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1

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

PubMed Central

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

Eggen, André; Gautier, Mathieu; Billaut, Alain; Petit, Élisabeth; Hayes, Hélène; Laurent, Pascal; Urban, Catherine; Pfister-Genskow, Martha; Eilertsen, Ken; Bishop, Michael D

2001-01-01

2

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

E-print Network

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

Markow, Therese

3

A genomic BAC library and a new BAC-GFP vector to study the holocentric pest Spodoptera frugiperda  

Microsoft Academic Search

Two genomic tools for the study of Lepidoptera and the holocentric structure of their chromosomes are presented in this paper.A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA partially digested with HindIII from eggs of Spodoptera frugiperda. The library contains a total of 36?864 clones with an average insert size of 125 kb, which corresponds to approximately 11.5

Emmanuelle d’Alençon; Pietro Piffanelli; Anne-Nathalie Volkoff; Xavier Sabau; Sylvie Gimenez; Janick Rocher; Pierre Cérutti; Philippe Fournier

2004-01-01

4

Toward an Integrated BAC Library Resource for Genome Sequencing and Analysis  

SciTech Connect

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.

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

2002-02-26

5

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

PubMed Central

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

2011-01-01

6

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

PubMed Central

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

2013-01-01

7

New genomic resources for switchgrass: a BAC library and comparative analysis of homoeologous genomic regions harboring bioenergy traits  

PubMed Central

Background Switchgrass, a C4 species and a warm-season grass native to the prairies of North America, has been targeted for development into an herbaceous biomass fuel crop. Genetic improvement of switchgrass feedstock traits through marker-assisted breeding and biotechnology approaches calls for genomic tools development. Establishment of integrated physical and genetic maps for switchgrass will accelerate mapping of value added traits useful to breeding programs and to isolate important target genes using map based cloning. The reported polyploidy series in switchgrass ranges from diploid (2X = 18) to duodecaploid (12X = 108). Like in other large, repeat-rich plant genomes, this genomic complexity will hinder whole genome sequencing efforts. An extensive physical map providing enough information to resolve the homoeologous genomes would provide the necessary framework for accurate assembly of the switchgrass genome. Results A switchgrass BAC library constructed by partial digestion of nuclear DNA with EcoRI contains 147,456 clones covering the effective genome approximately 10 times based on a genome size of 3.2 Gigabases (~1.6 Gb effective). Restriction digestion and PFGE analysis of 234 randomly chosen BACs indicated that 95% of the clones contained inserts, ranging from 60 to 180 kb with an average of 120 kb. Comparative sequence analysis of two homoeologous genomic regions harboring orthologs of the rice OsBRI1 locus, a low-copy gene encoding a putative protein kinase and associated with biomass, revealed that orthologous clones from homoeologous chromosomes can be unambiguously distinguished from each other and correctly assembled to respective fingerprint contigs. Thus, the data obtained not only provide genomic resources for further analysis of switchgrass genome, but also improve efforts for an accurate genome sequencing strategy. Conclusions The construction of the first switchgrass BAC library and comparative analysis of homoeologous harboring OsBRI1 orthologs present a glimpse into the switchgrass genome structure and complexity. Data obtained demonstrate the feasibility of using HICF fingerprinting to resolve the homoeologous chromosomes of the two distinct genomes in switchgrass, providing a robust and accurate BAC-based physical platform for this species. The genomic resources and sequence data generated will lay the foundation for deciphering the switchgrass genome and lead the way for an accurate genome sequencing strategy. PMID:21767393

2011-01-01

8

A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells  

PubMed Central

Cultured mouse or human embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Chemical or insertional mutagenesis of Blm-deficient mouse ES cells can be used to generate genome-wide libraries of homozygous mutant ES cells, which are the substrates for conducting phenotype-driven loss-of-function genetic screens. However, the existing insertional mutation libraries are limited by incomplete genomic coverage. In this study, we have explored the use of piggyBac (PB) transposon-mediated mutagenesis to extend the genomic coverage of mutation libraries in Blm-deficient ES cells. A library composed of 14,000 individual gene-trap clones was generated and a recessive genetic screen conducted to identify cells with defects in DNA mismatch repair (MMR) genes. Independent mutations in all known genes of the pathway Msh2, Msh6, Pms2, and Mlh1 were recovered in these screens. The genomic coverage in this library confirms its utility as a new genetic resource for conducting recessive genetic screens in mammalian cells. PMID:19233961

