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Sample records for genomic bac library

  1. CONSTRUCTION OF A WATERMELON BAC LIBRARY AND IDENTIFICATION OF SSRS ANCHORED TO MELON OR ARABIDOPSIS GENOMES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A bacterial artificial chromosome (BAC) library was constructed for watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] with an average insert-size of 106 kb, providing 21 haploid genome equivalents. The library was used to identify BAC clones that are anchored to probes evenly dis...

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

    PubMed

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

    2016-02-01

    The small abalone (Haliotis diversicolor) is one of the most important aquaculture species in East Asia. To facilitate gene cloning and characterization, genome analysis, and genetic breeding of it, we constructed a large-insert bacterial artificial chromosome (BAC) library, which is an important genetic tool for advanced genetics and genomics research. The small abalone BAC library includes 92,610 clones with an average insert size of 120 Kb, equivalent to approximately 7.6× of the small abalone genome. We set up three-dimensional pools and super pools of 18,432 BAC clones for target gene screening using PCR method. To assess the approach, we screened 12 target genes in these 18,432 BAC clones and identified 16 positive BAC clones. Eight positive BAC clones were then sequenced and assembled with the next generation sequencing platform. The assembled contigs representing these 8 BAC clones spanned 928 Kb of the small abalone genome, providing the first batch of genome sequences for genome evaluation and characterization. The average GC content of small abalone genome was estimated as 40.33 %. A total of 21 protein-coding genes, including 7 target genes, were annotated into the 8 BACs, which proved the feasibility of PCR screening approach with three-dimensional pools in small abalone BAC library. One hundred fifty microsatellite loci were also identified from the sequences for marker development in the future. The BAC library and clone pools provided valuable resources and tools for genetic breeding and conservation of H. diversicolor. PMID:26438131

  3. The Drosophila BAC resource The 19 Genomes of Drosophila: a BA C Library Resource for Genus-wide and1

    E-print Network

    Markow, Therese

    The Drosophila BAC resource 1 The 19 Genomes of Drosophila: a BA C Library Resource for Genus.126540 #12;The Drosophila BAC resource 2 Running Title: The Drosophila BAC resource22 Key Words: Drosophila;The Drosophila BAC resource 3 38 ABSTRA C T39 The genus Drosophila has been the subject of intense

  4. CONSTRUCTION AND CHARACTERIZATION OF TWO BAC LIBRARIES FROM BRACHYPODIUM DISTACHYPON, A NEW MODEL FOR GRASS GENOMICS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brachypodium is well suited to be a model system for temperate grasses because of its compact genome and a range of biological features. In an effort to develop resources for genome research in this emerging model species, we constructed two bacterial artificial chromosome (BAC) libraries from the d...

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

    PubMed Central

    2011-01-01

    Background Eucalyptus species are among the most planted hardwoods in the world because of their rapid growth, adaptability and valuable wood properties. The development and integration of genomic resources into breeding practice will be increasingly important in the decades to come. Bacterial artificial chromosome (BAC) libraries are key genomic tools that enable positional cloning of important traits, synteny evaluation, and the development of genome framework physical maps for genetic linkage and genome sequencing. Results We describe the construction and characterization of two deep-coverage BAC libraries EG_Ba and EG_Bb obtained from nuclear DNA fragments of E. grandis (clone BRASUZ1) digested with HindIII and BstYI, respectively. Genome coverages of 17 and 15 haploid genome equivalents were estimated for EG_Ba and EG_Bb, respectively. Both libraries contained large inserts, with average sizes ranging from 135 Kb (Eg_Bb) to 157 Kb (Eg_Ba), very low extra-nuclear genome contamination providing a probability of finding a single copy gene ? 99.99%. Libraries were screened for the presence of several genes of interest via hybridizations to high-density BAC filters followed by PCR validation. Five selected BAC clones were sequenced and assembled using the Roche GS FLX technology providing the whole sequence of the E. grandis chloroplast genome, and complete genomic sequences of important lignin biosynthesis genes. Conclusions The two E. grandis BAC libraries described in this study represent an important milestone for the advancement of Eucalyptus genomics and forest tree research. These BAC resources have a highly redundant genome coverage (> 15×), contain large average inserts and have a very low percentage of clones with organellar DNA or empty vectors. These publicly available BAC libraries are thus suitable for a broad range of applications in genetic and genomic research in Eucalyptus and possibly in related species of Myrtaceae, including genome sequencing, gene isolation, functional and comparative genomics. Because they have been constructed using the same tree (E. grandis BRASUZ1) whose full genome is being sequenced, they should prove instrumental for assembly and gap filling of the upcoming Eucalyptus reference genome sequence. PMID:21375742

  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. CONSTRUCTION OF AN ARABICA COFFEE BAC LIBRARY FOR MOLECULAR DISSECTION OF AN ALLOTETRAPLOID GENOME

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabica coffee (C. Arabica) is commercially the most important species of the genus Coffea. Arabica accounts for 70% of the world’s coffee production. We constructed a bacterial artificial chromosome (BAC) library using genomic DNA from the small bean, high-cupping quality, Arabica variety Mokka Hyb...

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

    PubMed Central

    2013-01-01

    Background The narrow-leafed lupin, Lupinus angustifolius L., is a grain legume species with a relatively compact genome. The species has 2n?=?40 chromosomes and its genome size is 960 Mbp/1C. During the last decade, L. angustifolius genomic studies have achieved several milestones, such as molecular-marker development, linkage maps, and bacterial artificial chromosome (BAC) libraries. Here, these resources were integratively used to identify and sequence two gene-rich regions (GRRs) of the genome. Results The genome was screened with a probe representing the sequence of a microsatellite fragment length polymorphism (MFLP) marker linked to Phomopsis stem blight resistance. BAC clones selected by hybridization were subjected to restriction fingerprinting and contig assembly, and 232 BAC-ends were sequenced and annotated. BAC fluorescence in situ hybridization (BAC-FISH) identified eight single-locus clones. Based on physical mapping, cytogenetic localization, and BAC-end annotation, five clones were chosen for sequencing. Within the sequences of clones that hybridized in FISH to a single-locus, two large GRRs were identified. The GRRs showed strong and conserved synteny to Glycine max duplicated genome regions, illustrated by both identical gene order and parallel orientation. In contrast, in the clones with dispersed FISH signals, more than one-third of sequences were transposable elements. Sequenced, single-locus clones were used to develop 12 genetic markers, increasing the number of L. angustifolius chromosomes linked to appropriate linkage groups by five pairs. Conclusions In general, probes originating from MFLP sequences can assist genome screening and gene discovery. However, such probes are not useful for positional cloning, because they tend to hybridize to numerous loci. GRRs identified in L. angustifolius contained a low number of interspersed repeats and had a high level of synteny to the genome of the model legume G. max. Our results showed that not only was the gene nucleotide sequence conserved between soybean and lupin GRRs, but the order and orientation of particular genes in syntenic blocks was homologous, as well. These findings will be valuable to the forthcoming sequencing of the lupin genome. PMID:23379841

  9. 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.

  10. A BAC library of the SP80-3280 sugarcane variety (saccharum sp.) and its inferred microsynteny with the sorghum genome

    PubMed Central

    2012-01-01

    Background Sugarcane breeding has significantly progressed in the last 30 years, but achieving additional yield gains has been difficult because of the constraints imposed by the complex ploidy of this crop. Sugarcane cultivars are interspecific hybrids between Saccharum officinarum and Saccharum spontaneum. S. officinarum is an octoploid with 2n?=?80 chromosomes while S. spontaneum has 2n?=?40 to 128 chromosomes and ploidy varying from 5 to 16. The hybrid genome is composed of 70-80%?S. officinaram and 5-20%?S. spontaneum chromosomes and a small proportion of recombinants. Sequencing the genome of this complex crop may help identify useful genes, either per se or through comparative genomics using closely related grasses. The construction and sequencing of a bacterial artificial chromosome (BAC) library of an elite commercial variety of sugarcane could help assembly the sugarcane genome. Results A BAC library designated SS_SBa was constructed with DNA isolated from the commercial sugarcane variety SP80-3280. The library contains 36,864 clones with an average insert size of 125 Kb, 88% of which has inserts larger than 90 Kb. Based on the estimated genome size of 760–930 Mb, the library exhibits 5–6 times coverage the monoploid sugarcane genome. Bidirectional BAC end sequencing (BESs) from a random sample of 192 BAC clones sampled genes and repetitive elements of the sugarcane genome. Forty-five per cent of the total BES nucleotides represents repetitive elements, 83% of which belonging to LTR retrotransposons. Alignment of BESs corresponding to 42 BACs to the genome sequence of the 10 sorghum chromosomes revealed regions of microsynteny, with expansions and contractions of sorghum genome regions relative to the sugarcane BAC clones. In general, the sampled sorghum genome regions presented an average 29% expansion in relation to the sugarcane syntenic BACs. Conclusion The SS_SBa BAC library represents a new resource for sugarcane genome sequencing. An analysis of insert size, genome coverage and orthologous alignment with the sorghum genome revealed that the library presents whole genome coverage. The comparison of syntenic regions of the sorghum genome to 42 SS_SBa BES pairs revealed that the sorghum genome is expanded in relation to the sugarcane genome. PMID:22524198

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

    PubMed Central

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

    2011-01-01

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

  12. 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 ...

  13. 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.

  14. Towards the physical map of the Trypanosoma cruzi nuclear genome: construction of YAC and BAC libraries of the reference clone T. cruzi CL-Brener.

    PubMed

    Ferrari, I; Lorenzi, H; Santos, M R; Brandariz, S; Requena, J M; Schijman, A; Vázquez, M; da Silveira, J F; Ben-Dov, C; Medrano, C; Ghío, S; López Bergami, P; Cano, I; Zingales, B; Urmenyi, T P; Rondinelli, E; González, A; Cortes, A; Lopez, M C; Thomas, M C; Alonso, C; Ramírez, J L; Chiurrillo, M A; Aldao, R R; Levin, M J

    1997-01-01

    Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease. PMID:9566217

  15. Construction of a BAC library and mapping BAC clones to the linkage map of Barramundi, Lates calcarifer

    PubMed Central

    Wang, Chun Ming; Lo, Loong Chueng; Feng, Felicia; Gong, Ping; Li, Jian; Zhu, Ze Yuan; Lin, Grace; Yue, Gen Hua

    2008-01-01

    Background Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its first generation linkage map has been applied to map QTL for growth traits. To identify genes located in QTL responsible for specific traits, genomic large insert libraries are of crucial importance. We reported herein a bacterial artificial chromosome (BAC) library and the mapping of BAC clones to the linkage map. Results This BAC library consisted of 49,152 clones with an average insert size of 98 kb, representing 6.9-fold haploid genome coverage. Screening the library with 24 microsatellites and 15 ESTs/genes demonstrated that the library had good genome coverage. In addition, 62 novel microsatellites each isolated from 62 BAC clones were mapped onto the first generation linkage map. A total of 86 BAC clones were anchored on the linkage map with at least one BAC clone on each linkage group. Conclusion We have constructed the first BAC library for L. calcarifer and mapped 86 BAC clones to the first generation linkage map. This BAC library and the improved linkage map with 302 DNA markers not only supply an indispensable tool to the integration of physical and linkage maps, the fine mapping of QTL and map based cloning genes located in QTL of commercial importance, but also contribute to comparative genomic studies and eventually whole genome sequencing. PMID:18366732

  16. 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...

  17. 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...

  18. 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 ...

  19. Construction of a BAC Library for a Defoliating Insect-Resistant Soybean and Identification of Candidate

    E-print Network

    Parrott, Wayne

    Construction of a BAC Library for a Defoliating Insect-Resistant Soybean and Identification-resistance QTL on linkage group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library with an average insert size 131 kb. This library represents a 6-fold soybean haploid genome equivalents, allowing

  20. 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...

  1. 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.

  2. 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...

  3. 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...

  4. BAC library of T. pallidum DNA in E. coli.

    PubMed

    Smajs, David; McKevitt, Matthew; Wang, Ling; Howell, Jerrilyn K; Norris, Steven J; Palzkill, Timothy; Weinstock, George M

    2002-03-01

    Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidum chromosome was missing in the BAC library, between bp 248727 and 264323. In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene. Altogether, 13 predicted T. pallidum ORFs (1.25% of the total) were incomplete or missing in the library. Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert. Of mapped clones, 19 were selected to represent the minimal set of E. coli BAC clones covering 1026 of the total 1040 (98.7%) predicted T. pallidum ORFs. Using this minimal set of clones, at least 12 T. pallidum proteins were shown to react with pooled sera from rabbits immunized with T. pallidum, indicating that at least some T. pallidum genes are transcribed and expressed in E. coli. PMID:11875041

  5. BAC Library of T. pallidum DNA in E. coli

    PubMed Central

    Šmajs, David; McKevitt, Matthew; Wang, Ling; Howell, Jerrilyn K.; Norris, Steven J.; Palzkill, Timothy; Weinstock, George M.

    2002-01-01

    Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidum chromosome was missing in the BAC library, between bp 248727 and 264323. In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene. Altogether, 13 predicted T. pallidum ORFs (1.25% of the total) were incomplete or missing in the library. Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert. Of mapped clones, 19 were selected to represent the minimal set of E. coli BAC clones covering 1026 of the total 1040 (98.7%) predicted T. pallidum ORFs. Using this minimal set of clones, at least 12 T. pallidum proteins were shown to react with pooled sera from rabbits immunized with T. pallidum, indicating that at least some T. pallidum genes are transcribed and expressed in E. coli. PMID:11875041

  6. AN INTEGRATED BAC PHYSICAL MAP OF THE PIG GENOME AND SELECTION OF THE MINIMUM TILEPATH

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a physical map of the pig genome by integrating restriction fingerprints and BAC end sequences generated from 4 BAC libraries with radiation hybrid markers, and contig alignments to the human genome. The map provides coverage across the 18 pig autosomes and the X chromosome in 17...

  7. SCREENING OF SUNFLOWER BAC LIBRARY FOR THE IDENTIFICATION OF SPECIFIC BAC CLONES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One BAC library and one BIBAC library from an inbred line HA 89 were constructed by using two restriction enzymes (BamH1, HindIII) and two vectors (pECBAC1, pCLD04541). Using the large-insert libraries, we identified a set of sunflower linkage group-specific BAC or BIBAC clones by overgo hybridizati...

  8. 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.

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

    PubMed Central

    2013-01-01

    Background Rice false smut caused by Villosiclava virens is a devastating fungal disease that spreads in major rice-growing regions throughout the world. However, the genomic information for this fungal pathogen is limited and the pathogenic mechanism of this disease is still not clear. To facilitate genetic, molecular and genomic studies of this fungal pathogen, we constructed the first BAC-based physical map and performed the first genome survey for this species. Results High molecular weight genomic DNA was isolated from young mycelia of the Villosiclava virens strain UV-8b and a high-quality, large-insert and deep-coverage Bacterial Artificial Chromosome (BAC) library was constructed with the restriction enzyme HindIII. The BAC library consisted of 5,760 clones, which covers 22.7-fold of the UV-8b genome, with an average insert size of 140 kb and an empty clone rate of lower than 1%. BAC fingerprinting generated successful fingerprints for 2,290 BAC clones. Using the fingerprints, a whole genome-wide BAC physical map was constructed that contained 194 contigs (2,035 clones) spanning 51.2 Mb in physical length. Bidirectional-end sequencing of 4,512 BAC clones generated 6,560 high quality BAC end sequences (BESs), with a total length of 3,030,658 bp, representing 8.54% of the genome sequence. Analysis of the BESs revealed general genome information, including 51.52% GC content, 22.51% repetitive sequences, 376.12/Mb simple sequence repeat (SSR) density and approximately 36.01% coding regions. Sequence comparisons to other available fungal genome sequences through BESs showed high similarities to Metarhizium anisopliae, Trichoderma reesei, Nectria haematococca and Cordyceps militaris, which were generally in agreement with the 18S rRNA gene analysis results. Conclusion This study provides the first BAC-based physical map and genome information for the important rice fungal pathogen Villosiclava virens. The BAC clones, physical map and genome information will serve as fundamental resources to accelerate the genetic, molecular and genomic studies of this pathogen, including positional cloning, comparative genomic analysis and whole genome sequencing. The BAC library and physical map have been opened to researchers as public genomic resources (http://gresource.hzau.edu.cn/resource/resource.html). PMID:24341590

  10. 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.

  11. BAC library for the amphipod crustacean, Parhyale hawaiensis Ronald J. Parchem a,1

    E-print Network

    Patel, Nipam H.

    BAC library for the amphipod crustacean, Parhyale hawaiensis Ronald J. Parchem a,1 , Francis Poulin Crustacean Arthropod Amphipod BAC library Bacterial artificial chromosomes (BACs) are capable of propagating generated a BAC library for the marine amphipod crustacean, Parhyale hawaiensis. The library was generated

  12. 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...

  13. 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.

  14. A BAC library of the East African haplochromine cichlid fish Astatotilapia burtoni.

    PubMed

    Lang, Michael; Miyake, Tsutomu; Braasch, Ingo; Tinnemore, Deborah; Siegel, Nicol; Salzburger, Walter; Amemiya, Chris T; Meyer, Axel

    2006-01-15

    A BAC library was constructed from Astatotilapia burtoni, a haplochromine cichlid that is found in Lake Tanganyika, East Africa, and its surrounding rivers. The library was generated from genomic DNA of blood cells and comprises 96,768 individual clones. Its median insert size is 150 kb and the coverage is expected to represent about 14 genome equivalents. The coverage evaluation was based on genome size estimates that were obtained by flow cytometry. In addition, hybridization screens with five probes largely corroborate the above coverage estimate, although the number of clones ranged from 5 to 22 authenticated clones per single copy probe. The BAC library described here is expected to be useful to the scientific community interested in cichlid genomics as an important resource to gain new insights into the rapid evolution of the great species diversity of haplochromine cichlid fishes. PMID:16254984

  15. The European sea bass Dicentrarchus labrax genome puzzle: comparative BAC-mapping and low coverage shotgun sequencing

    PubMed Central

    2010-01-01

    Background Food supply from the ocean is constrained by the shortage of domesticated and selected fish. Development of genomic models of economically important fishes should assist with the removal of this bottleneck. European sea bass Dicentrarchus labrax L. (Moronidae, Perciformes, Teleostei) is one of the most important fishes in European marine aquaculture; growing genomic resources put it on its way to serve as an economic model. Results End sequencing of a sea bass genomic BAC-library enabled the comparative mapping of the sea bass genome using the three-spined stickleback Gasterosteus aculeatus genome as a reference. BAC-end sequences (102,690) were aligned to the stickleback genome. The number of mappable BACs was improved using a two-fold coverage WGS dataset of sea bass resulting in a comparative BAC-map covering 87% of stickleback chromosomes with 588 BAC-contigs. The minimum size of 83 contigs covering 50% of the reference was 1.2 Mbp; the largest BAC-contig comprised 8.86 Mbp. More than 22,000 BAC-clones aligned with both ends to the reference genome. Intra-chromosomal rearrangements between sea bass and stickleback were identified. Size distributions of mapped BACs were used to calculate that the genome of sea bass may be only 1.3 fold larger than the 460 Mbp stickleback genome. Conclusions The BAC map is used for sequencing single BACs or BAC-pools covering defined genomic entities by second generation sequencing technologies. Together with the WGS dataset it initiates a sea bass genome sequencing project. This will allow the quantification of polymorphisms through resequencing, which is important for selecting highly performing domesticated fish. PMID:20105308

  16. 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...

  17. 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.

  18. 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.

  19. BACS: evolution of an integrated library system toward information management.

    PubMed

    Kelly, E A; Halbrook, B; Igielnik, S; Rueby, C

    1985-01-01

    The evolution of the Washington University School of Medicine BACS integrated library system toward information management functions is outlined. The creation of a machine-readable database and its extension through telecommunications have consequences that reach beyond the functions of the library as we have perceived them. It is argued that libraries are flexible institutions that, with automation, are likely to enlarge rather than to diminish. PMID:3978300

  20. 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...

  1. PERMANENT GENETIC RESOURCES ARTICLE BAC library construction, screening and clone sequencing of

    E-print Network

    Bernatchez, Louis

    PERMANENT GENETIC RESOURCES ARTICLE BAC library construction, screening and clone sequencing full-length assembly of candidate genes. The lake whitefish BAC library consists of 181 050 clones equivalents. Ten BAC clones were isolated following a quantitative real-time PCR screening approach

  2. Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

    PubMed Central

    Loit, Evelin; Melnyk, Charles W; MacFarlane, Amanda J; Scott, Fraser W; Altosaar, Illimar

    2009-01-01

    Background Exposure to dietary wheat proteins in genetically susceptible individuals has been associated with increased risk for the development of Type 1 diabetes (T1D). Recently, a wheat protein encoded by cDNA WP5212 has been shown to be antigenic in mice, rats and humans with autoimmune T1D. To investigate the genomic origin of the identified wheat protein cDNA, a hexaploid wheat genomic library from Glenlea cultivar was screened. Results Three unique wheat globulin genes, Glo-3A, Glo3-B and Glo-3C, were identified. We describe the genomic structure of these genes and their expression pattern in wheat seeds. The Glo-3A gene shared 99% identity with the cDNA of WP5212 at the nucleotide and deduced amino acid level, indicating that we have identified the gene(s) encoding wheat protein WP5212. Southern analysis revealed the presence of multiple copies of Glo-3-like sequences in all wheat samples, including hexaploid, tetraploid and diploid species wheat seed. Aleurone and embryo tissue specificity of WP5212 gene expression, suggested by promoter region analysis, which demonstrated an absence of endosperm specific cis elements, was confirmed by immunofluorescence microscopy using anti-WP5212 antibodies. Conclusion Taken together, the results indicate that a diverse group of globulins exists in wheat, some of which could be associated with the pathogenesis of T1D in some susceptible individuals. These data expand our knowledge of specific wheat globulins and will enable further elucidation of their role in wheat biology and human health. PMID:19615078

  3. 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 insight into characteristics of the melon genome such as gene density, average protein length, or microsatellite and transposon content. The annotation of the BAC sequences revealed a high degree of collinearity and protein sequence identity between melon and its close relative Cucumis sativus (cucumber). Transposon content analysis of the syntenic regions suggests that transposition activity after the split of both cucurbit species has been low in cucumber but very high in melon. Conclusions The results presented here show that the strategy followed, which combines shotgun and BAC-end sequencing together with anchored marker information, is an excellent method for sequencing specific genomic regions, especially from relatively compact genomes such as that of melon. However, in agreement with other results, this map-based, BAC approach is confirmed to be an expensive way of sequencing a whole plant genome. Our results also provide a partial description of the melon genome's structure. Namely, our analysis shows that the melon genome is highly collinear with the smaller one of cucumber, the size difference being mainly due to the expansion of intergenic regions and proliferation of transposable elements. PMID:21073723

  4. 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

  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. 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

  7. 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...

  8. 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.

  9. Construction and characterization of a BAC library from a gynogenetic channel catfish, Ictalurus punctatus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two bacterial artificial chromosome (BAC) libraries, designated CCBL1 and CCBL2, were constructed by cloning Hind III-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Approximately 53,500 clones from CCBL1 were arrayed in 384-we...

  10. Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

    PubMed Central

    Lin, Jinke; Kudrna, Dave; Wing, Rod A.

    2011-01-01

    We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135?kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers. PMID:21234344

  11. 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.

  12. CONSTRUCTION OF BAC AND BIBAC LIBRARIES FROM SUNFLOWER AND IDENTIFICATION OF LINKAGE GROUP-SPECIFIC CLONES BY OVERGO HYBRIDIZATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two complementary BAC and BIBAC libraries were constructed from nuclear DNA of sunflower cultivar HA 89. The BAC library was constructed with BamHI in the pECBAC1 vector. It contains 107,136 clones and has an average insert size of 140 kb. The BIBAC library was constructed with HindIII in the plant-...

  13. 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

  14. 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

  15. 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 development of the two potato BAC libraries provides a starting point, and landmarks for BAC contig construction and chromosome walking towards the map-based cloning of agronomically important target genes in the species. PMID:15067385

  16. 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...

  17. BACS: An Integrated Library Automation System with Patron Access to the Current Contents and Medline Databases

    PubMed Central

    Igielnik, S.; Kelly, E.

    1985-01-01

    BACS is a modular Standard MUMPS based integrated library automation system. The system has an on-line public catalog, complete serials, circulation and acquisitions control, and journal citation searching. The system has been in use since 1980 and runs on microcomputers or mainframe computers.

  18. Versatile P[acman] BAC libraries for transgenesis

    E-print Network

    Cai, Long

    an automated colony picking device. To create a resource for manipulation and analysis of D. melanogaster genes 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined. The approaches used in model systems rely on the identification of mutations in genes and the characterization

  19. High-Resolution Genomic Profiles of Breast Cancer Cell Lines Assessed by Tiling BAC Array

    E-print Network

    Lunds Universitet,

    High-Resolution Genomic Profiles of Breast Cancer Cell Lines Assessed by Tiling BAC Array Physics,Lund University,Lund,Sweden 6 Lund Strategic Research Center for Stem Cell Biologyand Cell Therapy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK-BR-3, L56Br-C1, ZR-75-1, JIMT1

  20. Technology at Washington University School of Medicine Library: BACS, PHILSOM, and OCTANET.

    PubMed Central

    Crawford, S; Johnson, M F; Kelly, E A

    1983-01-01

    A brief overview of the Bibliographic Access and Control System developed by the Washington University School of Medicine Library is presented. Because the system has been described in two previous reports, this paper focuses on its relationship to other automated programs (i.e., PHILSOM and OCTANET), education of users, evaluation of the system, and outreach to the medical center. In operation for more than two years, BACS represents the computerization of much of the managerial and operational functions of the library, and marks the completion of stage 1 of the three stages of library evolution described in the AAMC report Academic Information in the Academic Health Sciences Center: Roles for the Library in Information Management. PMID:6688750

  1. TOWARD DEVELOPMENT OF A WHOLE-GENOME, BAC/BIBAC-BASED INTEGRATED PHYSICAL/GENETIC MAP OF THE COTTON GENOME USING THE UPLAND COTTON GENETIC STANDARD TM-1: BAC FINGERPRINTING AND PHYSICAL MAP CONTIG CONSTRUCTION.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are developing a whole-genome, BAC/BIBAC-based integrated physical/genetic map of the cotton (Gossypium hirsutum L.) genome using its genetic standard line TM-1 as the reference genotype. Whole-genome physical maps integrated with genetic maps will provide revolutionized tools and platforms for a...

  2. Using comparative genomics to reorder the human genome sequence into a virtual sheep genome

    PubMed Central

    Dalrymple, Brian P; Kirkness, Ewen F; Nefedov, Mikhail; McWilliam, Sean; Ratnakumar, Abhirami; Barris, Wes; Zhao, Shaying; Shetty, Jyoti; Maddox, Jillian F; O'Grady, Margaret; Nicholas, Frank; Crawford, Allan M; Smith, Tim; de Jong, Pieter J; McEwan, John; Oddy, V Hutton; Cockett, Noelle E

    2007-01-01

    Background Is it possible to construct an accurate and detailed subgene-level map of a genome using bacterial artificial chromosome (BAC) end sequences, a sparse marker map, and the sequences of other genomes? Results A sheep BAC library, CHORI-243, was constructed and the BAC end sequences were determined and mapped with high sensitivity and low specificity onto the frameworks of the human, dog, and cow genomes. To maximize genome coverage, the coordinates of all BAC end sequence hits to the cow and dog genomes were also converted to the equivalent human genome coordinates. The 84,624 sheep BACs (about 5.4-fold genome coverage) with paired ends in the correct orientation (tail-to-tail) and spacing, combined with information from sheep BAC comparative genome contigs (CGCs) built separately on the dog and cow genomes, were used to construct 1,172 sheep BAC-CGCs, covering 91.2% of the human genome. Clustered non-tail-to-tail and outsize BACs located close to the ends of many BAC-CGCs linked BAC-CGCs covering about 70% of the genome to at least one other BAC-CGC on the same chromosome. Using the BAC-CGCs, the intrachromosomal and interchromosomal BAC-CGC linkage information, human/cow and vertebrate synteny, and the sheep marker map, a virtual sheep genome was constructed. To identify BACs potentially located in gaps between BAC-CGCs, an additional set of 55,668 sheep BACs were positioned on the sheep genome with lower confidence. A coordinate conversion process allowed us to transfer human genes and other genome features to the virtual sheep genome to display on a sheep genome browser. Conclusion We demonstrate that limited sequencing of BACs combined with positioning on a well assembled genome and integrating locations from other less well assembled genomes can yield extensive, detailed subgene-level maps of mammalian genomes, for which genomic resources are currently limited. PMID:17663790

  3. Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori.

