approx}400 bp. This high accuracy makes our BESs match the human finished sequences with an average identity of 99% and a match length of 450 bp, and a frequency of one match per 12.8 kb contig sequence. Our sample tracking has ensured a clone tracking accuracy of >90%, which gives researchers a high confidence in (1) retrieving the right clone from the BA C libraries based on the sequence matches; and (2) building a minimum tiling path of sequence-ready clones across the genome and genome assembly scaffolds.

  14. 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.

  15. 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...

  16. A deep coverage Dictyostelium discoideum genomic DNA library replicates stably in Escherichia coli.

    PubMed

    Rosengarten, Rafael D; Beltran, Pamela R; Shaulsky, Gad

    2015-10-01

    The natural history of the amoeba Dictyostelium discoideum has inspired scientific inquiry for seventy-five years. A genetically tractable haploid eukaryote, D. discoideum appeals as a laboratory model as well. However, certain rote molecular genetic tasks, such as PCR and cloning, are difficult due to the AT-richness and low complexity of its genome. Here we report on the construction of a ~20 fold coverage D. discoideum genomic library in Escherichia coli, cloning 4-10 kilobase partial restriction fragments into a linear vector. End-sequencing indicates that most clones map to the six chromosomes in an unbiased distribution. Over 70% of these clones contain at least one complete open reading frame. We demonstrate that individual clones and library composition are stable over multiple replication cycles. Our library will enable numerous molecular biological applications and the completion of additional species' genome sequences, and suggests a path towards the long-elusive goal of genetic complementation. PMID:26028264

  17. 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...

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

    PubMed

    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

  19. 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

  20. 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...

  1. Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

    NASA Astrophysics Data System (ADS)

    Zhang, Yang; Zhang, Xiaojun; Scheuring, Chantel F.; Zhang, Hongbin; Li, Fuhua; Xiang, Jianhai

    2008-05-01

    Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

  2. AIMS Library - A community resource for sorghum genomic studies and breeding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sorghum genome sequence is completed. A systematically mutagenized population linking gene function to sequence is becoming increasingly urgent. A project was initiated to develop an Annotated Individually-pedigreed Mutagenized Sorghum (AIMS) library using (EMS) ethyl methane sulfonate for sel...

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:25858960

  4. 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

    2015-06-01

    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. PMID:25858960

  5. Preparation of a phage DNA fragment library for whole genome shotgun sequencing.

    PubMed

    Summer, Elizabeth J

    2009-01-01

    The most efficient method to determine the genomic sequence of a dsDNA phage is to use a whole genome shotgun approach (WGSA). Preparation of a library where each genomic fragment has an equal chance of being represented is critical to the success of the WGSA. For many phages, there are regions of the genome likely to be under-represented in the shotgun library, which results in more gaps in the shotgun assembly than predicted by the Poisson distribution. However, as phage genomes are relatively small, this increased number of gaps does not present an insurmountable impediment to using the WGSA. This chapter will focus on construction of a high-quality random library and sequence analysis of this library in a 96-well format. Techniques are described for the mechanical fragmentation of genomic DNA into 2 kb average size fragments, preparation of the fragmented DNA for shotgun cloning, and advice on the choice of cloning vector for library preparation. Protocols for deepwell block culture, plasmid isolation, and sequencing in 96-well format are given. The rationale for determining the total number of random clones from a library to sequence for a 50 and 150 kb genome is explained. The steps involved in going from hundreds of shotgun sequencing traces to generating contigs will be outlined as well as how to close gaps in the sequence by primer walking on phage DNA and PCR-generated templates. Finally, examples will be given of how biological information about the phage genomic termini can be derived by analysis of the organization of individual clones in the shotgun sequence assembly. Specific examples are given for the circularly permuted termini of pac type phages, the direct terminal repeats found in most T7-like phages, variable host DNA at either end as in the Mu-like phages, and the 5' and 3' overhanging ends of cos type phages. The end result of these steps is the entire DNA sequence of a novel phage, ready for gene prediction. PMID:19082550

  6. Genome-wide screen for miRNA targets using the MISSION target ID library.

    PubMed

    Coussens, Matthew J; Forbes, Kevin; Kreader, Carol; Sago, Jack; Cupp, Carrie; Swarthout, John

    2012-01-01

    The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details). To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3Î"TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%). PMID:22508434

  7. Construction and characterization of a 10-genome equivalent yeast artificial chromosome library for the laboratory rat, Rattus norvegicus

    SciTech Connect

    Cai, L.; Zee, R.Y.L.; Schalkwyk, L.C.

    1997-02-01

    Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species. 35 refs., 4 figs.

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

    PubMed

    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

  9. 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

  10. Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus

    PubMed Central

    2010-01-01

    Background Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome. Results Mj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences. Conclusions Our results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the

  11. 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

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

    2016-03-01

    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

  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. 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

  14. Construction of a high-coverage bacterial artificial chromosome library and comprehensive genetic linkage map of yellowtail Seriola quinqueradiata

    PubMed Central

    2014-01-01

    Background Japanese amberjack/yellowtail (Seriola quinqueradiata) is a commonly cultured marine fish in Japan. For cost effective fish production, a breeding program that increases commercially important traits is one of the major solutions. In selective breeding, information of genetic markers is useful and sufficient to identify individuals carrying advantageous traits but if the aim is to determine the genetic basis of the trait, large insert genomic DNA libraries are essential. In this study, toward prospective understanding of genetic basis of several economically important traits, we constructed a high-coverage bacterial artificial chromosome (BAC) library, obtained sequences from the BAC-end, and constructed comprehensive female and male linkage maps of yellowtail using Simple Sequence Repeat (SSR) markers developed from the BAC-end sequences and a yellowtail genomic library. Results The total insert length of the BAC library we constructed here was estimated to be approximately 11 Gb and hence 16-times larger than the yellowtail genome. Sequencing of the BAC-ends showed a low fraction of repetitive sequences comparable to that in Tetraodon and fugu. A total of 837 SSR markers developed here were distributed among 24 linkage groups spanning 1,026.70 and 1,057.83 cM with an average interval of 4.96 and 4.32 cM in female and male map respectively without any segregation distortion. Oxford grids suggested conserved synteny between yellowtail and stickleback. Conclusions In addition to characteristics of yellowtail genome such as low repetitive sequences and conserved synteny with stickleback, our genomic and genetic resources constructed and revealed here will be powerful tools for the yellowtail breeding program and also for studies regarding the genetic basis of traits. PMID:24684753

  15. BAC transgenic zebrafish for transcriptional promoter and enhancer studies.

    PubMed

    Kraus, Petra; Winata, Cecilia L; Lufkin, Thomas

    2015-01-01

    With the advent of BAC recombineering techniques, transcriptional promoter and enhancer isolation studies have become much more feasible in zebrafish than in mouse given the easy access to large numbers of fertilized zebrafish eggs and offspring in general, the easy to follow ex-utero development of zebrafish, an overall less skill demand and a more cost-effective technique. Here we provide guidelines for the generation of BAC recombineering-based transgenic zebrafish for DNA transcriptional promoter and enhancer identification studies as well as protocols for their analysis, which have been successfully applied in our laboratories many times. BAC recombineering in zebrafish allows for economical functional genomics studies, for example by integrating developmental biology with comparative genomics approaches to validate potential enhancer elements of vertebrate transcription factors. PMID:25239750

  16. 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…

  17. The first insight into the Taxus genome via fosmid library construction and end sequencing.

    PubMed

    Hao, DaCheng; Yang, Ling; Xiao, PeiGen

    2011-03-01

    Taxus mairei is a critically endangered and commercially important cultured medicinal gymnosperm in China and forms an important medicinal resource, but the research of its genome is absent. In this study, we constructed a T. mairei fosmid library and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of one million clones with an average insert size of about 39 kb, amounting to 3.9 genome equivalents. Fosmid stability assays indicate that T. mairei DNA was stable during propagation in the fosmid system. End sequencing of both 5' and 3' ends of 968 individual clones generated 1,923 sequences after trimming, with an average sequence length of 839 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 560 (29.1%) significant hits (E < e(-5)). Repetitive sequences analysis revealed that 20.8% of end sequences are repetitive elements, which were composed of retroelements, DNA transposons, satellites, simple repeats, and low complexity sequences. The distribution pattern of various repeat types was found to be more similar to the gymnosperm Pinus and Picea than to the monocot and dicot. The satellites of T. mairei were significantly longer than those of P. taeda and P. glauca. The tetra-nucleotide repeats of T. mairei were much longer than those of P. glauca and P. taeda. The fosmid library and the fosmid end sequences, for the first time, will serve as a useful resource for large-scale genome sequencing, physical mapping, SSR marker development and positional cloning, and provide a better understanding of the Taxus genome. PMID:21207064

  18. 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. PMID:26909353

  19. 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

  20. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing

    PubMed Central

    Urich, Mark A; Nery, Joseph R; Lister, Ryan; Schmitz, Robert J; Ecker, Joseph R

    2015-01-01

    Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein–DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples. PMID:25692984

  1. Scribl: an HTML5 Canvas-based graphics library for visualizing genomic data over the web

    PubMed Central

    Miller, Chase A.; Anthony, Jon; Meyer, Michelle M.; Marth, Gabor

    2013-01-01

    Motivation: High-throughput biological research requires simultaneous visualization as well as analysis of genomic data, e.g. read alignments, variant calls and genomic annotations. Traditionally, such integrative analysis required desktop applications operating on locally stored data. Many current terabyte-size datasets generated by large public consortia projects, however, are already only feasibly stored at specialist genome analysis centers. As even small laboratories can afford very large datasets, local storage and analysis are becoming increasingly limiting, and it is likely that most such datasets will soon be stored remotely, e.g. in the cloud. These developments will require web-based tools that enable users to access, analyze and view vast remotely stored data with a level of sophistication and interactivity that approximates desktop applications. As rapidly dropping cost enables researchers to collect data intended to answer questions in very specialized contexts, developers must also provide software libraries that empower users to implement customized data analyses and data views for their particular application. Such specialized, yet lightweight, applications would empower scientists to better answer specific biological questions than possible with general-purpose genome browsers currently available. Results: Using recent advances in core web technologies (HTML5), we developed Scribl, a flexible genomic visualization library specifically targeting coordinate-based data such as genomic features, DNA sequence and genetic variants. Scribl simplifies the development of sophisticated web-based graphical tools that approach the dynamism and interactivity of desktop applications. Availability and implementation: Software is freely available online at http://chmille4.github.com/Scribl/ and is implemented in JavaScript with all modern browsers supported. Contact: gabor.marth@bc.edu Supplementary information: Supplementary data are available at Bioinformatics

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

    PubMed

    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

  4. Analysis of BAC-end sequences in rainbow trout: content characterization and assessment of synteny between trout and other fish genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Rainbow trout (Oncorhynchus mykiss) are cultivated worldwide for aquaculture production and used as a model species to gain knowledge of many aspects of fish biology. As a salmonid, the species experienced recent whole genome duplication, making it a model for studying the evolution of t...

  5. 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...

  6. A high-density physical map of Sinorhizobium meliloti 1021 chromosome derived from bacterial artificial chromosome library

    PubMed Central

    Capela, Delphine; Barloy-Hubler, Frédérique; Gatius, Marie-Thérèse; Gouzy, Jérôme; Galibert, Francis

    1999-01-01

    As part of the European Sinorhizobium meliloti (strain 1021) chromosome sequencing project, four genomic bacterial artificial chromosome (BAC) libraries have been constructed, one of which was mainly used for chromosome mapping. This library consists of 1,824 clones with an average insert size of 80 kilobases and represents approximately 20-fold total genome coverage [6.8 megabases (Mbs)]. PCR screening of 384 BAC clones with 447 chromosomal markers (PCR primer pairs), consisting of 73 markers representing 118 genes (40 individual genes and 78 genes clustered in 23 operons), two markers from the rrn operon (three loci), four markers from insertion sequences (≈16 loci) and 368 sequence-tagged sites allowed the identification of 252 chromosomal BAC clones and the construction of a high-density physical map of the whole 3.7-Mb chromosome of S. meliloti. An average of 5.5 overlapping and colinear BAC clones per marker, correlated with a low rate of deleted or rearranged clones (0.8%) indicate a solid BAC contigation and a correct mapping. Systematic blastx analysis of sequence-tagged site marker sequences allowed prediction of a biological function for a number of putative ORFs. Results are available at http://www-recomgen.univ-rennes1.fr/meliloti. This map, whose resolution averages one marker every 9 kilobases, should provide a valuable tool for further sequencing, functional analysis, and positional cloning. PMID:10430947

  7. From the ORFeome concept to highly comprehensive, full-genome screening libraries.

    PubMed

    Rid, Raphaela; Abdel-Hadi, Omar; Maier, Richard; Wagner, Martin; Hundsberger, Harald; Hintner, Helmut; Bauer, Johann; Onder, Kamil

    2013-02-01

    Recombination-based cloning techniques have in recent times facilitated the establishment of genome-scale single-gene ORFeome repositories. Their further handling and downstream application in systematic fashion is, however, practically impeded because of logistical plus economic challenges. At this juncture, simultaneously transferring entire gene collections in compiled pool format could represent an advanced compromise between systematic ORFeome (an organism's entire set of protein-encoding open reading frames) projects and traditional random library approaches, but has not yet been considered in great detail. In our endeavor to merge the comprehensiveness of ORFeomes with a basically simple, streamlined, and easily executable single-tube design, we have here produced five different pooled screening-ready libraries for both Staphylococcus aureus and Homo sapiens. By evaluating the parallel transfer efficiencies of differentially sized genes from initial polymerase chain reaction (PCR) product amplification to entry and final destination library construction via quantitative real-time PCR, we found that the complexity of the gene population is fairly stably maintained once an entry resource has been successfully established, and that no apparent size-selection bias loss of large inserts takes place. Recombinational transfer processes are hence robust enough for straightforwardly achieving such pooled screening libraries. PMID:22621725

  8. Next-generation libraries for robust RNA interference-based genome-wide screens

    PubMed Central

    Kampmann, Martin; Horlbeck, Max A.; Chen, Yuwen; Tsai, Jordan C.; Bassik, Michael C.; Gilbert, Luke A.; Villalta, Jacqueline E.; Kwon, S. Chul; Chang, Hyeshik; Kim, V. Narry; Weissman, Jonathan S.

    2015-01-01

    Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity. PMID:26080438

  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. PMID:27041357

  10. 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.

  11. 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

  12. Construction, complete sequence, and annotation of a BAC contig covering the silkworm chorion locus.

    PubMed

    Chen, Zhiwei; Nohata, Junko; Guo, Huizhen; Li, Shenglong; Liu, Jianqiu; Guo, Youbing; Yamamoto, Kimiko; Kadono-Okuda, Keiko; Liu, Chun; Arunkumar, Kallare P; Nagaraju, Javaregowda; Zhang, Yan; Liu, Shiping; Labropoulou, Vassiliki; Swevers, Luc; Tsitoura, Panagiota; Iatrou, Kostas; Gopinathan, Karumathil P; Goldsmith, Marian R; Xia, Qingyou; Mita, Kazuei

    2015-01-01

    The silkmoth chorion was studied extensively by F.C. Kafatos' group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis. PMID:26594380

  13. Construction, complete sequence, and annotation of a BAC contig covering the silkworm chorion locus

    PubMed Central

    Chen, Zhiwei; Nohata, Junko; Guo, Huizhen; Li, Shenglong; Liu, Jianqiu; Guo, Youbing; Yamamoto, Kimiko; Kadono-Okuda, Keiko; Liu, Chun; Arunkumar, Kallare P.; Nagaraju, Javaregowda; Zhang, Yan; Liu, Shiping; Labropoulou, Vassiliki; Swevers, Luc; Tsitoura, Panagiota; Iatrou, Kostas; Gopinathan, Karumathil P.; Goldsmith, Marian R.; Xia, Qingyou; Mita, Kazuei

    2015-01-01

    The silkmoth chorion was studied extensively by F.C. Kafatos’ group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis. PMID:26594380

  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. Antarctic Notothenioid Fishes: Genomic Resources and Strategies for Analyzing an Adaptive Radiation

    PubMed Central

    Detrich, H. W.; Amemiya, Chris T.

    2010-01-01

    The perciform suborder Notothenoidei provides a compelling opportunity to study the adaptive radiation of a marine species-flock in the cold Southern Ocean that surrounds Antarctica. To facilitate genome-level studies of the diversification of these fishes, we present estimates of the genome sizes of 11 Antarctic species and describe the production of high-quality bacterial artificial chromosome (BAC) libraries for two, the red-blooded notothen Notothenia coriiceps and the white-blooded icefish Chaenocephalus aceratus. Our results indicate that evolution of phylogenetically derived notothenioid families (e.g., the crown group Channichthyidae [icefishes]), was accompanied by genome expansion. Six species from the basal family Nototheniidae had C-values between 0.98 and 1.20 pg, a range that is consistent with the genome sizes of proposed outgroups (e.g., percids) of the notothenioid suborder. In contrast, four icefishes had C-values in the range 1.66–1.83 pg. The BAC libraries VMRC-19 (N. coriiceps) and VMRC-21 (C. aceratus) comprise 12× and 10× coverage of the respective genomes and have average insert sizes of 138 and 168 kb. Paired BAC-end reads representing ∼0.1% of each genome showed that the repetitive element landscapes of the two genomes (13.4% of the N. coriiceps genome and 14.5% for C. aceratus) were similar. The availability of these high-quality and well-characterized BAC libraries sets the stage for targeted genomic analyses of the unusual anatomical and physiological adaptations of the notothenioids, some of which mimic human diseases. Here we consider the evolution of secondary pelagicism by various taxa of the group and illustrate the utility of Antarctic icefishes as an evolutionary-mutant model of human osteopenia (low-mineral density of bones). PMID:21082069

  16. Identifying microbial fitness determinants by Insertion Sequencing (INSeq) using genome-wide transposon mutant libraries

    PubMed Central

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

    2012-01-01

    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 16–17bp 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 (18h), 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 multi-well format is provided. PMID:22094732

  17. 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. PMID:25836973

  18. 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

  19. Discovery of new Mycoplasma pneumoniae antigens by use of a whole-genome lambda display library.

    PubMed

    Beghetto, Elisa; De Paolis, Francesca; Montagnani, Francesca; Cellesi, Carla; Gargano, Nicola

    2009-01-01

    Mycoplasma pneumoniae is the leading cause of atypical pneumonia in children and young adults. Bacterial colonization can occur in both the upper and the lower respiratory tracts and take place both endemically and epidemically worldwide. Characteristically, the infection is chronic in onset and recovery and both humoral and cell-mediated mechanisms are involved in the response to bacterial colonization. To identify bacterial proteins recognized by host antibody responses, a whole-genome M. pneumoniae library was created and displayed on lambda bacteriophage. The challenge of such a library with sera from individuals hospitalized for mycoplasmal pneumonia allowed the identification of a panel of recombinant bacteriophages carrying B-cell epitopes. Among the already known M. pneumoniae B-cell antigens, our results confirmed the immunogenicity of P1 and P30 adhesins. Also, the data presented in this study localized, within their sequences, the immunodominant epitopes recognized by human immunoglobulins. Furthermore, library screening allowed the identification of four novel immunogenic polypeptides, respectively, encoded by fragments of the MPN152, MPN426, MPN456 and MPN-500 open reading frames, highlighting and further confirming the potential of lambda display technology in antigen and epitope discovery. PMID:18992837

  20. 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

  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. Functional Screening of Metagenome and Genome Libraries for Detection of Novel Flavonoid-Modifying Enzymes

    PubMed Central

    Rabausch, U.; Juergensen, J.; Ilmberger, N.; Böhnke, S.; Fischer, S.; Schubach, B.; Schulte, M.

    2013-01-01

    The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. PMID:23686272

  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. 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…

  5. A Low-Copy-Number Plasmid for Retrieval of Toxic Genes from BACs and Generation of Conditional Targeting Constructs

    PubMed Central

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

    2016-01-01

    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. PMID:22945876

  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. The piggyBac transposon displays local and distant reintegration preferences and can cause mutations at noncanonical integration sites.