Wang, Wei; Bradley, Allan; Huang, Yue

2009-01-01

9

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

PubMed

To support genomic analysis of the endangered California condor (Gymnogyps californianus), a BAC library (CHORI-262) was generated using DNA from the blood of a female. The library consists of 89,665 recombinant BAC clones providing approximately 14-fold coverage of the presumed approximately 1.48-Gb genome. Taking advantage of recent progress in chicken genomics, we developed a first-generation comparative chicken-condor physical map using an overgo hybridization approach. The overgos were derived from chicken (164 probes) and New World vulture (8 probes) sequences. Screening a 2.8x subset of the total library resulted in 236 BAC-gene assignments with 2.5 positive BAC clones per successful probe. A preliminary comparative chicken-condor BAC-based map included 93 genes. Comparison of selected condor BAC sequences with orthologous chicken sequences suggested a high degree of conserved synteny between the two avian genomes. This work will aid in identification and characterization of candidate loci for the chondrodystrophy mutation to advance genetic management of this disease. PMID:16884891

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

2006-12-01

10

Construction and characterization of a papaya BAC library as a foundation for molecular dissection of a tree-fruit genome  

Microsoft Academic Search

A bacterial artificial chromosome (BAC) library was constructed from high-molecular-weight DNA isolated from young leaves\\u000a of papaya (Carica papaya L.). This BAC library consists of 39168 clones from two separate ligation reactions. The average insert size of the library\\u000a is 132 kb; 96.5% of the 18700 clones from the first ligation contained inserts that averaged 86 kb in size, 95.7%

R. Ming; P. H. Moore; F. Zee; C. A. Abbey; H. Ma; A. H. Paterson

2001-01-01

11

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...

12

Library Resources for Bac End Sequencing. Final Technical Report  

SciTech Connect

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.

Pieter J. de Jong

2000-10-01

13

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...

14

Characterizing the walnut genome through analyses of BAC end sequences  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

15

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

Microsoft Academic Search

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 115kb. Based on the rice field eel haploid genome size of 600Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8%

Songhun Jang; Fang Zhou; Laixin Xia; Wei Zhao; Hanhua Cheng; Rongjia Zhou

2006-01-01

16

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

E-print Network

of lake whitefish (Coregonus clupeaformis, Salmonidae) towards the elucidation of adaptive species, characterization and screening of a nonar- rayed BAC library for lake whitefish (Coregonus clupeaformis). We of extensive sequence information and other genomic resources. In lake whitefish (Coregonus clupeaformis

Bernatchez, Louis

17

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...

18

Development of genomic resources for Citrus clementina: Characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences  

Microsoft Academic Search

BACKGROUND: Citrus species constitute one of the major tree fruit crops of the subtropical regions with great economic importance. However, their peculiar reproductive characteristics, low genetic diversity and the long-term nature of tree breeding mostly impair citrus variety improvement. In woody plants, genomic science holds promise of improvements and in the Citrus genera the development of genomic tools may be

Javier Terol; Miguel A Naranjo; Patrick Ollitrault; Manuel Talon

2008-01-01

19

End Sequencing and Finger Printing of Human & Mouse BAC Libraries  

SciTech Connect

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.

Fraser, C.

2005-09-27

20

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...

21

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

SciTech Connect

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.

Shizuya, Hiroaki

2002-08-23

22

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

SciTech Connect

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.

Jang Songhun [Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Zhou Fang [Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Xia Laixin [Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Zhao Wei [Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072 (China); Cheng Hanhua [Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072 (China)]. E-mail: hhcheng@whu.edu.cn; Zhou Rongjia [Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072 (China)]. E-mail: rjzhou@whu.edu.cn

2006-09-22

23

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...

24

Characterizing the walnut genome through analyses of BAC end sequences.  