    PubMed

    Xu, Han-Fu; Xia, Qing-You; Liu, Chun; Cheng, Ting-Cai; Zhao, Ping; Duan, Jun; Zha, Xing-Fu; Liu, Shi-Ping

    2006-07-01

    piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species. PMID:16685528

  4. 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 genetic markers for plant breeding. PMID:19619287

  5. 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...

  6. High-resolution mapping and chromosome landing at the root-know nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library.

    PubMed

    Claverie, M; Dirlewanger, E; Cosson, P; Bosselut, N; Lecouls, A C; Voisin, R; Kleinhentz, M; Lafargue, B; Caboche, M; Chalhoub, B; Esmenjaud, D

    2004-10-01

    The Ma gene for root-knot nematode (RKN)resistance from Myrobalan plum (Prunus cerasifera L.)confers a complete-spectrum and a heat-stable resistance to Meloidogvne spp., conversely to Mi-I from tomato,which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designatedSCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175,Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175,totaling 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing. In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species. PMID:15322755

  7. 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...

  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. AFLP-based study of genetic difference and mega-base DNA isolation for BAC library construction from greenbug Schizaphis graminum (Rondani) (Homoptera:Aphididae) 

    E-print Network

    Li, Haiwen

    2002-01-01

    This research focuses on the study of genetic differences among eight greenbug biotypes revealed by AFLP analyses and isolation of mega-base DNA for BAC library construction. Genetic relationship among greenbug biotypes is an old, but interesting...

  10. Tuatara (Sphenodon) Genomics: BAC Library Construction, Sequence Survey,

    E-print Network

    Edwards, Scott

    as sphenodontids that was contem- poraneous with early dinosaurs, around 220 million years ago, justifying cluster- ing of tuataras with archosaurs (crocodilians and birds) or turtles, rather than with squamates

  11. Isolation of BAC clones: Occasionally, more than one BAC clone may be represented in a single well of the BAC library. It is recommended that the following procedure for the

    E-print Network

    Isolation of BAC clones: Occasionally, more than one BAC clone may be represented in a single well of BAC clones. 1. Streak some of the BAC culture from the stab tube to a LB plate containing 12.5 µg at 4ºC. Verification of clones: All cones should be verified after isolation using Southern blot

  12. [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

  13. PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia

    PubMed Central

    Baudry, Céline; Malinsky, Sophie; Restituito, Matthieu; Kapusta, Aurélie; Rosa, Sarah; Meyer, Eric; Bétermier, Mireille

    2009-01-01

    Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase. PMID:19884254

  14. A Bacterial Artificial Chromosome Library for Sequencing the Complete Human Genome

    PubMed Central

    Osoegawa, Kazutoyo; Mammoser, Aaron G.; Wu, Chenyan; Frengen, Eirik; Zeng, Changjiang; Catanese, Joseph J.; de Jong, Pieter J.

    2001-01-01

    A 30-fold redundant human bacterial artificial chromosome (BAC) library with a large average insert size (178 kb) has been constructed to provide the intermediate substrate for the international genome sequencing effort. The DNA was obtained from a single anonymous volunteer, whose identity was protected through a double-blind donor selection protocol. DNA fragments were generated by partial digestion with EcoRI (library segments 1–4: 24-fold) and MboI (segment 5: sixfold) and cloned into the pBACe3.6 and pTARBAC1 vectors, respectively. The quality of the library was assessed by extensive analysis of 169 clones for rearrangements and artifacts. Eighteen BACs (11%) revealed minor insert rearrangements, and none was chimeric. This BAC library, designated as “RPCI-11,” has been used widely as the central resource for insert-end sequencing, clone fingerprinting, high-throughput sequence analysis and as a source of mapped clones for diagnostic and functional studies. The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AQ936150–AQ936491.] PMID:11230172

  15. Yeast Genomic Library Genomic DNA Sau3AI partial digestion

    E-print Network

    Pace, Norman

    Yeast Genomic Library Concept: Genomic DNA ­ Sau3AI partial digestion Vector DNA ­ BamHI full digestion partial Ligate and transform above products Vector Information: · use centromeric plasmid to avoid of the mcs Preparing Vector: 1) digest 3-4ug of library vector with BamHI for 2-4hrs in a total volume of 20

  16. Construction, characterization and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    White clover (Trifolium repens L.) is a forage legume widely used in combination with grass in pastures due to its ability for nitrogen fixation. We have constructed a bacterial artificial chromosome (BAC) library of an advanced breeding line of white clover. The library contains 37,248 clones wit...

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

    PubMed Central

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

    2012-01-01

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

  18. 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...

  19. BAC manipulations for making BAC transgene arrays.

    PubMed

    Khanna, Nimish; Bian, Qian; Plutz, Matt; Belmont, Andrew S

    2013-01-01

    Chromosome tagging using lac or tet operator repeats for in vivo visualization of chromosome dynamics has now become a standard methodology used in a range of organisms. One variation of this approach has been to build transgene arrays creating artificial chromosome blocks to study various aspects of chromatin structure, transcription, replication, or DNA repair. Previously, plasmid transgenes with or without subsequent gene amplification have been used to build these arrays. However, plasmid arrays typically show heterochromatic properties, while gene amplification typically results in chromosome instability of the amplified regions. To avoid these problems, we are now building transgene arrays from large genomic DNA inserts cloned in bacterial artificial chromosomes (BAC). These BAC transgenes show transcriptional levels within several fold of endogenous genes while also exhibiting targeting to specific nuclear compartments similar to the targeting of the endogenous genes. Here we describe Tn5 transposition and BAC recombineering methods used to retrofit BACs for their use in building BAC transgene arrays. This includes insertion of operator repeats and selectable markers into these BACs as well as targeted insertion or deletion of BAC sequences. PMID:23980009

  20. De novo 454 sequencing of barcoded BAC pools for comprehensive gene survey and genome analysis in the complex genome of barley

    PubMed Central

    2009-01-01

    Background De novo sequencing the entire genome of a large complex plant genome like the one of barley (Hordeum vulgare L.) is a major challenge both in terms of experimental feasibility and costs. The emergence and breathtaking progress of next generation sequencing technologies has put this goal into focus and a clone based strategy combined with the 454/Roche technology is conceivable. Results To test the feasibility, we sequenced 91 barcoded, pooled, gene containing barley BACs using the GS FLX platform and assembled the sequences under iterative change of parameters. The BAC assemblies were characterized by N50 of ~50 kb (N80 ~31 kb, N90 ~21 kb) and a Q40 of 94%. For ~80% of the clones, the best assemblies consisted of less than 10 contigs at 24-fold mean sequence coverage. Moreover we show that gene containing regions seem to assemble completely and uninterrupted thus making the approach suitable for detecting complete and positionally anchored genes. By comparing the assemblies of four clones to their complete reference sequences generated by the Sanger method, we evaluated the distribution, quality and representativeness of the 454 sequences as well as the consistency and reliability of the assemblies. Conclusion The described multiplex 454 sequencing of barcoded BACs leads to sequence consensi highly representative for the clones. Assemblies are correct for the majority of contigs. Though the resolution of complex repetitive structures requires additional experimental efforts, our approach paves the way for a clone based strategy of sequencing the barley genome. PMID:19930547

  1. 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

  2. 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...

  3. 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...

  4. 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...

  5. CONSTRUCTION OF CONTIGS OF AEGILOPS TAUSCHII GENOMIC DNA FRAGMENTS CLONED IN BAC AND BIBAC VECTORS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A high-throughput, fully automated, multi-color fluorescent fingerprinting technique for large-insert genomic DNA clones was developed. The technique was used to fingerprint 200,000 genomic DNA fragments of Aegilops tauschii line genetically closely related to the D genome of Chinese Spring wheat. T...

  6. Large insert environmental genomic library production.

    PubMed

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

    2009-01-01

    The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE http://www.epibio.com/item.asp?ID=385). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol. PMID:19776717

  7. Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening.

    PubMed

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

    2015-08-10

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

  8. Mapping genomic library clones using oligonucleotide arrays

    SciTech Connect

    Sapolsky, R.J.; Lipshutz, R.J.

    1996-05-01

    We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adaptors. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density olligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection. 28 refs., 9 figs., 2 tabs.

  9. BACs representing several-fold clone coverage of the genome were sequenced

    E-print Network

    Lazzaro, Brian

    and then mapped to the genome based on microsatellite marker position. A portion of the genome (two of the linkage groups) is shown as displayed in the Genboree browser (www.genboree.org). The top track (A_STS:Linkage) shows the position of markers (at this stage of map development there were 854 mapped markers

  10. TOWARD AN INTEGRATED PHYSICAL AND GENETIC MAP OF COTTON GEMONE: BAC FINGERPRINTING AND PHYSICAL MAP CONSTRUCTION.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are developing an integrated physical/genetic map of the cultivated allotetraploid cotton (AD genome) using three large-insert BAC and BIBAC libraries that were constructed from the genetic standard line TM-1. We have investigated all fingerprinting methods both previously developed and used in t...

  11. 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.

  12. 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...

  13. 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 (...

  14. 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

  15. BAC to the future

    SciTech Connect

    Anderson, C.

    1993-03-19

    The emerging problems with YACs may have been a blow to many genome researchers, but some scientists have long been advocating more funding for alternative DNA carriers. As the Human Genome Project focused on YACs in recent years, however, support for the development of non-YAC systems has been hard to come by. But as the community comes to grips with the limitations of YACs, researchers are starting to take another look at the alternatives. One of the most promising is the newest-the Bacteria Artificial Chromosome (BAC). Like a YAC, it is formed by inserting foreign DNA into a microorganism - E. coli in the case of the BAC - and letting the organism's genetic reproduction machinery take over to make copies. Mel Simon and his group at Caltech developed the BAC in 1990 specifically to try to get around some of the problems with YACs, and Simon says they have ben at least partially successful. BACs don't appear to result in unrelated pieces of DNA being spliced together and cloned, for example, and very preliminary analysis suggests that deletion of stretches of DNA will not be a significant problem. Best of all, he says, those regions of the human genome that are uncloneable in YACs will probably clone just fine in BACs, and vice-versa. BACs do, however, have one significant disadvantage: They can carry stretches of only 200,000 to 300,000 bases of foreign DNA - less than one-quarter as long as the best YACs. And other possible alternatives to YACs are limited to even shorter fragments. Another bacteria-based carrier known as a P1 can carry DNA insets of about 100,000 bases, and cosmids, which have been around the longest, are shorter yet - about 40,000 bases. While none of these systems is going to challenge the YAC for the size of their genome fragments, Simon says, they should provide a more precise complement to the YACs brute-force capabilities.

  16. Characterization of novel and complex genomic aberrations in glioblastoma using a 32K BAC array

    PubMed Central

    Nord, Helena; Hartmann, Christian; Andersson, Robin; Menzel, Uwe; Pfeifer, Susan; Piotrowski, Arkadiusz; Bogdan, Adam; Kloc, Wojciech; Sandgren, Johanna; Olofsson, Tommie; Hesselager, Göran; Blomquist, Erik; Komorowski, Jan; von Deimling, Andreas; Bruder, Carl E.G.; Dumanski, Jan P.; de Ståhl, Teresita Díaz

    2009-01-01

    Glioblastomas (GBs) are malignant CNS tumors often associated with devastating symptoms. Patients with GB have a very poor prognosis, and despite treatment, most of them die within 12 months from diagnosis. Several pathways, such as the RAS, tumor protein 53 (TP53), and phosphoinositide kinase 3 (PIK3) pathways, as well as the cell cycle control pathway, have been identified to be disrupted in this tumor. However, emerging data suggest that these aberrations represent only a fraction of the genetic changes involved in gliomagenesis. In this study, we have applied a 32K clone-based genomic array, covering 99% of the current assembly of the human genome, to the detailed genetic profiling of a set of 78 GBs. Complex patterns of aberrations, including high and narrow copy number amplicons, as well as a number of homozygously deleted loci, were identified. Amplicons that varied both in number (three on average) and in size (1.4 Mb on average) were frequently detected (81% of the samples). The loci encompassed not only previously reported oncogenes (EGFR, PDGFRA, MDM2, and CDK4) but also numerous novel oncogenes as GRB10, MKLN1, PPARGC1A, HGF, NAV3, CNTN1, SYT1, and ADAMTSL3. BNC2, PTPLAD2, and PTPRE, on the other hand, represent novel candidate tumor suppressor genes encompassed within homozygously deleted loci. Many of these genes are already linked to several forms of cancer; others represent new candidate genes that may serve as prognostic markers or even as therapeutic targets in the future. The large individual variation observed between the samples demonstrates the underlying complexity of the disease and strengthens the demand for an individualized therapy based on the genetic profile of the patient. PMID:19304958

  17. 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...

  18. 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.

  19. 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

  20. 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

  1. GenomeD3Plot: a library for rich, interactive visualizations of genomic data in web applications

    PubMed Central

    Laird, Matthew R.; Langille, Morgan G.I.; Brinkman, Fiona S.L.

    2015-01-01

    Motivation: A simple static image of genomes and associated metadata is very limiting, as researchers expect rich, interactive tools similar to the web applications found in the post-Web 2.0 world. GenomeD3Plot is a light weight visualization library written in javascript using the D3 library. GenomeD3Plot provides a rich API to allow the rapid visualization of complex genomic data using a convenient standards based JSON configuration file. When integrated into existing web services GenomeD3Plot allows researchers to interact with data, dynamically alter the view, or even resize or reposition the visualization in their browser window. In addition GenomeD3Plot has built in functionality to export any resulting genome visualization in PNG or SVG format for easy inclusion in manuscripts or presentations. Results: GenomeD3Plot is being utilized in the recently released Islandviewer 3 (www.pathogenomics.sfu.ca/islandviewer/) to visualize predicted genomic islands with other genome annotation data. However, its features enable it to be more widely applicable for dynamic visualization of genomic data in general. Availability and implementation: GenomeD3Plot is licensed under the GNU-GPL v3 at https://github.com/brinkmanlab/GenomeD3Plot/. Contact: brinkman@sfu.ca PMID:26093150

  2. A Multiway Analysis for Identifying High Integrity Bovine BACs

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. 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. Using the Potato Genome Sequence! Robin Buell!

    E-print Network

    Douches, David S.

    Using the Potato Genome Sequence! Robin Buell! Michigan State University! Department of Plant;· RHPOTKEY BAC library (78000 clones; 9-10 g.e.) · Library clones fingerprintedGen Sequencing? 10 #12;Initial Strategy heterozygous clone (RH89-039-16) Contig assembly

  5. WEEVIL GENOMICS: AN EST LIBRARY FROM THE DIAPREPES ROOT WEEVIL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have initiated a project to analyze expressed sequence tags (ESTs from the Diaprepes root weevil. Results of the first EST library for this species are presented, including total number of sequences, unique sequences, contigs, matches with genomic databases, and putative gene functions....

  6. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach.

    PubMed

    Chari, Raj; Mali, Prashant; Moosburner, Mark; Church, George M

    2015-09-01

    We developed an in vivo library-on-library methodology to simultaneously assess single guide RNA (sgRNA) activity across ?1,400 genomic loci. Assaying across multiple human cell types and end-processing enzymes as well as two Cas9 orthologs, we unraveled underlying nucleotide sequence and epigenetic parameters. Our results and software (http://crispr.med.harvard.edu/sgRNAScorer) enable improved design of reagents, shed light on mechanisms of genome targeting, and provide a generalizable framework to study nucleic acid-nucleic acid interactions and biochemistry in high throughput. PMID:26167643

  7. Three minimum tile paths from bacterial artificial chromosome libraries of the soybean (Glycine max cv. 'Forrest'): tools for structural and functional genomics

    PubMed Central

    Shultz, JL; Yesudas, C; Yaegashi, S; Afzal, AJ; Kazi, S; Lightfoot, DA

    2006-01-01

    Background The creation of minimally redundant tile paths (hereafter MTP) from contiguous sets of overlapping clones (hereafter contigs) in physical maps is a critical step for structural and functional genomics. Build 4 of the physical map of soybean (Glycine max L. Merr. cv. 'Forrest') showed the 1 Gbp haploid genome was composed of 0.7 Gbp diploid, 0.1 Gbp tetraploid and 0.2 Gbp octoploid regions. Therefore, the size of the unique genome was about 0.8 Gbp. The aim here was to create MTP sub-libraries from the soybean cv. Forrest physical map builds 2 to 4. Results The first MTP, named MTP2, was 14,208 clones (of mean insert size 140 kbp) picked from the 5,597 contigs of build 2. MTP2 was constructed from three BAC libraries (BamHI (B), HindIII (H) and EcoRI (E) inserts). MTP2 encompassed the contigs of build 3 that derived from build 2 by a series of contig merges. MTP2 encompassed 2 Gbp compared to the soybean haploid genome of 1 Gbp and does not distinguish regions by ploidy. The second and third MTPs, called MTP4BH and MTP4E, were each based on build 4. Each was semi-automatically selected from 2,854 contigs. MTP4BH was 4,608 B and H insert clones of mean size 173 kbp in the large (27.6 kbp) T-DNA vector pCLD04541. MTP4BH was suitable for plant transformation and functional genomics. MTP4E was 4,608 BAC clones with large inserts (mean 175 kbp) in the small (7.5 kbp) pECBAC1 vector. MTP4E was suitable for DNA sequencing. MTP4BH and MTP4E clones each encompassed about 0.8 Gbp, the 0.7 Gbp diploid regions and 0.05 Gbp each from the tetraploid and octoploid regions. MTP2 and MTP4BH were used for BAC-end sequencing, EST integration, micro-satellite integration into the physical map and high information content fingerprinting. MTP4E will be used for genome sequence by pooled genomic clone index. Conclusion Each MTP and associated BES will be useful to deconvolute and ultimately finish the whole genome shotgun sequence of soybean. PMID:16725032

  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 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

  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

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

    PubMed Central

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

    2009-01-01

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

  11. 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

  12. 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.

  13. Chromosome region-specific libraries for human genome analysis

    SciTech Connect

    Kao, Fa-Ten.

    1991-01-01

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

  14. Construction and phenotypic screening of mid-size insert marine microbial environmental genomic libraries

    E-print Network

    Braff, Jennifer C

    2008-01-01

    Functional screening of environmental genomic libraries permits the identification of clones expressing activities of interest without requiring prior knowledge of the genes responsible. In this study, protocols were ...

  15. Genome-scale loss-of-function screening with a lentiviral RNAi library

    E-print Network

    Cai, Long

    at the Broad Institute. Our human and mouse libraries currently contain >135,000 lentiviral clones targeting 27 on the genome scale. In model organisms, genome-scale loss-of- function genetic approaches have revealed rich of Technology, Cambr

  16. 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...

  17. HIGH DENSITY ESTS BASED BOVINE PHYSICAL MAPS AND THEIR USE TO CONSTRUCT COMPARATIVE MAPS AND INFORM THE BOVINE GENOME SEQUENCE ASSEMBLY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 3000 rad bovine whole genome radiation hybrid panel and bovine BAC libraries have been used to construct an integrated physical map of the bovine genome, which will contribute to the final assembly of the bovine genome sequence. The RH map now contains more than 4000 markers, the majority of whi...

  18. 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.

  19. 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

  20. 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.

  1. Acc homoeoloci and the evolution of wheat genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  2. Against a Whole-Genome Shotgun Philip Green1

    E-print Network

    Batzoglou, Serafim

    . These are screened against a bacterial artificial chromosome (BAC) (or other large bacterial clone) library (Kim et serve as a reference against which human variation can be cataloged, and conse- quently it should haveAgainst a Whole-Genome Shotgun Philip Green1 Department of Molecular Biotechnology, University

  3. 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...

  4. 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...

  5. The Drosophila BAC resource The 19 Genomes of Drosophila: a BA C Library Resource for Genus-wide and1

    E-print Network

    Matzkin, Luciano M.

    Division of Biological Sciences, University of California, San Diego, La Jolla, CA ,26 92093,USA.27 Phone, AZ 85721 USA32 Email: rwing@ag.arizona.edu; Phone: 520-626-9595; Fax: 520-621-1259.33 34 35 36 37 #12 characterized and shown to54 have: large insert sizes (125 to 168 kb), low non-recombinant clone content (0

  6. 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

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

    ERIC Educational Resources Information Center

    Pollack, Miriam

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

  8. 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.

  9. 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

  10. 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:26483808

  11. 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

  12. Video Article Large Insert Environmental Genomic Library Production

    E-print Network

    Leander, Brian

    picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the Copy library production and automated colony picking takes at least 7-10 days as there are many incubation

  13. 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.

  14. [Construction of Frankia genomic libraries and isolation of clones homologous to nodulation genes from Rhizobium leguminosarum].

    PubMed

    Cui, Y H; Qin, M; Wang, Y L; Ding, J; Ma, Q S

    1990-01-01

    High molecular genomic DNAs were isolated by using the lysozyme plus achromopeptidase system from Frankia strains At4, Ccol and Hr16, the root nodule endophytes of Alnus, Casuarina and Hippophae respectively, and used to construct genomic libraries in pLAFR1, a broad host range cosmid vector within many gram-negative hosts. The genomic libraries were screened by in situ colony hybridization to identify clones homologous to common nodulation genes of Rhizobium leguminosarum, based on the sequence homology of EcoRI-digested Frankia total DNA to nodABC from Rhizobium meliloti. Several clones showing relatively strong hybridization were found, the recombinant plasmid was isolated, and their homology with Rhizobium nodulation genes was confirmed by spot hybridization. Further work on these positive clones is now underway. PMID:2268450

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  17. SVGenes: a library for rendering genomic features in scalable vector graphic format

    PubMed Central

    Etherington, Graham J.; MacLean, Daniel

    2013-01-01

    Motivation: Drawing genomic features in attractive and informative ways is a key task in visualization of genomics data. Scalable Vector Graphics (SVG) format is a modern and flexible open standard that provides advanced features including modular graphic design, advanced web interactivity and animation within a suitable client. SVGs do not suffer from loss of image quality on re-scaling and provide the ability to edit individual elements of a graphic on the whole object level independent of the whole image. These features make SVG a potentially useful format for the preparation of publication quality figures including genomic objects such as genes or sequencing coverage and for web applications that require rich user-interaction with the graphical elements. Results: SVGenes is a Ruby-language library that uses SVG primitives to render typical genomic glyphs through a simple and flexible Ruby interface. The library implements a simple Page object that spaces and contains horizontal Track objects that in turn style, colour and positions features within them. Tracks are the level at which visual information is supplied providing the full styling capability of the SVG standard. Genomic entities like genes, transcripts and histograms are modelled in Glyph objects that are attached to a track and take advantage of SVG primitives to render the genomic features in a track as any of a selection of defined glyphs. The feature model within SVGenes is simple but flexible and not dependent on particular existing gene feature formats meaning graphics for any existing datasets can easily be created without need for conversion. Availability: The library is provided as a Ruby Gem from https://rubygems.org/gems/bio-svgenes under the MIT license, and open source code is available at https://github.com/danmaclean/bioruby-svgenes also under the MIT License. Contact: dan.maclean@tsl.ac.uk PMID:23749959

  18. 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

  19. 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

  20. 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

  1. 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...

  2. 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

  3. Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes

    PubMed Central

    2010-01-01

    Background Sugarcane (Saccharum spp.) has become an increasingly important crop for its leading role in biofuel production. The high sugar content species S. officinarum is an octoploid without known diploid or tetraploid progenitors. Commercial sugarcane cultivars are hybrids between S. officinarum and wild species S. spontaneum with ploidy at ~12×. The complex autopolyploid sugarcane genome has not been characterized at the DNA sequence level. Results The microsynteny between sugarcane and sorghum was assessed by comparing 454 pyrosequences of 20 sugarcane bacterial artificial chromosomes (BACs) with sorghum sequences. These 20 BACs were selected by hybridization of 1961 single copy sorghum overgo probes to the sugarcane BAC library with one sugarcane BAC corresponding to each of the 20 sorghum chromosome arms. The genic regions of the sugarcane BACs shared an average of 95.2% sequence identity with sorghum, and the sorghum genome was used as a template to order sequence contigs covering 78.2% of the 20 BAC sequences. About 53.1% of the sugarcane BAC sequences are aligned with sorghum sequence. The unaligned regions contain non-coding and repetitive sequences. Within the aligned sequences, 209 genes were annotated in sugarcane and 202 in sorghum. Seventeen genes appeared to be sugarcane-specific and all validated by sugarcane ESTs, while 12 appeared sorghum-specific but only one validated by sorghum ESTs. Twelve of the 17 sugarcane-specific genes have no match in the non-redundant protein database in GenBank, perhaps encoding proteins for sugarcane-specific processes. The sorghum orthologous regions appeared to have expanded relative to sugarcane, mostly by the increase of retrotransposons. Conclusions The sugarcane and sorghum genomes are mostly collinear in the genic regions, and the sorghum genome can be used as a template for assembling much of the genic DNA of the autopolyploid sugarcane genome. The comparable gene density between sugarcane BACs and corresponding sorghum sequences defied the notion that polyploidy species might have faster pace of gene loss due to the redundancy of multiple alleles at each locus. PMID:20416060

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. 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

  7. 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.

  8. Fosmid library end sequencing reveals a rarely known genome structure of marine shrimp Penaeus monodon

    PubMed Central

    2011-01-01

    Background The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses. Results We found that microsatellite sequences were highly abundant in the P. monodon genome, comprising 8.3% of the total length. The density and the average length of microsatellites were evidently higher in comparison to those of other taxa. AT-rich microsatellite motifs, especially poly (AT) and poly (AAT), were the most abundant. High abundance of microsatellite sequences were also found in the transcribed regions. Furthermore, via self-BlastN analysis we identified 103 novel repetitive element families which were categorized into four groups, i.e., 33 WSSV-like repeats, 14 retrotransposons, 5 gene-like repeats, and 51 unannotated repeats. Overall, various types of repeats comprise 51.18% of the P. monodon genome in length. Approximately 7.4% of the FESs contained protein-coding sequences, and the Inhibitor of Apoptosis Protein (IAP) gene and the Innexin 3 gene homologues appear to be present in high abundance in the P. monodon genome. Conclusions The redundancy of various repeat types in the P. monodon genome illustrates its highly repetitive nature. In particular, long and dense microsatellite sequences as well as abundant WSSV-like sequences highlight the uniqueness of genome organization of penaeid shrimp from those of other taxa. These results provide substantial improvement to our current knowledge not only for shrimp but also for marine crustaceans of large genome size. PMID:21575266

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

    PubMed

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

    2013-11-01

    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

  10. 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

  11. Genome-wide high-resolution analysis of DNA copy number alterations in NF1-associated malignant peripheral nerve sheath tumors using 32K BAC array.