    PubMed

    Li, Meng Amy; Pettitt, Stephen J; Eckert, Sabine; Ning, Zemin; Rice, Stephen; Cadiñanos, Juan; Yusa, Kosuke; Conte, Nathalie; Bradley, Allan

    2013-04-01

    The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome. PMID:23358416

  8. The piggyBac Transposon Displays Local and Distant Reintegration Preferences and Can Cause Mutations at Noncanonical Integration Sites

    PubMed Central

    Li, Meng Amy; Pettitt, Stephen J.; Eckert, Sabine; Ning, Zemin; Rice, Stephen; Cadiñanos, Juan; Yusa, Kosuke; Conte, Nathalie

    2013-01-01

    The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome. PMID:23358416

  9. Generation of knockout alleles by RFLP based BAC targeting of polymorphic embryonic stem cells.

    PubMed

    Barakat, Tahsin Stefan; Gribnau, Joost

    2015-01-01

    The isolation of germ line competent mouse Embryonic Stem (ES) cells and the ability to modify the genome by homologous recombination has revolutionized life science research. Since its initial discovery, several approaches have been introduced to increase the efficiency of homologous recombination, including the use of isogenic DNA for the generation of targeting constructs, and the use of Bacterial Artificial Chromosomes (BACs). BACs have the advantage of combining long stretches of homologous DNA, thereby increasing targeting efficiencies, with the possibilities delivered by BAC recombineering approaches, which provide the researcher with almost unlimited possibilities to efficiently edit the genome in a controlled fashion. Despite these advantages of BAC targeting approaches, a widespread use has been hampered, mainly because of the difficulties in identifying BAC-targeted knockout alleles by conventional methods like Southern Blotting. Recently, we introduced a novel BAC targeting strategy, in which Restriction Fragment Length Polymorphisms (RFLPs) are targeted in polymorphic mouse ES cells, enabling an efficient and easy PCR-based readout to identify properly targeted alleles. Here we provide a detailed protocol for the generation of targeting constructs, targeting of ES cells, and convenient PCR-based analysis of targeted clones, which enable the user to generate knockout ES cells of almost every gene in the mouse genome within a 2-month period. PMID:25239745

  10. Library preparation methodology can influence genomic and functional predictions in human microbiome research

    PubMed Central

    Jones, Marcus B.; Highlander, Sarah K.; Anderson, Ericka L.; Li, Weizhong; Dayrit, Mark; Klitgord, Niels; Fabani, Martin M.; Seguritan, Victor; Green, Jessica; Pride, David T.; Yooseph, Shibu; Biggs, William; Nelson, Karen E.; Venter, J. Craig

    2015-01-01

    Observations from human microbiome studies are often conflicting or inconclusive. Many factors likely contribute to these issues including small cohort sizes, sample collection, and handling and processing differences. The field of microbiome research is moving from 16S rDNA gene sequencing to a more comprehensive genomic and functional representation through whole-genome sequencing (WGS) of complete communities. Here we performed quantitative and qualitative analyses comparing WGS metagenomic data from human stool specimens using the Illumina Nextera XT and Illumina TruSeq DNA PCR-free kits, and the KAPA Biosystems Hyper Prep PCR and PCR-free systems. Significant differences in taxonomy are observed among the four different next-generation sequencing library preparations using a DNA mock community and a cell control of known concentration. We also revealed biases in error profiles, duplication rates, and loss of reads representing organisms that have a high %G+C content that can significantly impact results. As with all methods, the use of benchmarking controls has revealed critical differences among methods that impact sequencing results and later would impact study interpretation. We recommend that the community adopt PCR-free–based approaches to reduce PCR bias that affects calculations of abundance and to improve assemblies for accurate taxonomic assignment. Furthermore, the inclusion of a known-input cell spike-in control provides accurate quantitation of organisms in clinical samples. PMID:26512100

  11. Whole Genome Profiling provides a robust framework for physical mapping and sequencing in the highly complex and repetitive wheat genome

    PubMed Central

    2012-01-01

    Background Sequencing projects using a clone-by-clone approach require the availability of a robust physical map. The SNaPshot technology, based on pair-wise comparisons of restriction fragments sizes, has been used recently to build the first physical map of a wheat chromosome and to complete the maize physical map. However, restriction fragments sizes shared randomly between two non-overlapping BACs often lead to chimerical contigs and mis-assembled BACs in such large and repetitive genomes. Whole Genome Profiling (WGP™) was developed recently as a new sequence-based physical mapping technology and has the potential to limit this problem. Results A subset of the wheat 3B chromosome BAC library covering 230 Mb was used to establish a WGP physical map and to compare it to a map obtained with the SNaPshot technology. We first adapted the WGP-based assembly methodology to cope with the complexity of the wheat genome. Then, the results showed that the WGP map covers the same length than the SNaPshot map but with 30% less contigs and, more importantly with 3.5 times less mis-assembled BACs. Finally, we evaluated the benefit of integrating WGP tags in different sequence assemblies obtained after Roche/454 sequencing of BAC pools. We showed that while WGP tag integration improves assemblies performed with unpaired reads and with paired-end reads at low coverage, it does not significantly improve sequence assemblies performed at high coverage (25x) with paired-end reads. Conclusions Our results demonstrate that, with a suitable assembly methodology, WGP builds more robust physical maps than the SNaPshot technology in wheat and that WGP can be adapted to any genome. Moreover, WGP tag integration in sequence assemblies improves low quality assembly. However, to achieve a high quality draft sequence assembly, a sequencing depth of 25x paired-end reads is required, at which point WGP tag integration does not provide additional scaffolding value. Finally, we suggest that WGP

  12. Identification of repetitive elements in the genome of Oreochromis niloticus: Tilapia repeat-masker

    PubMed Central

    Shirak, A.; Grabherr, M.; Di Palma, F.; Lindblad-Toh, K.; Hulata, G.; Ron, M.; Kocher, TD.

    2011-01-01

    The large-scale BAC end-sequencing project of Nile tilapia (Oreochromis niloticus) has generated extensive sequence data that allowed the examination of the repeat content in this fish genome and building of a repeat library specific for this species. This library was established based on Tilapiini repeat sequences from GenBank; sequences orthologous to the repeat library of zebrafish in Repbase; and novel repeats detected by genome analysis using MIRA assembler. We estimate that repeats constitute about 14% of the tilapia genome and also give estimates for the occurrence of the different repeats based on the BLAST searches within the database of known tilapia sequences. The frequent occurrence of novel repeats in the tilapia genome indicates the importance of using the species-specific repeat masker prior to sequence analyses. A web tool based on the RepeatMasker software was designed to assist tilapia genomics. PMID:19936833

  13. 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. PMID:24406816

  14. Isolation of rare recombinants without using selectable markers for one-step seamless BAC mutagenesis

    PubMed Central

    Lyozin, George T.; Kosaka, Yasuhiro; Demarest, Bradley L.; Yost, H. Joseph; Kuehn, Michael R.; Brunelli, Luca

    2014-01-01

    Current laboratory methods to isolate rare (1:10,000 to 1:100,000) bacterial artificial chromosome (BAC) recombinants require selectable markers. Seamless BAC mutagenesis needs two steps: isolation of rare recombinants using selectable markers, followed by marker removal through counterselection. Here we illustrate founder principle-driven enrichment (FPE), a simple method developed to rapidly isolate rare recombinants without using selectable markers, allowing one-step seamless BAC mutagenesis. As proof-of-principle, we isolated 1:100,000 seamless fluorescent protein-modified Nodal BACs via FPE and confirmed BAC functionality by generating fluorescent reporter mice. We also isolated small indel P1-phage derived artificial chromosome (PAC) and BAC recombinants. Statistical analysis revealed that 1:100,000 recombinants can be isolated running <40 PCRs and we developed a web-based calculator to optimize FPE. By eliminating the need for selection-counterselection, this work highlights a straightforward and low-cost approach to BAC mutagenesis, providing a tool for seamless recombineering pipelines in functional genomics. PMID:25028895

  15. Precise marker excision system using an animal-derived piggyBac transposon in plants.

    PubMed

    Nishizawa-Yokoi, Ayako; Endo, Masaki; Osakabe, Keishi; Saika, Hiroaki; Toki, Seiichi

    2014-02-01

    Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth-derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase-expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T0 plants without concomitant re-integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection. PMID:24164672

  16. Precise marker excision system using an animal-derived piggyBac transposon in plants

    PubMed Central

    Nishizawa-Yokoi, Ayako; Endo, Masaki; Osakabe, Keishi; Saika, Hiroaki; Toki, Seiichi

    2014-01-01

    Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth-derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase-expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T0 plants without concomitant re-integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection. PMID:24164672

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

    PubMed

    Zeng, Qingdong; Yuan, Fengping; Xu, Xin; Shi, Xue; Nie, Xiaojun; Zhuang, Hua; Chen, Xianming; Wang, Zhonghua; Wang, Xiaojie; Huang, Lili; Han, Dejun; Kang, Zhensheng

    2014-01-01

    For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sublibraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from the HindIII sublibrary and 573 clones from the BamHI sublibrary, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, the HindIII sublibrary was estimated to have a 3.01-fold coverage and the BamHI sublibrary a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700 Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interest. PMID:24895618

  18. Construction and Characterization of a Bacterial Artificial Chromosome Library for the Hexaploid Wheat Line 92R137

    PubMed Central

    Yuan, Fengping; Xu, Xin; Shi, Xue; Zhuang, Hua; Wang, Zhonghua; Huang, Lili; Han, Dejun; Kang, Zhensheng

    2014-01-01

    For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sublibraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis (PFGE) analysis of 453 clones randomly selected from the HindIII sublibrary and 573 clones from the BamHI sublibrary, the average insert sizes were estimated as 129 and 113 kb, respectively. Thus, the HindIII sublibrary was estimated to have a 3.01-fold coverage and the BamHI sublibrary a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700 Mb. The 765,696 clones were arrayed in 1,994 384-well plates. All clones were also arranged into plate pools and further arranged into 5-dimensional (5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence (such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interest. PMID:24895618

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

    PubMed Central

    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.

    2008-01-01

    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. PMID:18838673

  20. Conversion of BAC clones into binary BAC (BIBAC) vectors and their delivery into basidiomycete fungal cells using Agrobacterium tumefaciens.

    PubMed

    Ali, Shawkat; Bakkeren, Guus

    2015-01-01

    The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi. PMID:25239747

  1. How Accurate are the Extremely Small P-values Used in Genomic Research: An Evaluation of Numerical Libraries

    PubMed Central

    Bangalore, Sai Santosh; Wang, Jelai; Allison, David B.

    2009-01-01

    In the fields of genomics and high dimensional biology (HDB), massive multiple testing prompts the use of extremely small significance levels. Because tail areas of statistical distributions are needed for hypothesis testing, the accuracy of these areas is important to confidently make scientific judgments. Previous work on accuracy was primarily focused on evaluating professionally written statistical software, like SAS, on the Statistical Reference Datasets (StRD) provided by National Institute of Standards and Technology (NIST) and on the accuracy of tail areas in statistical distributions. The goal of this paper is to provide guidance to investigators, who are developing their own custom scientific software built upon numerical libraries written by others. In specific, we evaluate the accuracy of small tail areas from cumulative distribution functions (CDF) of the Chi-square and t-distribution by comparing several open-source, free, or commercially licensed numerical libraries in Java, C, and R to widely accepted standards of comparison like ELV and DCDFLIB. In our evaluation, the C libraries and R functions are consistently accurate up to six significant digits. Amongst the evaluated Java libraries, Colt is most accurate. These languages and libraries are popular choices among programmers developing scientific software, so the results herein can be useful to programmers in choosing libraries for CDF accuracy. PMID:20161126

  2. 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.

  3. pileup.js: a JavaScript library for interactive and in-browser visualization of genomic data

    PubMed Central

    Aksoy, B. Arman; Hodes, Isaac; Perrone, Jaclyn; Hammerbacher, Jeff

    2016-01-01

    pileup.js is a new browser-based genome viewer. It is designed to facilitate the investigation of evidence for genomic variants within larger web applications. It takes advantage of recent developments in the JavaScript ecosystem to provide a modular, reliable and easily embedded library. Availability and implementation: The code and documentation for pileup.js is publicly available at https://github.com/hammerlab/pileup.js under the Apache 2.0 license. Contact: correspondence@hammerlab.org PMID:27153605

  4. 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

  5. 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.

  6. Integration of physical and genetic maps in apple confirms whole-genome and segmental duplications in the apple genome

    PubMed Central

    Han, Yuepeng; Zheng, Danman; Vimolmangkang, Sornkanok; Khan, Muhammad A.; Beever, Jonathan E.; Korban, Schuyler S.

    2011-01-01

    A total of 355 simple sequence repeat (SSR) markers were developed, based on expressed sequence tag (EST) and bacterial artificial chromosome (BAC)-end sequence databases, and successfully used to construct an SSR-based genetic linkage map of the apple. The consensus linkage map spanned 1143 cM, with an average density of 2.5 cM per marker. Newly developed SSR markers along with 279 SSR markers previously published by the HiDRAS project were further used to integrate physical and genetic maps of the apple using a PCR-based BAC library screening approach. A total of 470 contigs were unambiguously anchored onto all 17 linkage groups of the apple genome, and 158 contigs contained two or more molecular markers. The genetically mapped contigs spanned ∼421 Mb in cumulative physical length, representing 60.0% of the genome. The sizes of anchored contigs ranged from 97 kb to 4.0 Mb, with an average of 995 kb. The average physical length of anchored contigs on each linkage group was ∼24.8 Mb, ranging from 17.0 Mb to 37.73 Mb. Using BAC DNA as templates, PCR screening of the BAC library amplified fragments of highly homologous sequences from homoeologous chromosomes. Upon integrating physical and genetic maps of the apple, the presence of not only homoeologous chromosome pairs, but also of multiple locus markers mapped to adjacent sites on the same chromosome was detected. These findings demonstrated the presence of both genome-wide and segmental duplications in the apple genome and provided further insights into the complex polyploid ancestral origin of the apple. PMID:21743103

  7. 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

  8. Development of microsatellite markers for common bean (Phaseolus vulgaris L.) based on screening of non-enriched, small-insert genomic libraries.

    PubMed

    Blair, Matthew W; Torres, Monica Muñoz; Pedraza, Fabio; Giraldo, Martha C; Buendía, Hector F; Hurtado, Natalia

    2009-09-01

    Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their development is time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screening of non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motif frequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony picking and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by digestion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and a less frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libraries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to create high-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSR motifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were tested for polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched libraries should be useful additions to previous markers developed from enriched libraries. PMID:19935925

  9. Sequencing crop genomes: approaches and applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant genome sequencing methodology parrallels the sequencing of the human genome. The first projects were slow and very expensive. BAC by BAC approaches were utilized first and whole-genome shotgun sequencing rapidly replaced that approach. So called 'next generation' technologies such as short rea...

  10. Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.)

    PubMed Central

    Ritschel, Patricia Silva; Lins, Tulio Cesar de Lima; Tristan, Rodrigo Lourenço; Buso, Gláucia Salles Cortopassi; Buso, José Amauri; Ferreira, Márcio Elias

    2004-01-01

    Background Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species. Results Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups. Conclusions Genomic library microsatellite enrichment is an efficient procedure for marker

  11. 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...

  12. Hybrid nonviral/viral vector systems for improved piggyBac DNA transposon in vivo delivery.

    PubMed

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

    2015-04-01

    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

  13. The first generation of a BAC-based physical map of Brassica rapa

    PubMed Central

    Mun, Jeong-Hwan; Kwon, Soo-Jin; Yang, Tae-Jin; Kim, Hye-Sun; Choi, Beom-Soon; Baek, Seunghoon; Kim, Jung Sun; Jin, Mina; Kim, Jin A; Lim, Myung-Ho; Lee, Soo In; Kim, Ho-Il; Kim, Hyungtae; Lim, Yong Pyo; Park, Beom-Seok

    2008-01-01

    Background The genus Brassica includes the most extensively cultivated vegetable crops worldwide. Investigation of the Brassica genome presents excellent challenges to study plant genome evolution and divergence of gene function associated with polyploidy and genome hybridization. A physical map of the B. rapa genome is a fundamental tool for analysis of Brassica "A" genome structure. Integration of a physical map with an existing genetic map by linking genetic markers and BAC clones in the sequencing pipeline provides a crucial resource for the ongoing genome sequencing effort and assembly of whole genome sequences. Results A genome-wide physical map of the B. rapa genome was constructed by the capillary electrophoresis-based fingerprinting of 67,468 Bacterial Artificial Chromosome (BAC) clones using the five restriction enzyme SNaPshot technique. The clones were assembled into contigs by means of FPC v8.5.3. After contig validation and manual editing, the resulting contig assembly consists of 1,428 contigs and is estimated to span 717 Mb in physical length. This map provides 242 anchored contigs on 10 linkage groups to be served as seed points from which to continue bidirectional chromosome extension for genome sequencing. Conclusion The map reported here is the first physical map for Brassica "A" genome based on the High Information Content Fingerprinting (HICF) technique. This physical map will serve as a fundamental genomic resource for accelerating genome sequencing, assembly of BAC sequences, and comparative genomics between Brassica genomes. The current build of the B. rapa physical map is available at the B. rapa Genome Project website for the user community. PMID:18549474

  14. Use of in vitro OmniPlex libraries for high-throughput comparative genomics and molecular haplotyping

    NASA Astrophysics Data System (ADS)

    Kamberov, Emmanuel; Sleptsova, Irina; Suchyta, Stephen; Bruening, Eric D.; Ziehler, William; Seward Nagel, Julie; Langmore, John P.; Makarov, Vladimir

    2002-06-01

    OmniPlex Technology is a new approach to genome amplification and targeted analysis. Initially the entire genome is reformatted into small, amplifiable molecules called Plexisomes, which represent the entire genome as an OmniPlex Library. The whole genome can be amplified en masse using universal primers; using locus-specific primers, regions as large as 50 kb can be amplified. Amplified Plexisomes can be analyzed using conventional methods such as capillary sequencing and microarray hybridization. The advantages to using OmniPlex as the 'front-end' for conventional analytical instruments are that a) the initial copy number of the analytes can be increased to achieve better signal-to-noire ratio, b) only a single priming site is used and c) up to 20 times fewer biochemical reactions and oligonucleotides are necessary to amplify a large region, compared to conventional PCR. These factors make OmniPlex more flexible, faster, and less expensive than conventional technologies. OmniPlex has been applied to targeted sequencing of human, animal, plant, and microorganism genomes. In addition, OmniPlex is inherently able to haplotype large regions of human DNA to accelerate target discovery and pharmacogenomics. OmniPlex will be a key tool for delivery of improved crops and livestock, new pharmaceutical products, and personalized medicine.

  15. BAC modification through serial or simultaneous use of CRE/Lox technology.

    PubMed

    Parrish, Mark; Unruh, Jay; Krumlauf, Robb

    2011-01-01

    Bacterial Artificial Chromosomes (BACs) are vital tools in mouse genomic analyses because of their ability to propagate large inserts. The size of these constructs, however, prevents the use of conventional molecular biology techniques for modification and manipulation. Techniques such as recombineering and Cre/Lox methodologies have thus become heavily relied upon for such purposes. In this work, we investigate the applicability of Lox variant sites for serial and/or simultaneous manipulations of BACs. We show that Lox spacer mutants are very specific, and inverted repeat variants reduce Lox reaction rates through reducing the affinity of Cre for the site, while retaining some functionality. Employing these methods, we produced serial modifications encompassing four independent changes which generated a mouse HoxB BAC with fluorescent reporter proteins inserted into four adjacent Hox genes. We also generated specific, simultaneous deletions using combinations of spacer variants and inverted repeat variants. These techniques will facilitate BAC manipulations and open a new repertoire of methods for BAC and genome manipulation. PMID:21197414

  16. 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.

  17. 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...

  18. Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics

    PubMed Central

    Yuan, Hui; Peng, Li; Han, Zhong; Xie, Juan-Juan; Liu, Xi-Peng

    2015-01-01

    Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of P. furiosus proteins at whole genome level, we constructed expression plasmids of each P. furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using phosphorothioate-modified primers and digesting PCR products by λ exonuclease. Our cloning method had a positive clone percentage of ≥ 80% in 96-well plate cloning format. Small-scale expression experiment showed that 55 out of 80 genes were efficiently expressed in Escherichia coli Strain Rosetta 2(DE3)pLysS. In summary, this recombinant expression library of P. furiosus provides a platform for functional and structural studies, as well as developing novel industrial enzymes. Our cloning scheme is adaptable to constructing recombinant expression library of other sequenced organisms. PMID:26441878

  19. 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. PMID:25634499

  20. BacWGSTdb, a database for genotyping and source tracking bacterial pathogens

    PubMed Central

    Ruan, Zhi; Feng, Ye

    2016-01-01

    Whole genome sequencing has become one of the routine methods in molecular epidemiological practice. In this study, we present BacWGSTdb (http://bacdb.org/BacWGSTdb), a bacterial whole genome sequence typing database which is designed for clinicians, clinical microbiologists and hospital epidemiologists. This database borrows the population structure from the current multi-locus sequence typing (MLST) scheme and adopts a hierarchical data structure: species, clonal complex and isolates. When users upload the pre-assembled genome sequences to BacWGSTdb, it offers the functionality of bacterial genotyping at both traditional MLST and whole-genome levels. More importantly, users are told which isolates in the public database are phylogenetically close to the query isolate, along with their clinical information such as host, isolation source, disease, collection time and geographical location. In this way, BacWGSTdb offers a rapid and convenient platform for worldwide users to address a variety of clinical microbiological issues such as source tracking bacterial pathogens. PMID:26433226

  1. GISH and BAC-FISH study of apomictic Beta M14.

    PubMed

    Ge, Yan; He, GuangChun; Wang, ZhiWei; Guo, DeDong; Qin, Rui; Li, RongTian

    2007-04-01

    Apomixis is a means of asexual reproduction by which plants produce embryos without fertilization and meiosis, therefore the embryo is of clonal and maternal origin. Interspecific hybrids between diploid B. vulgaris (2n=2x=18) and tetraploid B. corolliflora (2n=4x=36) were established, and then back-crossed with B. vulgaris. Among their offspring, monosomic addition line M14 (2n=2x=18+1) was selected because of the apomictic phenotype. We documented chromosome transmission from B. corolliflora into M14 by using genomic in situ hybridization (GISH). Suppression of cross-hybridization by blocking DNA was not necessary, indicating that the investigated Beta genome contains sufficient species-specific DNA, thus enabling the determination of genomic composition of the hybrids. We analyzed BAC microarrays of B. corolliflora chromosome 9 by using fluorescence-specific mRNA of B. vulgaris and Beta M14. BAC clones 16-M11 and 26-L15 were detected as fluorescence-specifics of BAC DNA of Beta M14. Then both BAC clones 16-M11 and 26-L15 were in situ hybridized to M14 chromosomes. The two hybridized BAC clones were located close to the telomere region of the long arm of a single chromosome 9, and showed hemizygosity. The results of BAC microarrays showed that these developments of embryo and endosperm have conservative expression patterns, indicating that sexual reproduction and apomixis have an interrelated pathway with common regulatory components and that the induction of a modified sexual reproduction program may enable the manifestation of apomixis in Beta species. It would be sufficient for the expression of apomixes with those apomictic-specific genes on chromosome 9 of B. corolliflora. PMID:17447032

  2. BacDive—the Bacterial Diversity Metadatabase

    PubMed Central

    Söhngen, Carola; Bunk, Boyke; Podstawka, Adam; Gleim, Dorothea; Overmann, Jörg

    2014-01-01

    BacDive—the Bacterial Diversity Metadatabase (http://bacdive.dsmz.de) merges detailed strain-linked information on the different aspects of bacterial and archaeal biodiversity. Currently (release 9/2013), BacDive contains entries for 23 458 strains and provides information on their taxonomy, morphology, physiology, sampling and concomitant environmental conditions as well as molecular biology. Where available, links to access the respective biological resources are given. The majority of the BacDive data is manually annotated and curated. The BacDive portal offers an easy-to-use simple search and in addition powerful advanced search functionalities allowing to combine more than 30 search fields for text and numerical data. The user can compile individual sets of strains to a download selection that can easily be imported into nearly all spreadsheet applications. PMID:24214959

  3. BacDive--the Bacterial Diversity Metadatabase.

    PubMed

    Söhngen, Carola; Bunk, Boyke; Podstawka, Adam; Gleim, Dorothea; Overmann, Jörg

    2014-01-01

    BacDive-the Bacterial Diversity Metadatabase (http://bacdive.dsmz.de) merges detailed strain-linked information on the different aspects of bacterial and archaeal biodiversity. Currently (release 9/2013), BacDive contains entries for 23 458 strains and provides information on their taxonomy, morphology, physiology, sampling and concomitant environmental conditions as well as molecular biology. Where available, links to access the respective biological resources are given. The majority of the BacDive data is manually annotated and curated. The BacDive portal offers an easy-to-use simple search and in addition powerful advanced search functionalities allowing to combine more than 30 search fields for text and numerical data. The user can compile individual sets of strains to a download selection that can easily be imported into nearly all spreadsheet applications. PMID:24214959

  4. Local Assemblies of Paired-End Reduced Representation Libraries Sequenced with the Illumina Genome Analyzer in Maize

    PubMed Central

    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. PMID:23093955

  5. Construction of bacterial artificial chromosome libraries from the parasitic nematode Brugia malayi and physical mapping of the genome of its Wolbachia endosymbiont.