PubMed

Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots of J. regia cv. Chandler using the HindIII and MboI cloning sites. A total of 48,218 high-quality BAC end sequences (BESs) were generated, with an accumulated sequence length of 31.2 Mb, representing approximately 5.1% of the walnut genome. Analysis of repeat DNA content in BESs revealed that approximately 15.42% of the genome consists of known repetitive DNA, while walnut-unique repetitive DNA identified in this study constitutes 13.5% of the genome. Among the walnut-unique repetitive DNA, Julia SINE and JrTRIM elements represent the first identified walnut short interspersed element (SINE) and terminal-repeat retrotransposon in miniature (TRIM) element, respectively; both types of elements are abundant in the genome. As in other species, these SINEs and TRIM elements could be exploited for developing repeat DNA-based molecular markers in walnut. Simple sequence repeats (SSR) from BESs were analyzed and found to be more abundant in BESs than in expressed sequence tags. The density of SSR in the walnut genome analyzed was also slightly higher than that in poplar and papaya. Sequence analysis of BESs indicated that approximately 11.5% of the walnut genome represents a coding sequence. This study is an initial characterization of the walnut genome and provides the largest genomic resource currently available; as such, it will be a valuable tool in studies aimed at genetically improving walnut. PMID:22101470

Wu, Jiajie; Gu, Yong Q; Hu, Yuqin; You, Frank M; Dandekar, Abhaya M; Leslie, Charles A; Aradhya, Mallikarjuna; Dvorak, Jan; Luo, Ming-Cheng

2012-01-01

25

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

PubMed

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

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

2012-01-01

26

Construction and sequence sampling of deep-coverage, large-insert BAC libraries for three model lepidopteran species  

PubMed Central

Background Manduca sexta, Heliothis virescens, and Heliconius erato represent three widely-used insect model species for genomic and fundamental studies in Lepidoptera. Large-insert BAC libraries of these insects are critical resources for many molecular studies, including physical mapping and genome sequencing, but not available to date. Results We report the construction and characterization of six large-insert BAC libraries for the three species and sampling sequence analysis of the genomes. The six BAC libraries were constructed with two restriction enzymes, two libraries for each species, and each has an average clone insert size ranging from 152–175 kb. We estimated that the genome coverage of each library ranged from 6–9 ×, with the two combined libraries of each species being equivalent to 13.0–16.3 × haploid genomes. The genome coverage, quality and utility of the libraries were further confirmed by library screening using 6~8 putative single-copy probes. To provide a first glimpse into these genomes, we sequenced and analyzed the BAC ends of ~200 clones randomly selected from the libraries of each species. The data revealed that the genomes are AT-rich, contain relatively small fractions of repeat elements with a majority belonging to the category of low complexity repeats, and are more abundant in retro-elements than DNA transposons. Among the species, the H. erato genome is somewhat more abundant in repeat elements and simple repeats than those of M. sexta and H. virescens. The BLAST analysis of the BAC end sequences suggested that the evolution of the three genomes is widely varied, with the genome of H. virescens being the most conserved as a typical lepidopteran, whereas both genomes of H. erato and M. sexta appear to have evolved significantly, resulting in a higher level of species- or evolutionary lineage-specific sequences. Conclusion The high-quality and large-insert BAC libraries of the insects, together with the identified BACs containing genes of interest, provide valuable information, resources and tools for comprehensive understanding and studies of the insect genomes and for addressing many fundamental questions in Lepidoptera. The sample of the genomic sequences provides the first insight into the constitution and evolution of the insect genomes. PMID:19558662

Wu, Chengcang; Proestou, Dina; Carter, Dorothy; Nicholson, Erica; Santos, Filippe; Zhao, Shaying; Zhang, Hong-Bin; Goldsmith, Marian R

2009-01-01

27

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...

28

A BAC-based physical map of the Drosophila buzzatii genome  

SciTech Connect

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.

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

29

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...

30

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

PubMed Central

Positional cloning in bread wheat is a tedious task due to its huge genome size and hexaploid character. BAC libraries represent an essential tool for positional cloning. However, wheat BAC libraries comprise more than million clones, which makes their screening very laborious. Here, we present a targeted approach based on chromosome-specific BAC libraries. Such libraries were constructed from flow-sorted arms of wheat chromosome 7D. A library from the short arm (7DS) consisting of 49,152 clones with 113?kb insert size represented 12.1 arm equivalents whereas a library from the long arm (7DL) comprised 50,304 clones of 116?kb providing 14.9x arm coverage. The 7DS library was PCR screened with markers linked to Russian wheat aphid resistance gene DnCI2401, the 7DL library was screened by hybridization with a probe linked to greenbug resistance gene Gb3. The small number of clones combined with high coverage made the screening highly efficient and cost effective. PMID:21318113