    PubMed

    Mantripragada, Kiran K; Díaz de Ståhl, Teresita; Patridge, Chris; Menzel, Uwe; Andersson, Robin; Chuzhanova, Nadia; Kluwe, Lan; Guha, Abhijit; Mautner, Victor; Dumanski, Jan P; Upadhyaya, Meena

    2009-10-01

    Neurofibromatosis Type I (NF1) is an autosomal dominant disorder characterized by the development of both benign and malignant tumors. The lifetime risk for developing a malignant peripheral nerve sheath tumor (MPNST) in NF1 patients is approximately 10% with poor survival rates. To date, the molecular basis of MPNST development remains unclear. Here, we report the first genome-wide and high-resolution analysis of DNA copy number alterations in MPNST using the 32K bacterial artificial chromosome microarray on a series of 24 MPNSTs and three neurofibroma samples. In the benign neurofibromas, apart from loss of one copy of the NF1 gene and copy number polymorphisms, no other changes were found. The profiles of malignant samples, however, revealed specific loss of chromosomal regions including 1p35-33, 1p21, 9p21.3, 10q25, 11q22-23, 17q11, and 20p12.2 as well as gain of 1q25, 3p26, 3q13, 5p12, 5q11.2-q14, 5q21-23, 5q31-33, 6p23-p21, 6p12, 6q15, 6q23-q24, 7p22, 7p14-p13, 7q21, 7q36, 8q22-q24, 14q22, and 17q21-q25. Copy number gains were more frequent than deletions in the MPNST samples (62% vs. 38%). The genes resident within common regions of gain were NEDL1 (7p14), AP3B1 (5q14.1), and CUL1 (7q36.1) and these were identified in >63% MPNSTs. The most frequently deleted locus encompassed CDKN2A, CDKN2B, and MTAP genes on 9p21.3 (33% cases). These genes have previously been implicated in other cancer conditions and therefore, should be considered for their therapeutic, prognostic, and diagnostic relevance in NF1 tumorigenesis. PMID:19603524

  12. Mutator System Derivatives Isolated from Sugarcane Genome Sequence.

    PubMed

    Manetti, M E; Rossi, M; Cruz, G M Q; Saccaro, N L; Nakabashi, M; Altebarmakian, V; Rodier-Goud, M; Domingues, D; D'Hont, A; Van Sluys, M A

    2012-09-01

    Mutator-like transposase is the most represented transposon transcript in the sugarcane transcriptome. Phylogenetic reconstructions derived from sequenced transcripts provided evidence that at least four distinct classes exist (I-IV) and that diversification among these classes occurred early in Angiosperms, prior to the divergence of Monocots/Eudicots. The four previously described classes served as probes to select and further sequence six BAC clones from a genomic library of cultivar R570. A total of 579,352 sugarcane base pairs were produced from these "Mutator system" BAC containing regions for further characterization. The analyzed genomic regions confirmed that the predicted structure and organization of the Mutator system in sugarcane is composed of two true transposon lineages, each containing a specific terminal inverted repeat and two transposase lineages considered to be domesticated. Each Mutator transposase class displayed a particular molecular structure supporting lineage specific evolution. MUSTANG, previously described domesticated genes, are located in syntenic regions across Sacharineae and, as expected for a host functional gene, posses the same gene structure as in other Poaceae. Two sequenced BACs correspond to hom(eo)logous locus with specific retrotransposon insertions that discriminate sugarcane haplotypes. The comparative studies presented, add information to the Mutator systems previously identified in the maize and rice genomes by describing lineage specific molecular structure and genomic distribution pattern in the sugarcane genome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12042-012-9104-y) contains supplementary material, which is available to authorized users. PMID:22905278

  13. Pre-Nursing Post-Bac Info

    E-print Network

    Pre-Nursing Post-Bac Info Session Rachel Small Co-Director, SFSU Pre- Nursing Post-Bac Program January 31, 2013 #12;SFSU Pre-Nursing Post-Bac Program ! Highly structured program intended for students with a bachelor's degree ! Provides prerequisite and elective coursework for pre-nursing students ! Strong

  14. Integrating cytogenetics and genomics in comparative evolutionary studies of cichlid fish

    PubMed Central

    2012-01-01

    Background The availability of a large number of recently sequenced vertebrate genomes opens new avenues to integrate cytogenetics and genomics in comparative and evolutionary studies. Cytogenetic mapping can offer alternative means to identify conserved synteny shared by distinct genomes and also to define genome regions that are still not fine characterized even after wide-ranging nucleotide sequence efforts. An efficient way to perform comparative cytogenetic mapping is based on BAC clones mapping by fluorescence in situ hybridization. In this report, to address the knowledge gap on the genome evolution in cichlid fishes, BAC clones of an Oreochromis niloticus library covering the linkage groups (LG) 1, 3, 5, and 7 were mapped onto the chromosomes of 9 African cichlid species. The cytogenetic mapping data were also integrated with BAC-end sequences information of O. niloticus and comparatively analyzed against the genome of other fish species and vertebrates. Results The location of BACs from LG1, 3, 5, and 7 revealed a strong chromosomal conservation among the analyzed cichlid species genomes, which evidenced a synteny of the markers of each LG. Comparative in silico analysis also identified large genomic blocks that were conserved in distantly related fish groups and also in other vertebrates. Conclusions Although it has been suggested that fishes contain plastic genomes with high rates of chromosomal rearrangements and probably low rates of synteny conservation, our results evidence that large syntenic chromosome segments have been maintained conserved during evolution, at least for the considered markers. Additionally, our current cytogenetic mapping efforts integrated with genomic approaches conduct to a new perspective to address important questions involving chromosome evolution in fishes. PMID:22958299

  15. 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…

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

    PubMed

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

    2014-01-01

    Due to impressive achievements in genomic research, the number of genome sequences has risen quickly, followed by an increasing number of genes with unknown or hypothetical function. This strongly calls for development of high-throughput methods in the fields of transcriptomics, proteomics and metabolomics. Of these platforms, metabolic profiling has the strongest correlation with the phenotype. We previously published a high-throughput metabolic profiling method for C. glutamicum as well as the automatic GC/MS processing software MetaboliteDetector. Here, we added a high-throughput transposon insertion determination for our C. glutamicum mutant library. The combination of these methods allows the parallel analysis of genotype/phenotype correlations for a large number of mutants. In a pilot project we analyzed the insertion points of 722 transposon mutants and found that 36% of the affected genes have unknown functions. This underlines the need for further information gathered by high-throughput techniques. We therefore measured the metabolic profiles of 258 randomly chosen mutants. The MetaboliteDetector software processed this large amount of GC/MS data within a few hours with a low relative error of 11.5% for technical replicates. Pairwise correlation analysis of metabolites over all genotypes showed dependencies of known and unknown metabolites. For a first insight into this large data set, a screening for interesting mutants was done by a pattern search, focusing on mutants with changes in specific pathways. We show that our transposon mutant library is not biased with respect to insertion points. A comparison of the results for specific mutants with previously published metabolic results on a deletion mutant of the same gene confirmed the concept of high-throughput metabolic profiling. Altogether the described method could be applied to whole mutant libraries and thereby help to gain comprehensive information about genes with unknown, hypothetical and known functions. PMID:24504095

  17. 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.

  18. 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...

  19. 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

  20. Barcoding-free BAC Pooling Enables Combinatorial Selective Sequencing of the Barley Gene Space

    E-print Network

    Lonardi, Stefano; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Close, Timothy J

    2011-01-01

    We propose a new sequencing protocol that combines recent advances in combinatorial pooling design and second-generation sequencing technology to efficiently approach de novo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when dealing with hundreds or thousands of DNA samples, such as genome-tiling gene-rich BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundreds of million of short reads and assign them to the correct BAC clones so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is extremely accurate (99.57% of the deconvoluted reads are assigned to the correct BAC), and the resulting BAC assemblies have very high quality (BACs are covered by contigs over about 77% of their length, on average). Experimental results on real data for a gene-rich subset of the barley genome confir...

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

    SciTech Connect

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

    2009-01-01

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

  2. 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

  3. A sequence-ready BAC contig of the GABAA receptor gene cluster Gabrg1-Gabra2-Gabrb1 on mouse chromosome 5.

    PubMed

    Lengeling, A; Wiltshire, T; Otmani, C; Bucán, M

    1999-08-01

    The type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate postsynaptic inhibition. The functional diversity of these receptors comes from the use of a large repertoire of subunits encoded by separate genes, as well as from differences in subunit composition of individual receptors. In mammals, a majority of GABA(A) receptor subunit genes are located in gene clusters that may be important for their regulated expression and function. We have established a high-resolution physical map of the cluster of genes encoding GABA(A) receptor subunits alpha2 (Gabra2), beta1 (Gabrb1), and gamma(1) (Gabrg1) on mouse chromosome 5. Rat cDNA probes and specific sequence probes for all three GABA(A) receptor subunit genes have been used to initiate the construction of a sequence-ready contig of bacterial artificial chromosomes (BACs) encompassing this cluster. In the process of contig construction clones from 129/Sv and C57BL/6J BAC libraries were isolated. The assembled 1.3-Mb contig, consisting of 45 BACs, gives five- to sixfold coverage over the gene cluster and provides an average resolution of one marker every 32 kb. A number of BAC insert ends were sequenced, generating 30 new sequence tag sites (STS) in addition to 6 Gabr gene-based and 3 expressed sequence tag (EST)-based markers. STSs from, and surrounding, the Gabrg1-Gabra2-Gabrb1 gene cluster were mapped in the T31 mouse radiation hybrid panel. The integration of the BAC contig with a map of loci ordered by radiation hybrid mapping suggested the most likely genomic orientation of this cluster on mouse chromosome 5: cen-D5Mit151-Gabrg1-Gabra2-Gabrb1-D5Mit58- tel. This established contig will serve as a template for genomic sequencing and for functional analysis of the GABA(A) gene cluster on mouse chromosome 5 and the corresponding region on human chromosome 4. PMID:10447508

  4. Protective activity of Streptococcus pneumoniae Spr1875 protein fragments identified using a phage displayed genomic library.

    PubMed

    Cardaci, Angela; Papasergi, Salvatore; Midiri, Angelina; Mancuso, Giuseppe; Domina, Maria; Cariccio, Veronica Lanza; Mandanici, Francesca; Galbo, Roberta; Lo Passo, Carla; Pernice, Ida; Donato, Paolo; Ricci, Susanna; Biondo, Carmelo; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2012-01-01

    There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine. PMID:22570729

  5. Protective Activity of Streptococcus pneumoniae Spr1875 Protein Fragments Identified Using a Phage Displayed Genomic Library

    PubMed Central

    Cardaci, Angela; Papasergi, Salvatore; Midiri, Angelina; Mancuso, Giuseppe; Domina, Maria; Cariccio, Veronica Lanza; Mandanici, Francesca; Galbo, Roberta; Passo, Carla Lo; Pernice, Ida; Donato, Paolo; Ricci, Susanna; Biondo, Carmelo; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2012-01-01

    There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine. PMID:22570729

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

    PubMed

    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

    2015-11-10

    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

  7. 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

  8. 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

  9. 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...

  10. 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

  11. 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

  12. Construction of Signature-tagged Mutant Library in Mesorhizobium loti as a Powerful Tool for Functional Genomics

    PubMed Central

    Shimoda, Yoshikazu; Mitsui, Hisayuki; Kamimatsuse, Hiroko; Minamisawa, Kiwamu; Nishiyama, Eri; Ohtsubo, Yoshiyuki; Nagata, Yuji; Tsuda, Masataka; Shinpo, Sayaka; Watanabe, Akiko; Kohara, Mitsuyo; Yamada, Manabu; Nakamura, Yasukazu; Tabata, Satoshi; Sato, Shusei

    2008-01-01

    Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants. The completion of several rhizobial genome sequences provides opportunities for genome-wide functional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide phenotypic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were obtained. Along with the collection of transposon mutants, we have determined the transposon insertion sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome without any bias toward G+C contents of insertion target sites and transposon plasmids used for the mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable resource for researchers to further our understanding of rhizobial biology. PMID:18658183

  13. 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.

  14. 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

  15. 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

  16. A CYTOGENETIC MAP OF MAIZE IN OAT ADDITION LINES USING SORGHUM BACS AS FISH PROBES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are developing a pachytene cytogenetic FISH map of the maize genome using sorghum BACs corresponding to the 90 maize Core Bin Marker (CBM) loci. These loci were chosen because they are uniformly distributed and they delineate the genetic bins derived from the UMC98 (Genetic 2005) maize linkage ma...

  17. Sequence for BAC OSJNBa0044D15 from Rice Variety Nipponbare GenBank

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete DNA sequence of BAC OSJNa0044D15 from chromosome 11 of rice variety Nipponbare was deposited in Genbank as part of the International Rice Sequencing Project. The DNA sequence with syntenic (corresponding) region of the rice genome associated with disease resistance to rice blast has be...

  18. 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

  19. Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies

    PubMed Central

    Papa, Riccardo; Morrison, Clayton M; Walters, James R; Counterman, Brian A; Chen, Rui; Halder, Georg; Ferguson, Laura; Chamberlain, Nicola; ffrench-Constant, Richard; Kapan, Durrell D; Jiggins, Chris D; Reed, Robert D; McMillan, William O

    2008-01-01

    Background With over 20 parapatric races differing in their warningly colored wing patterns, the butterfly Heliconius erato provides a fascinating example of an adaptive radiation. Together with matching races of its co-mimic Heliconius melpomene, H. erato also represents a textbook case of Müllerian mimicry, a phenomenon where common warning signals are shared amongst noxious organisms. It is of great interest to identify the specific genes that control the mimetic wing patterns of H. erato and H. melpomene. To this end we have undertaken comparative mapping and targeted genomic sequencing in both species. This paper reports on a comparative analysis of genomic sequences linked to color pattern mimicry genes in Heliconius. Results Scoring AFLP polymorphisms in H. erato broods allowed us to survey loci at approximately 362 kb intervals across the genome. With this strategy we were able to identify markers tightly linked to two color pattern genes: D and Cr, which were then used to screen H. erato BAC libraries in order to identify clones for sequencing. Gene density across 600 kb of BAC sequences appeared relatively low, although the number of predicted open reading frames was typical for an insect. We focused analyses on the D- and Cr-linked H. erato BAC sequences and on the Yb-linked H. melpomene BAC sequence. A comparative analysis between homologous regions of H. erato (Cr-linked BAC) and H. melpomene (Yb-linked BAC) revealed high levels of sequence conservation and microsynteny between the two species. We found that repeated elements constitute 26% and 20% of BAC sequences from H. erato and H. melpomene respectively. The majority of these repetitive sequences appear to be novel, as they showed no significant similarity to any other available insect sequences. We also observed signs of fine scale conservation of gene order between Heliconius and the moth Bombyx mori, suggesting that lepidopteran genome architecture may be conserved over very long evolutionary time scales. Conclusion Here we have demonstrated the tractability of progressing from a genetic linkage map to genomic sequence data in Heliconius butterflies. We have also shown that fine-scale gene order is highly conserved between distantly related Heliconius species, and also between Heliconius and B. mori. Together, these findings suggest that genome structure in macrolepidoptera might be very conserved, and show that mapping and positional cloning efforts in different lepidopteran species can be reciprocally informative. PMID:18647405

  20. piggyBac transposon system modification of primary human T cells.

    PubMed

    Saha, Sunandan; Nakazawa, Yozo; Huye, Leslie E; Doherty, Joseph E; Galvan, Daniel L; Rooney, Cliona M; Wilson, Matthew H

    2012-01-01

    The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect, mammalian, and human cells6 including primary human T cells. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency. Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized. Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry. PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb). Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes. PMID:23149543

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

    PubMed

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

    2015-10-13

    Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA). However, the biological activities of many xenobiotics are masked by the pleiotropic drug resistance (PDR) network which effluxes xenobiotics that are PDR substrates. The PDR network in S. cerevisiae is almost entirely under the control of two functionally homologous transcription factors Pdr1p and Pdr3p. Herein we report the construction of a PDR-attenuated haploid non-essential DMA (PA-DMA), lacking PDR1 and PDR3, which permits the MoA elucidation of xenobiotics that are PDR substrates at low concentrations. The functionality of four key cellular processes commonly activated in response to xenobiotic stress: oxidative stress response, general stress response, unfolded stress response and calcium signalling pathways were assessed in the absence of PDR1 and PDR3 genes and were found to unaltered, therefore, these key chemogenomic signatures are not lost when using the PA-DMA. Efficacy of the PA-DMA was demonstrated using cycloheximide and latrunculin A at low nanomolar concentrations to attain chemical genetic profiles that were more specific to their known main mechanisms. We also found a two-fold increase in the number of compounds that are bioactive in the pdr1?pdr3? compared to the wild type strain in screening the commercially available LOPAC(1280) library. The PA-DMA should be particularly applicable to mechanism determination of xenobiotics that have limited availability, such as natural products. PMID:26381459

  2. High resolution physical map of porcine chromosome 7 QTL region and comparative mapping of this region among vertebrate genomes

    PubMed Central

    Demars, Julie; Riquet, Juliette; Feve, Katia; Gautier, Mathieu; Morisson, Mireille; Demeure, Olivier; Renard, Christine; Chardon, Patrick; Milan, Denis

    2006-01-01

    Background On porcine chromosome 7, the region surrounding the Major Histocompatibility Complex (MHC) contains several Quantitative Trait Loci (QTL) influencing many traits including growth, back fat thickness and carcass composition. Previous studies highlighted that a fragment of ~3.7 Mb is located within the Swine Leucocyte Antigen (SLA) complex. Internal rearrangements of this fragment were suggested, and partial contigs had been built, but further characterization of this region and identification of all human chromosomal fragments orthologous to this porcine fragment had to be carried out. Results A whole physical map of the region was constructed by integrating Radiation Hybrid (RH) mapping, BAC fingerprinting data of the INRA BAC library and anchoring BAC end sequences on the human genome. 17 genes and 2 reference microsatellites were ordered on the high resolution IMNpRH212000rad Radiation Hybrid panel. A 1000:1 framework map covering 550 cR12000 was established and a complete contig of the region was developed. New micro rearrangements were highlighted between the porcine and human genomes. A bovine RH map was also developed in this region by mapping 16 genes. Comparison of the organization of this region in pig, cattle, human, mouse, dog and chicken genomes revealed that 1) the translocation of the fragment described previously is observed only on the bovine and porcine genomes and 2) the new internal micro rearrangements are specific of the porcine genome. Conclusion We estimate that the region contains several rearrangements and covers 5.2 Mb of the porcine genome. The study of this complete BAC contig showed that human chromosomal fragments homologs of this heavily rearranged QTL region are all located in the region of HSA6 that surrounds the centromere. This work allows us to define a list of all candidate genes that could explain these QTL effects. PMID:16433907

  3. 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

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

    PubMed

    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 ? 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

  5. 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 ? 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

  6. Algorithms and software tools for ordering clone libraries: application to the mapping of the genome of Schizosaccharomyces pombe.

    PubMed Central

    Mott, R; Grigoriev, A; Maier, E; Hoheisel, J; Lehrach, H

    1993-01-01

    A complete set of software tools to aid the physical mapping of a genome has been developed and successfully applied to the genomic mapping of the fission yeast Schizosaccharomyces pombe. Two approaches were used for ordering single-copy hybridisation probes: one was based on the simulated annealing algorithm to order all probes, and another on inferring the minimum-spanning subset of the probes using a heuristic filtering procedure. Both algorithms produced almost identical maps, with minor differences in the order of repetitive probes and those having identical hybridisation patterns. A separate algorithm fitted the clones to the established probe order. Approaches for handling experimental noise and repetitive elements are discussed. In addition to these programs and the database management software, tools for visualizing and editing the data are described. The issues of combining the information from different libraries are addressed. Also, ways of handling multiple-copy probes and non-hybridisation data are discussed. PMID:8493107

  7. Wheat Genomics: Present Status and Future Prospects

    PubMed Central

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

    2008-01-01

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

  8. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Whole-genome disassembly Phil Green*

    E-print Network

    Batzoglou, Serafim

    of Washington, Seattle, WA 98195 The race to sequence the human ge- nome has garnered a level of popular phase. For the human genome, which com- prises some 3 billion base pairs, the public effort adopted first cloned into a bacterial host (as bac- terial artificial chromosomes or BACs) and then sequenced

  11. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  13. CONSTRUCTING A CYTOGENETIC MAP OF MAIZE CORE BIN MARKERS IN OAT ADDITION LINES USING SORGHUM BACS AS FISH PROBES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are developing a pachytene cytogenetic FISH map of the maize genome using sorghum BACs corresponding to the 90 maize Core Bin Marker (CBM) loci. These loci were chosen because they are uniformly distributed and they delineate the genetic bins derived from the UMC98 (Genetic 2005) maize linkage ma...

  14. 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

  15. 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...

  16. 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.

  17. BAC-Pool Sequencing and Analysis of Large Segments of A12 and D12 Homoeologous Chromosomes in Upland Cotton

    PubMed Central

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

    2013-01-01

    Although new and emerging next-generation sequencing (NGS) technologies have reduced sequencing costs significantly, much work remains to implement them for de novo sequencing of complex and highly repetitive genomes such as the tetraploid genome of Upland cotton (Gossypium hirsutum L.). Herein we report the results from implementing a novel, hybrid Sanger/454-based BAC-pool sequencing strategy using minimum tiling path (MTP) BACs from Ctg-3301 and Ctg-465, two large genomic segments in A12 and D12 homoeologous chromosomes (Ctg). To enable generation of longer contig sequences in assembly, we implemented a hybrid assembly method to process ~35x data from 454 technology and 2.8-3x data from Sanger method. Hybrid assemblies offered higher sequence coverage and better sequence assemblies. Homology studies revealed the presence of retrotransposon regions like Copia and Gypsy elements in these contigs and also helped in identifying new genomic SSRs. Unigenes were anchored to the sequences in Ctg-3301 and Ctg-465 to support the physical map. Gene density, gene structure and protein sequence information derived from protein prediction programs were used to obtain the functional annotation of these genes. Comparative analysis of both contigs with Arabidopsis genome exhibited synteny and microcollinearity with a conserved gene order in both genomes. This study provides insight about use of MTP-based BAC-pool sequencing approach for sequencing complex polyploid genomes with limited constraints in generating better sequence assemblies to build reference scaffold sequences. Combining the utilities of MTP-based BAC-pool sequencing with current longer and short read NGS technologies in multiplexed format would provide a new direction to cost-effectively and precisely sequence complex plant genomes. PMID:24116150

  18. Construction of a 2-Mb resolution BAC microarray for CGH analysis of canine tumors.

    PubMed

    Thomas, Rachael; Scott, Allyson; Langford, Cordelia F; Fosmire, Susan P; Jubala, Cristan M; Lorentzen, Travis D; Hitte, Christophe; Karlsson, Elinor K; Kirkness, Ewen; Ostrander, Elaine A; Galibert, Francis; Lindblad-Toh, Kerstin; Modiano, Jaime F; Breen, Matthew

    2005-12-01

    Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques. PMID:16339382

  19. 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.

  20. 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

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

    PubMed

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

    2011-09-01

    Sugarcane (Saccharum spp.) breeders in the early 20th century made remarkable progress in increasing yield and disease resistance by crossing Saccharum spontaneum L., a wild relative, to Saccharum officinarum L., a traditional cultivar. Modern sugarcane cultivars have approximately 71%-83% of their chromosomes originating from S. officinarum, approximately 10%-21% from S. spontaneum, and approximately 2%-13% recombinant or translocated chromosomes. In the present work, C(0)t-based cloning and sequencing (CBCS) was implemented to further explore highly repetitive DNA and to seek species-specific repeated DNA in both S. officinarum and S. spontaneum. For putatively species-specific sequences, overlappping oligonucleotide probes (overgos) were designed and hybridized to BAC filters from the interspecific hybrid sugarcane cultivar 'R570' to try to deduce parental origins of BAC clones. We inferred that 12?967 BACs putatively originated from S. officinarum and 5117 BACs from S. spontaneum. Another 1103 BACs were hybridized by both species-specific overgos, too many to account for by conventional recombination, thus suggesting ectopic recombination and (or) translocation of DNA elements. Constructing a low C(0)t library is useful to collect highly repeated DNA sequences and to search for potentially species-specific molecular markers, especially among recently diverged species. Even in the absence of repeat families that are species-specific in their entirety, the identification of localized variations within consensus sequences, coupled with the site specificity of short synthetic overgos, permits researchers to monitor species-specific or species-enriched variants. PMID:21883018

  2. 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

  3. GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION1

    E-print Network

    Borges, Rita

    NOTE GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE A method for isolating high-quality DNA is pre- sented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and in- vasive

  4. 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, through the removal of the toxic product acrylyl-CoA. PMID:24848004

  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. Hox complex analysis through BAC recombineering.

    PubMed

    Parrish, Mark; Ahn, Youngwook; Nolte, Christof; De Kumar, Bony; Krumlauf, Robb

    2014-01-01

    BAC transgenesis in mice has proved to be useful in exploring the regulatory mechanisms and functions of the Hox complexes. The large constructs used may include most of the relevant components of the cis-regulatory landscape. Manipulations can be accomplished without compromising the integrity of the endogenous complex which reduces the likelihood of producing confounding phenotypic abnormalities. The development of recombineering tools has been critical in providing the means necessary to make many types of precise and varied manipulations of these large constructs. Here, we will discuss the methodologies necessary to manipulate Hox complex BACs, generation of transgenic animals bearing these constructs and the utilization of these resources to address fundamental aspects of Hox biology. PMID:25151158

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

    PubMed

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

    2008-10-01

    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

  8. 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

  9. Rapid genomic DNA changes in allotetraploid fish hybrids

    PubMed Central

    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

    2015-01-01

    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

  10. 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

  11. 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...

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

    PubMed Central

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

    2003-01-01

    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

  13. BANK ACCOUNT FORM FOR EXTERNAL EXAMINERS (POSTGRADUATE) 1. The University prefers to make payment to external suppliers through BACS. This ensures that the

    E-print Network

    Bearhop, Stuart

    PD98 BANK ACCOUNT FORM FOR EXTERNAL EXAMINERS (POSTGRADUATE) 1. The University prefers to make payment to external suppliers through BACS. This ensures that the payment reaches your bank account more, The Old Library, Prince of Wales Road, Exeter, EX4 4SB. 4. If you change your bank account, you should

  14. 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...

  15. A New Normalizing Algorithm for BAC CGH Arrays with Quality Control Metrics

    PubMed Central

    Miecznikowski, Jeffrey C.; Gaile, Daniel P.; Liu, Song; Shepherd, Lori; Nowak, Norma

    2011-01-01

    The main focus in pin-tip (or print-tip) microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH) experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose “SmoothArray”, a new method to preprocess comparative genomic hybridization (CGH) bacterial artificial chromosome (BAC) arrays and we show the effects on a cancer dataset. As part of our R software package “aCGHplus,” this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology. PMID:21403910

  16. 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

  17. 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

  18. 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

  19. Kovach et al. BMC Genomics 2010, 11:420 http://www.biomedcentral.com/1471-2164/11/420

    E-print Network

    Yandell, Mark

    speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs

  20. 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

  1. Low-copy episomal vector pFY20 and high-saturation coverage genomic libraries for the fission yeast Schizosaccharomyces pombe

    PubMed Central

    Wahls, Wayne P.; Davidson, Mari K.

    2011-01-01

    In fission yeast, as in many organisms, episomally replicating plasmid DNA molecules can be used for a wide variety of applications. However, replicating plasmids described previously are each propagated at a high copy number per cell. Plasmid fission yeast twenty (pFY20) contains the ura4+ gene for positive and negative selection, an origin of replication (ars1 ) and a stability element (stb). Although this plasmid does not have a centromere, it is propagated with a copy number of about two plasmids per haploid genome equivalent and it is transmitted with relatively high fidelity in mitosis and meiosis. This low-copy vector is useful for screens and mutational studies where overexpression (e.g. from high copy plasmids) is undesirable. We therefore constructed multiple partial-digest, size-fractionated, fission yeast genomic DNA libraries in pFY20 and in the cloning vector pBluescript KS+. These libraries have sufficient complexity (average of 2100 genome equivalents each) for saturation screening by complementation, plasmid shuffle or hybridization. PMID:18613214

  2. Specific single-cell isolation and genomic amplification of uncultured microorganisms.

    PubMed

    Kvist, Thomas; Ahring, Birgitte K; Lasken, Roger S; Westermann, Peter

    2007-03-01

    We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism. PMID:17109170

  3. 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...

  4. Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq

    PubMed Central

    Rhodes, Johanna; Beale, Mathew A.; Fisher, Matthew C.