    PubMed

    Foster, Jeremy M; Kumar, Sanjay; Ganatra, Mehul B; Kamal, Ibrahim H; Ware, Jennifer; Ingram, Jessica; Pope-Chappell, Jesse; Guiliano, David; Whitton, Claire; Daub, Jennifer; Blaxter, Mark L; Slatko, Barton E

    2004-05-01

    The parasitic nematode, Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis. The parasite contains an endosymbiotic alpha-proteobacterium of the genus Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes. Bacterial artificial chromosome libraries were constructed from B. malayi DNA and provide over 11-fold coverage of the nematode genome. Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1 Mb bacterial genome. A physical framework for the Wolbachia genome was developed by construction of a plasmid library enriched for Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters. Bacterial artificial chromosome end sequencing provided additional Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome. The Wolbachia sequences provided a marker approximately every 10 kb. Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native Wolbachia genomic DNA. Comparison of Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69 Wolbachia peptide functions and two rRNA genes. Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the Wolbachia endosymbiont from Drosophila melanogaster, there was no evidence for long-range synteny. Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb. The molecular scaffold produced for the genome of the Wolbachia from B. malayi forms the basis of a genomic sequencing effort for

  6. Conserved microstructure of the Brassica B Genome of Brassica nigra in relation to homologous regions of Arabidopsis thaliana, B. rapa and B. oleracea

    PubMed Central

    2013-01-01

    Background The Brassica B genome is known to carry several important traits, yet there has been limited analyses of its underlying genome structure, especially in comparison to the closely related A and C genomes. A bacterial artificial chromosome (BAC) library of Brassica nigra was developed and screened with 17 genes from a 222 kb region of A. thaliana that had been well characterised in both the Brassica A and C genomes. Results Fingerprinting of 483 apparently non-redundant clones defined physical contigs for the corresponding regions in B. nigra. The target region is duplicated in A. thaliana and six homologous contigs were found in B. nigra resulting from the whole genome triplication event shared by the Brassiceae tribe. BACs representative of each region were sequenced to elucidate the level of microscale rearrangements across the Brassica species divide. Conclusions Although the B genome species separated from the A/C lineage some 6 Mya, comparisons between the three paleopolyploid Brassica genomes revealed extensive conservation of gene content and sequence identity. The level of fractionation or gene loss varied across genomes and genomic regions; however, the greatest loss of genes was observed to be common to all three genomes. One large-scale chromosomal rearrangement differentiated the B genome suggesting such events could contribute to the lack of recombination observed between B genome species and those of the closely related A/C lineage. PMID:23586706

  7. Advances in BAC-Based Physical Mapping and Map Integration Strategies in Plants

    PubMed Central

    Ariyadasa, Ruvini; Stein, Nils

    2012-01-01

    In the advent of next-generation sequencing (NGS) platforms, map-based sequencing strategy has been recently suppressed being too expensive and laborious. The detailed studies on NGS drafts alone indicated these assemblies remain far from gold standard reference quality, especially when applied on complex genomes. In this context the conventional BAC-based physical mapping has been identified as an important intermediate layer in current hybrid sequencing strategy. BAC-based physical map construction and its integration with high-density genetic maps have benefited from NGS and high-throughput array platforms. This paper addresses the current advancements of BAC-based physical mapping and high-throughput map integration strategies to obtain densely anchored well-ordered physical maps. The resulted maps are of immediate utility while providing a template to harness the maximum benefits of the current NGS platforms. PMID:22500080

  8. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.

    PubMed

    Poser, Ina; Sarov, Mihail; Hutchins, James R A; Hériché, Jean-Karim; Toyoda, Yusuke; Pozniakovsky, Andrei; Weigl, Daniela; Nitzsche, Anja; Hegemann, Björn; Bird, Alexander W; Pelletier, Laurence; Kittler, Ralf; Hua, Sujun; Naumann, Ronald; Augsburg, Martina; Sykora, Martina M; Hofemeister, Helmut; Zhang, Youming; Nasmyth, Kim; White, Kevin P; Dietzel, Steffen; Mechtler, Karl; Durbin, Richard; Stewart, A Francis; Peters, Jan-Michael; Buchholz, Frank; Hyman, Anthony A

    2008-05-01

    The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems. PMID:18391959

  9. PiggyBac transposon mutagenesis: a tool for cancer gene discovery in mice.

    PubMed

    Rad, Roland; Rad, Lena; Wang, Wei; Cadinanos, Juan; Vassiliou, George; Rice, Stephen; Campos, Lia S; Yusa, Kosuke; Banerjee, Ruby; Li, Meng Amy; de la Rosa, Jorge; Strong, Alexander; Lu, Dong; Ellis, Peter; Conte, Nathalie; Yang, Fang Tang; Liu, Pentao; Bradley, Allan

    2010-11-19

    Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here, we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon-transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed that PiggyBac is capable of genome-wide mutagenesis. The PiggyBac screen uncovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 21 mouse lines to be compatible with both transposon systems in constitutive, tissue- or temporal-specific mutagenesis. Mice with different transposon types, copy numbers, and chromosomal locations support wide applicability. PMID:20947725

  10. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    PubMed Central

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  11. BACFinder: genomic localisation of large insert genomic clones based on restriction fingerprinting

    PubMed Central

    Crowe, Mark L.; Rana, Debashis; Fraser, Fiona; Bancroft, Ian; Trick, Martin

    2002-01-01

    We have developed software that allows the prediction of the genomic location of a bacterial artificial chromosome (BAC) clone, or other large genomic clone, based on a simple restriction digest of the BAC. The mapping is performed by comparing the experimentally derived restriction digest of the BAC DNA with a virtual restriction digest of the whole genome sequence. Our trials indicate that this program identified the genomic regions represented by BAC clones with a degree of accuracy comparable to that of end-sequencing, but at considerably less cost. Although the program has been developed principally for use with Arabidopsis BACs, it should align large insert genomic clones to any fully sequenced genome. PMID:12409477

  12. Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives.

    PubMed

    Rana, Debashis; van den Boogaart, Tom; O'Neill, Carmel M; Hynes, Llewelyn; Bent, Elisabeth; Macpherson, Lee; Park, Jee Young; Lim, Yong Pyo; Bancroft, Ian

    2004-12-01

    The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting

  13. Remobilizing deleted piggyBac vector post-integration for transgene stability in silkworm.

    PubMed

    Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Xu, Hanfu; Yuan, Lin; Ding, Huan; Ma, Sanyuan; Zhou, You; Zhao, Ping; Xia, Qingyou

    2015-06-01

    Deletion of transposable elements post-genomic integration holds great promise for stability of the transgene in the host genome and has an essential role for the practical application of transgenic animals. In this study, a modified piggyBac vector that mediated deletion of the transposon sequence post-integration for transgene stability in the economically important silkworm Bombyx mori was constructed. The piggyBac vector architecture contains inversed terminal repeat sequences L1, L2 and R1, which can form L1/R1 and L2/R1 types of transposition cassettes. hsp70-PIG as the piggyBac transposase expression cassette for initial transposition, further remobilization and transgene stabilization test was transiently expressed in a helper vector or integrated into the modified vector to produce a transgenic silkworm. Shortening L2 increased the transformation frequency of L1/R1 into the silkworm genome compared to L2/R1. After the integration of L1/R1 into the genome, the remobilization of L2/R1 impaired the transposon structure and the resulting transgene linked with an impaired transposon was stable in the genome even in the presence of exogenously introduced transposase, whereas those flanked by the intact transposon were highly mobile in the genome. Our results demonstrated the feasibility of post-integration deletion of transposable elements to guarantee true transgene stabilization in silkworm. We suggest that the modified vector will be a useful resource for studies of transgenic silkworms and other piggyBac-transformed organisms. PMID:25589404

  14. 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. PMID:26607054

  15. 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.

  16. Recombineering BAC transgenes for protein tagging.

    PubMed

    Ciotta, Giovanni; Hofemeister, Helmut; Maresca, Marcello; Fu, Jun; Sarov, Mihail; Anastassiadis, Konstantinos; Stewart, A Francis

    2011-02-01

    Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression. PMID:20868752

  17. A PiggyBac-mediated approach for muscle gene transfer or cell therapy.

    PubMed

    Ley, Déborah; Van Zwieten, Ruthger; Puttini, Stefania; Iyer, Pavithra; Cochard, Alessia; Mermod, Nicolas

    2014-11-01

    An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies. PMID:25310255

  18. 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.

  19. 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. PMID:24610029

  20. Screening of Metagenomic and Genomic Libraries Reveals Three Classes of Bacterial Enzymes That Overcome the Toxicity of Acrylate

    PubMed Central

    Curson, Andrew R. J.; Burns, Oliver J.; Voget, Sonja; Daniel, Rolf; Todd, Jonathan D.; McInnis, Kathryn; Wexler, Margaret; Johnston, Andrew W. B.

    2014-01-01

    Acrylate is produced in significant quantities through the microbial cleavage of the highly abundant marine osmoprotectant dimethylsulfoniopropionate, an important process in the marine sulfur cycle. Acrylate can inhibit bacterial growth, likely through its conversion to the highly toxic molecule acrylyl-CoA. Previous work identified an acrylyl-CoA reductase, encoded by the gene acuI, as being important for conferring on bacteria the ability to grow in the presence of acrylate. However, some bacteria lack acuI, and, conversely, many bacteria that may not encounter acrylate in their regular environments do contain this gene. We therefore sought to identify new genes that might confer tolerance to acrylate. To do this, we used functional screening of metagenomic and genomic libraries to identify novel genes that corrected an E. coli mutant that was defective in acuI, and was therefore hyper-sensitive to acrylate. The metagenomic libraries yielded two types of genes that overcame this toxicity. The majority encoded enzymes resembling AcuI, but with significant sequence divergence among each other and previously ratified AcuI enzymes. One other metagenomic gene, arkA, had very close relatives in Bacillus and related bacteria, and is predicted to encode an enoyl-acyl carrier protein reductase, in the same family as FabK, which catalyses the final step in fatty-acid biosynthesis in some pathogenic Firmicute bacteria. A genomic library of Novosphingobium, a metabolically versatile alphaproteobacterium that lacks both acuI and arkA, yielded vutD and vutE, two genes that, together, conferred acrylate resistance. These encode sequential steps in the oxidative catabolism of valine in a pathway in which, significantly, methacrylyl-CoA is a toxic intermediate. These findings expand the range of bacteria for which the acuI gene encodes a functional acrylyl-CoA reductase, and also identify novel enzymes that can similarly function in conferring acrylate resistance, likely, again

  1. Characteristics of Highly Polymorphic Segmental Copy-Number Variations Observed in Japanese by BAC-Array-CGH

    PubMed Central

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

    2011-01-01

    Segmental copy-number variations (CNVs) may contribute to genetic variation in humans. Reports of the existence and characteristics of CNVs in a large Japanese cohort are quite limited. We report the data from a large Japanese population. We conducted population screening for 213 unrelated Japanese individuals using comparative genomic hybridization based on a bacterial artificial chromosome microarray (BAC-aCGH). We summarize the data by focusing on highly polymorphic CNVs in ≥5.0% of the individual, since they may be informative for demonstrating the relationships between genotypes and their phenotypes. We found a total of 680 CNVs at 16 different BAC-regions in the genome. The majority of the polymorphic CNVs presented on BAC-clones that overlapped with regions of segmental duplication, and the majority of the polymorphic CNVs observed in this population had been previously reported in other publications. Some of the CNVs contained genes which might be related to phenotypic heterogeneity among individuals. PMID:21197411

  2. SplinkBES - A Splinkerette-Based Method for Generating Long End Sequences From Large Insert DNA Libraries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report on the development of a novel splinkerette-based method for generating long end-sequences from large insert library clones, using a carrot (Daucus carota L.) BAC library as a model. The procedure involves digestion of the BAC DNA with a 6-bp restriction enzyme, followed by ligation of spli...

  3. SweetBac: Applying MultiBac Technology Towards Flexible Modification of Insect Cell Glycosylation.

    PubMed

    Palmberger, Dieter; Rendic, Dubravko

    2015-01-01

    Observed different glycosylation patterns of glycoconjugates (recombinantly) produced in various eukaryotic organisms are a direct consequence of differences in numerous proteins involved in biosynthesis of the relevant glycan chains in these species. The need for efficient, robust and flexible methods for recombinant expression of proteins is met by the recently described MultiBac technology, an advanced and optimized baculovirus-based system for simultaneous recombinant protein expression in insect cells. A derivative of MultiBac technology, the SweetBac system aims at the modification of the glycosylation potential of insect cells as expression hosts. The application of SweetBac, including the methods needed to investigate the glycosylation pattern of the purified recombinant protein, is described in this chapter. PMID:26082221

  4. From Human Monocytes to Genome-Wide Binding Sites - A Protocol for Small Amounts of Blood: Monocyte Isolation/ChIP-Protocol/Library Amplification/Genome Wide Computational Data Analysis

    PubMed Central

    Weiterer, Sebastian; Uhle, Florian; Bhuju, Sabin; Jarek, Michael; Weigand, Markus A.; Bartkuhn, Marek

    2014-01-01

    Chromatin immunoprecipitation in combination with a genome-wide analysis via high-throughput sequencing is the state of the art method to gain genome-wide representation of histone modification or transcription factor binding profiles. However, chromatin immunoprecipitation analysis in the context of human experimental samples is limited, especially in the case of blood cells. The typically extremely low yields of precipitated DNA are usually not compatible with library amplification for next generation sequencing. We developed a highly reproducible protocol to present a guideline from the first step of isolating monocytes from a blood sample to analyse the distribution of histone modifications in a genome-wide manner. Conclusion: The protocol describes the whole work flow from isolating monocytes from human blood samples followed by a high-sensitivity and small-scale chromatin immunoprecipitation assay with guidance for generating libraries compatible with next generation sequencing from small amounts of immunoprecipitated DNA. PMID:24732314

  5. 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.

  6. 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

  7. Coexisting/Coexpressing Genomic Libraries (CoGeL) identify interactions among distantly located genetic loci for developing complex microbial phenotypes

    PubMed Central

    Nicolaou, Sergios A.; Gaida, Stefan M.; Papoutsakis, Eleftherios T.

    2011-01-01

    In engineering novel microbial strains for biotechnological applications, beyond a priori identifiable pathways to be engineered, it is becoming increasingly important to develop complex, ill-defined cellular phenotypes. One approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs. Such libraries are limited by their inability to identify interactions among distant genetic loci. To solve this problem, we constructed plasmid- and fosmid-based Escherichia coli Coexisting/Coexpressing Genomic Libraries (CoGeLs). As a proof of principle, four sets of two genes of the l-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes demonstrating the interaction between the two libraries. CoGeLs were also screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. CoGeL screening identified combination of genes known to enhance acid tolerance (gadBC operon and adiC), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold. arcZ is a small RNA that we show increases pH tolerance alone and together with recA. PMID:21976725

  8. Chromosome evolution in Solanum traced by cross-species BAC-FISH.

    PubMed

    Szinay, Dóra; Wijnker, Erik; van den Berg, Ronald; Visser, Richard G F; de Jong, Hans; Bai, Yuling

    2012-08-01

    Chromosomal rearrangements are relatively rare evolutionary events and can be used as markers to study karyotype evolution. This research aims to use such rearrangements to study chromosome evolution in Solanum. Chromosomal rearrangements between Solanum crops and several related wild species were investigated using tomato and potato bacterial artificial chromosomes (BACs) in a multicolour fluorescent in situ hybridization (FISH). The BACs selected are evenly distributed over seven chromosomal arms containing inversions described in previous studies. The presence/absence of these inversions among the studied Solanum species were determined and the order of the BAC-FISH signals was used to construct phylogenetic trees.Compared with earlier studies, data from this study provide support for the current grouping of species into different sections within Solanum; however, there are a few notable exceptions, such as the tree positions of S. etuberosum (closer to the tomato group than to the potato group) and S. lycopersicoides (sister to S. pennellii). These apparent contradictions might be explained by interspecific hybridization events and/or incomplete lineage sorting. This cross-species BAC painting technique provides unique information on genome organization, evolution and phylogenetic relationships in a wide variety of species. Such information is very helpful for introgressive breeding. PMID:22686400

  9. 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. PMID:22831897

  10. Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids.

    PubMed

    Nováková, Jana; Izsáková, Anita; Grivalský, Tomáš; Ottmann, Christian; Farkašovský, Marian

    2014-01-01

    High transformation competency of Escherichia coli is one of the critical factors in the bacterial artificial chromosome (BAC)-based DNA library construction. Many electroporation protocols have been published until now, but the majority of them was optimized for transformation of small plasmids. Large plasmids with a size above 50 kbp display reduced transformation efficiency and thereby require specific conditions in the preparation and electroporation of electrocompetent cells. In the present work, we have optimized the parameters critical to the application of BAC DNA electrotransformation into E. coli. Systematic evaluation of electroporation variables has revealed several key factors like temperature of growth, media supplements, washing buffer, and cell concentration. Improvements made in the transformation protocol have led to electrocompetent cells with transformation efficiency up to 7 × 10(8) transformants per microgram of 120 kbp BAC plasmid DNA. We have successfully used in-house prepared competent cells, the quality of which is comparable with those produced by different companies, in the construction of metagenomic libraries from the soil. Our protocol can also be beneficial for other application with limited DNA source. PMID:23846555

  11. The MICHR Genomic DNA BioLibrary: An Empirical Study of the Ethics of Biorepository Development

    PubMed Central

    Roessler, Blake J.; Steneck, Nicholas H.; Powell, Lisa

    2015-01-01

    In this article, we report on an effort to study the development and usefulness of a large, broad-use, opt-in biorepository for genomic research, focusing on three ethical issues: providing appropriate understanding, recruiting in ways that do not comprise autonomous decisions, and assessing costs vs. benefits. We conclude: 1) Understanding can be improved by separating the task of informing subjects from documenting informed consent (Common Rule) and permission to use personal health information and samples for research (HIPAA); however, regulations might have to be changed to accommodate this approach. 2) Changing recruiting methods increases efficiency but can interfere with subject autonomy. 3) Finally, we propose a framework for the objective evaluation of the utility of biorepositories and suggest that more attention needs to be paid to use and sustainability. PMID:25742665

  12. The MICHR Genomic DNA BioLibrary: An Empirical Study of the Ethics of Biorepository Development.

    PubMed

    Roessler, Blake J; Steneck, Nicholas H; Connally, Lisa

    2015-02-01

    In this article, we report on an effort to study the development and usefulness of a large, broad-use, opt-in biorepository for genomic research, focusing on three ethical issues: providing appropriate understanding, recruiting in ways that do not comprise autonomous decisions, and assessing costs versus benefits. We conclude the following: (a) Understanding can be improved by separating the task of informing subjects from documenting informed consent (Common Rule) and permission to use personal health information and samples for research (Health Insurance Portability and Accountability Act [HIPAA]); however, regulations might have to be changed to accommodate this approach. (b) Changing recruiting methods increases efficiency but can interfere with subject autonomy. (c) Finally, we propose a framework for the objective evaluation of the utility of biorepositories and suggest that more attention needs to be paid to use and sustainability. PMID:25742665

  13. [Advanced Treatment of Incineration Leachate with O3-BAC and Double O3-BAC].