Šimková, Hana; Šafá?, Jan; Kubaláková, Marie; Suchánková, Pavla; ?íhalíková, Jarmila; Robert-Quatre, Heda; Azhaguvel, Perumal; Weng, Yiqun; Peng, Junhua; Lapitan, Nora L. V.; Ma, Yaqin; You, Frank M.; Luo, Ming-Cheng; Bartoš, Jan; Doležel, Jaroslav

2011-01-01

31

BAC library construction, screening and clone sequencing of lake whitefish (Coregonus clupeaformis, Salmonidae) towards the elucidation of adaptive species divergence.  

PubMed

Genomic DNA sequences and other genomic resources are essential towards the elucidation of the genomic bases of adaptive divergence and reproductive isolation. Here, we describe the construction, characterization and screening of a nonarrayed BAC library for lake whitefish (Coregonus clupeaformis). We then show how the combined use of BAC library screening and next-generation sequencing can lead to efficient full-length assembly of candidate genes. The lake whitefish BAC library consists of 181,050 clones derived from a single heterozygous fish. The mean insert size is 92 Kb, representing 5.2 haploid genome equivalents. Ten BAC clones were isolated following a quantitative real-time PCR screening approach that targeted five previously identified candidate genes. Sequencing of these clones on a 454 GS FLX system yielded 178,000 reads with a mean length of 358 bp, for a total of 63.8 Mb. De novo assembly and annotation then allowed retrieval of contigs corresponding to each candidate gene, which also contained up- and/or downstream noncoding sequences. These results suggest that the lake whitefish BAC library combined with next-generation sequencing technologies will be key resources to achieve a better understanding of both adaptive divergence and reproductive isolation in lake whitefish species pairs as well as salmonid evolution in general. PMID:21481212

Jeukens, J; Boyle, B; Kukavica-Ibrulj, I; St-Cyr, J; Lévesque, R C; Bernatchez, L

2011-05-01

32

Construction of a sugar beet BAC library from a hybrid with diverse traits  

Microsoft Academic Search

A bacterial artificial chromosome (BAC) library of the 750-Mbp sugar beet genome represented in hybrid US H20 was constructed\\u000a fromHind III-digested DNA, with an average insert size of 120 kbp. US H20 is a variety grown in the eastern United States. It exhibits\\u000a heterosis for emergence and yield, presumably because of its hybridity between eastern and western US germplasm sources.

J. Mitchell McGrath; R. Scott Shaw; Benildo G. de los Reyes; John J. Weiland

2004-01-01

33

piggyBac transposase tools for genome engineering  

PubMed Central

The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc+Int?) transposase. Our findings also suggest the position of a target DNA–transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc+Int? transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int? phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc+Int? transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase–target DNA interaction. PMID:23723351

Li, Xianghong; Burnight, Erin R.; Cooney, Ashley L.; Malani, Nirav; Brady, Troy; Sander, Jeffry D.; Staber, Janice; Wheelan, Sarah J.; Joung, J. Keith; McCray, Paul B.; Bushman, Frederic D.; Sinn, Patrick L.; Craig, Nancy L.

2013-01-01

34

piggyBac transposase tools for genome engineering.  

PubMed

The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc(+)Int(-)) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc(+)Int(-) transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int(-) phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc(+)Int(-) transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction. PMID:23723351

Li, Xianghong; Burnight, Erin R; Cooney, Ashley L; Malani, Nirav; Brady, Troy; Sander, Jeffry D; Staber, Janice; Wheelan, Sarah J; Joung, J Keith; McCray, Paul B; Bushman, Frederic D; Sinn, Patrick L; Craig, Nancy L

2013-06-18

35

Construction of a Coix BAC library and isolation of the 22 kDa ?-coixin gene cluster.  