    2014-01-01

    The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use. PMID:25409295

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

    PubMed Central

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

    2013-01-01

    Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations. PMID:23921635

  6. Library Regulations Library Regulations

    E-print Network

    Birmingham, University of

    -13 Library Regulations `Service' is deemed to include any system whereby Library Services provides accessLibrary Regulations 2012-13 Library Regulations UNIVERSITY OF BIRMINGHAM REGULATIONS LIBRARY REGULATIONS Preamble: The Library Regulations apply to all users of library facilities managed on behalf

  7. Leading Edge Genomic and Cellular Complexity

    E-print Network

    Bordenstein, Seth

    a cicada ancestor tens of millions of years ago and now supplements them with essential amino acids missing that coexist alongside one cicada genome and the co-symbiont Sulcia. Remarkably, the two Hodgkinia genomes observation teased out by Van Leuven et al. is that the cicada bacteriome, the organ that houses bac- terial

  8. Optimizing the BACEnd Strategy for Sequencing the Human Genome

    E-print Network

    Shamir, Ron

    University, Tel Aviv, 69978, Israel. 1 #12; 1 Introduction With the Human Genome Project moving from the map sequencing has become central. The classical strategy set forth by the founders of the Human Genome ProjectOptimizing the BAC­End Strategy for Sequencing the Human Genome Richard M. Karp \\Lambda Ron Shamir

  9. Framework for a physical map of the human 22q13 region using bacterial artificial chromosomes (BACs)

    SciTech Connect

    Schmitt, H.; Kim, Ung-Jin; Slepak, T.

    1996-04-01

    Detailed physical maps of entire chromosomes based on combined genetic, cytogenetic, and structural information are essential components for positional cloning and genomic sequencing. Despite the wealth of genetic information of the known diseases in the chromosome 22q13 region, the construction of a detailed physical map of the terminal region is difficult due to the sparsity of the genetic markers. We present here a map of bacterial artificial chromosome (BAC) contigs that cover a number of genetic loci in the 22q13 region. One hundred thirty-six BACs with an average insert size of 140 kb are assembled into 35 contigs defined by 64 markers in 22q13-qter. Twenty-three anonymous markers are now linked to the previously mapped genetic anchor points. 55 refs., 4 figs., 3 tabs.

  10. 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 in generating genomic resources provides a non-biased glimpse into the publicly available EST-sequences, yam genome, and GBS profiles with affirmation that the genomic complexity can be methodically unraveled and constitute a critical foundation for future studies in linkage mapping, germplasm analysis, and predictive breeding. PMID:26222616

  11. 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 in generating genomic resources provides a non-biased glimpse into the publicly available EST-sequences, yam genome, and GBS profiles with affirmation that the genomic complexity can be methodically unraveled and constitute a critical foundation for future studies in linkage mapping, germplasm analysis, and predictive breeding. PMID:26222616

  12. 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

    2015-01-01

    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

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

    PubMed Central

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

    2015-01-01

    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

  14. 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.

  15. 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

  16. 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.

  17. Application of BAC-probes to visualize copy number variants (CNVs).

    PubMed

    Weise, Anja; Othman, Moneeb A K; Bhatt, Samarth; Löhmer, Sharon; Liehr, Thomas

    2015-01-01

    Copy number variations (CNVs) are structural variations of the human genome. These alterations result in variant copy numbers of certain stretches of DNA. In other words, some regions may be present in more or less copies than in a reference genome; however, these copy number changes do not have any impact on the phenotype. Also, CNVs may be extremely large and cytogenetically detectable or submicroscopic but still spanning several megabasepairs (Mb). In the recent years, array technology has identified especially the latter ones as so-called copy number variant (CNV) polymorphisms. These CNVs are detected in ~12 % of the human genome sequences and may comprise several hundred kilobasepairs. CNVs contribute significantly to the inter-individual differences in humans, and can range between 0.5 and 1.5 Mb amongst different genomes, well within the level of detection using cytogenetics techniques. Thus, they can be visualized by FISH using bacterial artificial chromosomes (BACs) as probes. Here we describe a method that enables discrimination of individual homologous chromosomes at the single cell level based on CNVs in the genome, called parental origin determination fluorescence in situ hybridization (POD-FISH). Possible fields of applications of this single cell-directed approach are in analyses of the parental origin of single chromosomes in inherited and acquired chromosomal aberrations. PMID:25239754

  18. Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

    PubMed Central

    2010-01-01

    Background From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas. PMID:20507617

  19. Aerobic biological activated carbon (BAC) treatment of a phenolic wastewater

    SciTech Connect

    Wei Lin; Weber, A.S. )

    1992-05-01

    Organic removal rates achieved in the aerobic BAC process were comparable to rates typically reported for traditional aerobic fixed-film systems. When operated at organic loading rates lower than 0.03 g COD/g GAC-d and air as the oxygen source, greater than 90% COD removal and 99% phenol removal was achieved. At higher organic loading rates, oxygen limitations resulted in less than optimal performance. Observed oxygen limitations were mitigated by the use of pure oxygen. Long-term stability of operation of the BAC process was excellent with one aerobic BAC column operated under the same conditions in excess of 260 days. During that time, consistent column performance was achieved without the need to provide supplemental carbon or carbon regeneration. System biomass yields ranged from 0.05 to 0.30 g VSS/g COD removed and increased with effluent COD concentration.

  20. BacMam Platform for Vaccine Antigen Delivery.

    PubMed

    Keil, Günther M; Pollin, Reiko; Müller, Claudia; Giesow, Katrin; Schirrmeier, Horst

    2016-01-01

    Recombinant baculo viruses based on Autographa californica multiple nuclear polyhedrosis virus carrying vertebrate cell active expression cassettes, so-called BacMam viruses, are increasingly used as gene delivery vectors for vaccination of animals against pathogens. Different approaches for generation of BacMams exist and a variety of transfer vectors to improve target protein expression in vivo have been constructed. Here we describe a use of transfer vector which contains an insect cell-restricted expression cassette for the green fluorescent protein and thus enables easy monitoring of BacMam virus rescue, fast plaque purification of recombinants and their convenient titer determination and which has been proven to be efficacious for gene delivery in vaccination/challenge experiments. PMID:26458832

  1. 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

  2. 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

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

    MedlinePLUS

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

  4. 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 ...

  5. Supplementary Figure 1. Overview of BAC clones and targeting strategy for generation of cell-type specific ChR2(H134R)-EYFP BAC transgenic mice. (a) Diagram of BAC clones used in this study. All BAC clones are

    E-print Network

    Schnitzer, Mark

    #12;Supplementary Figure 1. Overview of BAC clones and targeting strategy for generation of cell-type specific ChR2(H134R)-EYFP BAC transgenic mice. (a) Diagram of BAC clones used in this study. All BAC clones modification strategy to introduce ChR2(H134R)-EYFP under the control of the cell-type specific promoter

  6. A re-assigned American mink (Neovison vison) map optimal for genome-wide studies.

    PubMed

    Anistoroaei, Razvan; Nielsen, Vivi; Markakis, Marios Nektarios; Karlskov-Mortensen, Peter; Jørgensen, Claus B; Christensen, Knud; Fredholm, Merete

    2012-12-10

    Our previously published second generation genetic map for the American mink (Neovison vison) has been used and redesigned in its best for genome-wide studies with maximum of efficiency. A number of 114 selected markers, including 33 newly developed microsatellite markers from the CHORI-231 mink Bacterial Artificial Chromosome (BAC) library, have been genotyped in a two generation population composed of 1200 individuals. The outcome reassigns the position of some markers on the chromosomes and it produces a more reliable map with a convenient distance between markers. A total of 104 markers mapped to 14 linkage groups corresponding to the mink autosomes. Six markers are unlinked and four markers are allocated to the X chromosome by homology but no linkage was detected. The sex-average linkage map spans 1192 centiMorgans (cM) with an average intermarker distance of 11.4cM and 1648cM when the ends of the linkage groups and the autosomal unlinked markers are added. Sex-specific genetic linkage maps were also generated. The male sex-specific map had a total length of 1014.6cM between the linked markers and an average inter-marker interval of 9.7cM. The female map has a corresponding length of 1378.6cM and an average inter-marker interval of 13.3cM. The study is complemented with additional anchorage for most of the chromosomes of the map by BAC in situ hybridization with clones containing microsatellites strategically selected from the various parts of the genome. This map provides an improved tool for genetic mapping and comparative genomics in mink, also useful for the future assembly of the mink genome sequence when this will be taken forward. PMID:22982743

  7. 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.

  8. Prokaryotic Genomes Eurkaryotic Genomes

    E-print Network

    Qiu, Weigang

    Prokaryotic Genomes Eurkaryotic Genomes Chapter 6. Genomics and Gene Identification Weigang Qiu Weigang Qiu Chapter 6. Genomics and Gene Identification #12;Prokaryotic Genomes Eurkaryotic Genomes Outline 1 Prokaryotic Genomes 2 Eurkaryotic Genomes Weigang Qiu Chapter 6. Genomics and Gene

  9. 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...

  10. The organization of genes tightly linked to the Ha locus in Aegilops tauschii, the D-genome donor to wheat.

    PubMed

    Turnbull, K M; Turner, M; Mukai, Y; Yamamoto, M; Morell, M K; Appels, R; Rahman, S

    2003-04-01

    The grain hardness locus, Ha, is located at the distal end of the short arm of chromosome 5D in wheat. Three polypeptides, puroindoline-a, puroindoline-b, and grain softness protein (GSP-1), have been identified as components of friabilin, a biochemical marker for grain softness, and the genes for these polypeptides are known to be tightly linked to the Ha locus. However, this region of the chromosome 5D has not been well characterized and the physical distance between the markers is not known. Separate lambda clones containing the puroindoline-a gene and the puroindoline-b gene have been isolated from an Aegilops tauschii (the donor of the D genome to wheat) genomic lambda library and investigated. Considerable variation appears to exist in the organization of the region upstream of the gene for puroindoline-b among species closely related to wheat. Using in situ hybridization the genes for puroindoline-a, -b, and GSP-1 were demonstrated to be physically located at the tip of the short arm of chromosome 5 of A. tauschii. Four overlapping clones were isolated from a large-insert BAC library constructed from A. tauschii and of these one contained genes for all of puroindoline-a, puroindoline-b, and GSP-1. The gene for puroindoline-a is located between the other two genes at a distance no greater than approximately 30 kb from either gene. The BAC clone containing all three known genes was used to screen a cDNA library constructed from hexaploid wheat and cDNAs that could encode novel polypeptides were isolated. PMID:12723049

  11. 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...

  12. 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

  13. 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.

  14. Library Histories.

    ERIC Educational Resources Information Center

    Fentress, Helen Woodward; Taylor, Carole R.; Register, Janet L.; Thomas, Susan E.

    1997-01-01

    These 13 articles provide histories of Georgia libraries that include junior college libraries, rural libraries, county and regional systems, private and special libraries, black universities, public libraries, and academic libraries. (LRW)

  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. Preparation of BAC DNA for Pronuclear Injection Drop Dialysis

    E-print Network

    Preparation of BAC DNA for Pronuclear Injection Drop Dialysis 1. Fill the bottom of a Petri dish with injection buffer (MIB)a. 2. Float the dialysis membrane (VSWP02500, Millipore) on the surface of MIB in determining the dialysis time required. Most samples are dialyzed in less than 30 minutes. 4. Place a tight

  17. VEHS BACS 3/2011 Principal Investigator Assurance

    E-print Network

    Wikswo, John

    VEHS BACS 3/2011 Principal Investigator Assurance for Department of Defense Funded Research Activities I assure that I have involved the Facility Safety Director/Manager in the planning and safety, and will help him/her prepare the annual Facility Safety Plan Status Report (FSPSR). I assure

  18. 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:25917908

  19. 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...

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

    SciTech Connect

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

    1994-09-01

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

  1. 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

  2. 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

  3. Library Helpsheet Cochrane Library

    E-print Network

    Wapstra, Erik

    or a selection of databases can be made. The 3 most commonly searched databases are: · Cochrane database of systematic reviews (Cochrane reviews) Reports of the best available evidence to support treatments · Database Helpsheet Cochrane Library February 09 Page 1 of 5 Cochrane Library About the Cochrane Library The Cochrane

  4. 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…

  5. 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.

  6. The piggyBac Transposon as a Platform Technology for Somatic Cell Reprogramming Studies in Mouse.

    PubMed

    Woltjen, Knut; Kim, Shin-Il; Nagy, Andras

    2016-01-01

    Somatic cell reprogramming to induced pluripotent stem cells (iPSCs) is a revolutionary technology, with repercussions affecting modern functional genomics and regenerative medicine. Still, relatively little is known about the processes underlying this dramatic cellular and molecular metamorphosis. Reprogramming technology based on the implementation of piggyBac (PB) transposons has enabled studies of iPSC reprogramming mechanisms, shedding an increasing light on these processes. Unique characteristics of PB transposons such as efficient genomic integration, unlimited cargo capacity, robust gene expression, and even seamless excision highlight the importance of this transgenic tool in advancing stem cell biology. In this chapter, we provide a detailed overview of versatile primary iPSC generation from mouse somatic cells using PB transposons, and the subsequent establishment of robust secondary reprogramming systems. These protocols are highlighted with examples from recent studies as to how PB has been, and continues to be, conducive to the dissection of reprogramming processes at the cellular and molecular levels. PMID:26126450

  7. Marking Embryonic Stem Cells with a 2A Self-Cleaving Peptide: A NKX2-5 Emerald GFP BAC Reporter

    PubMed Central

    Hsiao, Edward C.; Yoshinaga, Yuko; Nguyen, Trieu D.; Musone, Stacy L.; Kim, Judy E.; Swinton, Paul; Espineda, Isidro; Manalac, Carlota; deJong, Pieter J.; Conklin, Bruce R.

    2008-01-01

    Background Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available. Methodology Here, we describe a series of modular marker plasmids containing independent reporter, bacterial selection, and eukaryotic selection components, compatible with both Gateway recombination and lambda prophage bacterial artificial chromosome (BAC) recombineering techniques. A 2A self-cleaving peptide links the reporter to the native open reading frame. We use an emerald GFP marker cassette to create a human BAC reporter and ES cell reporter line for the early cardiac marker NKX2-5. NKX2-5 expression was detected in differentiating mouse ES cells and ES cell-derived mice. Conclusions Our results describe a NKX2-5 ES cell reporter line for studying early events in cardiomyocyte formation. The results also demonstrate that our modular marker plasmids could be used for generating reporters from unmodified BACs, potentially as part of an ES cell reporter library. PMID:18596956

  8. 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…

  9. 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

  10. The Genome Sequence of the Fungal Pathogen Fusarium virguliforme That Causes Sudden Death Syndrome in Soybean

    PubMed Central

    Srivastava, Subodh K.; Huang, Xiaoqiu; Brar, Hargeet K.; Fakhoury, Ahmad M.; Bluhm, Burton H.; Bhattacharyya, Madan K.

    2014-01-01

    Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease. Methodology/Principal Findings We have generated the draft genome sequence of F. virguliforme by conducting whole-genome shotgun sequencing on a 454 GS-FLX Titanium sequencer. Initially, single-end reads of a 400-bp shotgun library were assembled using the PCAP program. Paired end sequences from 3 and 20 Kb DNA fragments and approximately 100 Kb inserts of 1,400 BAC clones were used to generate the assembled genome. The assembled genome sequence was 51 Mb. The N50 scaffold number was 11 with an N50 Scaffold length of 1,263 Kb. The AUGUSTUS gene prediction program predicted 14,845 putative genes, which were annotated with Pfam and GO databases. Gene distributions were uniform in all but one of the major scaffolds. Phylogenic analyses revealed that F. virguliforme was closely related to the pea pathogen, Nectria haematococca. Of the 14,845 F. virguliforme genes, 11,043 were conserved among five Fusarium species: F. virguliforme, F. graminearum, F. verticillioides, F. oxysporum and N. haematococca; and 1,332 F. virguliforme-specific genes, which may include pathogenicity genes. Additionally, searches for candidate F. virguliforme pathogenicity genes using gene sequences of the pathogen-host interaction database identified 358 genes. Conclusions The F. virguliforme genome sequence and putative pathogenicity genes presented here will facilitate identification of pathogenicity mechanisms involved in SDS development. Together, these resources will expedite our efforts towards discovering pathogenicity mechanisms in F. virguliforme. This will ultimately lead to improvement of SDS resistance in soybean. PMID:24454689

  11. Creating Library Spaces: Libraries 2040.

    ERIC Educational Resources Information Center

    Bruijnzeels, Rob

    This paper suggests that by 2004, the traditional public libraries will have ceased to exist and new, attractive future libraries will have taken their place. The Libraries 2040 project of the Netherlands is initiating seven different libraries of the future. The Brabant library is the "ultimate library of the future" for the Dutch province of…

  12. The Library The Public Library

    E-print Network

    automated system The library services The automated circulation system Recalls Reservation Reference works regarding legal issues that are closely linked to the program's aims. The library uses an automated systemThe Library The Public Library The Library Mission The library Function Acquisition Section

  13. 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...

  14. 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...

  15. Optical Mapping of BAC Clones from the Human Y Chromosome DAZ Locus

    E-print Network

    Mishra, Bud

    Optical Mapping of BAC Clones from the Human Y Chromosome DAZ Locus Joseph Giacalone,1 Stephanie a set of 16 BAC clones derived from the DAZ locus of the human Y chromosome long arm, a locus in which to be Y- chromosome repetitive sequences may actually repre- sent a number of gene families (Lahn and Page

  16. 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...

  17. Microcolinearity and genome evolution in the AdhA region of diploid and polyploid cotton (Gossypium)

    E-print Network

    Wendel, Jonathan F.

    Microcolinearity and genome evolution in the AdhA region of diploid and polyploid cotton (Gossypium. Prior analysis of the CesA region in two cotton genomes that diverged 5­10 million years ago (Ma to include BAC sequences surrounding the gene encoding alcohol dehydrogenase A (AdhA) from four cotton

  18. 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

  19. 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...

  20. Sequencing a Genome by Walking With Clone-end Sequences

    E-print Network

    Sequencing a Genome by Walking With Clone-end Sequences: A Mathematical Analysis Serafim Batzoglou-insert clones (such as bacterial artificial chromosomes (BACs)) and then (ii) to take successive 'walking' steps by selecting and sequencing minimally overlapping clones, using information such as clone-end sequences

  1. 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...

  2. NEW PIGGYBAC VECTORS FOR FUNCTIONAL GENOMICS AND TRANSGENIC INSECT RELEASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our research focuses on creating transgenic fruit fly strains for improved biological control and functional genomics analysis. This has involved the development of the piggyBac transposon-based transformation system that is now used in four orders of insects. For biocontrol based on conditional l...

  3. 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...

  4. Library Learning.

    ERIC Educational Resources Information Center

    Hull, Barbara

    1999-01-01

    After years of cutbacks, the pivotal role of public libraries in providing lifelong learning resources is being recognized. In Britain, a consortium for public-library networking is extending libraries' ability to deliver knowledge-based services. (SK)

  5. Multimedia Library

    Cancer.gov

    Home News and Events Multimedia Library Multimedia Library Learn more about TCGA, its researchers, components and how it works from our multimedia library. Images Image: TCGA Pipeline for Comprehensive Characterization 72 DPI | 300 DPIThe path of TCGA

  6. Superconductivity in the Graphite Intercalation Compound BaC(6).

    PubMed

    Heguri, Satoshi; Kawade, Naoya; Fujisawa, Takumi; Yamaguchi, Akira; Sumiyama, Akihiko; Tanigaki, Katsumi; Kobayashi, Mototada

    2015-06-19

    Among many two-dimensional (2D) high T(C) superconductors, graphite intercalation compounds (GICs) are the most famous intercalation family, which are classified as typical electron-phonon mediated superconductors. We show unambiguous experimental facts that BaC(6), the superconductivity of which has been missing for many years so far among various alkaline earth metal (Ca, Sr, and Ba) intercalted GICs, exhibits superconductivity at T(C)=65??mK. By adding this finding as the additional experimental point, a complete figure displaying the relationship between T(C) and interlayer distance (d) for GICs is now provided, and their possible superconducting mechanisms raised so far are revisited. The present study settles a long-running debate between theories and experiments on the superconductivity in the first stage GICs. PMID:26197001

  7. 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.

  8. Pre-oxidation efficiency of BAC treatment for VOC removal

    SciTech Connect

    Yu, M.J.; Ahn, S.K.; Kim, Y.R.; Lee, S.H.

    1996-12-31

    Studies have been conducted to evaluate pretreatment for the removal of trichloroethylene(TCE), tetrachloroethylene(PCE) and 1,1,1-trichloroethane(1,1,1-TCA) using the combination of chemical oxidation and biological system with a selected microbial consortium (SMC). SMC were acclimated with the mixture of three chemicals as the sole carbon and energy source. The microbial species isolated from the soil contaminated by VOCs were gram-negative, rod-shaped bacteria, identified as Pseudomonas aeruginosa and Acinobacter calcoaceticus. The purposes of this research were to investigate pre-treatment efficiency for the removal of VOCs in groundwater and to develop design and operational parameters by using a pilot plant system. Simulated groundwater contaminated by VOCs was used to examine the efficiency of chemical oxidation using O{sub 3} alone, H{sub 2}O{sub 2}/O{sub 3}, following BAC column. The results showed the efficiency of pretreatment to remove VOCs through six processes composed of ozone generator, H{sub 2}O{sub 2}/O{sub 3} reactor and BAC column. Ozone and hydrogen peroxide pretreatment system was more effective to remove VOCs than non-pretreated system and subsequent biodegradation was influenced by pretreatment processes. The mixture of VOCs were removed in the order of TCE, PCE and TCA with H{sub 2}O{sub 2}/O{sub 3} and ozone alone. Results obtained show that chemically oxidized VOC can considerably decrease the adsorption capacity. Biological removal efficiency was in the order of TCE, TCA and PCE.

  9. 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.

  10. 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...

  11. Chicken genomics resource: sequencing and annotation of 35,407 ESTs from single and multiple tissue cDNA libraries and CAP3 assembly of a chicken gene index.

    PubMed

    Carre, Wilfrid; Wang, Xiaofei; Porter, Tom E; Nys, Yves; Tang, Jianshan; Bernberg, Erin; Morgan, Robin; Burnside, Joan; Aggrey, Samuel E; Simon, Jean; Cogburn, Larry A

    2006-05-16

    Its accessibility, unique evolutionary position, and recently assembled genome sequence have advanced the chicken to the forefront of comparative genomics and developmental biology research as a model organism. Several chicken expressed sequence tag (EST) projects have placed the chicken in 10th place for accrued ESTs among all organisms in GenBank. We have completed the single-pass 5'-end sequencing of 37,557 chicken cDNA clones from several single and multiple tissue cDNA libraries and have entered 35,407 EST sequences into GenBank. Our chicken EST sequences and those found in public databases (on July 1, 2004) provided a total of 517,727 public chicken ESTs and mRNAs. These sequences were used in the CAP3 assembly of a chicken gene index composed of 40,850 contigs and 79,192 unassembled singlets. The CAP3 contigs show a 96.7% match to the chicken genome sequence. The University of Delaware (UD) EST collection (43,928 clones) was assembled into 19,237 nonredundant sequences (13,495 contigs and 5,742 unassembled singlets). The UD collection contains 6,223 unique sequences that are not found in other public EST collections but show a 76% match to the chicken genome sequence. Our chicken contig and singlet sequences were annotated according to the highest BlastX and/or BlastN hits. The UD CAP3 contig assemblies and singlets are searchable by nucleotide sequence or key word (http://cogburn.dbi.udel.edu), and the cDNA clones are readily available for distribution from the chick EST website and clone repository (http://www.chickest.udel.edu). The present paper describes the construction and normalization of single and multiple tissue chicken cDNA libraries, high-throughput EST sequencing from these libraries, the CAP3 assembly of a chicken gene index from all public ESTs, and the identification of several nonredundant chicken gene sets for production of custom DNA microarrays. PMID:16554550

  12. Initial and residual activity of VectoBac 12 AS, VectoBac WDG, and VectoLex WDG for control of mosquitoes in Ararat Valley, Turkey.

    PubMed

    Aldemir, Adnan

    2009-03-01

    Two formulations, VectoBac 12 AS and VectoBac WDG, of Bacillus thuringiensis israelensis (Bti) and one formulation, VectoLex WDG, of Bacillus sphaericus were tested against Anopheles maculipennis, Culex pipiens, Culex theileri, Aedes caspius, and Aedes dorsalis larvae in drainage canals, a flooded plain, and a drainage well in the Igdir Plain of Arafat Valley, Turkey. VectoBac 12 AS applied at 0.5 and 1 liter/ha to a drainage canal provided complete control of Cx. theileri and 80% to 98% control of An. maculipennis. VectoLex WDG at 400 g/ha provided 100% larval control of Cx. pipiens and Cx. theileri in polluted drainage canals. High larval density of Ae. caspius decreased the efficacy of VectoBac 12 AS and VectoBac WDG in the flooded plain. Residual effects of Bti formulations lasted only a few days in the breeding areas. Residual activity of VectoLex WDG was negatively influenced by water depth, organic pollution, and sunlight and positively influenced by low water velocity and emergent vegetation. PMID:19432078

  13. 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 72h 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

  14. 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

  15. Genic regions of a large salamander genome contain long introns and novel genes

    PubMed Central

    Smith, Jeramiah J; Putta, Srikrishna; Zhu, Wei; Pao, Gerald M; Verma, Inder M; Hunter, Tony; Bryant, Susan V; Gardiner, David M; Harkins, Timothy T; Voss, S Randal

    2009-01-01

    Background The basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 × 109 bp) were isolated and sequenced to characterize the structure of genic regions. Results Annotation of genes within BACs showed that axolotl introns are on average 10× longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin metamorphosis. Unexpectedly, a third novel gene was also discovered while manually annotating BACs. Analysis of human-axolotl protein-coding sequences suggests there are 2% more lineage specific genes in the axolotl genome than the human genome, but the great majority (86%) of genes between axolotl and human are predicted to be 1:1 orthologs. Considering that axolotl genes are on average 5× larger than human genes, the genic component of the salamander genome is estimated to be incredibly large, approximately 2.8 gigabases! Conclusion This study shows that a large salamander genome has a correspondingly large genic component, primarily because genes have incredibly long introns. These intronic sequences may harbor novel coding and non-coding sequences that regulate biological processes that are unique to salamanders. PMID:19144141

  16. Library Skills.

    ERIC Educational Resources Information Center

    Bhullar, Pushpajit K., Ed.; Lawhorne, Anne R., Ed.

    This guide is designed to acquaint students of the University of Missouri-Columbia with the facilities and resources of the Ellis Library, and is intended for students enrolled in Library Science 105: Library Skills. The guide is organized into sections dealing with search strategies and types of library materials. It opens with an orientation to…

  17. Library Computing.

    ERIC Educational Resources Information Center

    Sloan, Bernard C.; And Others

    1986-01-01

    "Library Journal's" fourth semiannual section on library automation provides basic and introductory articles on: remote public access to automated library systems, electronic bulletin boards, the online integrated automation system at the Oklahoma City public library, and buying a phone system. Tables include statistics on various aspects of…

  18. Physical map-assisted whole-genome shotgun sequence assemblies

    PubMed Central

    Warren, René L.; Varabei, Dmitry; Platt, Darren; Huang, Xiaoqiu; Messina, David; Yang, Shiaw-Pyng; Kronstad, James W.; Krzywinski, Martin; Warren, Wesley C.; Wallis, John W.; Hillier, LaDeana W.; Chinwalla, Asif T.; Schein, Jacqueline E.; Siddiqui, Asim S.; Marra, Marco A.; Wilson, Richard K.; Jones, Steven J.M.