    PubMed

    Du, An-jing; Fan, Ju-hong; Liu, Rui; Qiu, Song-kai; Wen, Xiao-gang; Chen, Lü-jun

    2015-11-01

    Ozone-biological activated carbon (O3-BAC) process and double O3-BAC process were respectively used for advanced treatment of the biologically treated effluent of incineration leachate, and their pollutant removal performances were compared. The results showed that the double O3-BAC removed 75.9% ± 2.1% of chemical oxygen demand (COD), 78.8% ± 2.9% of UV254 and 96.8% ± 0.9% of color at ozone dosage of 200 mg x L(-1). The treated effluent was with COD of below 100 mg x L(-1) and color of below 40 times, meeting the emission requirements of GB 16889-2008. At the same ozone dosage, however, the O3-BAC removed 68.2% ± 1.3% of COD, 69.7% ± 0.5% of UV254 and 92.5% ± 1.1% of color. The treated effluent was with COD of around 150 mg x L(-1) and color of about 60 times, failing to meet the emission requirements. Namely, ozone of 290 mg x L(-1) was required by O3-BAC in order to achieve similar pollutant removals as those in double O3-BAC at O3 dosage of 200 mg x L(-1). In double O3-BAC at ozone dosage of 200 mg x L(-1), total phosphorus was removed by 63.5% ± 4.4%, and the phosphorus concentration in the effluent was remained 1 mg x L(-1) or less, directly meeting the emission requirement of GB 16889-2008. PMID:26911003

  14. Development of a Genomic Microsatellite Library in Perennial Ryegrass (Lolium perenne) and its Use in Trait Mapping

    PubMed Central

    King, J.; Thorogood, D.; Edwards, K. J.; Armstead, I. P.; Roberts, L.; Skøt, K.; Hanley, Z.; King, I. P.

    2008-01-01

    Background and Aims Perennial ryegrass (Lolium perenne) is one of the key forage and amenity grasses throughout the world. In the UK it accounts for 70 % of all agricultural land use with an estimated farm gate value of £6 billion per annum. However, in terms of the genetic resources available, L. perenne has lagged behind other major crops in Poaceae. The aim of this project was therefore the construction of a microsatellite-enriched genomic library for L. perenne to increase the number of genetic markers available for both marker-assisted selection in breeding programmes and gene isolation. Methods Primers for 229 non-redundant microsatellite markers were designed and used to screen two L. perenne genotypes, one amenity and one forage. Of the 229 microsatellites, 95 were found to show polymorphism between amenity and forage genotypes. A selection of microsatellite primers was selected from these 95 and used to screen two mapping populations derived from intercrossing and backcrossing the two forage and amenity grass genotypes. Key Results and Conclusions The utility of the resulting genetic maps for analysis of the genetic control of target traits was demonstrated by the mapping of genes associated with heading date to linkage groups 4 and 7. PMID:18281692

  15. 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.

  16. [Construction and analysis of rumen bacterial artificial chromosome library from a dairy cow rumen microflora].

    PubMed

    Zhu, Ya-xin; Wang, Jia-qi; Ma, Run-lin; Huang, Li; Dong, Zhi-yang

    2007-04-01

    The high molecular weight DNA was extracted and purified directly from rumen samples in the study by using culture-independent and pulsed field gel electrophoresis approaches. After digestion with Hind III, DNA fragments ranging from 50-100 kb was collected and ligated to pCC BAC vector. The ligation mixture was transformed into E. coli EPI300 and a rumen metagenomic BAC library with about 15360 clones was constructed. The average insert size is about 54.5 kb, mostly ranging from 50-70 kb, and the capacity of this BAC library is about 837Mb. Several BAC clones with activity of amylase, Cmcellulase had been screened from the BAC library. The clones with Cmcelluase activity were screened further for linchenase, xylanase, cellobioase activity and the result is that 25 of them have at least one kind of other enzyme activity. PMID:17552222

  17. Large-scale sequencing based on full-length-enriched cDNA libraries in pigs: contribution to annotation of the pig genome draft sequence

    PubMed Central

    2012-01-01

    Background Along with the draft sequencing of the pig genome, which has been completed by an international consortium, collection of the nucleotide sequences of genes expressed in various tissues and determination of entire cDNA sequences are necessary for investigations of gene function. The sequences of expressed genes are also useful for genome annotation, which is important for isolating the genes responsible for particular traits. Results We performed a large-scale expressed sequence tag (EST) analysis in pigs by using 32 full-length-enriched cDNA libraries derived from 28 kinds of tissues and cells, including seven tissues (brain, cerebellum, colon, hypothalamus, inguinal lymph node, ovary, and spleen) derived from pigs that were cloned from a sow subjected to genome sequencing. We obtained more than 330,000 EST reads from the 5′-ends of the cDNA clones. Comparison with human and bovine gene catalogs revealed that the ESTs corresponded to at least 15,000 genes. cDNA clones representing contigs and singlets generated by assembly of the EST reads were subjected to full-length determination of inserts. We have finished sequencing 31,079 cDNA clones corresponding to more than 12,000 genes. Mapping of the sequences of these cDNA clones on the draft sequence of the pig genome has indicated that the clones are derived from about 15,000 independent loci on the pig genome. Conclusions ESTs and cDNA sequences derived from full-length-enriched libraries are valuable for annotation of the draft sequence of the pig genome. This information will also contribute to the exploration of promoter sequences on the genome and to molecular biology-based analyses in pigs. PMID:23150988

  18. Single haplotype assembly of the human genome from a hydatidiform mole.

    PubMed

    Steinberg, Karyn Meltz; Schneider, Valerie A; Graves-Lindsay, Tina A; Fulton, Robert S; Agarwala, Richa; Huddleston, John; Shiryev, Sergey A; Morgulis, Aleksandr; Surti, Urvashi; Warren, Wesley C; Church, Deanna M; Eichler, Evan E; Wilson, Richard K

    2014-12-01

    A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. PMID:25373144

  19. Single haplotype assembly of the human genome from a hydatidiform mole

    PubMed Central

    Steinberg, Karyn Meltz; Schneider, Valerie A.; Graves-Lindsay, Tina A.; Fulton, Robert S.; Agarwala, Richa; Huddleston, John; Shiryev, Sergey A.; Morgulis, Aleksandr; Surti, Urvashi; Warren, Wesley C.; Church, Deanna M.; Eichler, Evan E.; Wilson, Richard K.

    2014-01-01

    A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. PMID:25373144

  20. Herpesvirus mutagenesis facilitated by infectious bacterial artificial chromosomes (iBACs).

    PubMed

    Robinson, Karl E; Mahony, Timothy J

    2015-01-01

    A critical factor in the study of herpesviruses, their genes and gene functions is the capacity to derive mutants that harbor deletions, truncations, or insertions within the genetic elements of interest. Once constructed the impact of the introduced mutation on the phenotypic properties of the rescued virus can be determined in either in vitro or in vivo systems. However, the construction of such mutants by traditional virological mutagenesis techniques can be a difficult and laborious undertaking. The maintenance of a viral genome as an infectious bacterial artificial chromosome (iBAC), however, endows the capacity to manipulate the viral genome for mutagenesis studies with relative ease. Here, the construction and characterization of two gene deletion mutants of an alphaherpesvirus maintained as iBAC in combination with an inducible homologous recombination system in Escherichia coli is detailed. The methodology is generally applicable to any iBAC and is demonstrated to be a highly efficient and informative approach for mutant virus construction. PMID:25239746

  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. PMID:25434926

  2. Karyotyping of Brachypodium pinnatum (2n = 18) chromosomes using cross-species BAC-FISH.

    PubMed

    Wolny, Elzbieta; Fidyk, Wojciech; Hasterok, Robert

    2013-04-01

    Identification of individual chromosomes in a complement is usually a difficult task in the case of most plant species, especially for those with small, numerous, and morphologically uniform chromosomes. In this paper, we demonstrate that the landmarks produced by cross-species fluorescence in situ hybridisation (FISH) of Brachypodium distachyon derived bacterial artificial chromosome (BAC) clones can be used for discrimination of Brachypodium pinnatum (2n = 18) chromosomes. Selected sets of clones were hybridised in several sequential experiments performed on exactly the same chromosome spreads, using reprobing of cytological preparations. Analysis of the morphometric features of B. pinnatum chromosomes was performed to establish their total length, the position of centromeres, and the position of BAC-based landmarks in relation to the centromere, thereby enabling their effective karyotyping, which is a prerequisite for more complex study of the grass genome structure and evolution at the cytomolecular level. PMID:23706077

  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. 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. PMID:21969025

  5. Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome

    PubMed Central

    Quinn, Nicole L; Levenkova, Natasha; Chow, William; Bouffard, Pascal; Boroevich, Keith A; Knight, James R; Jarvie, Thomas P; Lubieniecki, Krzysztof P; Desany, Brian A; Koop, Ben F; Harkins, Timothy T; Davidson, William S

    2008-01-01

    Background With a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library. Results An initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30× coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (~0.09× coverage) were incorporated. The addition of paired end sequencing reads (additional ~26× coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (~10.5× coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly. Conclusion These results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS

  6. piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila.

    PubMed

    Hacker, Udo; Nystedt, Sverker; Barmchi, Mojgan Padash; Horn, Carsten; Wimmer, Ernst A

    2003-06-24

    P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms. PMID:12802016

  7. 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

  8. Generation of BAC Transgenic Epithelial Organoids

    PubMed Central

    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. PMID:24204693

  9. Generation of a complete single-gene knockout bacterial artificial chromosome library of cowpox virus and identification of its essential genes.

    PubMed

    Xu, Zhiyong; Zikos, Dimitrios; Osterrieder, Nikolaus; Tischer, B Karsten

    2014-01-01

    Cowpox virus (CPXV) belongs to the genus Orthopoxvirus in the Poxviridae family. It infects a broad range of vertebrates and can cause zoonotic infections. CPXV has the largest genome among the orthopoxviruses and is therefore considered to have the most complete set of genes of all members of the genus. Since CPXV has also become a model for studying poxvirus genetics and pathogenesis, we created and characterized a complete set of single gene knockout bacterial artificial chromosome (BAC) clones of the CPXV strain Brighton Red. These mutants allow a systematic assessment of the contribution of single CPXV genes to the outcome of virus infection and replication, as well as to the virus host range. A full-length BAC clone of CPXV strain Brighton Red (pBRF) harboring the gene expressing the enhanced green fluorescent protein under the control of a viral late promoter was modified by introducing the mrfp1 gene encoding the monomeric red fluorescent protein driven by a synthetic early vaccinia virus promoter. Based on the modified BAC (pBRFseR), a library of targeted knockout mutants for each single viral open reading frame (ORF) was generated. Reconstitution of infectious virus was successful for 109 of the 183 mutant BAC clones, indicating that the deleted genes are not essential for virus replication. In contrast, 74 ORFs were identified as essential because no virus progeny was obtained upon transfection of the mutant BAC clones and in the presence of a helper virus. More than 70% of all late CPXV genes belonged to this latter group of essential genes. PMID:24155400

  10. Repetitive genome elements in a European corn borer, Ostrinia nubilalis, bacterial artificial chromosome library were indicated by bacterial artificial chromosome end sequencing and development of sequence tag site markers: implications for lepidopteran genomic research.

    PubMed

    Coates, Brad S; Sumerford, Douglas V; Hellmich, Richard L; Lewis, Leslie C

    2009-01-01

    The European corn borer, Ostrinia nubilalis, is a serious pest of food, fiber, and biofuel crops in Europe, North America, and Asia and a model system for insect olfaction and speciation. A bacterial artificial chromosome library constructed for O. nubilalis contains 36 864 clones with an estimated average insert size of >or=120 kb and genome coverage of 8.8-fold. Screening OnB1 clones comprising approximately 2.76 genome equivalents determined the physical position of 24 sequence tag site markers, including markers linked to ecologically important and Bacillus thuringiensis toxin resistance traits. OnB1 bacterial artificial chromosome end sequence reads (GenBank dbGSS accessions ET217010 to ET217273) showed homology to annotated genes or expressed sequence tags and identified repetitive genome elements, O. nubilalis miniature subterminal inverted repeat transposable elements (OnMITE01 and OnMITE02), and ezi-like long interspersed nuclear elements. Mobility of OnMITE01 was demonstrated by the presence or absence in O. nubilalis of introns at two different loci. A (GTCT)n tetranucleotide repeat at the 5' ends of OnMITE01 and OnMITE02 are evidence for transposon-mediated movement of lepidopteran microsatellite loci. The number of repetitive elements in lepidopteran genomes will affect genome assembly and marker development. Single-locus sequence tag site markers described here have downstream application for integration within linkage maps and comparative genomic studies. PMID:19132072

  11. Whitefly (Bemisia tabaci) genome project: analysis of sequenced clones from egg, instar, and adult (viruliferous and non-viruliferous) cDNA libraries

    PubMed Central

    Leshkowitz, Dena; Gazit, Shirley; Reuveni, Eli; Ghanim, Murad; Czosnek, Henryk; McKenzie, Cindy; Shatters, Robert L; Brown, Judith K

    2006-01-01

    Background The past three decades have witnessed a dramatic increase in interest in the whitefly Bemisia tabaci, owing to its nature as a taxonomically cryptic species, the damage it causes to a large number of herbaceous plants because of its specialized feeding in the phloem, and to its ability to serve as a vector of plant viruses. Among the most important plant viruses to be transmitted by B. tabaci are those in the genus Begomovirus (family, Geminiviridae). Surprisingly, little is known about the genome of this whitefly. The haploid genome size for male B. tabaci has been estimated to be approximately one billion bp by flow cytometry analysis, about five times the size of the fruitfly Drosophila melanogaster. The genes involved in whitefly development, in host range plasticity, and in begomovirus vector specificity and competency, are unknown. Results To address this general shortage of genomic sequence information, we have constructed three cDNA libraries from non-viruliferous whiteflies (eggs, immature instars, and adults) and two from adult insects that fed on tomato plants infected by two geminiviruses: Tomato yellow leaf curl virus (TYLCV) and Tomato mottle virus (ToMoV). In total, the sequence of 18,976 clones was determined. After quality control, and removal of 5,542 clones of mitochondrial origin 9,110 sequences remained which included 3,843 singletons and 1,017 contigs. Comparisons with public databases indicated that the libraries contained genes involved in cellular and developmental processes. In addition, approximately 1,000 bases aligned with the genome of the B. tabaci endosymbiotic bacterium Candidatus Portiera aleyrodidarum, originating primarily from the egg and instar libraries. Apart from the mitochondrial sequences, the longest and most abundant sequence encodes vitellogenin, which originated from whitefly adult libraries, indicating that much of the gene expression in this insect is directed toward the production of eggs. Conclusion This

  12. Identification of a Mitochondrial DNA Polymerase Affecting Cardiotoxicity of Sunitinib Using a Genome-Wide Screening on S. pombe Deletion Library.

    PubMed

    Kim, Dong-Myung; Kim, Hanna; Yeon, Ji-Hyun; Lee, Ju-Hee; Park, Han-Oh

    2016-01-01

    Drug toxicity is a key issue for drug R&D, a fundamental challenge of which is to screen for the targets genome-wide. The anticancer tyrosine kinase inhibitor sunitinib is known to induce cardiotoxicity. Here, to understand the molecular insights of cardiotoxicity by sunitinib at the genome level, we used a genome-wide drug target screening technology (GPScreen) that measures drug-induced haploinsufficiency (DIH) in the fission yeast Schizosaccharomyces pombe genome-wide deletion library and found a mitochondrial DNA polymerase (POG1). In the results, sunitinib induced more severe cytotoxicity and mitochondrial damage in POG1-deleted heterozygous mutants compared to wild type (WT) of S. pombe. Furthermore, knockdown of the human ortholog POLG of S. pombe POG1 in human cells significantly increased the cytotoxicity of sunitinib. Notably, sunitinib dramatically decreased the levels of POLG mRNAs and proteins, of which downregulation was already known to induce mitochondrial damage of cardiomyocytes, causing cardiotoxicity. These results indicate that POLG might play a crucial role in mitochondrial damage as a gene of which expressional pathway is targeted by sunitinib for cardiotoxicity, and that genome-wide drug target screening with GPScreen can be applied to drug toxicity target discovery to understand the molecular insights regarding drug toxicity. PMID:26385865

  13. 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

  14. Generation of bacterial artificial chromosome (BAC) transgenic mice.

    PubMed

    Beil, Jane; Buch, Thorsten

    2014-01-01

    Transgenic mice are among the most helpful tools to study the role of genes in physiological conditions. In this protocol, we describe the generation of bacterial artificial chromosome (BACs) constructs, which are used to express a gene of interest under a particular promoter. BACs as driver of transgenes have the advantage that a characterization of transcriptional control elements is unnecessary and the construct's size usually reduces position effects from random integration. In the following, we firstly explain in detail the amplification of the BAC, the generation of the targeting construct as well as the recombination by ET-cloning, and the analysis of the recombined clones by Southern blot analysis. Finally, we also describe the preparation of the BACs for oocyte injection. In total, the construction of such BAC transgenes needs around 6-8 weeks. PMID:25064102

  15. 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. PMID:25669608

  16. New resources inform study of genome size, content, and organization in nonavian reptiles

    PubMed Central

    Janes, Daniel E.; Organ, Christopher; Valenzuela, Nicole

    2008-01-01

    Genomic resources for studies of nonavian reptiles have recently improved and will reach a new level of access once the genomes of the painted turtle (Chrysemys picta) and the green anole (Anolis carolinensis) have been published. Eleven speakers gathered for a symposium on reptilian genomics and evolutionary genetics at the 2008 meeting of the Society for Integrative and Comparative Biology in San Antonio, Texas. Presentations described results of reptilian genetic studies concerning molecular evolution, chromosomal evolution, genomic architecture, population dynamics, endocrinology and endocrine disruption, and the evolution of developmental mechanisms. The presented studies took advantage of the recent generation of genetic and genomic tools and resources. Novel findings demonstrated the positive impact made by the improved availability of resources like genome annotations and bacterial artificial chromosomes (BACs). The symposium was timely and important because it provided a vehicle for the dissemination of novel findings that advance the field. Moreover, this meeting fostered the synergistic interaction of the participants as a group, which is anticipated to encourage the funding and creation of further resources such as additional BAC libraries and genomic projects. Novel data have already been collected and studies like those presented in this symposium promise to shape and improve our understanding of overall amniote evolution. Additional reptilian taxa such as the American alligator (Alligator mississippiensis), tuatara (Sphenodon punctatus), and garter snake (Thamnophis sirtalis) should be the foci of future genomic projects. We hope that the following articles in this volume will help promote these efforts by describing the conclusions and the potential that the improvement of genomic resources for nonavian reptiles can continue having in this important area of integrative and comparative biology. PMID:21669805

  17. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon

    PubMed Central

    Yusa, Kosuke; Rad, Roland; Takeda, Junji; Bradley, Allan

    2009-01-01

    Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4, Sox2, Klf4, and cMyc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we show a highly efficient piggyBac transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. Transposons were removed from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be easily identified by HSVtk-FIAU selection. piggyBac excises without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as expression of embryonic stem cell-specific markers, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs. PMID:19337237

  18. 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

  19. GENE DUPLICATION AND PALEOPOLYPLOIDY IN SOYBEAN AND THE IMPLICATIONS FOR WHOLE GENOME SEQUENCING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seventeen BACs representing ~ 2.03 Mb were sequenced as representative homeologous regions from the paleopolyploid soybean (Glycine max (L.) Merr.) genome. Sequence identity comparisons between homeologous BACs shows that the soybean genome is a mosaic of retained paleopolyploid regions. some havin...

  20. Olefin Isomerization Regiochemistries during Tandem Action of BacA and BacB on Prephenate in Bacilysin Biosynthesis†

    PubMed Central

    Parker, Jared B.; Walsh, Christopher T.

    2012-01-01

    BacA and BacB, the first two enzymes of the bacilysin pathway, convert prephenate to an exocylic regioisomer of dihydrohydroxyphenylpyruvate (ex-H2HPP) on the way to the epoxycyclohexanone warhead in the dipeptide antibiotic, bacilysin. BacA decarboxylates prephenate without aromatization, converting the 1,4-diene in prephenate to the endocyclic 1,3 diene in Δ4Δ8-dihydrohydroxyphenylpyruvate (en-H2HPP). BacB then performs an allylic isomerization to bring the diene into conjugation with the 2-ketone in the product Δ3Δ5-dihydrohydroxyphenylpyruvate (ex-H2HPP). To prove that BacA acts regiospecifically on one of the two prochiral olefins in prephenate, we generated 1,5,8-[13C]-chorismate from bacterial fermentation of 5-[13C]-glucose and in turn produced 2,4,6-[13C]-prephenate via chorismate mutase. Tandem action of BacA and BacB gave 2,4,8-[13C]-7R-ex-H2HPP, showing that BacA isomerizes only the pro-R double bond in prephenate. Nonenzymatic isomerization of the BacA product into conjugation gives only the Δ3 E-geometric isomer of Δ3Δ5-ex-H2HPP. On the other hand, acceleration of the allylic isomerization by BacB gives a mixture of the E- and Z-geometric isomers of the 7R-product, indicating some rerouting of the flux, likely through dienolate geometric isomers. PMID:22483065

  1. Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis.

    PubMed

    Marh, Joel; Stoytcheva, Zoia; Urschitz, Johann; Sugawara, Atsushi; Yamashiro, Hideaki; Owens, Jesse B; Stoytchev, Ilko; Pelczar, Pawel; Yanagimachi, Ryuzo; Moisyadi, Stefan

    2012-11-20

    We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community. PMID:23093669

  2. 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.

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

    PubMed

    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

  4. Poultry Genome Sequences: Progress and Outstanding Challenges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The first build of the chicken genome sequence appeared in March 2004 – the first genome sequence of any animal agriculture species. That sequence was done primarily by whole genome shotgun Sanger sequencing, along with the use of an extensive BAC contig-based physical map to assemble the sequence ...

  5. 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

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

    PubMed Central

    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

  7. PiggyBac:A flexible and highly active transposon as compared to Sleeping Beauty, Tol2, and Mos1 in mammalian cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A non-viral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study, we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty(SB11), Tol2, and Mos1 in four mammalia...

  8. Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome.

    PubMed

    Arii, Jun; Hushur, Orkash; Kato, Kentaro; Kawaguchi, Yasushi; Tohya, Yukinobu; Akashi, Hiroomi

    2006-04-01

    Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals. PMID:16515874

  9. A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells.

    PubMed

    Hong, Jin-Bon; Chou, Fu-Ju; Ku, Amy T; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

    2014-01-01

    PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells. PMID:24586748

  10. A Nucleolus-Predominant piggyBac Transposase, NP-mPB, Mediates Elevated Transposition Efficiency in Mammalian Cells

    PubMed Central

    Ku, Amy T.; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

    2014-01-01

    PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3–4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells. PMID:24586748

  11. From raw materials to validated system: the construction of a genomic library and microarray to interpret systemic perturbations in Northern bobwhite

    PubMed Central

    Rawat, Arun; Deng, Youping; Garcia-Reyero, Natàlia; Quinn, Michael J.; Johnson, Mark S.; Indest, Karl J.; Elasri, Mohamed O.; Perkins, Edward J.

    2010-01-01

    The limited availability of genomic tools and data for nonmodel species impedes computational and systems biology approaches in nonmodel organisms. Here we describe the development, functional annotation, and utilization of genomic tools for the avian wildlife species Northern bobwhite (Colinus virginianus) to determine the molecular impacts of exposure to 2,6-dinitrotoluene (2,6-DNT), a field contaminant of military concern. Massively parallel pyrosequencing of a normalized multitissue library of Northern bobwhite cDNAs yielded 71,384 unique transcripts that were annotated with gene ontology (GO), pathway information, and protein domain analysis. Comparative genome analyses with model organisms revealed functional homologies in 8,825 unique Northern bobwhite genes that are orthologous to 48% of Gallus gallus protein-coding genes. Pathway analysis and GO enrichment of genes differentially expressed in livers of birds exposed for 60 days (d) to 10 and 60 mg/kg/d 2,6-DNT revealed several impacts validated by RT-qPCR including: prostaglandin pathway-mediated inflammation, increased expression of a heme synthesis pathway in response to anemia, and a shift in energy metabolism toward protein catabolism via inhibition of control points for glucose and lipid metabolic pathways, PCK1 and PPARGC1, respectively. This research effort provides the first comprehensive annotated gene library for Northern bobwhite. Transcript expression analysis provided insights into the metabolic perturbations underlying several observed toxicological phenotypes in a 2,6-DNT exposure case study. Furthermore, the systemic impact of dinitrotoluenes on liver function appears conserved across species as PPAR signaling is similarly affected in fathead minnow liver tissue after exposure to 2,4-DNT. PMID:20406850

  12. Second-Generation Genetic Linkage Map of Catfish and Its Integration with the BAC-Based Physical Map

    PubMed Central

    Ninwichian, Parichart; Peatman, Eric; Liu, Hong; Kucuktas, Huseyin; Somridhivej, Benjaporn; Liu, Shikai; Li, Ping; Jiang, Yanliang; Sha, Zhenxia; Kaltenboeck, Ludmilla; Abernathy, Jason W.; Wang, Wenqi; Chen, Fei; Lee, Yoona; Wong, Lilian; Wang, Shaolin; Lu, Jianguo; Liu, Zhanjiang

    2012-01-01

    Construction of high-density genetic linkage maps is crucially important for quantitative trait loci (QTL) studies, and they are more useful when integrated with physical maps. Such integrated maps are valuable genome resources for fine mapping of QTL, comparative genomics, and accurate and efficient whole-genome assembly. Previously, we established both linkage maps and a physical map for channel catfish, Ictalurus punctatus, the dominant aquaculture species in the United States. Here we added 2030 BAC end sequence (BES)-derived microsatellites from 1481 physical map contigs, as well as markers from singleton BES, ESTs, anonymous microsatellites, and SNPs, to construct a second-generation linkage map. Average marker density across the 29 linkage groups reached 1.4 cM/marker. The increased marker density highlighted variations in recombination rates within and among catfish chromosomes. This work effectively anchored 44.8% of the catfish BAC physical map contigs, covering ∼52.8% of the genome. The genome size was estimated to be 2546 cM on the linkage map, and the calculated physical distance per centimorgan was 393 Kb. This integrated map should enable comparative studies with teleost model species as well as provide a framework for ordering and assembling whole-genome scaffolds. PMID:23050234

  13. A genomic-scale artificial microRNA library as a tool to investigate the functionally redundant gene space in Arabidopsis.

    PubMed

    Hauser, Felix; Chen, Wenxiao; Deinlein, Ulrich; Chang, Kenneth; Ossowski, Stephan; Fitz, Joffrey; Hannon, Gregory J; Schroeder, Julian I

    2013-08-01

    Traditional forward genetic screens are limited in the identification of homologous genes with overlapping functions. Here, we report the analyses and assembly of genome-wide protein family definitions that comprise the largest estimate for the potentially redundant gene space in Arabidopsis thaliana. On this basis, a computational design of genome-wide family-specific artificial microRNAs (amiRNAs) was performed using high-performance computing resources. The amiRNA designs are searchable online (http://phantomdb.ucsd.edu). A computationally derived library of 22,000 amiRNAs was synthesized in 10 sublibraries of 1505 to 4082 amiRNAs, each targeting defined functional protein classes. For example, 2964 amiRNAs target annotated DNA and RNA binding protein families and 1777 target transporter proteins, and another sublibrary targets proteins of unknown function. To evaluate the potential of an amiRNA-based screen, we tested 122 amiRNAs targeting transcription factor, protein kinase, and protein phosphatase families. Several amiRNA lines showed morphological phenotypes, either comparable to known phenotypes of single and double/triple mutants or caused by overexpression of microRNAs. Moreover, novel morphological and abscisic acid-insensitive seed germination mutants were identified for amiRNAs targeting zinc finger homeodomain transcription factors and mitogen-activated protein kinase kinase kinases, respectively. These resources provide an approach for genome-wide genetic screens of the functionally redundant gene space in Arabidopsis. PMID:23956262

  14. 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...

  15. 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

  16. 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. PMID:24773339

  17. Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System

    PubMed Central

    Yazdani, Yaghoub; Azari, Shahram; Kalhor, Hamid Reza

    2016-01-01

    Purpose: Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer’s disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Results: Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Conclusion: Human TG2 was expressed efficiently in the active biological form in the Bac-to-Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system. PMID:27123417

  18. Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System

    PubMed Central

    Bire, Solenne; Ley, Déborah; Casteret, Sophie; Mermod, Nicolas; Bigot, Yves; Rouleux-Bonnin, Florence

    2013-01-01

    Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors. PMID:24312663

  19. Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian Pooling-Coordinate Sequencing

    PubMed Central

    Vandewalle, Kristof; Festjens, Nele; Plets, Evelyn; Vuylsteke, Marnik; Saeys, Yvan; Callewaert, Nico

    2015-01-01

    Reverse genetics research approaches require the availability of methods to rapidly generate specific mutants. Alternatively, where these methods are lacking, the construction of pre-characterized libraries of mutants can be extremely valuable. However, this can be complex, expensive and time consuming. Here, we describe a robust, easy to implement parallel sequencing-based method (Cartesian Pooling-Coordinate Sequencing or CP-CSeq) that reports both on the identity as well as on the location of sequence-tagged biological entities in well-plate archived clone collections. We demonstrate this approach using a transposon insertion mutant library of the Mycobacterium bovis BCG vaccine strain, providing the largest resource of mutants in any strain of the M. tuberculosis complex. The method is applicable to any entity for which sequence-tagged identification is possible. PMID:25960123

  20. 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.

  1. 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. PMID:27565868

  2. A genomic library-based amplification approach (GL-PCR) for the mapping of multiple IS6110 insertion sites and strain differentiation of Mycobacterium tuberculosis.

    PubMed

    Namouchi, Amine; Mardassi, Helmi

    2006-11-01

    Evidence suggests that insertion of the IS6110 element is not without consequence to the biology of Mycobacterium tuberculosis complex strains. Thus, mapping of multiple IS6110 insertion sites in the genome of biomedically relevant clinical isolates would result in a better understanding of the role of this mobile element, particularly with regard to transmission, adaptability and virulence. In the present paper, we describe a versatile strategy, referred to as GL-PCR, that amplifies IS6110-flanking sequences based on the construction of a genomic library. M. tuberculosis chromosomal DNA is fully digested with HincII and then ligated into a plasmid vector between T7 and T3 promoter sequences. The ligation reaction product is transformed into Escherichia coli and selective PCR amplification targeting both 5' and 3' IS6110-flanking sequences are performed on the plasmid library DNA. For this purpose, four separate PCR reactions are performed, each combining an outward primer specific for one IS6110 end with either T7 or T3 primer. Determination of the nucleotide sequence of the PCR products generated from a single ligation reaction allowed mapping of 21 out of the 24 IS6110 copies of two 12 banded M. tuberculosis strains, yielding an overall sensitivity of 87,5%. Furthermore, by simply comparing the migration pattern of GL-PCR-generated products, the strategy proved to be as valuable as IS6110 RFLP for molecular typing of M. tuberculosis complex strains. Importantly, GL-PCR was able to discriminate between strains differing by a single IS6110 band. PMID:16725220

  3. 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, "LJ" launched…

  4. Sequencing and comparative genomics analysis in Senecio scandens Buch.-Ham. Ex D. Don, based on full-length cDNA library

    PubMed Central

    Qian, Gang; Ping, Junjiao; Zhang, Zhen; Xu, Delin

    2014-01-01

    Senecio scandens Buch.-Ham. ex D. Don, an important antibacterial source of Chinese traditional medicine, has a widespread distribution in a few ecological habitats of China. We generated a full-length complementary DNA (cDNA) library from a sample of elite individuals with superior antibacterial properties, with satisfactory parameters such as library storage (4.30 × 106 CFU), efficiency of titre (1.30 × 106 CFU/mL), transformation efficiency (96.35%), full-length ratio (64.00%) and redundancy ratio (3.28%). The BLASTN search revealed the facile formation of counterparts between the experimental sample and Arabidopsis thaliana in view of high-homology cDNA sequence (90.79%) with e-values <1e – 50. Sequence similarities to known proteins indicate that the entire sequences of the full-length cDNA clones consist of the major of functional genes identified by a large set of microarray data from the present experimental material. For other Compositae species, a large set of full-length cDNA clones reported in the present article will serve as a useful resource to facilitate further research on the transferability of expressed sequence tag-derived simple sequence repeats (EST-SSR) development, comparative genomics and novel transcript profiles. PMID:26740776

  5. New glucosidase activities identified by functional screening of a genomic DNA library from the gut microbiota of the termite Reticulitermes santonensis.

    PubMed

    Mattéotti, Christel; Thonart, Philippe; Francis, Frédéric; Haubruge, Eric; Destain, Jacqueline; Brasseur, Catherine; Bauwens, Julien; De Pauw, Edwin; Portetelle, Daniel; Vandenbol, Micheline

    2011-12-20

    β-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying β-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose. PMID:21324659

  6. 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...

  7. "Cre/loxP plus BAC": a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens.

    PubMed

    Hu, Shengbiao; Liu, Zhengqiang; Zhang, Xu; Zhang, Guoyong; Xie, Yali; Ding, Xuezhi; Mo, Xiangtao; Stewart, A Francis; Fu, Jun; Zhang, Youming; Xia, Liqiu

    2016-01-01

    Heterologous expression has been proven to be a valid strategy for elucidating the natural products produced by gene clusters uncovered by genome sequencing projects. Efforts have been made to efficiently clone gene clusters directly from genomic DNA and several approaches have been developed. Here, we present an alternative strategy based on the site-specific recombinase system Cre/loxP for direct cloning gene clusters. A type three secretion system (T3SS) gene cluster (~32 kb) from Photorhabdus luminescens TT01 and DNA fragment (~78 kb) containing the siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58 have been successfully cloned into pBeloBAC11 with "Cre/loxP plus BAC" strategy. Based on the fact that Cre/loxP system has successfully used for genomic engineering in a wide range of organisms, we believe that this strategy could be widely used for direct cloning of large DNA fragment. PMID:27364376

  8. 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. PMID:17095691

  9. The Genome of the Sea Urchin Strongylocentrotus purpuratus

    PubMed Central

    2011-01-01

    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. PMID:17095691

  10. 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

  11. BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.

    PubMed

    Placantonakis, Dimitris G; Tomishima, Mark J; Lafaille, Fabien; Desbordes, Sabrina C; Jia, Fan; Socci, Nicholas D; Viale, Agnes; Lee, Hyojin; Harrison, Neil; Tabar, Viviane; Studer, Lorenz

    2009-03-01

    Human embryonic stem cells (hESCs) have enormous potential for applications in basic biology and regenerative medicine. However, harnessing the potential of hESCs toward generating homogeneous populations of specialized cells remains challenging. Here we describe a novel technology for the genetic identification of defined hESC-derived neural cell types using bacterial artificial chromosome (BAC) transgenesis. We generated hESC lines stably expressing Hes5::GFP, Dll1::GFP, and HB9::GFP BACs that yield green fluorescent protein (GFP)(+) neural stem cells, neuroblasts, and motor neurons, respectively. Faithful reporter expression was confirmed by cell fate analysis and appropriate transgene regulation. Prospective isolation of HB9::GFP(+) cells yielded purified human motor neurons with proper marker expression and electrophysiological activity. Global mRNA and microRNA analyses of Hes5::GFP(+) and HB9::GFP(+) populations revealed highly specific expression signatures, suggesting that BAC transgenesis will be a powerful tool for establishing expression libraries that define the human neural lineage and for accessing defined cell types in applications of human disease. PMID:19074416

  12. 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

  13. Establishment of the BacMam system using silkworm baculovirus.

    PubMed

    Imai, Atsutoshi; Tadokoro, Takashi; Kita, Shunsuke; Horiuchi, Masataka; Fukuhara, Hideo; Maenaka, Katsumi

    2016-09-16

    The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate-buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy. PMID:27480929

  14. BacMet: antibacterial biocide and metal resistance genes database

    PubMed Central

    Pal, Chandan; Bengtsson-Palme, Johan; Rensing, Christopher; Kristiansson, Erik; Larsson, D. G. Joakim

    2014-01-01

    Antibiotic resistance has become a major human health concern due to widespread use, misuse and overuse of antibiotics. In addition to antibiotics, antibacterial biocides and metals can contribute to the development and maintenance of antibiotic resistance in bacterial communities through co-selection. Information on metal and biocide resistance genes, including their sequences and molecular functions, is, however, scattered. Here, we introduce BacMet (http://bacmet.biomedicine.gu.se)—a manually curated database of antibacterial biocide- and metal-resistance genes based on an in-depth review of the scientific literature. The BacMet database contains 470 experimentally verified resistance genes. In addition, the database also contains 25 477 potential resistance genes collected from public sequence repositories. All resistance genes in the BacMet database have been organized according to their molecular function and induced resistance phenotype. PMID:24304895

  15. BacDive--The Bacterial Diversity Metadatabase in 2016.

    PubMed

    Söhngen, Carola; Podstawka, Adam; Bunk, Boyke; Gleim, Dorothea; Vetcininova, Anna; Reimer, Lorenz Christian; Ebeling, Christian; Pendarovski, Cezar; Overmann, Jörg

    2016-01-01

    BacDive-the Bacterial Diversity Metadatabase (http://bacdive.dsmz.de) provides strain-linked information about bacterial and archaeal biodiversity. The range of data encompasses taxonomy, morphology, physiology, sampling and concomitant environmental conditions as well as molecular biology. The majority of data is manually annotated and curated. Currently (with release 9/2015), BacDive covers 53 978 strains. Newly implemented RESTful web services provide instant access to the content in machine-readable XML and JSON format. Besides an overall increase of data content, BacDive offers new data fields and features, e.g. the search for gene names, plasmids or 16S rRNA in the advanced search, as well as improved linkage of entries to external life science web resources. PMID:26424852

  16. 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...

  17. 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

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

    PubMed

    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

  19. 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 ...

  20. As Blood Alcohol Content (BAC) Increases, So Does Impairment | NIH MedlinePlus the Magazine

    MedlinePlus

    ... on. Feature: Rethinking Drinking As Blood Alcohol Content (BAC) Increases, So Does Impairment Past Issues / Spring 2014 ... For purposes of law enforcement, blood alcohol content (BAC) is used to define intoxication and provides a ...

  1. 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...

  2. 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

    2015-11-01

    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. PMID:26401030

  3. Library Cooperation.

    ERIC Educational Resources Information Center

    Lund, Patricia; And Others

    1993-01-01

    Includes nine articles that discuss cooperative library networking in Illinois. Highlights include library systems as cooperative agencies; PALI (Private Academic Libraries of Illinois); rural school and public library development; systemwide users; regional medical libraries; virtual libraries and the Coalition for Networked Information; a…

  4. High-throughput generation of an activation-tagged mutant library for functional genomic analyses in tobacco.

    PubMed

    Liu, Feng; Gong, Daping; Zhang, Qian; Wang, Dawei; Cui, Mengmeng; Zhang, Zhiguo; Liu, Guanshan; Wu, Jinxia; Wang, Yuanying

    2015-03-01

    Tobacco (Nicotiana tabacum L.) is an ideal model system for molecular biological and genetic studies. In this study, activation tagging was used to generate approximately 100,000 transgenic tobacco plants. Southern blot analysis indicated that there were 1.6 T-DNA inserts per line on average in our transformed population. The phenotypes observed include abnormalities in leaf and flower morphology, plant height, flowering time, branching, and fertility. Among 6,000 plants in the T0 generation, 57 displayed obvious phenotypes. Among 4,105 lines in the T1 generation, 311 displayed abnormal phenotypes. Fusion primer and nested integrated PCR was used to identify 963 independent genomic loci of T-DNA insertion sites in 1,257 T1 lines. The distribution of T-DNA insertions was non-uniform and correlated well with the predicted gene density along each chromosome. The insertions were biased toward genic regions and noncoding regions within 5 kb of a gene. Fifteen plants that showed the same phenotype as their parent with a dominant pattern in the T2 generation were chosen randomly to detect the expression levels of genes adjacent to the T-DNA integration sites by semi-quantitative RT-PCR. Fifteen candidate genes were identified. Activation was observed in 7 out of the 15 adjacent genes, including one that was located 13.1 kb away from the enhancer sequence. The activation-tagged population described in this paper will be a highly valuable resource for tobacco functional genomics research using both forward and reverse genetic approaches. PMID:25408504

  5. In Vitro Synthesis, Delivery, and Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac Transposition.

    PubMed

    Bire, Solenne; Ishac, Nicole; Rouleux-Bonnin, Florence

    2016-01-01

    Nowadays, nonviral gene transfer is currently of great importance for introducing exogenous genes into genomes and for ensuring that transgene expression is suitable for therapeutic and bioproduction purposes. The piggyBac transposon-based system is particularly interesting since it is easy to engineer and has a large cargo capacity, up to 100 kb. In its setup, the system requires only the piggyBac transposase protein and the transgene delineated by the two piggyBac-specific inverted terminal repeats. Usually the source of transposase is carried by a DNA plasmid. However, the principal drawback of this method is the lasting presence of the transposase, due to episomal persistence or possible integration of the transposase gene vector into the cell's genome. This can lead to genotoxic effects such as multiple genomic integration events and remobilization of the transposon vector once it has been integrated. One alternative to improve the safety of the system is to deliver the transposase as in vitro-synthesized messenger RNA in order to define a very narrow expression window during which a one-shot transposition process would occur. Issues that can be encountered when working on mRNA cell transfer are related to the quality of the synthetic mRNA, the system used to introduce mRNA into the cells and the bioavailability of the mRNA molecules. Here we describe a method to produce mRNA, verify its quality, determine which transfecting reagents can be used and how this mRNA is available to promote the transposition process in HeLa cells. Additionally, we illustrate this method in stromal mesenchymal cell lines in order to support hematopoiesis. PMID:27236801

  6. 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.