    2006-01-01

    We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the layout phase of WGS assemblies. This process is facilitated by FASSI, a stand-alone application that calculates BAC end and BAC overlap length constraints from clone fingerprint map contigs created by the FPC package. FASSI is designed to work with the assembly tool PCAP, but its output can be formatted to work with other WGS assembly algorithms able to use length constraints for individual clones. The FASSI method is simple to implement, potentially cost-effective, and has resulted in the increase of scaffold contiguity for both the Drosophila melanogaster and Cryptococcus gattii genomes when compared to a control assembly without map-derived constraints. A 6.5-fold coverage draft DNA sequence of the Pan troglodytes (chimpanzee) genome was assembled using map-derived constraints and resulted in a 26.1% increase in scaffold contiguity. PMID:16741162

  19. Physical map-assisted whole-genome shotgun sequence assemblies.

    PubMed

    Warren, René L; Varabei, Dmitry; Platt, Darren; Huang, Xiaoqiu; Messina, David; Yang, Shiaw-Pyng; Kronstad, James W; Krzywinski, Martin; Warren, Wesley C; Wallis, John W; Hillier, LaDeana W; Chinwalla, Asif T; Schein, Jacqueline E; Siddiqui, Asim S; Marra, Marco A; Wilson, Richard K; Jones, Steven J M

    2006-06-01

    We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the layout phase of WGS assemblies. This process is facilitated by FASSI, a stand-alone application that calculates BAC end and BAC overlap length constraints from clone fingerprint map contigs created by the FPC package. FASSI is designed to work with the assembly tool PCAP, but its output can be formatted to work with other WGS assembly algorithms able to use length constraints for individual clones. The FASSI method is simple to implement, potentially cost-effective, and has resulted in the increase of scaffold contiguity for both the Drosophila melanogaster and Cryptococcus gattii genomes when compared to a control assembly without map-derived constraints. A 6.5-fold coverage draft DNA sequence of the Pan troglodytes (chimpanzee) genome was assembled using map-derived constraints and resulted in a 26.1% increase in scaffold contiguity. PMID:16741162

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

    E-print Network

    Feng, Guoping

    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 ...

  1. Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes

    PubMed Central

    2013-01-01

    Background Small supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA). To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms. Results By FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a lack of information about the covered region in the reference sequence (1/19) or sample insufficiency (6/19). Conclusions Our results demonstrate that this pericentromeric clone set is useful as an alternative tool for sSMC characterization, primarily in cases of very small SMCs that contain either heterochromatin exclusively or a tiny amount of euchromatic sequence, and also in cases of low-level or cryptic mosaicism. The resulting data will foster knowledge of human proximal euchromatic regions involved in chromosomal imbalances, thereby improving genotype–phenotype correlations. PMID:24171812

  2. 23 CFR 1225.4 - Adoption of 0.08 BAC per se law.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 23 Highways 1 2013-04-01 2013-04-01 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... committed a per se offense of driving while intoxicated or an equivalent per se offense. The law must:...

  3. 23 CFR 1225.4 - Adoption of 0.08 BAC per se law.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 23 Highways 1 2014-04-01 2014-04-01 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... committed a per se offense of driving while intoxicated or an equivalent per se offense. The law must:...

  4. 23 CFR 1225.4 - Adoption of 0.08 BAC per se law.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 23 Highways 1 2012-04-01 2012-04-01 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... committed a per se offense of driving while intoxicated or an equivalent per se offense. The law must:...

  5. 23 CFR 1225.4 - Adoption of 0.08 BAC per se law.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 23 Highways 1 2011-04-01 2011-04-01 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... committed a per se offense of driving while intoxicated or an equivalent per se offense. The law must:...

  6. 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... committed a per se offense of driving while intoxicated or an equivalent per se offense. The law must:...

  7. Region-specific deficits in dopamine, but not norepinephrine, signaling in a novel A30P ?-synuclein BAC transgenic mouse.

    PubMed

    Taylor, Tonya N; Potgieter, Dawid; Anwar, Sabina; Senior, Steven L; Janezic, Stephanie; Threlfell, Sarah; Ryan, Brent; Parkkinen, Laura; Deltheil, Thierry; Cioroch, Milena; Livieratos, Achilleas; Oliver, Peter L; Jennings, Katie A; Davies, Kay E; Ansorge, Olaf; Bannerman, David M; Cragg, Stephanie J; Wade-Martins, Richard

    2014-02-01

    Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of ?-synuclein. Approximately 5-10% of PD patients have a familial form of Parkinsonism, including mutations in ?-synuclein. To better understand the cell-type specific role of ?-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human ?-synuclein in a spatially-relevant manner from the 111kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca-/-) background. The BAC transgenic mice expressed ?-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P ?-synuclein or wildtype ?-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca-/- animals. A30P ?-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca-/- mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca-/- mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P ?-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD. PMID:24121116

  8. Expression of green fluorescent protein in the chicken using in vivo transfection of the piggyBac transposon.

    PubMed

    Jordan, Brian J; Vogel, Seth; Stark, Michael R; Beckstead, Robert B

    2014-03-10

    The chicken is a well-established model system for studying developmental biology and is recognized as one of the top food production animals in the world. For this reason the chicken is an excellent candidate for transgenic applications, as the technology can be applied to both areas of research. Transgenic technology has not been broadly utilized in the chicken model, however, primarily due to difficulties in targeting germ cells and establishing germ line transmission. Transgenic technologies using non-replicating viral particles have been used in the chick, but are unsuitable for many applications because of size and sequence restraints and low efficiency. To create a more versatile method to target chick germ line stem cells, we utilized the transposable element system piggyBac paired with an in vivo transfection reagent, JetPEI. piggyBac has been previously shown to be highly active in mammalian cells and will transpose into the chicken genome. Here, we show that JetPEI can transfect multiple chick cell types, most notably germline stem cells. We also show that pairing these two reagents is a viable and reproducible method for long-term expression of a transgene in the chicken. Stable expression of the green fluorescent protein (GFP) transgene was seen in multiple tissue types including heart, brain, liver, intestine, kidney and gonad. Combining an in vivo transfection strategy with the PB system provides a simple and flexible method for efficiently producing stable chimeric birds and could be used for production of germ line transgenics. PMID:24452099

  9. Integrated Genomic Characterization of Pheochromocytoma and Paraganglioma - Matthew Wilkerson, TCGA Scientific Symposium 2015

    Cancer.gov

    Home News and Events Multimedia Library Videos Integrated Genomic Characterization of Pheochromocytoma and Paraganglioma - Matthew Wilkerson, TCGA Integrated Genomic Characterization of Pheochromocytoma and Paraganglioma - Matthew Wilkerson, TCGA

  10. 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.

  11. National Libraries: An Analysis

    ERIC Educational Resources Information Center

    Burston, Godfrey

    1973-01-01

    The title National Library'' means different things in different countries. It can encompass parliamentary libraries, public libraries, special interest libraries (museums, etc.), and reference and lending libraries. (DH)

  12. Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids1

    PubMed Central

    Li, Zicong; Zeng, Fang; Meng, Fanming; Xu, Zhiqian; Zhang, Xianwei; Huang, Xiaoling; Tang, Fei; Gao, Wenchao; Shi, Junsong; He, Xiaoyan; Liu, Dewu; Wang, Chong; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2014-01-01

    ABSTRACT The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis. PMID:24671876

  13. Expression Analysis of CB2-GFP BAC Transgenic Mice

    PubMed Central

    Schrage, Hanna; Beins, Eva; Krämer, Alexandra; Zimmer, Till; Limmer, Andreas; Zimmer, Andreas; Otte, David-Marian

    2015-01-01

    The endocannabinoid system (ECS) is a retrograde messenger system, consisting of lipid signaling molecules that bind to at least two G-protein-coupled receptors, Cannabinoid receptor 1 and 2 (CB1 and 2). As CB2 is primarily expressed on immune cells such as B cells, T cells, macrophages, dendritic cells, and microglia, it is of great interest how CB2 contributes to immune cell development and function in health and disease. Here, understanding the mechanisms of CB2 involvement in immune-cell function as well as the trafficking and regulation of CB2 expressing cells are crucial issues. Up to now, CB2 antibodies produce unclear results, especially those targeting the murine protein. Therefore, we have generated BAC transgenic GFP reporter mice (CB2-GFPTg) to trace CB2 expression in vitro and in situ. Those mice express GFP under the CB2 promoter and display GFP expression paralleling CB2 expression on the transcript level in spleen, thymus and brain tissue. Furthermore, by using fluorescence techniques we show that the major sources for GFP-CB2 expression are B cells in spleen and blood and microglia in the brain. This novel CB2-GFP transgenic reporter mouse line represents a powerful resource to study CB2 expression in different cell types. Furthermore, it could be used for analyzing CB2-mediated mobilization and trafficking of immune cells as well as studying the fate of recruited immune cells in models of acute and chronic inflammation. PMID:26406232

  14. 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

  15. Ataxin-2 Regulates RGS8 Translation in a New BAC-SCA2 Transgenic Mouse Model

    PubMed Central

    Figueroa, Karla P.; Rinehart, Marc D.; Wiest, Shaina; Pflieger, Lance T.; Scoles, Daniel R.; Pulst, Stefan M.

    2015-01-01

    Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder with progressive degeneration of cerebellar Purkinje cells (PCs) and other neurons caused by expansion of a glutamine (Q) tract in the ATXN2 protein. We generated BAC transgenic lines in which the full-length human ATXN2 gene was transcribed using its endogenous regulatory machinery. Mice with the ATXN2 BAC transgene with an expanded CAG repeat (BAC-Q72) developed a progressive cellular and motor phenotype, whereas BAC mice expressing wild-type human ATXN2 (BAC-Q22) were indistinguishable from control mice. Expression analysis of laser-capture microdissected (LCM) fractions and regional expression confirmed that the BAC transgene was expressed in PCs and in other neuronal groups such as granule cells (GCs) and neurons in deep cerebellar nuclei as well as in spinal cord. Transcriptome analysis by deep RNA-sequencing revealed that BAC-Q72 mice had progressive changes in steady-state levels of specific mRNAs including Rgs8, one of the earliest down-regulated transcripts in the Pcp2-ATXN2[Q127] mouse line. Consistent with LCM analysis, transcriptome changes analyzed by deep RNA-sequencing were not restricted to PCs, but were also seen in transcripts enriched in GCs such as Neurod1. BAC-Q72, but not BAC-Q22 mice had reduced Rgs8 mRNA levels and even more severely reduced steady-state protein levels. Using RNA immunoprecipitation we showed that ATXN2 interacted selectively with RGS8 mRNA. This interaction was impaired when ATXN2 harbored an expanded polyglutamine. Mutant ATXN2 also reduced RGS8 expression in an in vitro coupled translation assay when compared with equal expression of wild-type ATXN2-Q22. Reduced abundance of Rgs8 in Pcp2-ATXN2[Q127] and BAC-Q72 mice supports our observations of a hyper-excitable mGluR1-ITPR1 signaling axis in SCA2, as RGS proteins are linked to attenuating mGluR1 signaling. PMID:25902068

  16. A highly conserved gene island of three genes on chromosome 3B of hexaploid wheat: diverse gene function and genomic structure maintained in a tightly linked block

    PubMed Central

    2010-01-01

    Background The complexity of the wheat genome has resulted from waves of retrotransposable element insertions. Gene deletions and disruptions generated by the fast replacement of repetitive elements in wheat have resulted in disruption of colinearity at a micro (sub-megabase) level among the cereals. In view of genomic changes that are possible within a given time span, conservation of genes between species tends to imply an important functional or regional constraint that does not permit a change in genomic structure. The ctg1034 contig completed in this paper was initially studied because it was assigned to the Sr2 resistance locus region, but detailed mapping studies subsequently assigned it to the long arm of 3B and revealed its unusual features. Results BAC shotgun sequencing of the hexaploid wheat (Triticum aestivum cv. Chinese Spring) genome has been used to assemble a group of 15 wheat BACs from the chromosome 3B physical map FPC contig ctg1034 into a 783,553 bp genomic sequence. This ctg1034 sequence was annotated for biological features such as genes and transposable elements. A three-gene island was identified among >80% repetitive DNA sequence. Using bioinformatics analysis there were no observable similarity in their gene functions. The ctg1034 gene island also displayed complete conservation of gene order and orientation with syntenic gene islands found in publicly available genome sequences of Brachypodium distachyon, Oryza sativa, Sorghum bicolor and Zea mays, even though the intergenic space and introns were divergent. Conclusion We propose that ctg1034 is located within the heterochromatic C-band region of deletion bin 3BL7 based on the identification of heterochromatic tandem repeats and presence of significant matches to chromodomain-containing gypsy LTR retrotransposable elements. We also speculate that this location, among other highly repetitive sequences, may account for the relative stability in gene order and orientation within the gene island. Sequence data from this article have been deposited with the GenBank Data Libraries under accession no. GQ422824 PMID:20507561

  17. 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 ?-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 attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC. PMID:23604115

  18. 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.

  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. Mouse and Human BAC Transgenes Recapitulate Tissue-Specific Expression of the Vitamin D Receptor in Mice and Rescue the VDR-Null Phenotype

    PubMed Central

    Lee, Seong Min; Bishop, Kathleen A.; Goellner, Joseph J.; O'Brien, Charles A.

    2014-01-01

    The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo. PMID:24693968

  1. Deficiency of a Sinorhizobium meliloti bacA Mutant in Alfalfa Symbiosis Correlates with Alteration of the Cell Envelope

    PubMed Central

    Ferguson, Gail P.; Roop II, R. Martin; Walker, Graham C.

    2002-01-01

    The BacA protein is essential for the long-term survival of Sinorhizobium meliloti and Brucella abortus within acidic compartments in plant and animal cells, respectively. Since both the S. meliloti and B. abortus bacA mutants have an increased resistance to bleomycin, it was hypothesized that BacA was a transporter of bleomycin and bleomycin-like compounds into the bacterial cell. However, our finding that the S. meliloti bacA mutant also has an increased sensitivity to detergents, a hydrophobic dye, ethanol, and acid pH supported a model in which BacA function affects the bacterial cell envelope. In addition, an S. meliloti lpsB mutant that is defective at a stage in infection of the host similar to that found for a bacA mutant is also sensitive to the same agents, and the carbohydrate content of its lipopolysaccharide (LPS) is altered. However, analysis of crude preparations of the bacA mutant LPS suggested that, unlike that for LpsB, BacA function did not affect the carbohydrate composition of the LPS. Rather, we found that at least one function of BacA is to affect the distribution of LPS fatty acids, including a very-long-chain fatty acid thought to be unique to the ?-proteobacteria, including B. abortus. PMID:12270820

  2. 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;…

  3. BCH222 -Sidechain Rotamer-plot Data Lovell et al. (2000) Proteins: Struc Funct Genomics 40, 389 on the "penultimate library" of

    E-print Network

    Richardson, David

    2000-01-01

    BCH222 - Sidechain Rotamer-plot Data Reading Lovell et al. (2000) Proteins: Struc Funct Genomics 40NG. In the initial view, note that the overall , plot for Gln looks a lot like the general case. Turn on the p, t, t, and m 1 values match the order in Lovell 2000? _______ BCH 222: Ramachandran-plot Data http

  4. 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

  5. LTC: a novel algorithm to improve the efficiency of contig assembly for physical mapping in complex genomes

    PubMed Central

    2010-01-01

    Background Physical maps are the substrate of genome sequencing and map-based cloning and their construction relies on the accurate assembly of BAC clones into large contigs that are then anchored to genetic maps with molecular markers. High Information Content Fingerprinting has become the method of choice for large and repetitive genomes such as those of maize, barley, and wheat. However, the high level of repeated DNA present in these genomes requires the application of very stringent criteria to ensure a reliable assembly with the FingerPrinted Contig (FPC) software, which often results in short contig lengths (of 3-5 clones before merging) as well as an unreliable assembly in some difficult regions. Difficulties can originate from a non-linear topological structure of clone overlaps, low power of clone ordering algorithms, and the absence of tools to identify sources of gaps in Minimal Tiling Paths (MTPs). Results To address these problems, we propose a novel approach that: (i) reduces the rate of false connections and Q-clones by using a new cutoff calculation method; (ii) obtains reliable clusters robust to the exclusion of single clone or clone overlap; (iii) explores the topological contig structure by considering contigs as networks of clones connected by significant overlaps; (iv) performs iterative clone clustering combined with ordering and order verification using re-sampling methods; and (v) uses global optimization methods for clone ordering and Band Map construction. The elements of this new analytical framework called Linear Topological Contig (LTC) were applied on datasets used previously for the construction of the physical map of wheat chromosome 3B with FPC. The performance of LTC vs. FPC was compared also on the simulated BAC libraries based on the known genome sequences for chromosome 1 of rice and chromosome 1 of maize. Conclusions The results show that compared to other methods, LTC enables the construction of highly reliable and longer contigs (5-12 clones before merging), the detection of "weak" connections in contigs and their "repair", and the elongation of contigs obtained by other assembly methods. PMID:21118513

  6. OHSU Library Using Library Materials

    E-print Network

    Chapman, Michael S.

    OHSU Library Using Library Materials · All journals and books can be used in the library. Photocopy those databases that are free to everyone. · Journals ­ All electronic journals are available to anyone of free electronic journals is available at http://atoz.ebsco. com/titles.asp?Id=sfohs&sid=216520571&Tab

  7. Draft Genome Sequence of Weissella koreensis KCTC 3621T Je Hee Lee,a

    E-print Network

    Bae, Jin-Woo

    -shaped, nonmotile, and facultative anaerobic species belonging to the lactic acid bac- teria (LAB). The members of this species have been repeatedly isolated from kimchi (a traditional Korean fermented food) and are known amino acids such as -aminobutyric acid and ornithine. Here we report the genome sequence of the type

  8. GENOME-WIDE PHYSICAL MAPPING OF PATHOGEN-RESPONSE GENES IN BARLEY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genes involved in resistance to pathogens are an intricated network of cell signaling events. A genome-wide search was undertaken to identify gene-rich BAC clones from barley that contained defense-related genes. HarvEST Triticeae (http://harvest.ucr.edu/) was utilized to identify ESTs uniquely asso...

  9. ToxoDB: accessing the Toxoplasma gondii genome Jessica C. Kissinger, Bindu Gajria1

    E-print Network

    Kissinger, Jessica

    . Paulsen2 and David S. Roos1,* Department of Genetics/Center for Tropical and Emerging Global Diseases or are in progress: an EST project (http://genome.wustl.edu/est/index. php?toxoplasma=1), a BAC clone endDB, we took advantage of existing database software and tools developed for the Allgenes (10

  10. 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)

  11. Application of the BacT/ALERTR 3D system for sterility testing of injectable products

    PubMed Central

    Bugno, Adriana; Lira, Rodolfo Santos; Oliveira, Wesley Anderson; Almodovar, Adriana Aparecida Buzzo; Saes, Deborah Pita Sanches; de Jesus Andreoli Pinto, Terezinha

    2015-01-01

    Sterility testing as described in the pharmacopoeia compendia requires a 14-day incubation period to obtain an analytical result. Alternative methods that could be applied to evaluating product sterility are especially interesting due to the possibility of reducing this incubation period and thus the associated costs. The aims of this study were to evaluate the performance of the BacT/ALERTR 3D system in detecting microorganisms in large-volume parenteral solutions that were intentionally contaminated and to compare this system to pharmacopoeia sterility testing using the membrane filtration method. The results indicated that there were no significant differences between the methods regarding the ability to detect microbial contamination; however, detection with the BacT/ALERTR 3D system was faster compared to the pharmacopoeia method. Therefore, the BacT/ALERTR 3D system is a viable alternative for assessing the sterility of injectable products. PMID:26413055

  12. Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool.

    PubMed

    Kim, Adele; Pyykko, Ilmari

    2011-08-01

    Transposons have been promising elements for gene integration, and the Sleeping Beauty (SB) system has been the major one for many years, although there have been several other transposon systems available, for example, Tol2. However, recently another system known as PiggyBac (PB) has been introduced and developed for fulfilling the same purposes, for example, mutagenesis, transgenesis and gene therapy and in some cases with improved transposition efficiency and advantages over the Sleeping Beauty transposon system, although improved hyperactive transposase has highly increased the transposition efficacy for SB. The PB systems have been used in many different scientific research fields; therefore, the purpose of this review is to describe some of these versatile uses of the PiggyBac system to give readers an overview on the usage of PiggyBac system. PMID:21516337

  13. A Simple and Effective Procedure for Discovering Microsatellites from Small Insert Libraries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite loci are useful markers for studying genetic diversity and for creating linkage maps in plants and animals. However, microsatellite discovery from a genomic library is tedious and costly. We have constructed a small insert genomic library and six repeats-enriched libraries. Instead of...

  14. Databases and information integration for the Medicago truncatula genome and transcriptome.

    PubMed

    Cannon, Steven B; Crow, John A; Heuer, Michael L; Wang, Xiaohong; Cannon, Ethalinda K S; Dwan, Christopher; Lamblin, Anne-Francoise; Vasdewani, Jayprakash; Mudge, Joann; Cook, Andrew; Gish, John; Cheung, Foo; Kenton, Steve; Kunau, Timothy M; Brown, Douglas; May, Gregory D; Kim, Dongjin; Cook, Douglas R; Roe, Bruce A; Town, Chris D; Young, Nevin D; Retzel, Ernest F

    2005-05-01

    An international consortium is sequencing the euchromatic genespace of Medicago truncatula. Extensive bioinformatic and database resources support the marker-anchored bacterial artificial chromosome (BAC) sequencing strategy. Existing physical and genetic maps and deep BAC-end sequencing help to guide the sequencing effort, while EST databases provide essential resources for genome annotation as well as transcriptome characterization and microarray design. Finished BAC sequences are joined into overlapping sequence assemblies and undergo an automated annotation process that integrates ab initio predictions with EST, protein, and other recognizable features. Because of the sequencing project's international and collaborative nature, data production, storage, and visualization tools are broadly distributed. This paper describes databases and Web resources for the project, which provide support for physical and genetic maps, genome sequence assembly, gene prediction, and integration of EST data. A central project Web site at medicago.org/genome provides access to genome viewers and other resources project-wide, including an Ensembl implementation at medicago.org, physical map and marker resources at mtgenome.ucdavis.edu, and genome viewers at the University of Oklahoma (www.genome.ou.edu), the Institute for Genomic Research (www.tigr.org), and Munich Information for Protein Sequences Center (mips.gsf.de). PMID:15888676

  15. 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...

  16. ALPHABET SOUP FOR BEETS: STATUS OF ESTS, BACS, RILS, AND OTHER GENOMIC SUNDRIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dogma holds that phenotype = genotype + environment; DNA makes RNA makes protein; and form follows function. What does this mean? The beet's work is accomplished in large part by proteins, these proteins (i.e. gene products) are inherited from the parents, and expression of genes is influenced by ...

  17. 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...

  18. 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,…

  19. Minnesota: Library Automation and Technology in Libraries.

    ERIC Educational Resources Information Center

    Feye-Stukas, Jan

    1996-01-01

    Provides an overview of library automation in Minnesota. Topics include regional public library systems; library automation vendors; multitype library systems; postsecondary and academic libraries; state government libraries; the Internet; telecommunications and statewide online system legislation and funding; and state library agency involvement…

  20. 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.

  1. A Hybrid Adenoviral Vector System Achieves Efficient Long-Term Gene Expression in the Liver via piggyBac Transposition.

    PubMed

    Smith, Ryan P; Riordan, Jesse D; Feddersen, Charlotte R; Dupuy, Adam J

    2015-06-01

    Much research has gone into the development of hybrid gene delivery systems that combine the broad tropism and efficient transduction of adenoviral vectors with the ability to achieve stable expression of cargo genes. In addition to gene therapy applications, such a system has considerable advantages for studies of gene function in vivo, permitting fine-tuned genetic manipulation with higher throughput than can be achieved using standard transgenic and DNA targeting techniques. Existing strategies are limited, however, by low integration efficiencies, small cargo capacity, and/or a dependence on target cell division. The utility of this approach could be enhanced by a system that provides all of the following: (1) efficient delivery, (2) stable expression in a high percentage of target cells (whether mitotic or not), (3) large cargo capacity, (4) flexibility to use with a wide range of additional experimental conditions, and (5) simple experimental technique. Here we report the initial characterization of a hybrid system that meets these criteria by utilizing piggyBac (PB) transposition to achieve genomic integration from adenoviral vectors. We demonstrate stable expression of an adenovirus (Ad)-PB-delivered reporter gene in ?20-40% of hepatocytes following standard tail vein injection. Its high efficiency and flexibility relative to existing hybrid adenoviral gene delivery approaches indicate a considerable potential utility of the Ad-PB system for therapeutic gene delivery and in vivo studies of gene function. PMID:25808258

  2. 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…

  3. 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…

  4. 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…

  5. 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....

  6. 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...

  7. 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...

  8. 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)

  9. Measuring brain activity cycling (BAC) in long term EEG monitoring of preterm babies.

    PubMed

    Stevenson, Nathan J; Palmu, Kirsi; Wikström, Sverre; Hellström-Westas, Lena; Vanhatalo, Sampsa

    2014-07-01

    Measuring fluctuation of vigilance states in early preterm infants undergoing long term intensive care holds promise for monitoring their neurological well-being. There is currently, however, neither objective nor quantitative methods available for this purpose in a research or clinical environment. The aim of this proof-of-concept study was, therefore, to develop quantitative measures of the fluctuation in vigilance states or brain activity cycling (BAC) in early preterm infants. The proposed measures of BAC were summary statistics computed on a frequency domain representation of the proportional duration of spontaneous activity transients (SAT%) calculated from electroencephalograph (EEG) recordings. Eighteen combinations of three statistics and six frequency domain representations were compared to a visual interpretation of cycling in the SAT% signal. Three high performing measures (band energy/periodogram: R = 0.809, relative band energy/nonstationary frequency marginal: R = 0.711, g-statistic/nonstationary frequency marginal: R = 0.638) were then compared to a grading of sleep wake cycling based on the visual interpretation of the amplitude-integrated EEG trend. These measures of BAC are conceptually straightforward, correlate well with the visual scores of BAC and sleep wake cycling, are robust enough to cope with the technically compromised monitoring data available in intensive care units, and are recommended for further validation in prospective studies. PMID:24901751

  10. 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

  11. 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

  12. The Library of Maynard-Smith: My Search for Meaning in the Protein

    E-print Network

    Arnold, Frances H.

    glorious creatures Mendel's library contains! But the vast majority of possible genomes does not encode in a hope- less search for these magnificent, but extremely rare books? Maynard-Smith's library is different

  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. On-line intergrated library system: bibliographic access and control system of Washington University School of Medicine.

    PubMed

    Kelly, E A; Yedlin, D K; Crawford, S Y; Igielnik, S

    1982-07-01

    The on-line integrated library system is a relatively simple and logical concept. The perception that many library functions can be incorporated within one system, using a single data base, has led a number of practitioners to independently develop such systems. This paper describes the Bibliographic Access and Control System (BACS), developed by the Washington University School of Medicine Library, and identifies some of the underlying principles, components, and capabilities of this system from the vantage point of operational experience over one year. PMID:6896835

  15. On-line intergrated library system: bibliographic access and control system of Washington University School of Medicine.

    PubMed Central

    Kelly, E A; Yedlin, D K; Crawford, S Y; Igielnik, S

    1982-01-01

    The on-line integrated library system is a relatively simple and logical concept. The perception that many library functions can be incorporated within one system, using a single data base, has led a number of practitioners to independently develop such systems. This paper describes the Bibliographic Access and Control System (BACS), developed by the Washington University School of Medicine Library, and identifies some of the underlying principles, components, and capabilities of this system from the vantage point of operational experience over one year. PMID:6896835

  16. THE FAD2 GENE FAMILY OF SOYBEAN:INSIGHTS INTO THE STRUCTURAL AND FUNCTIONAL DIVERGENCE OF A PALEOPOLYPLOID GENOME

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The omega-6 fatty acid desaturase (FAD2) gene family in soybean consists of at least five members in four regions of the genome. These desaturases are responsible for the conversion of oleic acid to linoleic acid. Bacterial artificial chromosomes (BACs) corresponding to these loci were sequenced to ...