  7. PiggyBac transposon vectors: the tools of the human gene encoding

    PubMed Central

    Zhao, Shuang; Jiang, Enze; Chen, Shuangshuang; Gu, Yuan; Shangguan, Anna Junjie; Lv, Tangfeng

    2016-01-01

    A transposon is a DNA segment, which is able to change its relative position within the entire genome of a cell. The piggyBac (PB) transposon is a movable genetic element that efficiently transposes between vectors and chromosomes through a “cut-and-paste” mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeats (ITRs) sequences located on both ends of the transposon vector and eight efficiently moves the contents from its original positions and efficiently integrates them into TTAA chromosomal sites. PB has drawn much attention because of its transposition efficiency, safety and stability. Due to its priorities, PB can be used as a new genetic vehicle, a new tool for oncogene screening and a new method for gene therapy. PB has created a new outlook for human gene encoding. PMID:26958506

  8. Bacteriocin Protein BacL1 of Enterococcus faecalis Is a Peptidoglycan d-Isoglutamyl-l-lysine Endopeptidase*

    PubMed Central

    Kurushima, Jun; Hayashi, Ikue; Sugai, Motoyuki; Tomita, Haruyoshi

    2013-01-01

    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. PMID:24235140

  9. Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1

    PubMed Central

    Song, Jinlong; Shi, Yanhua; Li, Kang; Zhao, Bin; Yan, Yanchun

    2013-01-01

    A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L−1 and 56.33 nmol min−1, respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments. PMID:24155944

  10. Microcollinearity in an ethylene receptor coding gene region of the Coffea canephora genome is extensively conserved with Vitis vinifera and other distant dicotyledonous sequenced genomes

    PubMed Central

    Guyot, Romain; de la Mare, Marion; Viader, Véronique; Hamon, Perla; Coriton, Olivier; Bustamante-Porras, José; Poncet, Valérie; Campa, Claudine; Hamon, Serge; de Kochko, Alexandre

    2009-01-01

    Background Coffea canephora, also called Robusta, belongs to the Rubiaceae, the fourth largest angiosperm family. This diploid species (2x = 2n = 22) has a fairly small genome size of ≈ 690 Mb and despite its extreme economic importance, particularly for developing countries, knowledge on the genome composition, structure and evolution remain very limited. Here, we report the 160 kb of the first C. canephora Bacterial Artificial Chromosome (BAC) clone ever sequenced and its fine analysis. Results This clone contains the CcEIN4 gene, encoding an ethylene receptor, and twenty other predicted genes showing a high gene density of one gene per 7.8 kb. Most of them display perfect matches with C. canephora expressed sequence tags or show transcriptional activities through PCR amplifications on cDNA libraries. Twenty-three transposable elements, mainly Class II transposon derivatives, were identified at this locus. Most of these Class II elements are Miniature Inverted-repeat Transposable Elements (MITE) known to be closely associated with plant genes. This BAC composition gives a pattern similar to those found in gene rich regions of Solanum lycopersicum and Medicago truncatula genomes indicating that the CcEIN4 regions may belong to a gene rich region in the C. canephora genome. Comparative sequence analysis indicated an extensive conservation between C. canephora and most of the reference dicotyledonous genomes studied in this work, such as tomato (S. lycopersicum), grapevine (V. vinifera), barrel medic M. truncatula, black cottonwood (Populus trichocarpa) and Arabidopsis thaliana. The higher degree of microcollinearity was found between C. canephora and V. vinifera, which belong respectively to the Asterids and Rosids, two clades that diverged more than 114 million years ago. Conclusion This study provides a first glimpse of C. canephora genome composition and evolution. Our data revealed a remarkable conservation of the microcollinearity between C. canephora and V

  11. 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

    2016-02-01

    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. PMID:26742580

  12. A physical map of the human genome

    SciTech Connect

    McPherson, J.D.; Marra, M.; Hillier, L.; Waterston, R.H.; Chinwalla, A.; Wallis, J.; Sekhon, M.; Wylie, K.; Mardis, E.R.; Wilson, R.K.; Fulton, R.; Kucaba, T.A.; Wagner-McPherson, C.; Barbazuk, W.B.; Gregory, S.G.; Humphray, S.J.; French, L.; Evans, R.S.; Bethel, G.; Whittaker, A.; Holden, J.L.; McCann, O.T.; Dunham, A.; Soderlund, C.; Scott, C.E.; Bentley, D.R.; Schuler, G.; Chen, H.-C.; Jang, W.; Green, E.D.; Idol, J.R.; Maduro, V.V. Braden; Montgomery, K.T.; Lee, E.; Miller, A.; Emerling, S.; Kucherlapati; Gibbs, R.; Scherer, S.; Gorrell, J.H.; Sodergren, E.; Clerc-Blankenburg, K.; Tabor, P.; Naylor, S.; Garcia, D.; de Jong, P.J.; Catanese, J.J.; Nowak, N.; Osoegawa, K.; Qin, S.; Rowen, L.; Madan, A.; Dors, M.; Hood, L.; Trask, B.; Friedman, C.; Massa, H.; Cheung, V.G.; Kirsch, I.R.; Reid, T.; Yonescu, R.; Weissenbach, J.; Bruls, T.; Heilig, R.; Branscomb, E.; Olsen, A.; Doggett, N.; Cheng, J.F.; Hawkins, T.; Myers, R.M.; Shang, J.; Ramirez, L.; Schmutz, J.; Velasquez, O.; Dixon, K.; Stone, N.E.; Cox, D.R.; Haussler, D.; Kent, W.J.; Furey, T.; Rogic, S.; Kennedy, S.; Jones, S.; Rosenthal, A.; Wen, G.; Schilhabel, M.; Gloeckner, G.; Nyakatura, G.; Siebert, R.; Schlegelberger, B.; Korenberg, J.; Chen, X.N.; Fujiyama, A.; Hattori, M.; Toyoda, A.; Yada, T.; Park, H.S.; Sakaki, Y.; Shimizu, N.; Asakawa, S.; Kawasaki, K.; Sasaki, T.; Shintani, A.; Shimizu, A.; Shibuya, K.; Kudoh, J.; Minoshima, S.; Ramser, J.; Seranski, P.; Hoff, C.; Poustka, A.; Reinhardt, R.; Lehrach, H.

    2001-01-01

    The human genome is by far the largest genome to be sequenced, and its size and complexity present many challenges for sequence assembly. The International Human Genome Sequencing Consortium constructed a map of the whole genome to enable the selection of clones for sequencing and for the accurate assembly of the genome sequence. Here we report the construction of the whole-genome bacterial artificial chromosome (BAC) map and its integration with previous landmark maps and information from mapping efforts focused on specific chromosomal regions. We also describe the integration of sequence data with the map.

  13. VI. Genome structure and cognitive map of Williams syndrome.

    PubMed

    Korenberg, J R; Chen, X N; Hirota, H; Lai, Z; Bellugi, U; Burian, D; Roe, B; Matsuoka, R

    2000-01-01

    Williams syndrome (WMS) is a most compelling model of human cognition, of human genome organization, and of evolution. Due to a deletion in chromosome band 7q11.23, subjects have cardiovascular, connective tissue, and neurodevelopmental deficits. Given the striking peaks and valleys in neurocognition including deficits in visual-spatial and global processing, preserved language and face processing, hypersociability, and heightened affect, the goal of this work has been to identify the genes that are responsible, the cause of the deletion, and its origin in primate evolution. To do this, we have generated an integrated physical, genetic, and transcriptional map of the WMS and flanking regions using multicolor metaphase and interphase fluorescence in situ hybridization (FISH) of bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs), BAC end sequencing, PCR gene marker and microsatellite, large-scale sequencing, cDNA library, and database analyses. The results indicate the genomic organization of the WMS region as two nested duplicated regions flanking a largely single-copy region. There are at least two common deletion breakpoints, one in the centromeric and at least two in the telomeric repeated regions. Clones anchoring the unique to the repeated regions are defined along with three new pseudogene families. Primate studies indicate an evolutionary hot spot for chromosomal inversion in the WMS region. A cognitive phenotypic map of WMS is presented, which combines previous data with five further WMS subjects and three atypical WMS subjects with deletions; two larger (deleted for D7S489L) and one smaller, deleted for genes telomeric to FZD9, through LIMK1, but not WSCR1 or telomeric. The results establish regions and consequent gene candidates for WMS features including mental retardation, hypersociability, and facial features. The approach provides the basis for defining pathways linking genetic underpinnings with the neuroanatomical

  14. BacT/Alert: an automated colorimetric microbial detection system.

    PubMed Central

    Thorpe, T C; Wilson, M L; Turner, J E; DiGuiseppi, J L; Willert, M; Mirrett, S; Reller, L B

    1990-01-01

    BacT/Alert (Organon Teknika Corp., Durham, N.C.) is an automated microbial detection system based on the colorimetric detection of CO2 produced by growing microorganisms. Results of an evaluation of the media, sensor, detection system, and detection algorithm indicate that the system reliably grows and detects a wide variety of bacteria and fungi. Results of a limited pilot clinical trial with a prototype research instrument indicate that the system is comparable to the radiometric BACTEC 460 system in its ability to grow and detect microorganisms in blood. On the basis of these initial findings, large-scale clinical trials comparing BacT/Alert with other commercial microbial detection systems appear warranted. PMID:2116451

  15. [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.

  16. Individual chromosome assignment and chromosomal collinearity in Gossypium thurberi, G. trilobum and D subgenome of G. barbadense revealed by BAC-FISH.

    PubMed

    Gan, Yimei; Chen, Dan; Liu, Fang; Wang, Chunying; Li, Shaohui; Zhang, Xiangdi; Wang, Yuhong; Peng, Renhai; Wang, Kunbo

    2011-01-01

    The experiment on individual chromosome assignments and chromosomal diversity was conducted using a multi-probe fluorescence in situ hybridization (FISH) system in D subgenome of tetraploid Gossypium barbadense (D(b)), G. thurberi (D(1)) and G. trilobum (D(8)), which the later two were the possible subgenome donors of tetraploid cottons. The FISH probes contained a set of bacterial artificial chromosome (BAC) clones specific to 13 individual chromosomes from D subgenome of G. hirsutum (D(h)), a D genome centromere-specific BAC clone 150D24, 45S and 5S ribosomal DNA (rDNA) clones, respectively. All tested chromosome orientations were confirmed by the centromere-specific BAC probe. In D(1) and D(8), four 45S rDNA loci were found assigning at the end of the short arm of chromosomes 03, 07, 09 and 11, while one 5S rDNA locus was successfully marked at pericentromeric region of the short arm of chromosome 09. In D(b), three 45S rDNA loci and two 5S rDNA loci were found out. Among them, two 45S rDNA loci were located at the terminal of the short arm of chromosomes D(b)07 and D(b)09, whilst one 5S rDNA locus was found situating near centromeric region of the short arm of chromosome D(b)09. The positions of the BAC clones specific to the 13 individual chromosomes from D(h) were compared between D(1), D(8) and D(b). The result showed the existence of chromosomal collinearity within D(1) and D(8), and as well between them and D(b). The results will serve as a base for understanding chromosome structure of cotton and polyploidy evolution of cotton genome and will provide bio-information for assembling the sequences of finished and the on-going cotton whole genome sequencing projects. PMID:21952206

  17. 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.

  18. 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. PMID:26025620

  19. Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates

    PubMed Central

    Borenstein, Ronen; Frenkel, Niza

    2009-01-01

    Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constraints. We introduced the 160-kb human herpes virus 6A (HHV-6A) genome into BACs by digesting the viral DNA replicative intermediates with the Sfil enzyme that cleaves the viral genome in a single site. The recombinant BACs contained also the puromycin selection gene, GFP, and LoxP sites flanking the BAC sequences. The HHV-6A-BAC vectors were retained stably in puromycin selected 293T cells. In the presence of irradiated helper virus, supplying most likely proteins enhancing gene expression they expressed early and late genes in SupT1 T cells. The method is especially attractive for viruses that replicate inefficiently and for viruses propagated in suspension cells. We have used the fact that the BAC cloning “freezes” the viral DNA replication intermediates to analyze their structure. The results revealed that HHV-6A-BACs contained a single direct repeat (DR) rather than a DR-DR sequence, predicted to arise by circularization of parental genomes with a DR at each terminus. HHV-6A DNA molecules prepared from the infected cells also contained DNA molecules with a single DR. Such forms were not previously described for HHV-6 DNA. PMID:19858479

  20. 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

  1. 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...

  2. Time to Detection with BacT/Alert FA Plus Compared to BacT/Alert FA Blood Culture Media.

    PubMed

    Nutman, A; Fisher Even-Tsur, S; Shapiro, G; Braun, T; Schwartz, D; Carmeli, Y

    2016-09-01

    Rapid identification of the causative pathogen in patients with bacteremia allows adjustment of antibiotic therapy and improves patient outcomes. We compared in vitro and real-life time to detection (TTD) of two blood culture media, BacT/Alert FA (FA) and BacT/Alert FA Plus (FA Plus), for the nine most common species of bacterial pathogens recovered from blood samples. Experimental data from simulated cultures was compared with microbiology records of TTD for both culture media with growth of the species of interest in clinical blood cultures. In the experimental conditions, median TTD was 3.8 hours (23.9 %) shorter using FA Plus media. The magnitude of reduction differed between species. Similarly, in real life data, FA Plus had shorter TTD than FA media; however, the difference between culture media was smaller, and median TTD was only 1 hour (8.5 %) less. We found shorter TTD with BacT/Alert FA Plus culture media, both experimentally and in real-life conditions and unrelated to antibiotic neutralization, highlighting the importance of appropriate blood culture media selection. PMID:27272123

  3. Cloning of the Koi Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome Demonstrates That Disruption of the Thymidine Kinase Locus Induces Partial Attenuation in Cyprinus carpio koi▿

    PubMed Central

    Costes, B.; Fournier, G.; Michel, B.; Delforge, C.; Raj, V. Stalin; Dewals, B.; Gillet, L.; Drion, P.; Body, A.; Schynts, F.; Lieffrig, F.; Vanderplasschen, A.

    2008-01-01

    Koi herpesvirus (KHV) is the causative agent of a lethal disease in koi and common carp. In the present study, we describe the cloning of the KHV genome as a stable and infectious bacterial artificial chromosome (BAC) clone that can be used to produce KHV recombinant strains. This goal was achieved by the insertion of a loxP-flanked BAC cassette into the thymidine kinase (TK) locus. This insertion led to a BAC plasmid that was stably maintained in bacteria and was able to regenerate virions when permissive cells were transfected with the plasmid. Reconstituted virions free of the BAC cassette but carrying a disrupted TK locus (the FL BAC-excised strain) were produced by the transfection of Cre recombinase-expressing cells with the BAC. Similarly, virions with a wild-type revertant TK sequence (the FL BAC revertant strain) were produced by the cotransfection of cells with the BAC and a DNA fragment encoding the wild-type TK sequence. Reconstituted recombinant viruses were compared to the wild-type parental virus in vitro and in vivo. The FL BAC revertant strain and the FL BAC-excised strain replicated comparably to the parental FL strain. The FL BAC revertant strain induced KHV infection in koi carp that was indistinguishable from that induced by the parental strain, while the FL BAC-excised strain exhibited a partially attenuated phenotype. Finally, the usefulness of the KHV BAC for recombination studies was demonstrated by the production of an ORF16-deleted strain by using prokaryotic recombination technology. The availability of the KHV BAC is an important advance that will allow the study of viral genes involved in KHV pathogenesis, as well as the production of attenuated recombinant candidate vaccines. PMID:18337580

  4. 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,…

  5. 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

  6. 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

  7. The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As a major step toward understanding the biology and evolution of ruminants, the cattle genome was sequenced to ~7x coverage using a combined whole genome shotgun and BAC skim approach. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs found in seven mammalian...

  8. 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. PMID:25662411

  9. 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

  10. A new genomic tool, ultra-frequently cleaving TaqII/sinefungin endonuclease with a combined 2.9-bp recognition site, applied to the construction of horse DNA libraries

    PubMed Central

    2013-01-01

    Background Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases). We have shown previously that class-IIS/IIC/IIG TspGWI REase, the prototype member of the Thermus sp. enzyme family, can be chemically relaxed by a cofactor analogue, allowing it to recognize very short DNA sequences of 3-bp combined frequency. Such frequently cleaving REases are extremely rare, with CviJI/CviJI*, SetI and FaiI the only other ones found in nature. Their unusual features make them very useful molecular tools for the development of representative DNA libraries. Results We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) – an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5′-GACCGA-3′ [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation. Conclusions

  11. Genomic evolution in Barrett’s adenocarcinoma cells: critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome

    PubMed Central

    Pal, J; Bertheau, R; Buon, L; Qazi, A; Batchu, RB; Bandyopadhyay, S; Ali-Fehmi, R; Beer, DG; Weaver, DW; Reis, RJ Shmookler; Goyal, RK; Huang, Q; Munshi, NC; Shammas, MA

    2012-01-01

    A prominent feature of most cancers including Barrett’s adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease. PMID:21423218

  12. 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.

  13. Utility of array comparative genomic hybridization in cytogenetic analysis.

    PubMed

    Singh, Rashmi R; Cheung, K-John J; Horsman, Douglas E

    2011-01-01

    Conventional comparative genomic hybridization (CGH), high-resolution oligonucleotide, and BAC array CGH have modernized the field of cytogenetics to enable access to unbalanced genomic aberrations such as whole or partial chromosomal gains and losses. The basic principle of array CGH involves hybridizing differentially labeled proband/test (e.g., tumor) and normal reference DNA on an array of oligonucleotide or BAC clones instead of normal metaphases as in conventional CGH. The sub-megabase resolution tiling BAC arrays are extremely useful for the analysis of acquired aberrations in cancer genomes. Array CGH can be extremely useful to identify the chromosomal makeup of marker and ring chromosomes, to define/delineate the precise location/bands involved in structural aberrations and the accurate localization of translocation breakpoints in both simple and complex karyotypes either alone or in combination with standard karyotype analysis. PMID:21431645

  14. 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)

  15. 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.

  16. 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...

  17. Protective efficacy of a recombinant BAC clone of Marek's disease virus containing REV-LTR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insertion of reticuloendotheliosis virus (REV) long-terminal repeat (LTR) into a bacterial artificial chromosome (BAC) clone of a very virulent strain of Marek’s disease (MD) virus (MDV), Md5 (Kim et al, 2011) rendered the resultant recombinant virus termed rMd5 REV-LTR BAC fully attenuated at passa...

  18. 76 FR 44055 - BAC Home Loans Servicing, LP, et al.; Notice of Application and Temporary Order

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-22

    ... COMMISSION BAC Home Loans Servicing, LP, et al.; Notice of Application and Temporary Order July 18, 2011... Act, with respect to an injunction entered against BAC Home Loans Servicing, LP (``HLS'') on May 31... entered the Injunction against HLS, formerly Countrywide Home Loans Servicing LP, in a matter brought...

  19. Recombineering strategies for developing next generation BAC transgenic tools for optogenetics and beyond

    PubMed Central

    Ting, Jonathan T.; Feng, Guoping

    2014-01-01

    The development and application of diverse BAC transgenic rodent lines has enabled rapid progress for precise molecular targeting of genetically-defined cell types in the mammalian central nervous system. These transgenic tools have played a central role in the optogenetic revolution in neuroscience. Indeed, an overwhelming proportion of studies in this field have made use of BAC transgenic Cre driver lines to achieve targeted expression of optogenetic probes in the brain. In addition, several BAC transgenic mouse lines have been established for direct cell-type specific expression of Channelrhodopsin-2 (ChR2). While the benefits of these new tools largely outweigh any accompanying challenges, many available BAC transgenic lines may suffer from confounds due in part to increased gene dosage of one or more “extra” genes contained within the large BAC DNA sequences. Here we discuss this under-appreciated issue and propose strategies for developing the next generation of BAC transgenic lines that are devoid of extra genes. Furthermore, we provide evidence that these strategies are simple, reproducible, and do not disrupt the intended cell-type specific transgene expression patterns for several distinct BAC clones. These strategies may be widely implemented for improved BAC transgenesis across diverse disciplines. PMID:24772073

  20. 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...

  1. 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;…

  2. High-Throughput SNP Discovery through Deep Resequencing of a Reduced Representation Library to Anchor and Orient Scaffolds in the Soybean Whole Genome Sequence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The soybean Consensus Map 4.0 facilitated the anchoring of 95.6% of the soybean whole genome sequence developed by the Joint Genome Institute, Department of Energy but only properly oriented 66% of the sequence scaffolds. To find additional single nucleotide polymorphism (SNP) markers for additiona...

  3. 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. PMID:25944057

  4. Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon.

    PubMed

    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

  5. Rapid recombinant protein production from piggyBac transposon-mediated stable CHO cell pools.

    PubMed

    Balasubramanian, Sowmya; Matasci, Mattia; Kadlecova, Zuzana; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2015-04-20

    Heterogeneous populations of stably transfected cells (cell pools) can serve for the rapid production of moderate amounts of recombinant proteins. Here, we propose the use of the piggyBac (PB) transposon system to improve the productivity and long-term stability of cell pools derived from Chinese hamster ovary (CHO) cells. PB is a naturally occurring genetic element that has been engineered to facilitate the integration of a transgene into the genome of the host cell. In this report PB-derived cell pools were generated after 10 days of selection with puromycin. The resulting cell pools had volumetric productivities that were 3-4 times higher than those achieved with cell pools generated by conventional plasmid transfection even though the number of integrated transgene copies per cell was similar in the two populations. In 14-day batch cultures, protein levels up to 600 and 800 mg/L were obtained for an Fc-fusion protein and a monoclonal antibody, respectively, at volumetric scales up to 1L. In general, the volumetric protein yield from cell pools remained constant for up to 3 months in the absence of selection. In conclusion, transfection of CHO cells with the PB transposon system is a simple, efficient, and reproducible approach to the generation of cell pools for the rapid production of recombinant proteins. PMID:25758242

  6. piggyBac transposon-based insertional mutagenesis in mouse haploid embryonic stem cells.

    PubMed

    Pettitt, Stephen J; Tan, E-Pien; Yusa, Kosuke

    2015-01-01

    Forward genetic screening is a powerful non-hypothesis-driven approach to unveil the molecular mechanisms and pathways underlying phenotypes of interest. In this approach, a genome-wide mutant library is first generated and then screened for a phenotype of interest. Subsequently, genes responsible for the phenotype are identified. There have been a number of successful screens in yeasts, Caenorhabditis elegans and Drosophila. These model organisms all allow loss-of-function mutants to be generated easily on a genome-wide scale: yeasts have a haploid stage in their reproductive cycles and the latter two organisms have short generation times, allowing mutations to be systematically bred to homozygosity. However, in mammals, the diploid genome and long generation time have always hampered rapid and efficient production of homozygous mutant cells and animals. The recent discovery of several haploid mammalian cell lines promises to revolutionize recessive genetic screens in mammalian cells. In this protocol, we describe an overview of insertional mutagenesis, focusing on DNA transposons, and provide a method for an efficient generation of genome-wide mutant libraries using mouse haploid embryonic stem cells. PMID:25408399

  7. 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...

  8. 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.

  9. Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome

    PubMed Central

    Wussow, Felix; Fickenscher, Helmut; Tischer, B. Karsten

    2009-01-01

    Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors. PMID:19997639

  10. 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

  11. Post-integration stability of piggyBac in Aedes aegypti.

    PubMed

    Sethuraman, Nagaraja; Fraser, Malcolm J; Eggleston, Paul; O'Brochta, David A

    2007-09-01

    The post-integration activity of piggyBac transposable element gene vectors in Aedes aegypti mosquitoes was tested under a variety of conditions. The embryos from five independent transgenic lines of Ae. aegypti, each with a single integrated non-autonomous piggyBac transposable element gene vector, were injected with plasmids containing the piggyBac transposase open-reading frame under the regulatory control of the Drosophila melanogaster hsp70 promoter. No evidence for somatic remobilization was detected in the subsequent adults whereas somatic remobilization was readily detected when similar lines of transgenic D. melanogaster were injected with the same piggyBac transposase-expressing plasmid. Ae. aegypti heterozygotes of piggyBac reporter-containing transgenes and piggyBac transposase-expressing transgenes showed no evidence of somatic and germ-line remobilization based on phenotypic and molecular detection methods. The post-integration mobility properties of piggyBac in Ae. aegypti enhance the utility of this gene vector for certain applications, particularly those where any level of vector remobilization is unacceptable. PMID:17681233

  12. 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.

  13. BacPP: a web-based tool for Gram-negative bacterial promoter prediction.

    PubMed

    de Avila E Silva, S; Notari, D L; Neis, F A; Ribeiro, H G; Echeverrigaray, S

    2016-01-01

    Bacterial Promoter Prediction (BacPP) is a tool used to predict given sequences as promoters of Gram-negative bacteria according to the σ factor that recognizes it. The first version of BacPP was implemented in Python language in a desktop version without a friendly interface. For this reason, a web version of BacPP is now available with the purpose of improving its usability and availability. The present paper describes the implementation of the web version of this tool, focusing on its software architecture and user functionalities. The software is available at www.bacpp.bioinfoucs.com/home. PMID:27173187

  14. 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,…

  15. A Contiguous 3-Mb Sequence-Ready Map in the S3–MX Region on 21q22.2 Based on High- Throughput Nonisotopic Library Screenings

    PubMed Central

    Hildmann, Thomas; Kong, Xianging; O’Brien, John; Riesselman, Lisa; Christensen, Hoang-My; Dagand, Emilie; Lehrach, Hans; Yaspo, Marie-Laure

    1999-01-01

    Progress in complete genomic sequencing of human chromosome 21 relies on the construction of high-quality bacterial clone maps spanning large chromosomal regions. To achieve this goal, we have applied a strategy based on nonradioactive hybridizations to contig building. A contiguous sequence-ready map was constructed in the Down syndrome congenital heart disease (DS-CHD) region in 21q22.2, as a framework for large-scale genomic sequencing and positional candidate gene approach. Contig assembly was performed essentially by high throughput nonisotopic screenings of genomic libraries, prior to clone validation by (1) restriction digest fingerprinting, (2) STS analysis, (3) Southern hybridizations, and (4) FISH analysis. The contig contains a total of 50 STSs, of which 13 were newly isolated. A minimum tiling path (MTP) was subsequently defined that consists of 20 PACs, 2 BACs, and 5 cosmids covering 3 Mb between D21S3 and MX1. Gene distribution in the region includes 9 known genes (c21–LRP, WRB, SH3BGR, HMG14, PCP4, DSCAM, MX2, MX1, and TMPRSS2) and 14 new additional gene signatures consisting of cDNA selection products and ESTs. Forthcoming genomic sequence information will unravel the structural organization of potential candidate genes involved in specific features of Down syndrome pathogenesis. PMID:10207158

  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 legislation…

  17. 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…

  18. Macintoshed Libraries.

    ERIC Educational Resources Information Center

    Valauskas, Edward J., Ed.; John, Nancy R., Ed.

    Contributed by librarians from public, academic, school, and special libraries, the 17 essays in this collection describe ways in which the Apple Macintosh is used in their libraries: (1) "Workstations and the Apple Macintosh" (Edward J. Valauskas); (2) "The Macintosh Experience at Chesapeake College" (Liz Cooper); (3) "ANSEL Character Set for the…

  19. Library Lighting.

    ERIC Educational Resources Information Center

    Metcalf, Keyes D.

    Chapter I provides a background and explains pertinent library lighting problems such as quality, function, aesthetics, intensity, and costs. Emphasis is on the quality and function of lighting for library users. Chapter II deals with the comments and answers to questions by persons who have a special interest and competence in the field of…

  20. 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…

  1. 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.

  2. Analysis of a genomic DNA expression library of Mycobacterium tuberculosis using tuberculosis patient sera: evidence for modulation of host immune response.

    PubMed Central

    Amara, R R; Satchidanandam, V

    1996-01-01

    DNA obtained from a human sputum isolate of Mycobacterium tuberculosis, NTI-64719, which showed extensive dissemination in the guinea pig model resulting in a high score for virulence was used to construct an expression library in the lambda ZAP vector. The size of DNA inserts in the library ranged from 1 to 3 kb, and recombinants represented 60% of the total plaques obtained. When probed with pooled serum from chronically infected tuberculosis patients, the library yielded 176 recombinants with a range of signal intensities. Among these, 93 recombinants were classified into 12 groups on the basis of DNA hybridization experiments. The polypeptides synthesized by the recombinants were predominantly LacZ fusion proteins. Serum obtained from patients who were clinically diagnosed to be in the early phase of M. tuberculosis infection was used to probe the 176 recombinants obtained. Interestingly, some recombinants that gave very strong signals in the original screen did not react with early-phase serum; conversely, other whose signals were extremely weak in the original screen gave very intense signals with serum from recently infected patients. This indicates the differential nature of either the expression of these antigens or the immune response elicited by them as a function of disease progression. PMID:8751927

  3. 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...

  4. Optimal pooling for genome re-sequencing with ultra-high-throughput short-read technologies

    PubMed Central

    Hajirasouliha, Iman; Hormozdiari, Fereydoun; Sahinalp, S. Cenk; Birol, Inanc

    2008-01-01

    New generation sequencing technologies offer unique opportunities and challenges for re-sequencing studies. In this article, we focus on re-sequencing experiments using the Solexa technology, based on bacterial artificial chromosome (BAC) clones, and address an experimental design problem. In these specific experiments, approximate coordinates of the BACs on a reference genome are known, and fine-scale differences between the BAC sequences and the reference are of interest. The high-throughput characteristics of the sequencing technology makes it possible to multiplex BAC sequencing experiments by pooling BACs for a cost-effective operation. However, the way BACs are pooled in such re-sequencing experiments has an effect on the downstream analysis of the generated data, mostly due to subsequences common to multiple BACs. The experimental design strategy we develop in this article offers combinatorial solutions based on approximation algorithms for the well-known max n-cut problem and the related max n-section problem on hypergraphs. Our algorithms, when applied to a number of sample cases give more than a 2-fold performance improvement over random partitioning. Contact: cenk@cs.sfu.ca PMID:18586730

  5. Strain-Specific Regulation of Striatal Phenotype in Drd2-eGFP BAC Transgenic Mice

    PubMed Central

    Chan, C. Savio; Peterson, Jayms D.; Gertler, Tracy S.; Glajch, Kelly E.; Quintana, Ruth E.; Cui, Qiaoling; Sebel, Luke E.; Plotkin, Joshua L.; Shen, Weixing; Heiman, Myriam; Heintz, Nathaniel; Greengard, Paul; Surmeier, D. James

    2012-01-01

    Mice carrying bacterial artificial chromosome (BAC) transgenes have become important tools for neuroscientists, providing a powerful means of dissecting complex neural circuits in the brain. Recently, it was reported that one popular line of these mice – mice possessing a BAC transgene with a D2 dopamine receptor (Drd2) promoter construct coupled to an enhanced green fluorescent protein (eGFP) reporter – had abnormal striatal gene expression, physiology and motor behavior. Unlike most of the work using BAC mice, this interesting study relied upon mice backcrossed on the outbred Swiss Webster strain that were homozygous for the Drd2-eGFP BAC transgene.The experiments reported here were conducted to determine whether mouse strain or zygosity was a factor in the reported abnormalities. As reported, SW mice were very sensitive to transgene expression. However, in more commonly used inbred strains of mice (C57BL/6, FVB/N) that were hemizygous for the transgene, the Drd2-eGFP BAC transgene did not alter striatal gene expression, physiology or motor behavior. Thus, the use of inbred strains of mice which are hemizygous for the Drd2 BAC transgene provide a reliable tool for studying basal ganglia function. PMID:22764222

  6. Measurement Properties of the Low Back Activity Confidence Scale (LoBACS).

    PubMed

    Davenport, Todd E; Cleland, Joshua A; Yamada, Kimiko A; Kulig, Kornelia

    2016-06-01

    The purpose of this study was to determine the measurement properties of the Low Back Activity Confidence Scale (LoBACS) in individuals with post-acute low back pain (LBP) receiving nonsurgical intervention, including construct validity, factorial validity, and internal consistency reliability. Data were analyzed from an existing randomized clinical trial involving 112 patients with LBP. Evidence for convergent validity was observed through significant correlations between LoBACS subscale scores and other function, pain, and psychobehavioral measures. LoBACS subscales accounted for 36% of the unique variance in dependent variable measurements, suggesting a satisfactory level of statistical divergence between the LoBACS and other psychobehavioral measurements in this study. Cronbach's α ranged from .88 to .92 for LoBACS subscales, and item-total correlations exceeded .6, indicating high internal consistency reliability. Principal axis factoring confirmed the hypothesized three-subscale structure by correctly classifying 14 of the 15 items. These findings indicate the LoBACS is valid and internally consistent to measure domain-specific self-efficacy beliefs. PMID:24686745

  7. 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...

  8. 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...

  9. 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

  10. 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. PMID:26478540

  11. 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)

  12. 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.

  13. 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.

  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. 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;…

  16. Ataxin-2 regulates RGS8 translation in a new BAC-SCA2 transgenic mouse model.

    PubMed

    Dansithong, Warunee; Paul, Sharan; Figueroa, Karla P; Rinehart, Marc D; Wiest, Shaina; Pflieger, Lance T; Scoles, Daniel R; Pulst, Stefan M

    2015-04-01

    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

  17. Standards for British Libraries.

    ERIC Educational Resources Information Center

    Vaughan, Anthony

    1982-01-01

    Reviews developments in British library standards since 1971, highlighting types of standards, public libraries, academic libraries (university, polytechnic, college), school libraries, and special libraries (hospital and health sciences, prison, subject specializations). Thirty-nine references are cited. (EJS)

  18. 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...

  19. Survey of 42,000 Gossypium hirsutum cv. Maxxa BAC-End Sequences and Frequency, Type, and Annotation of BAC-derived SSRs.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The quest for more molecular markers is a major initiative in cotton, which lags behind crops such as soybean, maize, and rice in this type of research. In an effort to increase the number of microsatellite markers in Gossypium, BAC-end sequences from a publicly available Gossypium hirsutum cv. Maxx...

  20. Analysis of BAC-end sequences in common bean (Phaseolus vulgaris L.) towards the development and characterization of long motifs SSRs.

    PubMed

    Müller, Bárbara Salomão de Faria; Sakamoto, Tetsu; de Menezes, Ivandilson Pessoa Pinto; Prado, Guilherme Souza; Martins, Wellington Santos; Brondani, Claudio; de Barros, Everaldo Gonçalves; Vianello, Rosana Pereira

    2014-11-01

    The increasing volume of genomic data on the Phaseolus vulgaris species have contributed to its importance as a model genetic species and positively affected the investigation of other legumes of scientific and economic value. To expand and gain a more in-depth knowledge of the common bean genome, the ends of a number of bacterial artificial chromosome (BAC) were sequenced, annotated and the presence of repetitive sequences was determined. In total, 52,270 BESs (BAC-end sequences), equivalent to 32 Mbp (~6 %) of the genome, were processed. In total, 3,789 BES-SSRs were identified, with a distribution of one SSR (simple sequence repeat) per 8.36 kbp and 2,000 were suitable for the development of SSRs, of which 194 were evaluated in low-resolution screening. From 40 BES-SSRs based on long motifs SSRs (≥ trinucleotides) analyzed in high-resolution genotyping, 34 showed an equally good amplification for the Andean and for the Mesoamerican genepools, exhibiting an average gene diversity (H E) of 0.490 and 5.59 alleles/locus, of which six classified as Class I showed a H E ≥ 0.7. The PCoA and structure analysis allowed to discriminate the gene pools (K = 2, FST = 0.733). From the 52,270 BESs, 2 % corresponded to transcription factors and 3 % to transposable elements. Putative functions for 24,321 BESs were identified and for 19,363 were assigned functional categories (gene ontology). This study identified highly polymorphic BES-SSRs containing tri- to hexanucleotides motifs and bringing together relevant genetic characteristics useful for breeding programs. Additionally, the BESs were incorporated into the international genome-sequencing project for the common bean. PMID:25164100

  1. The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.

    PubMed

    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

  2. Use of Baculovirus BacMam Vectors for Expression of ABC Drug Transporters in Mammalian Cells

    PubMed Central

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul

    2012-01-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  3. Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.

    PubMed

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul; Ambudkar, Suresh V

    2012-02-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  4. Efficient production of transgenic chickens based on piggyBac.

    PubMed

    Liu, Xiaojuan; Li, Ning; Hu, Xiaoxiang; Zhang, Ran; Li, Qingyuan; Cao, Dainan; Liu, Tongxin; Zhang, Yaqiong; Liu, Xiaofang

    2013-04-01

    Transgenic techniques in chickens have been developed much more slowly than in mammals due to chickens' unique reproduction mechanism. Retroviral methods have been the most successful. piggyBac (PB) is a transposon that has a 13 bp perfect terminal invert repeat sequence. PB can be inserted into TTAA sites and can also be precisely excised in mammals. Therefore, we have selected PB as a candidate to establish a new method to produce transgenic chickens. We constructed three donor vectors (ZGl-neo, ZGm-neo and ZGs-neo) expressing a GFP marker-gene and a neomycin resistant gene based on PB. We co-transfected each donor vector with a helper vector (CAG-PBase). We found that ZGl-neo was the most efficient PB vector. This vector could insert into TTAA sites in DF-1 cells. PB vectors were microinjected into sub-germinal cavity of newly laid eggs, and electroporation was then performed with a 20-V pulse for 5 cycles of 50 ms on and 100 ms off. GFP was expressed in different tissues of the embryos, including the gonads. Twenty-two chickens hatched after microinjection with compounds ZGl-neo and CAG-PBase (3:1). When we screened the blood DNA, 73 % (16/22) of the individuals were positive. Thirteen of the chickens grew to adulthood, 11 of which were males. 40 % (4/10) of the individuals were semen positive, and their copy numbers ranged from 0.05 to 0.21 (0.11, 0.21, 0.05, 0.06). No G1 offspring containing the integrated transposon were produced. We conclude that the PB transposon system is a novel useful tool for the efficient production of transgenic chickens. PMID:22918673

  5. 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

  6. Computational analysis of ordering in non-liquid crystalline versus liquid crystalline materials with special reference to nBAC

    SciTech Connect

    Lakshmi Praveen, P.; Veera Bhadra Reddy, K.; Ajeetha, N.; Ojha, D. P.

    2009-12-15

    A computational analysis of ordering in non-liquid crystalline p-n-alkyl benzoic acid, having 1 (1BAC), 2 (2BAC) and 3(3BAC) carbon atoms in the alkyl chain has been carried out with respect to translatory and orientational motions, but detailed results are reported only for 3BAC. The evaluation of net atomic charges and dipole moments at each atomic center has been carried out using complete neglect differential overlap (CNDO/2) method. The modified Rayleigh-Schrodinger perturbation theory along with the multicentered-multipole expansion method has been employed to evaluate long-range interactions, while a '6-exp' potential function has been assumed for short-range interactions. On the basis of stacking, in-plane and terminal interaction energy calculations, all possible arrangements of a molecular pair have been considered. A comparative picture of molecular parameters, such as total energy, binding energy, and total dipole moment of 3BAC with higher homologous series liquid crystalline compounds having 4(4BAC), 5(5BAC), and 6(6BAC) alkyl chain carbon atoms, has been given. It is found that, if a suitable functional group is attached to 3BAC, so that the length to breadth ratio is increased, the molecule will show a change in the long-range order, the phase transition temperature and other liquid crystalline properties.

  7. Development and characterization of BAC-end sequence derived SSRs, and their incorporation into a new higher density genetic map for cultivated peanut (Arachis hypogaea L.)

    PubMed Central

    2012-01-01

    Background Cultivated peanut (Arachis hypogaea L.) is an important crop worldwide, valued for its edible oil and digestible protein. It has a very narrow genetic base that may well derive from a relatively recent single polyploidization event. Accordingly molecular markers have low levels of polymorphism and the number of polymorphic molecular markers available for cultivated peanut is still limiting. Results Here, we report a large set of BAC-end sequences (BES), use them for developing SSR (BES-SSR) markers, and apply them in genetic linkage mapping. The majority of BESs had no detectable homology to known genes (49.5%) followed by sequences with similarity to known genes (44.3%), and miscellaneous sequences (6.2%) such as transposable element, retroelement, and organelle sequences. A total of 1,424 SSRs were identified from 36,435 BESs. Among these identified SSRs, dinucleotide (47.4%) and trinucleotide (37.1%) SSRs were predominant. The new set of 1,152 SSRs as well as about 4,000 published or unpublished SSRs were screened against two parents of a mapping population, generating 385 polymorphic loci. A genetic linkage map was constructed, consisting of 318 loci onto 21 linkage groups and covering a total of 1,674.4 cM, with an average distance of 5.3 cM between adjacent loci. Two markers related to resistance gene homologs (RGH) were mapped to two different groups, thus anchoring 1 RGH-BAC contig and 1 singleton. Conclusions The SSRs mined from BESs will be of use in further molecular analysis of the peanut genome, providing a novel set of markers, genetically anchoring BAC clones, and incorporating gene sequences into a linkage map. This will aid in the identification of markers linked to genes of interest and map-based cloning. PMID:22260238

  8. 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…

  9. A first generation BAC-based physical map of the Rainbow trout genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 effici...

  10. PiggyBac Transgene Insertion Site GIZA Strain

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetically modified New World screwworms were analyzed by inverse PCR and several sequences of genomic DNA flanking the transposon insertion sites were obtained. One transgenic strain, designated GIZA, exhibited the properties of a lethal genetic mutation. Classic genetic crosses of these insects r...

  11. Public Libraries

    ERIC Educational Resources Information Center

    Library Journal, 1972

    1972-01-01

    Building data is given for the following public libraries: New York, New York; Blue Island, Illinois; Corte Madera, California; Muskogee, Oklahoma: Charlotte, North Carolina; Washington, D.C.; Houston, Texas; Albermarle, North Carolina; Spokane, Washington; and Hemet, California. (Author/NH)

  12. Library Venturing.

    ERIC Educational Resources Information Center

    Wilson, H. Donald

    1986-01-01

    There is opportunity for service and profit to imaginative libraries organizing to provide new forms of knowledge. Librarians as entrepreneurs must learn venture management and finance. Available assistance includes growing entrepreneural understanding in large institutions; family and friends; private wealth-seeking investment; new business…

  13. Library Handbook.

    ERIC Educational Resources Information Center

    Cupp, Christian M., Ed.

    The purpose of this handbook is to help students, staff, and community patrons attain a reasonable degree of skill in using the Southeastern Community College Library effectively. The first section instructs the user on how to find information. A discussion of the card catalog and its use provides examples of catalog cards, reviews the…

  14. Library Environment.

    ERIC Educational Resources Information Center

    Computers in Libraries, 1993

    1993-01-01

    This special section includes two articles that review products and services for the automated library environment. Highlights include ergonomic products; products for visually, hearing-, and speech-impaired users; analog film recorders; computer filters; document imaging systems; electric filing systems; and printers. A list of vendors is…

  15. BacDive – The Bacterial Diversity Metadatabase in 2016

    PubMed Central

    Söhngen, Carola; Podstawka, Adam; Bunk, Boyke; Gleim, Dorothea; Vetcininova, Anna; Reimer, Lorenz Christian; Ebeling, Christian; Pendarovski, Cezar; Overmann, Jörg

    2016-01-01

    BacDive–the Bacterial Diversity Metadatabase (http://bacdive.dsmz.de) provides strain-linked information about bacterial and archaeal biodiversity. The range of data encompasses taxonomy, morphology, physiology, sampling and concomitant environmental conditions as well as molecular biology. The majority of data is manually annotated and curated. Currently (with release 9/2015), BacDive covers 53 978 strains. Newly implemented RESTful web services provide instant access to the content in machine-readable XML and JSON format. Besides an overall increase of data content, BacDive offers new data fields and features, e.g. the search for gene names, plasmids or 16S rRNA in the advanced search, as well as improved linkage of entries to external life science web resources. PMID:26424852

  16. Construction and Characterization of New piggyBac Vectors for Constitutive or Inducible Expression of Heterologous Gene Pairs and the Identification of a Previously Unrecognized Activator Sequence in piggyBac

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The piggyBac transposable element was first identified when it inserted itself into baculovirus genes during infection, causing a mutant plaque morphology phenotype. Since then, piggyBac has been developed into a germline tranformation vector usable with a wide variety of organisms. In this study...