  17. Public Libraries

    ERIC Educational Resources Information Center

    Library Journal, 1970

    1970-01-01

    Building data is given for the following public libraries: (1) San Jose, California (2) Davenport, Iowa; (3) Louisville, Kentucky; (4) Gainesville, Georgia; (5) New Albany-Floyd County, Indiana and (6) Uniontown, Pennsylvania. (MF)

  18. 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;…

  19. TcruziDB: an integrated Trypanosoma cruzi genome resource.

    PubMed

    Luchtan, Michael; Warade, Chetna; Weatherly, D Brent; Degrave, Wim M; Tarleton, Rick L; Kissinger, Jessica C

    2004-01-01

    TcruziDB (http://TcruziDB.org) is an integrated genome database for the parasitic organism Trypanosoma cruzi, the causative agent of Chagas' disease. The database currently incorporates all available sequence data (Genomic, BAC, EST) in a single user-friendly location. The database contains a variety of tools specifically designed for searching unannotated draft sequence via BLAST, keyword searches of pre-computed BLAST results, and protein motif searches. Release 1.0 of the database contains nearly 730 million bp of genome sequence from 1.1 million sequence reads generated by the TIGR-Karolinska-SBRI Trypanosoma cruzi Genome Consortium and 15 million bp of clustered EST and genomic sequence obtained from other sources. As annotation, microarray and proteomic data become available, the database will incorporate and integrate these data using the GUS (http://www.gusdb. org) relational framework. PMID:14681430

  20. 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…

  1. The Construction of a Gossypium AD-genome-wide Comprehensive Reference Map Based on Diverse Data Resources 

    E-print Network

    Yu, Jing

    2010-07-14

    Yu Head of Department, David D. Baltensperger May 2009 Major Subject: Plant Breeding iii ABSTRACT The Construction of a Gossypium AD-genome-wide Comprehensive Reference Map Based on Diverse Data Resources. (May 2009) Jing Yu, B... At or Dt A or D subgenome of the tetraploid AD genome BAC Bacterial Artificial Chromosome CMap Comparative Map Viewer DAG Directed Acyclic Graph EST Expressed Sequence Tag FISH Fluorescence In Situ Hybridization NP Nondeterministic Polynomial time...

  2. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer.

    PubMed

    Wu, Zhenfang; Xu, Zhiqian; Zou, Xian; Zeng, Fang; Shi, Junsong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Li, Zicong

    2013-12-01

    The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT. PMID:23857557

  3. SFU LIBRARY The SFU Library serves

    E-print Network

    SFU LIBRARY HIGHLIGHTS 2013-2014 #12;The SFU Library serves the University through the W.A.C. Bennett Library (Burnaby), the Fraser Valley Real Estate Board Library (Surrey) and the Samuel and Frances Belzberg Library (Vancouver), as well as through extensive online resources and services. Guided

  4. Combined BAC and MIEX pre-treatment of secondary wastewater effluent to reduce fouling of nanofiltration membranes.

    PubMed

    Aryal, Ashok; Sathasivan, Arumugam; Heitz, Anna; Zheng, Gang; Nikraz, Hamid; Ginige, Maneesha P

    2015-03-01

    Biological activated carbon (BAC) and magnetic ion exchange resin (MIEX) were used to pre-treat secondary wastewater effluent (SWWE) and assessed for their capacity to reduce fouling of a nanofiltration membrane. BAC pre-treated water facilitated a lower but a steady flux while MIEX treated water resulted in a higher but a rapidly declining flux. Their combined use increased average flux from 58 to 89%. MIEX combined with BAC, in that order, was superior in reducing membrane fouling. Measurement of average Stokes radius (m) and apparent molecular weight distribution of dissolved organic matter (DOM), by nuclear magnetic resonance (NMR) and liquid chromatography organic carbon detection (LC-OCD), respectively, revealed that the microbial activity of BAC changed the nature of organic matter, probably by increasing the size of DOM molecules. BAC generally decreased the lower apparent molecular weight (LMW) fraction of dissolved organic carbon (DOC). Hence, the removal of LMW DOC and an increase of average Stokes radius (m) of DOM appeared to be important in facilitating a longer steady flux. Specifically, the combined MIEX/BAC pre-treatments appeared to target and reduce the foulants in SWWE that are largely responsible for the reduction of flux in nanofiltration membranes. PMID:25540835

  5. [Construction of a general piggyBac transposon inducible cell immortalization vector and verification of its basic properties].

    PubMed

    Huang, Hui; Hu, Guangdong; Kang, Jian; Qing, Suzhu; Zhang, Yong

    2014-08-01

    In order to construct generally efficient cell immortalization vector, pTP-hTERT, we modified the traditional piggyBac (PB) transposon using artificial synthesis, PCR and enzyme digestion. The modified vector contained the necessary transposon elements, a PB transposase expression cassette, a co-expression selectable element and a human telomerase reverse transcriptase (hTERT) expression cassette. The co-expression selectable element had two markers, enhanced green fluorescent protein (EGFP) gene and puromycin-resistance (Puro) gene, linked by porcine teschovirus-1 2A peptide (P2A). To validate the functionality of vector elements, we transfected pTP-hTERT into HEK293 cell, selected the positive cell clones and then conducted RT-PCR, Western blotting (WB) and Tail-PCR, methylene blue staining and statistic analysis on selected cells. The results of sequencing and cell culture show that the pTP-hTERT was constructed successfully and the positive cell could be selected by puromycin. The WB results, P2A cutting EGFP and Puro fusion protein with high efficiency, reflected the selectable element worked. The sequencing result of Tail-PCR confirmed the vector integrated into the genome through transposition. The results of methylene blue staining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERT significantly increased (P < 0.01) compared with control group. The construction of pTP-hTERT provides an efficient tool for establishing immortalized cell lines and a demonstration for building other eukaryotic plasmids. PMID:25507471

  6. [Construction of a general piggyBac transposon inducible cell immortalization vector and verification of its basic properties].

    PubMed

    Huang, Hui; Hu, Guangdong; Kang, Jian; Qing, Suzhu; Zhang, Yong

    2014-08-01

    In order to construct generally efficient cell immortalization vector, pTP-hTERT, we modified the traditional piggyBac (PB) transposon using artificial synthesis, PCR and enzyme digestion. The modified vector contained the necessary transposon elements, a PB transposase expression cassette, a co-expression selectable element and a human telomerase reverse transcriptase (hTERT) expression cassette. The co-expression selectable element had two markers, enhanced green fluorescent protein (EGFP) gene and puromycin-resistance (Puro) gene, linked by porcine teschovirus-1 2A peptide (P2A). To validate the functionality of vector elements, we transfected pTP-hTERT into HEK293 cell, selected the positive cell clones and then conducted RT-PCR, Western blotting (WB) and Tail-PCR, methylene blue staining and statistic analysis on selected cells. The results of sequencing and cell culture show that the pTP-hTERT was constructed successfully and the positive cell could be selected by puromycin. The WB results, P2A cutting EGFP and Puro fusion protein with high efficiency, reflected the selectable element worked. The sequencing result of Tail-PCR confirmed the vector integrated into the genome through transposition. The results of methylene blue staining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERT significantly increased (P < 0.01) compared with control group. The construction of pTP-hTERT provides an efficient tool for establishing immortalized cell lines and a demonstration for building other eukaryotic plasmids. PMID:25423748

  7. Cloning of equine herpesvirus type 1 438/77 strain genome as a bacterial artificial chromosome.

    PubMed

    Sun, Xueqiang; Yao, Huochun; Zhang, Cun; Lu, Chengping

    2011-01-01

    Equine herpesvirus type 1 (EHV-1) is a major cause of respiratory and reproductive diseases in horses worldwide. The genome of EHV-1 strain 438/77 (isolated from an aborted equine fetus) was cloned as a bacterial artificial chromosome (BAC) in E. coli without any gene deletions. The mini-F plasmid sequence was inserted in the middle of ORF19 and 20 via homologous recombination following co-transfection of viral DNA and plasmid pE19_20/HA into RK13 cells. Circular viral DNA was extracted from RK13 cells infected with purified recombinant virus expressing green fluorescent protein (GFP) and electrophorated into E. coli DH10B cells. The clone harboring the BAC was screened and analyzed by PCR and RFLP. Reconstitution of the recombinant virus was achieved successfully by transfection of the BAC DNA into RK13 cells. The mini-F sequence in the reconstituted virus was subsequently removed by homologous recombination between virus DNA and plasmid pE1920XM, inducing a point mutation in the Xbal site in ORF19. Comparison of RFLP profiles of the rescued, recovered and the wild-type viral genome demonstrated that no unexpected changes occurred during mutagenesis. In vitro replication assays showed that BAC-reconstituted virus mutant growth kinetics and plaque formation morphology/size were indistinguishable to those measured for wild-type virus. PMID:22059290

  8. QU Library http://library.queensu.ca Education Library http://library,queensu.ca/webedu/ January 2015 Your Education Library

    E-print Network

    Fletcher, Robin

    Keyword Anywhere Location: Education Library Children's & Young Adult Books Find the books you want: QULQU Library http://library.queensu.ca Education Library http://library,queensu.ca/webedu/ January 2015 Your Education Library To find your Education Library "teacher" passwords go to http

  9. 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…

  10. Modification of the GS LT Paired-end Library Protocol for Constructing Longer Insert Size Libraries

    SciTech Connect

    Peng, Ze; Peng, Ze; Hamilton, Matthew; Ting, Sara; Tu, Hank; Goltsman, Eugene; Lapidus, Alla; Lucas, Susan; Cheng, Jan-Fang

    2008-05-22

    Paired-end library sequencing has been proven useful in scaffold construction during de novo assembly of genomic sequences. The ability of generating mate pairs with 8 Kb or greater insert sizes is especially important for genomes containing long repeats. While the current 454 GS LT Paired-end library preparation protocol can successfully construct libraries with 3 Kb insert size, it fails to generate longer insert sizes because the protocol is optimized to purify shorter fragments. We have made several changes in the protocol in order to increase the fragment length. These changes include the use of Promega column to increase the yield of large size DNA fragments, two gel purification steps to remove contaminated short fragments, and a large reaction volume in the circularization step to decrease the formation of chimeras. We have also made additional changes in the protocol to increase the overall quality of the libraries. The quality of the libraries are measured by a set of metrics, which include levels of redundant reads, linker positive, linker negative, half linker reads, and driver DNA contamination, and read length distribution, were used to measure the primary quality of these libraries. We have also assessed the quality of the resulted mate pairs including levels of chimera, distribution of insert sizes, and genome coverage after the assemblies are completed. Our data indicated that all these changes have improved the quality of the longer insert size libraries.

  11. Virtual Library on Genetics from Oak Ridge National Laboratory

    DOE Data Explorer

    The World Wide Web (WWW) Virtual Library is a collaborative effort to provide topic indices that break down into many subtopics guiding users to vast resources of information around the world. ORNL hosts the Virtual Library on Genetics as part of the WWWVL's Biosciences topic area. The VL on Genetics is also a collection of links to information resources that supported the DOE Human Genome Project. That project has now evolved into Genomics: GTL. GTL is DOE's next step in genomics--builds on data and resources from the Human Genome Project, the Microbial Genome Program, and systems biology. GTL will accelerate understanding of dynamic living systems for solutions to DOE mission challenges in energy and the environment. The section of the Virtual Library on Genetics that is titled Organisms guides users to genetic information resources and gene sequences for animals, insects, microbes, and plant life.

  12. An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: The Adh region

    SciTech Connect

    Ashburner, M.; Misra, S.; Roote, J.; Lewis, S.E.; Blazej, R.; Davis, T.; Doyle, C.; Galle, R.; George, R.; Harris, N.; Hartzell, G.; Harvey, D.; Hong, L.; Houston, K.; Hoskins, R.; Johnson, G.; Martin, C.; Moshrefi, A.; Palazzolo, M.; Reese, M.G.; Spradling, A.; Tsang, G.; Wan, K.; Whitelaw, K.; Kimmel, B.; Celniker, S.; Rubin, G.M.

    1999-03-24

    A contiguous sequence of nearly 3 Mb from the genome of Drosophila melanogaster has been sequenced from a series of overlapping P1 and BAC clones. This region covers 69 chromosome polytene bands on chromosome arm 2L, including the genetically well-characterized

  13. GENOMIC ANALYSIS OF A 1 MB REGION NEAR THE TELOMERE OF HESSIAN FLY CHROMOSOME X2 AND AVIRULENCE GENE VH13

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosome walking and FISH were utilized to identify a contig of 50 BAC clones near the telomere of the short arm of Hessian fly chromosome X2 and near the avirulence gene vH13. These clones enabled us to correlate physical and genetic distance in this region of the Hessian fly genome. Sequence da...

  14. Pilot Sequencing of Onion Genomic DNA Reveals Fragments of Transposable Elements, Low Gene Densities, and Significant Gene Enrichment After Methyl Filtration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Onion (Allium cepa) is a diploid (2n=2x=16) monocot with one of the largest nuclear genomes among cultivated plants, over 6 and 16 times that of maize and rice, respectively. In this study, we sequenced onion BACs to estimate gene densities and investigate the nature and distribution of repetitive ...

  15. Articledoi:10.1006/geno.2001.6616, available online at http://www.idealibrary.com on IDEAL INTRODUCTION

    E-print Network

    Cross, George

    sequences of bacte- ria, yeast, fruit fly, worm (Caenorhabditis elegans), mouse, and human genomes chromo- some (BAC) cloning system and the construction of repre- sentative genomic BAC libraries [1 of random clones. Furthermore, the routine gene-cloning strategy cannot be applied to the study

  16. Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified approximately 13000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel ...

  17. Association Between Pachytene Chromosomes and Linkage Groups in Carrot

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The genome of carrot (Daucus carota L.) consists of ~ 480 Mb/1C organized in 9 chromosome pairs. The importance of carrots in human nutrition is triggering the development of genomic resources, including carrot linkage maps, a bacterial artificial chromosome (BAC) clone library and BAC end sequence...

  18. Human Cytomegalovirus pUL93 Is Required for Viral Genome Cleavage and Packaging.

    PubMed

    DeRussy, Bernadette M; Tandon, Ritesh

    2015-12-01

    Human cytomegalovirus (HCMV) pUL93 is essential for virus growth, but its precise function in the virus life cycle is unknown. Here, we characterize a UL93 stop mutant virus (UL93st-TB40/E-BAC) to demonstrate that the absence of this protein does not restrict viral gene expression; however, cleavage of viral DNA into unit-length genomes as well as genome packaging is abolished. Thus, pUL93 is required for viral genome cleavage and packaging. PMID:26401033

  19. New Zealand Libraries.

    ERIC Educational Resources Information Center

    Woodhouse, Helen; And Others

    1993-01-01

    Special section on libraries in New Zealand addresses the implications of the two cultures (i.e., Maori and British), public library service, innovation in teaching library and information studies, library services for Maori people, library services to schools, services to children, and key strategic issues facing the library and information…

  20. Oregon: Library Automation Developments.

    ERIC Educational Resources Information Center

    Brandis, Rushton

    1996-01-01

    Discusses Oregon library automation projects, including Internet connectivity and a statewide multitype library network; a bibliographic information system with college and university libraries, including a union catalog; a Portland Area Library System that connects multitype libraries; and library staff training for the Internet. (LRW)

  1. PUBLICATIONS LIBRARY INFORMATION

    E-print Network

    ) is an integrated library system available on the Internet at www.lib.noaa.gov. NOAALINC represents the libraryPUBLICATIONS LIBRARY INFORMATION The NOAA Library and Information Network Catalog (NOAALINC holdings of the NOAA Central Library in Silver Spring, Maryland and 27 other NOAA libraries located

  2. Library Automation in Pakistan.

    ERIC Educational Resources Information Center

    Haider, Syed Jalaluddin

    1998-01-01

    Examines the state of library automation in Pakistan. Discusses early developments; financial support by the Netherlands Library Development Project (Pakistan); lack of automated systems in college/university and public libraries; usage by specialist libraries; efforts by private-sector libraries and the National Library in Pakistan; commonly used…

  3. 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)

  4. Bortezomib for Patients with Advanced Stage Bronchioloalveolar Carcinoma (BAC): A California Cancer Consortium Phase II Study (NCI # 7003)

    PubMed Central

    Ramalingam, Suresh S.; Davies, Angela M.; Longmate, Jeffrey; Edelman, Martin J.; Lara, Primo N.; Vokes, Everett E.; Villalona-Calero, Miguel; Gitlitz, Barbara; Reckamp, Karen; Salgia, Ravi; Wright, John J.; Belani, Chandra P.; Gandara, David R.

    2011-01-01

    Background Bronchioloalveolar carcinoma (BAC), a subtype of non-small cell lung cancer (NSCLC), is a difficult disease to treat with low response rates with cytotoxic chemotherapy. Bortezomib, a proteasome inhibitor, has demonstrated objective responses in BAC patients in early phase clinical trials. We conducted a phase II study of bortezomib inpatients with advanced stage BAC. Methods Patients with advanced BAC, adenocarcinoma with BAC features or BAC with adenocarcinoma features and less than two prior regimens were eligible. Prior epidermal growth factor receptor (EGFR) inhibitor therapy was allowed. Bortezomib was administered intravenously at 1.6 mg/m2 on days 1 and 8 of every 21 days cycle until disease progression or unacceptable toxicity. The primary endpoint was response rate. The Simon two-stage design was utilized. Results Forty-two patients were enrolled and the study was halted early for slow accrual. Patient characteristics were: female 55%, median age 68 years, and ECOG performance status of 0 and 1 in 31 and 11 patients respectively. Twenty-six(62%)patients had received prior therapy with an EGFR inhibitor. A median of 4 cycles of therapy were administered. Objective responses were noted in 5% while 57% had disease stabilization. The median progression-free survival and overall survival were 5.5 months and 13.6 months respectively. Grade 3 diarrhea and fatigue were noted in 3 and 5 patients respectively. Conclusions Bortezomib is tolerated well and is associated with modest anti-cancer activity in advanced BAC, including inpatients that progressed on EGFR inhibitor therapy. PMID:21716143

  5. Computational Molecular Biology Biochem 218 BioMedical Informatics 231

    E-print Network

    .edu/ Doug Brutlag Professor Emeritus Biochemistry & Medicine (by courtesy) The Human Genome Project #12 Gibson & Muse, A Primer of Genome Science http://www.sinauer.com/genomics/ #12;The Human Genome Project?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=3294162 #12;Public Genome Assembly Process #12;BAC and PAC Libraries in Public Human Genome Project http

  6. LIBRARY GUIDE WWW.LIBRARY.OKSTATE.EDU

    E-print Network

    LIBRARY GUIDE WWW.LIBRARY.OKSTATE.EDU GET HELP Text: 405-592-4128 facebook.com/OkStateLibrary Call) ____________________ (Date) #12;GET HELP Text: 405-592-4128 facebook.com/OkStateLibrary Call: 405-744-9775 twitter

  7. Library Use, Library Instruction, and User Success.

    ERIC Educational Resources Information Center

    Shapiro, Beth J.; Marcus, Philip M.

    1987-01-01

    Describes the methodology and findings of a study that examined the reasons that people use academic libraries and the relationship between library use, library instruction, and user success. The conclusions identify library services and physical facilities that may increase user satisfaction. (Author/CLB)

  8. A galectin related protein from Oplegnathus fasciatus: Genomic, molecular, transcriptional features and biological responses against microbial pathogens.

    PubMed

    Thulasitha, William Shanthakumar; Whang, Ilson; Umasuthan, Navaneethaiyer; Kang, Hyun-Sil; Mothishri, M S; Lee, Seongdo; Qiang, Wan; Noh, Jae Koo; Lee, Jehee

    2016-03-01

    Galectins, a family of ?-galactoside-binding lectins, are pattern recognition receptors that recognize pathogen-associated molecular patterns and are subsequently involved in the opsonization, phagocytosis, complement activation, and killing of microbes. Here, we report a novel galectin related protein (GRP) identified from rock bream (Oplegnathus fasciatus), designated OfGal like B. The cDNA of OfGal like B is 517 bp with an open reading frame (ORF) of 438 bp, encoding 145 amino acids, with a single carbohydrate recognition domain (CRD). However, only two of the seven critical residues responsible for carbohydrate recognition were identified in the CRD. There was no signal peptide identified in the OfGal like B protein. The genomic structure of OfGal like B, determined using a bacterial artificial chromosome (BAC) genomic library, consists of four exons and three introns. Homology assessment, multiple sequence alignment, and phylogenetic analysis indicated that OfGal like B is an evolutionarily conserved lectin that is closely related to the proto-type galectins. OfGal like B mRNA was constitutively expressed in a wide range of tissues in healthy rock breams. When challenged with bacterial or viral stimulants, OfGal like B was up-regulated in the gills and spleen of rock breams, indicating that it likely plays an important role during bacterial and viral infections. Furthermore, recombinant OfGal like B (rOfGal like B) lacked carbohydrate-binding activity but was able to recognize and agglutinate bacteria, including Streptococcus iniae, Listeria monocytogenes, Vibrio tapetis, Escherichia coli, and Edwardsiella tarda, and a ciliate parasite, Miamiensis avidus. These results collectively suggest that OfGal like B is involved in pathogen recognition and plays a significant role(s) in the innate defense mechanism of rock bream. PMID:26615008

  9. Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

    PubMed Central

    Schmouth, Jean-François; Bonaguro, Russell J.; Corso-Diaz, Ximena; Simpson, Elizabeth M.

    2012-01-01

    An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and ?-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation. PMID:22396661

  10. Scaling up the 454 Titanium Library Construction and Pooling of Barcoded Libraries

    SciTech Connect

    Phung, Wilson; Hack, Christopher; Shapiro, Harris; Lucas, Susan; Cheng, Jan-Fang

    2009-03-23

    We have been developing a high throughput 454 library construction process at the Joint Genome Institute to meet the needs of de novo sequencing a large number of microbial and eukaryote genomes, EST, and metagenome projects. We have been focusing efforts in three areas: (1) modifying the current process to allow the construction of 454 standard libraries on a 96-well format; (2) developing a robotic platform to perform the 454 library construction; and (3) designing molecular barcodes to allow pooling and sorting of many different samples. In the development of a high throughput process to scale up the number of libraries by adapting the process to a 96-well plate format, the key process change involves the replacement of gel electrophoresis for size selection with Solid Phase Reversible Immobilization (SPRI) beads. Although the standard deviation of the insert sizes increases, the overall quality sequence and distribution of the reads in the genome has not changed. The manual process of constructing 454 shotgun libraries on 96-well plates is a time-consuming, labor-intensive, and ergonomically hazardous process; we have been experimenting to program a BioMek robot to perform the library construction. This will not only enable library construction to be completed in a single day, but will also minimize any ergonomic risk. In addition, we have implemented a set of molecular barcodes (AKA Multiple Identifiers or MID) and a pooling process that allows us to sequence many targets simultaneously. Here we will present the testing of pooling a set of selected fosmids derived from the endomycorrhizal fungus Glomus intraradices. By combining the robotic library construction process and the use of molecular barcodes, it is now possible to sequence hundreds of fosmids that represent a minimal tiling path of this genome. Here we present the progress and the challenges of developing these scaled-up processes.

  11. Toward positional cloning of everblooming gene (evb) in plants: a BAC library of Rosa chinensis cv. old blush 

    E-print Network

    Hess, Gregory

    2006-10-30

    flowering mutation named everblooming (evb). The mutation is recessive to once blooming and is found in the rose species Rosa chinensis. Although several molecular maps have been developed for rose, little is known about the evb gene, except for its classic...

  12. CONSTRUCTION OF A RYE CV. BLANCO BAC LIBRARY, AND PROGRESS TOWARDS CLONING THE RYE ALT3 ALUMINUM TOLERANCE GENE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In addition to being an important cereal crop, rye (Secale cereale L.) provides valuable traits for other crops, as a parent of the amphidiploid triticale, and as a donor of translocated chromosome segments in wheat. Rye possesses excellent tolerance to many biotic and abiotic stresses that could p...

  13. Construction of a Rye BAC Library for Utilization in Physical Mapping of an Aluminum Tolerance Gene Complex

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminium, the most abundant metal on earth, is highly toxic to plant growth and is found in about 2.5 billion hectares of acid soils, including more than 130 million hectares in the United States. Many of the world’s farmers are living on marginal soils that offer a stressful environment for plant ...

  14. Mapping of genes using a bovine BAC Library to determine their effects on economically important traits in cattle 

    E-print Network

    Herring, Kimberly Lynn

    1995-01-01

    and height (WWT and "T), slaughter weight and height (SWT and SLHT), hot carcass weight (HCWT), rib-eye area (REA), kidneypelvic-heart fat (KPH), adjusted fat thickness (ADJ), marbling (MARB) and quality and yield grade (QG and YG). The GH microsatellite...

  15. SCALEs: multiscale analysis of library enrichment Michael D Lynch, Tanya Warnecke & Ryan T Gill

    E-print Network

    Cai, Long

    growth selections on mix- tures of several plasmid libraries containing defined, yet different, insert of the different, distinct-sized libraries (Fig. 1). We hypothesized that selections performed on such mixed region of genomic DNA (that is, an operon), enrichment would occur only in those libraries containing

  16. Initial and Continuous Commissioning of Building Automation and Control Systems (BACS) -Preview EN ISO 16484- 

    E-print Network

    Kranz, H. R.

    2008-01-01

    stream_source_info ESL-IC-08-10-65.pdf.txt stream_content_type text/plain stream_size 3954 Content-Encoding ISO-8859-1 stream_name ESL-IC-08-10-65.pdf.txt Content-Type text/plain; charset=ISO-8859-1 8th International... 21 hans@kranz.com INITIAL AND CONTINUOUS COMMISSIONING OF BUILDING AUTOMATION AND CONTROL SYSTEMS (BACS) - PREVIEW EN ISO 16484 - Did you ever think about: ?why are our buildings so dumb?? The simple answer might be: because we have a...

  17. New Genomic Resources for Orchardgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One of the initial requirements of utilizing genomic approaches in plant improvement is the availability of DNA sequence information. Toward the goal of generating sequence information for forage and pasture grasses, we are developing an EST library from orchardgrass, or cocksfoot (Dactylis glomera...

  18. Cell Biology of Genomes Group

    Cancer.gov

    Contact Us Dr. Tom Misteli Email: mistelit@mail.nih.gov Tel: 301.402.3959 Fax: 301.496.4951 Address: Cell Biology of Genomes Group Laboratory of Receptor Biology and Gene Expression National Cancer Institute Building 41, Room B610 41 Library Drive Bethesd

  19. Baculovirus Superinfection: A Probable Restriction Factor on the Surface Display of Proteins for Library Screening

    PubMed Central

    Xu, Xiaodong; Chen, Yuanrong; Zhao, Yu; Liu, Xiaofen; Dong, Beitao; Jones, Ian M.; Chen, Hongying

    2013-01-01

    In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells co-expressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the re-infection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides. PMID:23365677

  20. Integrated Genomic Characterization of Endometrial Carcinoma - Douglas Levine, TCGA Scientific Symposium 2012

    Cancer.gov

    Home News and Events Multimedia Library Videos Integrated Genomic Characterization of Endometrial Carcinoma - Douglas Levine Integrated Genomic Characterization of Endometrial Carcinoma - Douglas Levine, TCGA Scientific Symposium 2012 You will need

  1. Integrated Genomic Characterization of Papillary Thyroid Carcinoma - Thomas J. Giordano, TCGA Scientific Symposium 2014

    Cancer.gov

    Home News and Events Multimedia Library Videos Integrated Genomic Characterization of Papillary Thyroid Carcinoma - Thomas J. Giordano Integrated Genomic Characterization of Papillary Thyroid Carcinoma - Thomas J. Giordano, TCGA Scientific Symposium

  2. The Somatic Genomic Landscape of Glioblastoma Multiforme - Roel Verhaak, TCGA Scientific Symposium 2012

    Cancer.gov

    Home News and Events Multimedia Library Videos The Somatic Genomic Landscape of Glioblastoma Multiforme - Roel Verhaak The Somatic Genomic Landscape of Glioblastoma Multiforme - Roel Verhaak, TCGA Scientific Symposium 2012 You will need Adobe Flash

  3. Correlating Protein Phosphorylation With Genomic Alterations in Cancer - Jianjiong Gao, TCGA Scientific Symposium 2011

    Cancer.gov

    Home News and Events Multimedia Library Videos Correlating Protein Phosphorylation With Genomic Alterations in Cancer - Jianjiong Gao Correlating Protein Phosphorylation With Genomic Alterations in Cancer - Jianjiong Gao, TCGA Scientific Symposium

  4. Lessons Learned From 24 Completely Sequenced AML Genomes - Timothy Ley, TCGA Scientific Symposium 2011

    Cancer.gov

    Home News and Events Multimedia Library Videos Lessons Learned From 24 Completely Sequenced AML Genomes - Timothy Ley Lessons Learned From 24 Completely Sequenced AML Genomes - Timothy Ley, TCGA Scientific Symposium 2011 You will need Adobe Flash

  5. Comprehensive and Integrative Genomic Characterization of Diffuse Lower Grade Gliomas - Daniel Brat, TCGA Scientific Symposium 2014

    Cancer.gov

    Home News and Events Multimedia Library Videos Comprehensive and Integrative Genomic Characterization of Diffuse Lower Grade Gliomas - Daniel Brat Comprehensive and Integrative Genomic Characterization of Diffuse Lower Grade Gliomas - Daniel Brat,

  6. Genomic Characterization of Invasive Lobular Breast Carcinoma - Michael L. Gatza, TCGA Scientific Symposium 2014

    Cancer.gov

    Home News and Events Multimedia Library Videos Genomic Characterization of Invasive Lobular Breast Carcinoma - Michael L. Gatza Genomic Characterization of Invasive Lobular Breast Carcinoma - Michael L. Gatza, TCGA Scientific Symposium 2014 You will

  7. Multi-Cancer Mutual Exclusivity Analysis of Genomic Alterations - Giovanni Ciriello, TCGA Scientific Symposium 2011

    Cancer.gov

    Home News and Events Multimedia Library Videos Multi-Cancer Mutual Exclusivity Analysis of Genomic Alterations - Giovanni Ciriello Multi-Cancer Mutual Exclusivity Analysis of Genomic Alterations - Giovanni Ciriello, TCGA Scientific Symposium 2011 You

  8. Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma - Tom Giordano, TCGA Scientific Symposium 2015

    Cancer.gov

    Home News and Events Multimedia Library Videos Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma - Tom Giordano, TCGA Scientif Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma - Tom Giordano, TCGA Scientific

  9. Individual Patient Cancer Profiles in The Cancer Genome Atlas - Jianjiong Gao, TCGA Scientific Symposium 2012

    Cancer.gov

    Home News and Events Multimedia Library Videos Individual Patient Cancer Profiles in The Cancer Genome Atlas - Jianjiong Gao Individual Patient Cancer Profiles in The Cancer Genome Atlas - Jianjiong Gao, TCGA Scientific Symposium 2012 You will need

  10. Comprehensive Genomic Characterization of Cutaneous Melanoma - Ian R. Watson, TCGA Scientific Symposium 2014

    Cancer.gov

    Home News and Events Multimedia Library Videos Comprehensive Genomic Characterization of Cutaneous Melanoma - Ian R. Watson Comprehensive Genomic Characterization of Cutaneous Melanoma - Ian R. Watson, TCGA Scientific Symposium 2014 You will need

  11. Control of KirBac3.1 Potassium Channel Gating at the Interface between Cytoplasmic Domains*

    PubMed Central

    Zubcevic, Lejla; Bavro, Vassiliy N.; Muniz, Joao R. C.; Schmidt, Matthias R.; Wang, Shizhen; De Zorzi, Rita; Venien-Bryan, Catherine; Sansom, Mark S. P.; Nichols, Colin G.; Tucker, Stephen J.

    2014-01-01

    KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating. PMID:24257749

  12. 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.

  13. 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…

  14. West Virginia: Library Automation.

    ERIC Educational Resources Information Center

    Brown, Thomas M., Ed.

    1996-01-01

    Reviews library automation efforts in West Virginia. Topics include information equity and rural areas, library systems and resource sharing, statewide connectivity, a higher education telecomputing network that has expanded to include elementary and secondary school libraries, multitype library cooperation and staff training, and academic library

  15. PUBLICATIONS LIBRARY INFORMATION

    E-print Network

    ) resides on Stilas, an integrated library system provided by the Sirsi Corporation. NOAALINC representsPUBLICATIONS LIBRARY INFORMATION The NOAA Library and Information Network Catalog (NOAALINC the automated holdings of the NOAA Central Library in Silver Spring, Maryland and 22 other NOAA libraries

  16. University Library Operating figures

    E-print Network

    Reyle, Uwe

    Berlin library system, comprising TU University Library and further departmental libraries, supports allUniversity Library Operating figures · Printed Volumes: 1.5 million · E-Books: 118,000 · Printed to 360 days per year at the major branch libraries · Budget: 4,717,431 Euro · Staff: 101.5 FTE Profile

  17. Library Buildings Section. Papers.

    ERIC Educational Resources Information Center

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

    Papers on library architecture, which were presented at the 1982 International Federation of Library Associations (IFLA) conference focus on the effect of library networks on library design. Topics include: (1) "Some Problems in Designing of the University Library Buildings in China: A Developing Country University Librarian's View Based on His…

  18. Efficacy of a BAC clone of a recombinant strain of Marek’s disease virus containing reticuloendotheliosis virus LTR following in ovo Vaccination at 18 days of embryonation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously reported on the pathogenicity of various passage levels of a bacterial artificial chromosome (BAC) clone of a recombinant Marek’s disease virus (MDV) strain rMd5 containing reticuloendotheliosis virus (REV) long terminal repeat (LTR) termed rMd5 REV LTR BAC. In this study, we eval...

  19. Controlled Clinical Comparison of Plastic versus Glass Bottles of BacT/ALERT PF Medium for Culturing Blood from Children

    PubMed Central

    Petti, Cathy A.; Mirrett, Stanley; Woods, Christopher W.; Reller, L. Barth

    2005-01-01

    The plastic pediatric BacT/ALERT (bioMérieux, Durham, N.C.) PF (PPF) is a new nonvented aerobic culture medium in a clear plastic bottle designed to prevent breakage. We compared the performance of the new PPF bottle to that of the present glass BacT/ALERT PF bottle for the recovery of microorganisms as well as for the time to detection of growth in samples of blood obtained for culture from children. We found that the PPF and PF bottles were comparable for recovery of microorganisms and that the safety advantage of plastic bottles can be achieved without compromising performance. PMID:15635011

  20. piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens

    PubMed Central

    Park, Tae Sub; Han, Jae Yong

    2012-01-01

    Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line–competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBac transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications. PMID:22645326

  1. Deciphering the genomic structure, function and evolution of carotenogenesis related phytoene synthases in grasses

    PubMed Central

    2012-01-01

    Background Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY) plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY1-3) that have been widely characterized in rice, maize and sorghum. Results In wheat, for which yellow pigment content is extremely important for flour colour, only PSY1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY2 has been partially characterized. Here, we report the isolation of bread wheat PSY3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA), since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY3 during abiotic stress. Conclusions We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses. PMID:22672222

  2. The Libraries in 2005 Smithsonian Institution Libraries 2005 annual report

    E-print Network

    Miller, Scott

    The Libraries in 2005 Smithsonian Institution Libraries 2005 annual report Knowledge Lives Here #12;Smithsonian Institution Libraries Annual Report 2005 The Smithsonian Libraries adopted this strong declaration resides in the Smithsonian's library collections, which supply the curatorial and scientific staff

  3. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

    PubMed

    Tamura, T; Thibert, C; Royer, C; Kanda, T; Abraham, E; Kamba, M; Komoto, N; Thomas, J L; Mauchamp, B; Chavancy, G; Shirk, P; Fraser, M; Prudhomme, J C; Couble, P; Toshiki, T; Chantal, T; Corinne, R; Toshio, K; Eappen, A; Mari, K; Natuo, K; Jean-Luc, T; Bernard, M; Gérard, C; Paul, S; Malcolm, F; Jean-Claude, P; Pierre, C

    2000-01-01

    We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications. PMID:10625397

  4. Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in

    E-print Network

    Monteiro, Antónia

    and Molecular Biology, Chongqing Normal University, Chongqing, People's Republic of China, 3 Department transformation by the piggyBac transposable element. Pogostick can be found at www. addgene.org, a non-profit, and partially supported by the National Natural Science Foundation of China (Grant No. 30870340

  5. Tuberculosis (TB) is caused by gram-positive bac-teria known as the Mycobacterium tuberculosis complex

    E-print Network

    Tuberculosis (TB) is caused by gram-positive bac- teria known as the Mycobacterium tuberculosis and, more rarely, wild animal species. We report an M. tuberculosis strain isolated from a wild of the biology and evolutionary history of this widespread infectious disease. Tuberculosis (TB) is caused

  6. Monochloramine disinfection kinetics of Nitrosomonas europaea by propidium monoazide quantitative PCR and Live/Dead BacLight Methods

    EPA Science Inventory

    Monochloramine disinfection kinetics were determined for the pure culture ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) by two culture independent methods: (1) LIVE/DEAD® BacLight™ (LD) and (2) propidium monoazide quantitative PCR (PMA-qPCR). Both methods were f...

  7. Development of a Set of Chromosome-Specific Cytogenetic DNA Markers in Sunflower Using BAC-FISH

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In diploid sunflower (2n=34), conventional karyotypes and various genetic linkage maps have been established. However, the relationship between genetic linkage groups and individual chromosomes of sunflower remains unknown. Recently, a set of linkage group-specific BAC and BIBAC clones were identifi...

  8. A draft physical map of a D-genome cotton species (Gossypium raimondii)

    PubMed Central

    2010-01-01

    Background Genetically anchored physical maps of large eukaryotic genomes have proven useful both for their intrinsic merit and as an adjunct to genome sequencing. Cultivated tetraploid cottons, Gossypium hirsutum and G. barbadense, share a common ancestor formed by a merger of the A and D genomes about 1-2 million years ago. Toward the long-term goal of characterizing the spectrum of diversity among cotton genomes, the worldwide cotton community has prioritized the D genome progenitor Gossypium raimondii for complete sequencing. Results A whole genome physical map of G. raimondii, the putative D genome ancestral species of tetraploid cottons was assembled, integrating genetically-anchored overgo hybridization probes, agarose based fingerprints and 'high information content fingerprinting' (HICF). A total of 13,662 BAC-end sequences and 2,828 DNA probes were used in genetically anchoring 1585 contigs to a cotton consensus genetic map, and 370 and 438 contigs, respectively to Arabidopsis thaliana (AT) and Vitis vinifera (VV) whole genome sequences. Conclusion Several lines of evidence suggest that the G. raimondii genome is comprised of two qualitatively different components. Much of the gene rich component is aligned to the Arabidopsis and Vitis vinifera genomes and shows promise for utilizing translational genomic approaches in understanding this important genome and its resident genes. The integrated genetic-physical map is of value both in assembling and validating a planned reference sequence. PMID:20569427

  9. The Release 6 reference sequence of the Drosophila melanogaster genome.

    PubMed

    Hoskins, Roger A; Carlson, Joseph W; Wan, Kenneth H; Park, Soo; Mendez, Ivonne; Galle, Samuel E; Booth, Benjamin W; Pfeiffer, Barret D; George, Reed A; Svirskas, Robert; Krzywinski, Martin; Schein, Jacqueline; Accardo, Maria Carmela; Damia, Elisabetta; Messina, Giovanni; Méndez-Lago, María; de Pablos, Beatriz; Demakova, Olga V; Andreyeva, Evgeniya N; Boldyreva, Lidiya V; Marra, Marco; Carvalho, A Bernardo; Dimitri, Patrizio; Villasante, Alfredo; Zhimulev, Igor F; Rubin, Gerald M; Karpen, Gary H; Celniker, Susan E

    2015-03-01

    Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads. PMID:25589440

  10. The Release 6 reference sequence of the Drosophila melanogaster genome

    PubMed Central

    Carlson, Joseph W.; Wan, Kenneth H.; Park, Soo; Mendez, Ivonne; Galle, Samuel E.; Booth, Benjamin W.; Pfeiffer, Barret D.; George, Reed A.; Svirskas, Robert; Krzywinski, Martin; Schein, Jacqueline; Accardo, Maria Carmela; Damia, Elisabetta; Messina, Giovanni; Méndez-Lago, María; de Pablos, Beatriz; Demakova, Olga V.; Andreyeva, Evgeniya N.; Boldyreva, Lidiya V.; Marra, Marco; Carvalho, A. Bernardo; Dimitri, Patrizio; Villasante, Alfredo; Zhimulev, Igor F.; Rubin, Gerald M.; Karpen, Gary H.

    2015-01-01

    Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads. PMID:25589440

  11. Libraries and the Environment.

    ERIC Educational Resources Information Center

    LaRue, James; And Others

    1991-01-01

    Three articles address issues that relate to libraries and the environment. Highlights include recycling projects; buying recycled paper products and other ecology-minded purchasing ideas; energy-efficient libraries; indoor pollution problems; a list of environmental information sources; designing library buildings; and activities that libraries

  12. The Library of Congress.

    ERIC Educational Resources Information Center

    Goodrum, Charles A.

    From its earliest beginnings to the post war information explosion, this text traces the history of the world's largest library, the Library of Congress (LC). The library's creation in 1800 for use by members of Congress is comprehensively discussed along with its development into a world wide service. Library methodology is covered in chapters on…

  13. Growing Competition for Libraries.

    ERIC Educational Resources Information Center

    Gibbons, Susan

    2001-01-01

    Describes the Questia subscription-based online academic digital books library. Highlights include weaknesses of the collection; what college students want from a library; importance of marketing; competition for traditional academic libraries that may help improve library services; and the ability of Questia to overcome barriers and…

  14. College and University Libraries.

    ERIC Educational Resources Information Center

    Shubert, Joseph F., Ed.; Josey, E. J., Ed.

    1986-01-01

    Following an introductory discussion by E. J. Josey that provides a perspective on college and university libraries, the following essays are presented: (1) "Academic Library Planning--Definitions and Early Planning Studies in Academic Libraries" (Stanton F. Biddle); (2) "Academic Libraries and Academic Computing--Rationale for a Modern Marriage"…

  15. Special Library Services.

    ERIC Educational Resources Information Center

    Ensley, Robert F., Ed.

    1975-01-01

    The September 1975 issue of Illinois Libraries focuses on the needs of the developmentally disabled, physically handicapped, and emotionally disturbed. Articles on library services to the blind and physically handicapped cover standards, services of local public libraries, Library of Congress programs, braille books and sound recordings,…

  16. Balfour (Zoology) Library Guide

    E-print Network

    to locate books across the university, using LibrarySearch 7 Classification of books in the Balfour LibraryBalfour (Zoology) Library Guide for Zoology postgraduate students #12;2 Contents Content Page number Introduction 4 History 4 Opening times 4 Out of hours access 4 Contact details 4 Library

  17. Balfour (Zoology) Library Guide

    E-print Network

    LibrarySearch 7 Classification of books in the Balfour Library 8 Loan periods 8 RegistrationBalfour (Zoology) Library Guide for NST Part II Zoology, Neuroscience and Biomedical and Biological of hours access 4 Contact details 4 Library facilities 5 Computers 5 Use of laptops and UniOfCam wireless

  18. HELMKE LIBRARY FUNDING CONCERNS

    E-print Network

    Hamburger, Peter

    of libraries within the IU regional campus system, IPFW library ranks last in total staff per student FTEHELMKE LIBRARY FUNDING CONCERNS AND RECOMMENDATIONS PREPARED BY CHERYL TRUESDELL, DEAN SUBMITTED BY IPFW SENATE LIBRARY SUBCOMMITTEE FEBRUARY 26, 2010 I P F W W A L T E R E . H E L M K E L I B R A R Y

  19. INTERCONTEXTUAL THEOLOGY LIBRARY INTRODUCTION

    E-print Network

    Gerhardy, Philipp

    loan system · a library catalogue, not a bibliography · a book and journal catalogue : printed booksINTERCONTEXTUAL THEOLOGY LIBRARY INTRODUCTION Svein Helge Birkeflet September 2014 #12;UBO AT BLINDERN - the most important academic library in Norway Humanities & Social Sciences Library / Main

  20. Helpful Websites Ebling Library

    E-print Network

    Bohnhoff, David

    Helpful Websites · Ebling Library o http://ebling.library.wisc.edu/ · Ebling Library NIH Public Access Policy Help o http://ebling.library.wisc.edu/help/nih.cfm · PubMed o http://www.pubmed.gov · NIH Public Access Homepage o http://publicaccess.nih.gov/ · NIH Manuscript Submission System o http

  1. Economics of Academic Libraries.

    ERIC Educational Resources Information Center

    Baumol, William J.; Marcus, Matityahu

    An analysis is conducted of economic issues pertinent to library planning in higher education in the face of rising costs and diminishing financial support. The individual chapters deal with: 1) growth rates in large university libraries; 2) library costs in colleges and universities; 3) cost trends and long-range plans; 4) library data; and 5) a…

  2. Digital Libraries and Mobility

    E-print Network

    Marshall, Cathy

    Digital Libraries and Mobility Digital libraries offer the promise of anywhere, anytime access to dig- ital resources come to light. Digital library patrons can: COMMUNICATIONS OF THE ACM May 2001/Vol or organizing materials. People may use mobile digital library technolo- gies to cross physical and digital

  3. Balfour (Zoology) Library Guide

    E-print Network

    Balfour (Zoology) Library Guide for NST Part II Zoology, Neuroscience and Biomedical and Biological Committee 16 #12;3 Libraries Gateway 16 #12;4 Balfour & Newton Libraries, Department of Zoology Web http three years of their study. The Library is located on the first floor of the Department of Zoology

  4. Balfour (Zoology) Library Guide

    E-print Network

    Balfour (Zoology) Library Guide for NST Part 1A students doing Zoology-taught courses #12 Libraries Gateway 11 #12;3 Balfour & Newton Libraries, Department of Zoology Web http three years of their study. The Library is located on the first floor of the Department of Zoology

  5. Balfour (Zoology) Library Guide

    E-print Network

    Balfour (Zoology) Library Guide for NST Part IA students doing Zoology-taught courses #12 Libraries Gateway 12 #12;3 Balfour & Newton Libraries, Department of Zoology Web http three years of their study. The Library is located on the first floor of the Department of Zoology

  6. Balfour (Zoology) Library Guide

    E-print Network

    Balfour (Zoology) Library Guide for Zoology postgraduate students #12;2 Contents Content Page Committee 16 Libraries Gateway 16 #12;4 Balfour & Newton Libraries, Department of Zoology Web http three years of their study. The Library is located on the first floor of the Department of Zoology

  7. Surveying School Libraries.

    ERIC Educational Resources Information Center

    Ptacek, William; Peterson, Susan

    1995-01-01

    Describes a survey administered by the King County Library System (Washington) that assessed the resources of school libraries and media centers in its service areas in order to determine the public library's appropriate role and level of service in supporting students. Results indicated that school libraries in King County are not well funded.…

  8. The "Integrated Library System."

    ERIC Educational Resources Information Center

    Dowlin, Kenneth E.

    1985-01-01

    Reviews internal and external dimensions of library environment that must be taken into account by library managers when choosing an integrated library system. The selection, acquisition, and implementation stages of Maggie III--a computerized library system sensitive to the internal and external organizational environment--are described. (MBR)

  9. Microbial Community Structures and Dynamics in the O3/BAC Drinking Water Treatment Process

    PubMed Central

    Tian, Jian; Lu, Jun; Zhang, Yu; Li, Jian-Cheng; Sun, Li-Chen; Hu, Zhang-Li

    2014-01-01

    Effectiveness of drinking water treatment, in particular pathogen control during the water treatment process, is always a major public health concern. In this investigation, the application of PCR-DGGE technology to the analysis of microbial community structures and dynamics in the drinking water treatment process revealed several dominant microbial populations including: ?-Proteobacteria, ?-Proteobacteria, ?-Proteobacteria, Bacteroidetes, Actinobacteria Firmicutes and Cyanobacteria. ?-Proteobacteria and ?-Proteobacteria were the dominant bacteria during the whole process. Bacteroidetes and Firmicutes were the dominant bacteria before and after treatment, respectively. Firmicutes showed season-dependent changes in population dynamics. Importantly, ?-Proteobacteria, which is a class of medically important bacteria, was well controlled by the O3/biological activated carbon (BAC) treatment, resulting in improved effluent water bio-safety. PMID:24937529

  10. The Mechanism of Voltage Dependent Gating of the NaChBac Prokaryotic Sodium Channel

    NASA Astrophysics Data System (ADS)

    Decaen, Paul G.

    Electrical signaling in cells depends on selective conductance of ions through membrane proteins called 'voltage gated ion channels'. These channels are characterized by their ability turn on and off the flow of ionic current by opening and closing their conductive pore in response to changes in membrane potential. The opening and closing of the pore is a mechanically linked to conformational movement of the positively charged fourth transmembrane segment (S4) in 'the voltage sensor' region. How the S4 moves in response to membrane potential is a controversial subject. In this thesis, we used the prokaryotic sodium channel NaChBac as our model sodium channel to study voltage dependent movement of the S4 in the voltage sensor. We use a disulfide-locking method where we introduced pairs of cysteines in the voltage sensor that crosslink and trap the S4 in its path after depolarization. We screened over one hundred mutations of the NaChBac channel in the whole cell patch clamp assay and demonstrated discrete and sequential voltage dependent ion pair interactions that occur in at least three states between the positively charged residues of the S4 segment and the acidic residues in the S1, S2 and S3 segments. In conjunction with structural modeling of the voltage sensor and our disulfide locking data, we propose that the S4 moves in and out of the plane of the membrane 8-13 A, forming distinct gating charge interactions with counter charges of the voltage sensor and adopts a 310 helix over a portion of its structure during activation. These findings are compatible with the sliding helix model and refine our understanding of the structural determinates of voltage sensor function in voltage gated ion channels.

  11. Isolation and characterization of microsatellite markers in the equine genome 

    E-print Network

    Mathiason, Kelli Janae

    1997-01-01

    Nine microsatellite markers were isolated from a horse genomic library in lambda bacteriophage. The markers were typed by PCR in 72 horses representing 14 breeds and in 36 parental animals of two Arabian half-sib families. Number of alleles...

  12. Extreme genomes

    PubMed Central

    DeLong, Edward F

    2000-01-01

    The complete genome sequence of Thermoplasma acidophilum, an acid- and heat-loving archaeon, has recently been reported. Comparative genomic analysis of this 'extremophile' is providing new insights into the metabolic machinery, ecology and evolution of thermophilic archaea. PMID:11178269

  13. America's Star Libraries, 2010: Top-Rated Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2010-01-01

    The "LJ" Index of Public Library Service 2010, "Library Journal"'s national rating of public libraries, identifies 258 "star" libraries. Created by Ray Lyons and Keith Curry Lance, and based on 2008 data from the IMLS, it rates 7,407 public libraries. The top libraries in each group get five, four, or three stars. All included libraries, stars or…

  14. The Pan-Cancer Proteomic Landscape of The Cancer Genome Atlas Projects - Rehan Akbani, TCGA Scientific Symposium 2014

    Cancer.gov

    Home News and Events Multimedia Library Videos The Pan-Cancer Proteomic Landscape of The Cancer Genome Atlas Projects - Rehan Akbani The Pan-Cancer Proteomic Landscape of The Cancer Genome Atlas Projects - Rehan Akbani, TCGA Scientific Symposium

  15. Genome-Wide Analysis of Expression Quantitative Trait Loci in Breast Cancer - Nicholas Knoblauch, TCGA Scientitifc Symposium 2012

    Cancer.gov

    Home News and Events Multimedia Library Videos Genome-Wide Analysis of Expression Quantitative Trait Loci in Breast Cancer - Nicholas Knoblauch Genome-Wide Analysis of Expression Quantitative Trait Loci in Breast Cancer - Nicholas Knoblauch, TCGA

  16. Profiling Long Intergenic Non-Coding RNA Interactions In The Cancer Genome - Samir B. Amin, TCGA Scientific Symposium 2014

    Cancer.gov

    Home News and Events Multimedia Library Videos Profiling Long Intergenic Non-Coding RNA Interactions In The Cancer Genome - Samir B. Amin Profiling Long Intergenic Non-Coding RNA Interactions In The Cancer Genome - Samir B. Amin, TCGA Scientific

  17. Genomic Characterization of Cancer-Adjacent Tissue: Evidence of Field Effects and Expression Subtypes - Melissa Troester, TCGA Scientific Symposium 2012

    Cancer.gov

    Home News and Events Multimedia Library Videos Genomic Characterization of Cancer-Adjacent Tissue - Melissa Troester Genomic Characterization of Cancer-Adjacent Tissue: Evidence of Field Effects and Expression Subtypes - Melissa Troester, TCGA Scientific

  18. Prediction of Individualized Therapeutic Vulnerabilities In Cancer from Genomic Profiles - Bulent Arman Aksoy, TCGA Scientific Symposium 2014

    Cancer.gov

    Home News and Events Multimedia Library Videos Prediction of Individualized Therapeutic Vulnerabilities In Cancer from Genomic Profiles - Bulent Ar Prediction of Individualized Therapeutic Vulnerabilities In Cancer from Genomic Profiles - Bulent

  19. TCGA Benchmark 4: Evaluating SV and SNV Calls Using Cell Line Genomes - Adam Ewing, TCGA Scientific Symposium 2012

    Cancer.gov

    Home News and Events Multimedia Library Videos TCGA Benchmark 4: Evaluating SV and SNV Calls Using Cell Line Genomes - Adam Ewing TCGA Benchmark 4: Evaluating SV and SNV Calls Using Cell Line Genomes - Adam Ewing, TCGA Scientific Symposium 2012 You

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

    E-print Network

    Lonardi, Stefano

    ,38 and Timothy J. Close1,* 1 Department of Botany and Plant Sciences, University of California, Riverside, CA of the Region Hana for Biotechnological and Agricultural Research, Institute of Experimental Botany, Sokolovskiz, Oregon State University, Corvallis, OR 97331, USA, 31 Department of Botany and Plant Pathology, Oregon

  1. Global Genomic Diversity of Oryza sativa Varieties Revealed by Comparative Physical Mapping

    PubMed Central

    Wang, Xiaoming; Kudrna, David A.; Pan, Yonglong; Wang, Hao; Liu, Lin; Lin, Haiyan; Zhang, Jianwei; Song, Xiang; Goicoechea, Jose Luis; Wing, Rod A.; Zhang, Qifa; Luo, Meizhong

    2014-01-01

    Bacterial artificial chromosome (BAC) physical maps embedding a large number of BAC end sequences (BESs) were generated for Oryza sativa ssp. indica varieties Minghui 63 (MH63) and Zhenshan 97 (ZS97) and were compared with the genome sequences of O. sativa spp. japonica cv. Nipponbare and O. sativa ssp. indica cv. 93-11. The comparisons exhibited substantial diversities in terms of large structural variations and small substitutions and indels. Genome-wide BAC-sized and contig-sized structural variations were detected, and the shared variations were analyzed. In the expansion regions of the Nipponbare reference sequence, in comparison to the MH63 and ZS97 physical maps, as well as to the previously constructed 93-11 physical map, the amounts and types of the repeat contents, and the outputs of gene ontology analysis, were significantly different from those of the whole genome. Using the physical maps of four wild Oryza species from OMAP (http://www.omap.org) as a control, we detected many conserved and divergent regions related to the evolution process of O. sativa. Between the BESs of MH63 and ZS97 and the two reference sequences, a total of 1532 polymorphic simple sequence repeats (SSRs), 71,383 SNPs, 1767 multiple nucleotide polymorphisms, 6340 insertions, and 9137 deletions were identified. This study provides independent whole-genome resources for intra- and intersubspecies comparisons and functional genomics studies in O. sativa. Both the comparative physical maps and the GBrowse, which integrated the QTL and molecular markers from GRAMENE (http://www.gramene.org) with our physical maps and analysis results, are open to the public through our Web site (http://gresource.hzau.edu.cn/resource/resource.html). PMID:24424778

  2. IN SILICO DISCOVERY, MAPPING, AND GENOTYPING OF 1,039 CATTLE SNPS ON A PANEL OF EIGHTEEN BREEDS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To contribute to cattle haplotype map construction we discovered ~3,000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences (BESs) from the cattle RPCI-42 BAC library with the cattle whole-genome shotgun (WGS) contigs. For the sequence alignment, the Time...