  17. 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.

  18. 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

  19. 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. PMID:20140028

  20. 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. PMID:25240849

  1. BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes.

    PubMed

    Staňková, Helena; Hastie, Alex R; Chan, Saki; Vrána, Jan; Tulpová, Zuzana; Kubaláková, Marie; Visendi, Paul; Hayashi, Satomi; Luo, Mingcheng; Batley, Jacqueline; Edwards, David; Doležel, Jaroslav; Šimková, Hana

    2016-07-01

    The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC-by-BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high-resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high-resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome-scale analysis of repetitive sequences and revealed a ~800-kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone-by-clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC-contig physical map and validate sequence assembly on a chromosome-arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome-by-chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules. PMID:26801360

  2. BacM, an N-terminally processed bactofilin of Myxococcus xanthus, is crucial for proper cell shape

    PubMed Central

    Koch, Matthias K.; McHugh, Colleen A.; Hoiczyk, Egbert

    2011-01-01

    Summary Bactofilins are fibre-forming bacterial cytoskeletal proteins. Here, we report the structural and biochemical characterization of MXAN_7475 (BacM), one of the four bactofilins of Myxococcus xanthus. Absence of BacM leads to a characteristic ‘crooked’ cell morphology and an increased sensitivity to antibiotics targeting cell wall biosynthesis. The absence of the other three bactofilins MXAN_4637–4635 (BacN-P) has no obvious phenotype. In M. xanthus, BacM exists as a 150-amino-acid full-length version and as a version cleaved before Ser28. In the cell, native BacM forms 3 nm wide fibres, which assemble into bundles forming helix-like cytoplasmic cables throughout the cell, and in a subset of cells additionally a polarly arranged lateral rod-like structure. Isolated fibres consist almost completely of the N-terminally truncated version, suggesting that the proteolytic cleavage occurs before or during fibre formation. Fusion of BacM to mCherry perturbs BacM function and cellular fibre arrangement, resulting for example in the formation of one prominent polar corkscrew-like structure per cell. Immunofluorescence staining of BacM and MreB shows that their cellular distributions are not matching. Taken together, these data suggest that rod-shaped bacteria like M. xanthus use bactofilin fibres to achieve and maintain their characteristic cell morphology and cell wall stability. PMID:21414039

  3. Suppression of the toxicity of Bac7 (1-35), a bovine peptide antibiotic, and its production in E. coli.

    PubMed

    Ishida, Yojiro; Inouye, Masayori

    2016-03-01

    Bac7 (1-35) is an Arg- and Pro-rich peptide antibiotic, produced in bovine cells to protect them from microbial infection. It has been demonstrated to inhibit the protein synthesis in E. coli, leading to cell death. Because of its toxicity, no cost effective methods have been developed for Bac7 production in Escherichia coli for its potential clinical use. Here, we found a method to suppress Bac7 (1-35) toxicity in E. coli to establish its high expression system, in which Bac7 (1-35) was fused to the C-terminal end of protein S, a major spore-coat protein from Myxococcus xanthus, using a linker containing a Factor Xa cleavage site. The resulting His6-PrS2-Bac7 (1-35) (PrS2 is consisted of two N-terminal half domains of protein S connected in tandem) was well expressed using the Single-Protein Production (SPP) system at low temperature and subsequently purified in a single step by using a Ni column. The combination of protein S fusion and its expression in the SPP system at low temperature appeared to suppress Bac7 (1-35) toxicity. Both the purified His6-PrS2-Bac7 (1-35) and His6-PrS2-Bac7 (1-35) treated by Factor Xa were proven to be a potent inhibitor for cell-free protein synthesis. PMID:26936849

  4. BacM, an N-terminally processed bactofilin of Myxococcus xanthus, is crucial for proper cell shape.

    PubMed

    Koch, Matthias K; McHugh, Colleen A; Hoiczyk, Egbert

    2011-05-01

    Bactofilins are fibre-forming bacterial cytoskeletal proteins. Here, we report the structural and biochemical characterization of MXAN_7475 (BacM), one of the four bactofilins of Myxococcus xanthus. Absence of BacM leads to a characteristic 'crooked' cell morphology and an increased sensitivity to antibiotics targeting cell wall biosynthesis. The absence of the other three bactofilins MXAN_4637-4635 (BacN-P) has no obvious phenotype. In M. xanthus, BacM exists as a 150-amino-acid full-length version and as a version cleaved before Ser28. In the cell, native BacM forms 3 nm wide fibres, which assemble into bundles forming helix-like cytoplasmic cables throughout the cell, and in a subset of cells additionally a polarly arranged lateral rod-like structure. Isolated fibres consist almost completely of the N-terminally truncated version, suggesting that the proteolytic cleavage occurs before or during fibre formation. Fusion of BacM to mCherry perturbs BacM function and cellular fibre arrangement, resulting for example in the formation of one prominent polar corkscrew-like structure per cell. Immunofluorescence staining of BacM and MreB shows that their cellular distributions are not matching. Taken together, these data suggest that rod-shaped bacteria like M. xanthus use bactofilin fibres to achieve and maintain their characteristic cell morphology and cell wall stability. PMID:21414039

  5. Library Automation and Library Education.

    ERIC Educational Resources Information Center

    Drabenstott, Jon, Ed.

    1987-01-01

    Several consultants address the issue of competencies required of professional librarians for the effective management of the automation process. Highlights include formal and professional ongoing education and the need for technical training and problem solving skills to enable librarians to evaluate and develop library systems effectively.…

  6. 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

  7. Targeting homologous recombination and telomerase in Barrett’s adenocarcinoma: Impact on telomere maintenance, genomic instability, and tumor growth

    PubMed Central

    Lu, Renquan; Pal, Jagannath; Buon, Leutz; Nanjappa, Puru; Shi, Jialan; Fulciniti, Mariateresa; Tai, Yu-Tzu; Guo, Lin; Yu, Min; Gryaznov, Sergei; Munshi, Nikhil C.; Shammas, Masood A.

    2014-01-01

    Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in BAC. The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contribute to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase, makes telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore

  8. Targeting homologous recombination and telomerase in Barrett's adenocarcinoma: impact on telomere maintenance, genomic instability and tumor growth.

    PubMed

    Lu, R; Pal, J; Buon, L; Nanjappa, P; Shi, J; Fulciniti, M; Tai, Y-T; Guo, L; Yu, M; Gryaznov, S; Munshi, N C; Shammas, M A

    2014-03-20

    Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett's esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for β-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their

  9. A physical map of the papaya genome with integrated genetic map and genome sequence

    PubMed Central

    2009-01-01

    Background Papaya is a major fruit crop in tropical and subtropical regions worldwide and has primitive sex chromosomes controlling sex determination in this trioecious species. The papaya genome was recently sequenced because of its agricultural importance, unique biological features, and successful application of transgenic papaya for resistance to papaya ringspot virus. As a part of the genome sequencing project, we constructed a BAC-based physical map using a high information-content fingerprinting approach to assist whole genome shotgun sequence assembly. Results The physical map consists of 963 contigs, representing 9.4× genome equivalents, and was integrated with the genetic map and genome sequence using BAC end sequences and a sequence-tagged high-density genetic map. The estimated genome coverage of the physical map is about 95.8%, while 72.4% of the genome was aligned to the genetic map. A total of 1,181 high quality overgo (overlapping oligonucleotide) probes representing conserved sequences in Arabidopsis and genetically mapped loci in Brassica were anchored on the physical map, which provides a foundation for comparative genomics in the Brassicales. The integrated genetic and physical map aligned with the genome sequence revealed recombination hotspots as well as regions suppressed for recombination across the genome, particularly on the recently evolved sex chromosomes. Suppression of recombination spread to the adjacent region of the male specific region of the Y chromosome (MSY), and recombination rates were recovered gradually and then exceeded the genome average. Recombination hotspots were observed at about 10 Mb away on both sides of the MSY, showing 7-fold increase compared with the genome wide average, demonstrating the dynamics of recombination of the sex chromosomes. Conclusion A BAC-based physical map of papaya was constructed and integrated with the genetic map and genome sequence. The integrated map facilitated the draft genome assembly

  10. Symposium on Presidential Libraries.

    ERIC Educational Resources Information Center

    Relyea, Harold C.; And Others

    1994-01-01

    Includes five articles that discuss presidential libraries. Highlights include an overview of the development of the federal presidential library system; the Ronald Reagan library; the Richard Nixon library archives; access at the Gerald Ford library; and computerizing the Jimmy Carter library. (LRW)

  11. 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)

  12. 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…

  13. 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

  14. FISH applications for genomics and plant breeding strategies in tomato and other solanaceous crops.

    PubMed

    Szinay, D; Bai, Y; Visser, R; de Jong, H

    2010-07-01

    This paper describes the use of advanced fluorescence in situ hybridization (FISH) technologies for genomics and breeding of tomato and related Solanum species. The first part deals with the major determinants of FISH technology: (1) spatial resolution, which depends on the diffraction limit of the microscope and the type of chromosome, chromatin or isolated DNA fibres as target for the hybridisation; (2) the detection sensitivity, which is limited by the sensitivity and dynamic range of the CCD camera and the quality of the microscope, and the amplification system of the weak signals from tiny probe molecules; (3) simultaneous detection of multiple probes labelled directly or indirectly with up to 5 different fluorophores, whether or not in different combinations and/or mixed at different ratios. The power and usability of such multicolour FISH is indispensable when large numbers of bacterial artificial chromosomes (BACs) or other vectors with genomic DNA are available. Mapping of multiple BACs on chromosomes are powerful instruments confirming their assumed genetic position, whereas pooled BACs for a given chromosome arm will reveal the gaps between the BACs or derived contigs of their physical maps. Tandem and dispersed repeats, which are abundant in the genomes of most species, can be analysed in repeat bar coding FISH, showing the major blocks of repeats in heterochromatin and euchromatin areas. Repeat-rich areas of the chromosomes can also be demonstrated by hybridisation of probed Cot fractions of sheared genomic DNA; a powerful method to elucidate the heterochromatin domains for genomic studies. In addition, unlabelled Cot DNA, as blocking agent in BAC-FISH painting, suppresses repetitive sequences from the BACs to hybridise on the chromosomes. Cross-species BAC-FISH painting with labelled probes from tomato and potato BACs and hybridised on the chromosomes of related species, under appropriate conditions, is a powerful instrument to demonstrate chromosomal

  15. piggyBac Transposon plus Insulators Overcome Epigenetic Silencing to Provide for Stable Signaling Pathway Reporter Cell Lines

    PubMed Central

    Mossine, Valeri V.; Waters, James K.; Hannink, Mark; Mawhinney, Thomas P.

    2013-01-01

    Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other “transgene unfriendly” cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5’- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular. PMID:24376882

  16. Characterization of trichobakin, a type I ribosome-inactivating protein from Trichosanthes sp. Bac Kan 8-98.

    PubMed

    Chi, P V; Truong, H Q; Ha, N T; Chung, W I; Binh, L T

    2001-10-01

    We have isolated a genomic clone encoding trichobakin (TBK), a type I ribosome-inactivating protein from the plant Trichosanthes sp. Bac Kan 8-98 (family Cucurbitaceae), by PCR using specific primers designed from the cDNA sequences of alpha-trichosanthin. The sequence encoding mature TBK was constructed in the pET-21d(+) vector for overexpression in Escherichia coli strain BL21(DE3). The recombinant protein was purified to homogeneity by CM-Sepharose chromatography on FPLC with a final yield of about 55 mg/l of culture. The protein has a molecular mass of about 27 kDa, as shown by SDS/PAGE and matrix-assisted laser-desorption ionization MS. It was found that the protein inhibited luciferase mRNA translation in the rabbit reticulocyte cell-free system with an IC(50) value (that which causes a 50% reduction of residual translational activity) of about 3.5 pM. The rRNA N-glycosidase activity of the protein was also proved at the above-mentioned concentration after rRNAs were treated with acid aniline. PMID:11592913

  17. piggyBac transposon plus insulators overcome epigenetic silencing to provide for stable signaling pathway reporter cell lines.

    PubMed

    Mossine, Valeri V; Waters, James K; Hannink, Mark; Mawhinney, Thomas P

    2013-01-01

    Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other "transgene unfriendly" cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5'- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular. PMID:24376882

  18. Peaks of linkage are localized by a BAC/PAC contig of the 6p reading disability locus.

    PubMed

    Ahn, J; Won, T W; Zia, A; Reutter, H; Kaplan, D E; Sparks, R; Gruen, J R

    2001-11-01

    A gene for reading disability has been localized by nonparametric linkage to 6p21.3-p22 in several published reports. However, the lack of an uninterrupted genomic clone contig has made it difficult to determine accurate intermarker distances, precise marker order, and genetic boundaries and hinders direct comparisons of linkage. The search and discovery of the hemochromatosis gene (HFE) led to the creation of a bacterial artificial chromosome (BAC) and P-1 derived artificial chromosome (PAC) contig that extended physical maps 4 Mb from the MHC toward pter and localized new markers in that region [10-12]. Using this contig, we localized 124 sequence tagged sites, expressed sequence tags, and short tandem repeats including most of the markers in linkage with reading disability phenotypes, succinic semialdehyde dehydrogenase, GPLD1, prolactin, and 18 uncharacterized genes. This new contig joins and extends previously published physical maps to span the entire chromosome 6 reading disability genetic locus. Physical mapping data from the complete contig show overlap of the published linkage peaks for reading disability, provide accurate intermarker distances and order, and offer resources for generating additional markers and candidate genes for high resolution genetic studies in this region. PMID:11707069

  19. 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...

  20. Insect transformation with piggyBac: getting the number of injections just right.

    PubMed

    Gregory, M; Alphey, L; Morrison, N I; Shimeld, S M

    2016-06-01

    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

  1. C9ORF72-ALS/FTD: Transgenic Mice Make a Come-BAC.

    PubMed

    Hayes, Lindsey R; Rothstein, Jeffrey D

    2016-05-01

    For five years, since the landmark discovery of the C9ORF72 hexanucleotide repeat expansion in ALS/FTD, a transgenic mouse model has remained elusive. Now, two laboratories (Liu et al., 2016; Jiang et al., 2016) report the development of BAC transgenic mice that recapitulate features of the human disease. PMID:27151634

  2. Genetic transformation mediated by piggyBac in the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Asian corn borer, Ostrinia furnacalis, is a serious pest of corn, sorghum and cotton in China and other Asian countries. The present study is the first attempt to establish the transgenic line in O. furnacalis using a piggyBac transposon, which will shed light on the future genetic control of O....

  3. 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)

  4. Genetic Transformation of the Codling Moth, Cydia pomonella L., with piggyBac EGFP

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct results in identification o...

  5. 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

  6. 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…

  7. Library Instruction Assessment in Academic Libraries

    ERIC Educational Resources Information Center

    Tancheva, Kornelia; Andrews, Camille; Steinhart, Gail

    2007-01-01

    Determining the best methods of assessment for a library instruction program in a large research university can be a challenging task. Albert R. Mann Library at Cornell University Library has pilot-tested three methods of formative and summative assessment for its library instruction program--attitudinal, outcomes-based, and gap-measure--and…

  8. Culture-independent Community Genomics Study of Microogranisms Associated with Acid Mine Drainage

    NASA Astrophysics Data System (ADS)

    Tyson, G. W.; Hugenholtz, P.; Detter, C.; Richardson, P. M.; Banfield, J. F.

    2002-12-01

    gene content and metabolic pathways of the majority of community members (especially Leptospirillum spp.), permit evaluation of the genomic heterogeneity within each species population, and potentially enable reconstruction of genome fragments and whole genomes. We have constructed small (3-4 Kb; shotgun) and large-insert (>40 Kb; fosmid, BAC) libraries from community DNA. We will use sequencing results from the small insert library to determine whether it will be possible to reassemble parts of genomes using shotgun data. The large insert libraries will ensure links between genes and host organisms and provide information about gene order. Using the first 2 Mb of small insert library data we have established reasonable coverage of the major microbial groups but more data is required before the overall sequencing strategy can be established. Organism-resolved metabolic pathway information will be used to develop methods to monitor microbial activity in the environment. Ultimately, our goal is to develop a predictive analytical ecological model that resolves the linkages, feedbacks, and controlling variables that underpin acid mine drainage generation.

  9. 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. PMID:26305472

  10. 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

  11. 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.

  12. [Identification of chromosomal aberration in esophageal cancer cells by mixed BAC DNA probes of chromosome arms and regions].

    PubMed

    Jiajie, Hao; Chunli, Wang; Wenyue, Gu; Xiaoyu, Cheng; Yu, Zhang; Xin, Xu; Yan, Cai; Mingrong, Wang

    2014-06-01

    Chromosomal aberration is an important genetic feature of malignant tumor cells. This study aimed to clarify whether BAC DNA could be used to identify chromosome region and arm alterations. For each chromosome region, five to ten 1 Mb BAC DNA clones were selected to construct mixed BAC DNA clones for the particular region. All of the mixed clones from regions which could cover the whole chromosome arm were then mixed to construct mixed BAC DNA clones for the arms. Mixed BAC DNA probes of arms and regions were labeled by degenerate oligonucleotide primed PCR (DOP-PCR) and Nick translation techniques, respectively. The specificities of these probes were validated by fluorescence in situ hybridization (FISH) on the metaphase chromosomes of normal human peripheral blood lymphocytes. FISH with arm-specific mixed BAC DNA probes showed that chromosomal rearrangements and involved chromosome arms were confirmed in several esophageal cancer cells. By using region-specific mixed probes, the breakpoint on 1q from the derivative chromosome t(1q;7q) was identified in 1q32-q41 in esophageal KYSE140 cells. In conclusion, we established an effective labeling method for 1 Mb BAC DNA mixed clone probes, and chromosome arm and region rearrangements could be identified in several esophageal cancer cells by using these probes. Our study provides a more precise method for identification of chromosomal aberration by M-FISH, and the established method may also be applied to the karyotype analysis of hematological malignancies and prenatal diagnosis. PMID:24929514

  13. Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

    PubMed Central

    Chen, Xiang; Cui, Jing; Yan, Zhengjian; Zhang, Hongmei; Chen, Xian; Wang, Ning; Shah, Palak; Deng, Fang; Zhao, Chen; Geng, Nisha; Li, Melissa; Denduluri, Sahitya K.; Haydon, Rex C.; Luu, Hue H.; Reid, Russell R.; He, Tong-Chuan

    2015-01-01

    Sustained, high level transgene expression in mammalian cells, especially stem cells, may be desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The piggyBac transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, piggyBac system has not been widely used, at least in part due to the limited availability of piggyBac vectors with manipulation flexibilities. Here, we seek to optimize piggyBac-mediated transgene expression and generate a more efficient, user-friendly piggyBac system. By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained, high transgene expression. PMID:25815368

  14. 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...

  15. POLYMORPHISMS IN THE GENOMES OF ONCOGENIC AND ATTENUATED PATHOTYPES OF MAREK'S DISEASE VIRUS SEROTYPE 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete DNA sequence of a bacterial artificial chromosome (BAC) clone of Marek’s disease virus serotype 1 (MDV-1) vaccine strain CVI988 was determined. The genome consists of 178,311 base pairs, with an overall G/C content of 44%, and represents the largest among the MDV strains sequenced so fa...

  16. 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. PMID:26801048

  17. Complete Genome Sequence of a Human Cytomegalovirus Strain AD169 Bacterial Artificial Chromosome Clone

    PubMed Central

    Ostermann, Eleonore; Spohn, Michael; Indenbirken, Daniela

    2016-01-01

    The complete sequence of the human cytomegalovirus strain AD169 (variant ATCC) cloned as a bacterial artificial chromosome (AD169-BAC, also known as HB15 or pHB15) was determined. The viral genome has a length of 230,290 bp and shows 52 nucleotide differences compared to a previously sequenced AD169varATCC clone. PMID:27034483

  18. Library Services. Miscellaneous Papers.

    ERIC Educational Resources Information Center

    International Federation of Library Associations, The Hague (Netherlands).

    Papers on library journal cooperation, interlibrary lending, library services to minorities, and school library media centers, which were presented at the 1983 International Federation of Library Associations (IFLA) conference, include: (1) "The Co-operation between Editors of Library Journals in Socialist Countries," in which Wolfgang Korluss…

  19. 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…

  20. Library Research and Statistics.

    ERIC Educational Resources Information Center

    Lynch, Mary Jo; Brier, David J.; Lebbin, Vickery K.; Halstead, Kent; Fox, Bette-Lee; Kremen, Maya L.; Miller, Marilyn L.; Shontz, Marilyn L.

    1998-01-01

    Provides nine articles: research on libraries and librarianship, 1997; changing faces of library education (ALA-accredited graduate program title changes); number of libraries in the U.S., Canada, and Mexico; highlights of NCES surveys; library acquisition expenditures; price indexes for public and academic libraries; state rankings of selected…