  3. Sorghum Ma5 and Ma6 maturity genes 

    E-print Network

    Brady, Jeffrey Alan

    2009-05-15

    of an integrated genetic, physical, and cytogenetic map of the sorghum genome. Genetic markers have been linked to both the Ma5 and Ma6 loci on chromosomes 2 and 6, respectively. BAC libraries have been screened to identify numerous BACs associated with each locus...

  4. Library construction for next-generation sequencing: Overviews and challenges

    PubMed Central

    Head, Steven R.; Komori, H. Kiyomi; LaMere, Sarah A.; Whisenant, Thomas; Van Nieuwerburgh, Filip; Salomon, Daniel R.; Ordoukhanian, Phillip

    2014-01-01

    High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed. PMID:24502796

  5. Predicting the molecular complexity of sequencing libraries.

    PubMed

    Daley, Timothy; Smith, Andrew D

    2013-04-01

    Predicting the molecular complexity of a genomic sequencing library is a critical but difficult problem in modern sequencing applications. Methods to determine how deeply to sequence to achieve complete coverage or to predict the benefits of additional sequencing are lacking. We introduce an empirical bayesian method to accurately characterize the molecular complexity of a DNA sample for almost any sequencing application on the basis of limited preliminary sequencing. PMID:23435259

  6. A dramatic increase in the positive blood culture rates of Helicobacter cinaedi: the evidence of differential detection abilities between the Bactec and BacT/Alert systems.

    PubMed

    Miyake, Noriko; Chong, Yong; Nishida, Ruriko; Nagasaki, Yoji; Kibe, Yasushi; Kiyosuke, Makiko; Shimomura, Takeshi; Shimono, Nobuyuki; Shimoda, Shinji; Akashi, Koichi

    2015-11-01

    In our hospital, positive blood culture rates of Helicobacter cinaedi dramatically increased after introducing the Bactec system. A simulated culture model of H. cinaedi bacteremia demonstrated no positive signals using the BacT/Alert system, despite efficient growth in bottles. Clinically suspected H. cinaedi bacteremia should be monitored more closely when using the BacT/Alert system, preferably with subcultivation after 7days of incubation. PMID:26298818

  7. Half-metallicity and stability of the rock salt BaC and SrC (111) surfaces: A density functional study

    SciTech Connect

    Tabatabaeifar, A. H.; Davatolhagh, S. Foroughpour, M.

    2013-12-07

    The electronic structure and magnetic properties of relaxed (111) surfaces of the alkaline-earth monocarbides BaC and SrC in the stable rock salt structure, are calculated on the basis of first principle density functional theory within the framework of self-consistent field plane wave pseudo-potential method, using the generalized gradient approximation for the exchange-correlation functional. The results of this study reveal that the C-terminated (111) surfaces retain the bulk half-metallic property in both BaC and SrC. The half-metallicity of the C-terminated BaC surface is found to be more robust compared to the bulk BaC due to the larger half-metallic energy gap. In contrast, the half-metallic energy gap of the C-terminated SrC surface is found to be smaller than that of the bulk. The Ba-terminated surface of BaC and the Sr-terminated surface of SrC, however, lose their bulk half-metallicity due to the formation of surface states in the majority spin band gap. The calculations also show that the atomic magnetic moments at the half-metallic C-terminated surfaces in both BaC and SrC increase considerably with respect to the corresponding bulk values, which is explained in terms of an increase in the number of unpaired 2p electrons of the carbon atom at the surface. We also discuss the stability of the surfaces via the calculated bulk formation energies. The bulk formation energies for both BaC and SrC in the rock salt structure are found to be positive, which indicate that the surfaces are not stable at normal pressure and temperature conditions, and non-equilibrium growth techniques may be required for the realization of BaC and SrC thin films.

  8. INTEGRATED KARYOTYPING OF SORGHUM BY IN SITU HYBRIDIZATION OF LANDED BAC'S

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The reliability of genome analysis and proficiency of genetic manipulation are increased by assignment of linkage groups to specific chromosomes, placement of centromeres, and orientation with respect to telomeres. We have endeavored to establish means to enable these steps in sorghum, the genome o...

  9. Modular Design of the Selectivity Filter Pore Loop in a Novel Family of Prokaryotic ‘Inward Rectifier’ (NirBac) channels

    PubMed Central

    Zubcevic, Lejla; Wang, Shizhen; Bavro, Vassiliy N.; Lee, Sun-Joo; Nichols, Colin G.; Tucker, Stephen J.

    2015-01-01

    Potassium channels exhibit a modular design with distinct structural and functional domains; in particular, a highly conserved pore-loop sequence that determines their ionic selectivity. We now report the functional characterisation of a novel group of functionally non-selective members of the prokaryotic ‘inward rectifier’ subfamily of K+ channels. These channels share all the key structural domains of eukaryotic and prokaryotic Kir/KirBac channels, but instead possess unique pore-loop selectivity filter sequences unrelated to any other known ionic selectivity filter. The strikingly unusual architecture of these ‘NirBac’ channels defines a new family of functionally non-selective ion channels, and also provides important insights into the modular design of ion channels, as well as the evolution of ionic selectivity within this superfamily of tetrameric cation channels. PMID:26470642

  10. Decoding the fine-scale structure of a breast cancer genome and transcriptome

    PubMed Central

    Volik, Stanislav; Raphael, Benjamin J.; Huang, Guiqing; Stratton, Michael R.; Bignel, Graham; Murnane, John; Brebner, John H.; Bajsarowicz, Krystyna; Paris, Pamela L.; Tao, Quanzhou; Kowbel, David; Lapuk, Anna; Shagin, Dmitri A.; Shagina, Irina A.; Gray, Joe W.; Cheng, Jan-Fang; de Jong, Pieter J.; Pevzner, Pavel; Collins, Colin

    2006-01-01

    A comprehensive understanding of cancer is predicated upon knowledge of the structure of malignant genomes underlying its many variant forms and the molecular mechanisms giving rise to them. It is well established that solid tumor genomes accumulate a large number of genome rearrangements during tumorigenesis. End Sequence Profiling (ESP) maps and clones genome breakpoints associated with all types of genome rearrangements elucidating the structural organization of tumor genomes. Here we extend the ESP methodology in several directions using the breast cancer cell line MCF-7. First, targeted ESP is applied to multiple amplified loci, revealing a complex process of rearrangement and coamplification in these regions reminiscent of breakage/fusion/bridge cycles. Second, genome breakpoints identified by ESP are confirmed using a combination of DNA sequencing and PCR. Third, in vitro functional studies assign biological function to a rearranged tumor BAC clone, demonstrating that it encodes antiapoptotic activity. Finally, ESP is extended to the transcriptome identifying four novel fusion transcripts and providing evidence that expression of fusion genes may be common in tumors. These results demonstrate the distinct advantages of ESP including: (1) the ability to detect all types of rearrangements and copy number changes; (2) straightforward integration of ESP data with the annotated genome sequence; (3) immortalization of the genome; (4) ability to generate tumor-specific reagents for in vitro and in vivo functional studies. Given these properties, ESP could play an important role in a tumor genome project. PMID:16461635

  11. Construction and characterization of a peach binary bacterial artificial chromosome library 

    E-print Network

    Wakefield, Laura

    2002-01-01

    Peach [Prunus persica (L.) Batch] has been proposed as a model organism for woody perennials. To facilitate genomic research in peach, a binary bacterial artificial chromosome (BIBAC) library of var. Texking was constructed using a plant...

  12. Construction of highly extensive polymorphic DNA libraries by in-gel competitive reassociation procedure

    SciTech Connect

    Inoue, Shinichi; Kiyama, Ryoiti; Oishi, Michio

    1996-02-01

    Differential genomic DNA libraries between two mouse strains and from two human individuals were constructed by means of the in-gel competitive reassociation (IGCR) procedure, a procedure developed for cloning altered anonymous restriction fragments. The libraries were highly enriched fragments, approximately 60 and 40% for the mouse and human libraries, respectively, and, more importantly, maintained most of the original complexities of the RFLP fragments. Therefore, differential genomic DNA libraries constructed by the IGCR procedure, particularly for human genomic DNA, should offer highly extensive sources for polymorphic DNA sequences necessary for a variety of genome analyses, including studies on the origin and mechanism of biological diversity among the same species. 19 refs., 4 figs.

  13. Genomic sequencing of Pleistocene cave bears

    SciTech Connect

    Noonan, James P.; Hofreiter, Michael; Smith, Doug; Priest, JamesR.; Rohland, Nadin; Rabeder, Gernot; Krause, Johannes; Detter, J. Chris; Paabo, Svante; Rubin, Edward M.

    2005-04-01

    Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of {approx}1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome, the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.

  14. The Connection between Persistent, Disinfectant-Resistant Listeria monocytogenes Strains from Two Geographically Separate Iberian Pork Processing Plants: Evidence from Comparative Genome Analysis.

    PubMed

    Ortiz, Sagrario; López-Alonso, Victoria; Rodríguez, Pablo; Martínez-Suárez, Joaquín V

    2015-01-01

    The aim of this study was to investigate the basis of the putative persistence of Listeria monocytogenes in a new industrial facility dedicated to the processing of ready-to-eat (RTE) Iberian pork products. Quaternary ammonium compounds, which included benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance to disinfectants was associated with persistence. Of the 14 isolates obtained from product contact and non-product contact surfaces, only five different pulsed-field gel electrophoresis (PFGE) types were identified during the 27-month study period. Two of these PFGE types (S1 and S10-1) were previously identified to be persistent and BAC-resistant (BAC(r)) strains in a geographically separate slaughterhouse belonging to the same company. The remaining three PFGE types, which were first identified in this study, were also BAC(r). Whole-genome sequencing and in silico multilocus sequence typing (MLST) analysis of five BAC(r) isolates of the different PFGE types identified in this study showed that the isolate of the S1 PFGE type belonged to MLST sequence type 31 (ST31), a low-virulence type characterized by mutations in the inlA and prfA genes. The isolates of the remaining four PFGE types were found to belong to MLST ST121, a persistent type that has been isolated in several countries. The ST121 strains contained the BAC resistance transposon Tn6188. The disinfection-resistant L. monocytogenes population in this RTE pork product plant comprised two distinct genotypes with different multidrug resistance phenotypes. This work offers insight into the L. monocytogenes subtypes associated with persistence in food processing environments. PMID:26497458

  15. Complete genome sequence of the moderate thermophile Anaerobaculum mobile type strain (NGAT)

    SciTech Connect

    Mavromatis, K; Stackebrandt, Erko; Held, Brittany; Lapidus, Alla L.; Nolan, Matt; Lucas, Susan; Hammon, Nancy; Deshpande, Shweta; Cheng, Jan-Fang; Tapia, Roxanne; Goodwin, Lynne A.; Pitluck, Sam; Liolios, Konstantinos; Pagani, Ioanna; Ivanova, N; Mikhailova, Natalia; Huntemann, Marcel; Pati, Amrita; Chen, Amy; Palaniappan, Krishna; Land, Miriam L; Rohde, Manfred; Spring, Stefan; Goker, Markus; Woyke, Tanja; Detter, J. Chris; Bristow, James; Eisen, Jonathan; Markowitz, Victor; Hugenholtz, Philip; Klenk, Hans-Peter; Kyrpides, Nikos C

    2013-01-01

    Anaerobaculum mobile Menes and Muxi 2002 is one of three described species of the genus Anaerobaculum, family Synergistaceae, phylum Synergistetes. This anaerobic and motile bac- terium ferments a range of carbohydrates and mono- and dicarboxylic acids with acetate, hy- drogen and CO2 as end products. A. mobile NGAT is the first member of the genus Anaerobaculum and the sixth member of the phylum Synergistetes with a completely se- quenced genome. Here we describe the features of this bacterium, together with the com- plete genome sequence, and annotation. The 2,160,700 bp long single replicon genome with its 2,053 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

  16. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

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

    Papers on art libraries and information services for the arts, which were presented at the 1983 International Federation of Library Associations (IFLA) conference, include: (1) "'I See All': Information Technology and the Universal Availability of Images" by Philip Pacey (United Kingdom); (2) "Online Databases in the Fine Arts" by Michael Rinehart…

  17. Community, Library, and Revolution

    ERIC Educational Resources Information Center

    Freiser, Leonard H.

    1970-01-01

    Discusses the need for greatly expanded community library services, for librarians to accept social responsibility for education and information, and for the library to become a program for getting information to where it is needed. (JS)

  18. Selecting Library Furniture & Equipment.

    ERIC Educational Resources Information Center

    Media & Methods, 1997

    1997-01-01

    Offers suggestions for selecting school library furniture and equipment. Describes various models of computer workstations; reading tables and chairs; and shelving. Sidebar lists names and addresses of library furniture manufactures and distributors. (AEF)

  19. Library Strategic 2015 -2020

    E-print Network

    Tobar, Michael

    J Marshall Library atrium 2 The University of Western Australia #12;`Overall UWA libraries provide. · Expenditure on information resources is around $10 million annually. Cover Image: Bird's-eye view of the Barry

  20. Israeli Special Libraries

    ERIC Educational Resources Information Center

    Foster, Barbara

    1974-01-01

    Israel is sprinkled with a noteworthy representation of special libraries which run the gamut from modest kibbutz efforts to highly technical scientific and humanities libraries. A few examples are discussed here. (Author/CH)

  1. Library for the future

    E-print Network

    Kaufman, Julie Hui-Guang

    2002-01-01

    The library is intended to be an egalitarian institution for the dissemination of knowledge to the public. With the advent of the internet, information has been further democratized and the status of the library has been ...

  2. Driving Performance on the Descending Limb of Blood Alcohol Concentration (BAC) in Undergraduate Students: A Pilot Study

    PubMed Central

    Silvey, Dustin; Behm, David; Albert, Wayne J.

    2015-01-01

    Young drivers are overrepresented in collisions resulting in fatalities. It is not uncommon for young drivers to socially binge drink and decide to drive a vehicle a few hours after consumption. To better understand the risks that may be associated with this behaviour, the present study has examined the effects of a social drinking bout followed by a simulated drive in undergraduate students on the descending limb of their BAC (blood alcohol concentration) curve. Two groups of eight undergraduate students (n = 16) took part in this study. Participants in the alcohol group were assessed before drinking, then at moderate and low BAC as well as 24 hours post-acute consumption. This group consumed an average of 5.3 ± 1.4 (mean ± SD) drinks in an hour in a social context and were then submitted to a driving and a predicted crash risk assessment. The control group was assessed at the same time points without alcohol intake or social context.; at 8 a.m., noon, 3 p.m. and 8 a.m. the next morning. These multiple time points were used to measure any potential learning effects from the assessment tools (i.e. driving simulator and useful field of view test (UFOV)). Diminished driving performance at moderate BAC was observed with no increases in predicted crash risk. Moderate correlations between driving variables were observed. No association exists between driving variables and UFOV variables. The control group improved measures of selective attention after the third asessement. No learning effect was observed from multiple sessions with the driving simulator. Our results show that a moderate BAC, although legal, increases the risky behaviour. Effects of alcohol expectancy could have been displayed by the experimental group. UFOV measures and predicted crash risk categories were not sentitive enough to predict crash risk for young drivers, even when intoxicated. PMID:25723618

  3. Genomic analysis of sorghum by fluorescence in situ hybridization 

    E-print Network

    Kim, Jeong-Soon

    2004-11-15

    are likely to be broad. In the first study, I developed a FISH-based karyotyping system for Sorghum bicolor Moench. I used integrated structural genomic resources, including linkage maps and large-insert clonal libraries of sorghum genomic DNA to develop a...

  4. LSU Libraries Office of the Dean of Libraries

    E-print Network

    Stephens, Jacqueline

    LSU Libraries Office of the Dean of Libraries Library Policy Memorandum 25 Revision 2 Approved by Dean's Council, May 24, 2000 Subject: Use of the Libraries' Internet Servers Purpose: The purpose of the LSU Libraries presence on the Internet is to support the mission of the Libraries to collect

  5. Hesburgh Libraries and St. Joseph County Public Library partner

    E-print Network

    Pfrender, Michael

    partnership between Notre Dame's main Hesburgh Library and the county library system. Access current bestHesburgh Libraries and St. Joseph County Public Library partner to offer new delivery services. Joseph County Public Library (SJCPL) materials at the Hesburgh Library. Individuals can also obtain

  6. Open Digital Libraries Hussein Suleman

    E-print Network

    Suleman, Hussein

    Open Digital Libraries Hussein Suleman Dissertation submitted to the faculty of the Virginia Varadarajan Gail McMillan November 19, 2002 Blacksburg, Virginia Tech Keywords: digital library Libraries Hussein Suleman (ABSTRACT) Digital Libraries (DLs) are software systems specifically designed

  7. The Bactofilin Cytoskeleton Protein BacM of Myxococcus xanthus Forms an Extended ?-Sheet Structure Likely Mediated by Hydrophobic Interactions

    PubMed Central

    Xie, Kefang; Engelhardt, Harald; Bosch, Jürgen; Hoiczyk, Egbert

    2015-01-01

    Bactofilins are novel cytoskeleton proteins that are widespread in Gram-negative bacteria. Myxococcus xanthus, an important predatory soil bacterium, possesses four bactofilins of which one, BacM (Mxan_7475) plays an important role in cell shape maintenance. Electron and fluorescence light microscopy, as well as studies using over-expressed, purified BacM, indicate that this protein polymerizes in vivo and in vitro into ~3 nm wide filaments that further associate into higher ordered fibers of about 10 nm. Here we use a multipronged approach combining secondary structure determination, molecular modeling, biochemistry, and genetics to identify and characterize critical molecular elements that enable BacM to polymerize. Our results indicate that the bactofilin-determining domain DUF583 folds into an extended ?-sheet structure, and we hypothesize a left-handed ?-helix with polymerization into 3 nm filaments primarily via patches of hydrophobic amino acid residues. These patches form the interface allowing head-to-tail polymerization during filament formation. Biochemical analyses of these processes show that folding and polymerization occur across a wide variety of conditions and even in the presence of chaotropic agents such as one molar urea. Together, these data suggest that bactofilins are comprised of a structure unique to cytoskeleton proteins, which enables robust polymerization. PMID:25803609

  8. The bactofilin cytoskeleton protein BacM of Myxococcus xanthus forms an extended ?-sheet structure likely mediated by hydrophobic interactions.

    PubMed

    Zuckerman, David M; Boucher, Lauren E; Xie, Kefang; Engelhardt, Harald; Bosch, Jürgen; Hoiczyk, Egbert

    2015-01-01

    Bactofilins are novel cytoskeleton proteins that are widespread in Gram-negative bacteria. Myxococcus xanthus, an important predatory soil bacterium, possesses four bactofilins of which one, BacM (Mxan_7475) plays an important role in cell shape maintenance. Electron and fluorescence light microscopy, as well as studies using over-expressed, purified BacM, indicate that this protein polymerizes in vivo and in vitro into ~3 nm wide filaments that further associate into higher ordered fibers of about 10 nm. Here we use a multipronged approach combining secondary structure determination, molecular modeling, biochemistry, and genetics to identify and characterize critical molecular elements that enable BacM to polymerize. Our results indicate that the bactofilin-determining domain DUF583 folds into an extended ?-sheet structure, and we hypothesize a left-handed ?-helix with polymerization into 3 nm filaments primarily via patches of hydrophobic amino acid residues. These patches form the interface allowing head-to-tail polymerization during filament formation. Biochemical analyses of these processes show that folding and polymerization occur across a wide variety of conditions and even in the presence of chaotropic agents such as one molar urea. Together, these data suggest that bactofilins are comprised of a structure unique to cytoskeleton proteins, which enables robust polymerization. PMID:25803609

  9. BAC transgenic zebrafish reveal hypothalamic enhancer activity around obesity associated SNP rs9939609 within the human FTO gene.

    PubMed

    Rinkwitz, Silke; Geng, Fan-Suo; Manning, Elizabeth; Suster, Maximiliano; Kawakami, Koichi; Becker, Thomas S

    2015-10-01

    Single Nucleotide Polymorphisms in FTO intron 1 have been associated with obesity risk, leading to the hypothesis that FTO is the obesity-related gene. However, other studies have shown that the FTO gene is part of the regulatory domain of the neighboring IRX3 gene and that enhancers in FTO intron 1 regulate IRX3. While Irx3 activity was shown to be necessary in the hypothalamus for the metabolic function of Irx3 in mouse, no enhancers with hypothalamic activity have been demonstrated in the risk-associated region within FTO. In order to identify potential enhancers at the human FTO locus in vivo, we tested regulatory activity in FTO intron 1 using BAC transgenesis in zebrafish. A minimal gata2 promoter-GFP cassette was inserted 1.3 kb upstream of the obesity associated SNP rs9939609 in a human FTO BAC plasmid. In addition to the previously identified expression domains in notochord and kidney, human FTO BAC:GFP transgenic zebrafish larvae expressed GFP in the ventral posterior tuberculum, the posterior hypothalamus and the anterior brainstem, which are also expression domains of zebrafish irx3a. In contrast, an in-frame insertion of a GFP cassette at the FTO start codon resulted in weak ubiquitous GFP expression indicating that the promoter of FTO does likely not react to enhancers located in the obesity risk-associated region. genesis 53:640-651, 2015. © 2015 The Authors. genesis Published by Wiley Periodicals, Inc. PMID:26271004

  10. Validation of the BacT/ALERT(R) 3D System for Rapid Sterility Testing of Biopharmaceutical Samples.

    PubMed

    Jimenez, Luis; Rana, Narendra; Amalraj, Joseph; Walker, Kimberly; Travers, Kasey

    2012-01-01

    The BacT/ALERT® 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were completed and verified. For the performance qualification, 11 microorganisms were evaluated, six compendial (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger, Clostridium sporogenes), one representing the number one microbial species in sterile product recalls (Burkholderia cepacia), and four environmental isolates (Kocuria rhizophila, Staphylococcus haemolyticus, Methylobacterium radiotolerans, and Penicillium spp.). Nine of the microorganisms were spiked into three different types of biopharmaceutical samples by three different analysts on different days to ascertain the equivalence, ruggedness, sensitivity, time of detection, and repeatability. In all samples, the BacT/ALERT® exhibited equivalent or better detection than the standard test. With the exception of M. radiotolerans, all 11 microorganisms were detected within 2.5 days using the BacT/ALERT® system and the standard test. The detection times for M. radiotolerans in the three sample types averaged 5.77 days. The minimum detectable level of cells for all the microorganisms tested was found to be within 1 to 2 CFU. The system optimized sterility testing by the simultaneous on-line, non-destructive incubation and detection of microbial growth. PMID:22307661

  11. Cognitive effects of antipsychotic dosage and polypharmacy: a study with the BACS in patients with schizophrenia and schizoaffective disorder.

    PubMed

    Elie, D; Poirier, M; Chianetta, Jm; Durand, M; Grégoire, Ca; Grignon, S

    2010-07-01

    Antipsychotic polypharmacy and high doses have been associated with poorer outcome, longer hospital stays, and increased side effects. The present naturalistic study assessed the cognitive effects of antipsychotics in 56 patients with a diagnosis of schizophrenia or schizoaffective disorder, using the Brief Assessment of Cognition in Schizophrenia (BACS). Antipsychotic daily dose (ADD) was expressed as mg risperidone equivalents/day (RIS eq), using a model based on drug doses from the Clinical Antipsychotic Trials in Intervention Effectiveness (CATIE) study for second generation antipsychotics (SGA) and chlorpromazine equivalents for first generation antipsychotics (FGA), with a 1/1 equivalence between haloperidol and risperidone. Increasing age was associated with polypharmacy, FGA prescription and decreasing BACS score. FGA prescription, in turn, predicted a poorer cognitive functioning, independently of age, PANSS subscores and ADD. ADD was associated with decreasing cognitive scores, an effect that remained significant after controlling for age, PANSS or polypharmacy. The detrimental cognitive effects of polypharmacy, in turn, appeared to be mediated by ADD. Different methods of data fitting suggested that ADD above 5-6 mg RIS eq/day were associated with lower BACS scores. Overall, these results show that increasing antipsychotic daily dose is associated with poorer cognitive functioning at doses lower than previously thought, independently of the number of antipsychotic drugs. PMID:19164494

  12. Technostress and Library Values.

    ERIC Educational Resources Information Center

    Gorman, Michael

    2001-01-01

    Discusses information overload and society's and libraries' responses to technology. Considers eight values that libraries should focus on and how they relate to technology in libraries: democracy, stewardship, service, intellectual freedom, privacy, rationalism, equity of access, and building harmony and balance. (LRW)

  13. School Libraries and Innovation

    ERIC Educational Resources Information Center

    McGrath, Kevin G.

    2015-01-01

    School library programs have measured success by improved test scores. But how do next-generation school libraries demonstrate success as they strive to be centers of innovation and creativity? These libraries offer solutions for school leaders who struggle to restructure existing systems built around traditional silos of learning (subjects and…

  14. A Truly Bookless Library

    ERIC Educational Resources Information Center

    Kolowich, Steve

    2011-01-01

    The difference between the University of Texas at San Antonio's Applied Engineering and Technology Library and other science-focused libraries is not that its on-site collection is also available electronically. It is that its on-site collection is only available electronically. The idea of libraries with no bound books has been a recurring theme…

  15. A new math library

    NASA Astrophysics Data System (ADS)

    Beebe, Nelson H. F.

    The design and development of a new mathematical function library is described. The library extends programming support to decimal, as well as binary, arithmetic, and aims to influence the future evolution of floating-point support in programming languages, libraries, and hardware. It encourages and facilitates research in new computer-arithmetic designs, and the analysis and comparison of historical ones.

  16. Staffing the College Library

    ERIC Educational Resources Information Center

    Thomas, Bruce

    1973-01-01

    College libraries use a variety of methods to identify and select professional library personnel. 70 libraries responded to a questionnaire about their methods and the results are presented. The effectiveness of advertisements, employment services, testing and reference checks are among the topics discussed. (DH)

  17. The Library Morphs

    ERIC Educational Resources Information Center

    Waters, John K.

    2008-01-01

    As campus renovation projects go, the Ohio State University's plan to turn its main library into "a library for the 21st century" is ambitious. The author describes the decade-long, $109 million transformation of the William Oxley Thompson Memorial Library. The overhaul calls for a complete replacement of all mechanical and electrical systems,…

  18. Merchandising Your Library.

    ERIC Educational Resources Information Center

    Sivulich, Kenneth G.

    1989-01-01

    Discusses library circulation figures as a reflection of the success of library services and describes merchandising techniques that have produced a 137 percent circulation increase at Queens Borough Public Library over the past seven years. Merchandising techniques such as minibranches, displays, signage, dumps, and modified shelving are…

  19. Library Building and Design.

    ERIC Educational Resources Information Center

    Jones, David J.; Gordon, Heather; Caddy, Julie; Kahlert, Maureen; Johnson, Carolyn; Holdstock, Fiona

    1997-01-01

    More frequently, community connections are being expressed in library design briefs and reflected in the completed buildings. This collection of brief articles discusses community involvement in library design and services and describes library construction projects in Australia and Malaysia. Also, discusses community art programs, integrating…

  20. The Honor System Library

    ERIC Educational Resources Information Center

    Marie, Kristen L.

    2005-01-01

    An honor system library can be created inside the library media center (LMC). Where students can access free books and magazines that require no formal checkouts. The honor library system at Washington High School, Fremont, California, has become self-sustaining. As many students, parents and teachers donate quality material. No student is ever…