PubMed

Coix lacryma-jobi L. (Coix) is a close relative of maize and is considered a valuable genetic resource for crop improvement. Here we report the construction of the first Coix bacterial artificial chromosome (BAC) library using accession PI 324059. This BAC library contains about 230 400 clones with an average insert size of 113 kb, has low organellar DNA contamination, and provides 16.3-fold coverage of the genome. The library was stored in 12 × 96 pools that could be screened with a PCR protocol. Library screening was performed for the 22 kDa ?-coixin gene family. A total of 57 positive pools were identified, and single clones were isolated from 19 of these pools. Based on DNA fingerprinting and Southern blot analysis, these 19 BAC clones form a single contig of about 340 kb in length, indicating that the 22 kDa ?-coixin genes occur in a cluster. These results demonstrated the suitability of this BAC library for gene isolation and comparative genomics studies of the Coix genome. PMID:20924416

Meng, Xiangzong; Huang, Binbin; Zhou, Liangliang; He, Yunxia; Chen, Qi; Yuan, Yiwei; Xu, Zhengkai; Song, Rentao

2010-09-01

36

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

PubMed Central

A bacterial artificial chromosome (BAC) library was constructed by cloning HindIII-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Approximately 53 500 clones were arrayed in 384-well plates and stored at -80°C (CCBL1), while clones from a smaller insert size fraction were stored at -80°C without arraying (CCBL2). Pulsed-field gel electrophoresis of 100 clones after NotI digestion revealed an average insert size of 165 kb for CCBL1 and 113 kb for CCBL2. Further characterization of CCBL1 demonstrated that 10% of the clones did not contain an insert. CCBL1 provides a 7.2-fold coverage of the channel catfish haploid genome. PCR-based screening demonstrated that 68 out of 74 unique loci were present in the library. This represents a 92% chance to find a unique sequence. These libraries will be useful for physical mapping of the channel catfish genome, and identification of genes controlling major traits in this economically important species. PMID:14604514

Quiniou, Sylvie MA; Katagiri, Takayuki; Miller, Norman W; Wilson, Melanie; Wolters, William R; Waldbieser, Geoffrey C

2003-01-01

37

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

PubMed Central

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

Wu, Shao-Ying; Wan, Qiu-Hong

2012-01-01

38

Bac clones generated from sheared dna  

SciTech Connect

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.

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

39

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...

40

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

PubMed Central

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

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

2011-01-01

41

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

PubMed

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

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

2010-07-01

42

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

NASA Astrophysics Data System (ADS)

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

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

2012-05-01

43

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

PubMed Central

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

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

2014-01-01

44

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

PubMed

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

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

2014-01-01

45

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-...

46

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

SciTech Connect

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.

Hubert, R.S.; Chen, X.N.; Mitchell, S. [Univ. of Los Angeles, CA (United States)] [and others

1994-09-01

47

Comparative BAC-based mapping in the white-throated sparrow, a novel behavioral genomics model, using interspecies overgo hybridization  

PubMed Central

Background The genomics era has produced an arsenal of resources from sequenced organisms allowing researchers to target species that do not have comparable mapping and sequence information. These new "non-model" organisms offer unique opportunities to examine environmental effects on genomic patterns and processes. Here we use comparative mapping as a first step in characterizing the genome organization of a novel animal model, the white-throated sparrow (Zonotrichia albicollis), which occurs as white or tan morphs that exhibit alternative behaviors and physiology. Morph is determined by the presence or absence of a complex chromosomal rearrangement. This species is an ideal model for behavioral genomics because the association between genotype and phenotype is absolute, making it possible to identify the genomic bases of phenotypic variation. Findings We initiated a genomic study in this species by characterizing the white-throated sparrow BAC library via filter hybridization with overgo probes designed for the chicken, turkey, and zebra finch. Cross-species hybridization resulted in 640 positive sparrow BACs assigned to 77 chicken loci across almost all macro-and microchromosomes, with a focus on the chromosomes associated with morph. Out of 216 overgos, 36% of the probes hybridized successfully, with an average number of 3.0 positive sparrow BACs per overgo. Conclusions These data will be utilized for determining chromosomal architecture and for fine-scale mapping of candidate genes associated with phenotypic differences. Our research confirms the utility of interspecies hybridization for developing comparative maps in other non-model organisms. PMID:21693052

2011-01-01

48

Construction and Preliminary Characterization Analysis of Wuzhishan Miniature Pig Bacterial Artificial Chromosome Library with Approximately 8-Fold Genome Equivalent Coverage  

PubMed Central

Bacterial artificial chromosome (BAC) libraries have been invaluable tools for the genome-wide genetic dissection of complex organisms. Here, we report the construction and characterization of a high-redundancy BAC library from a very valuable pig breed in China, Wuzhishan miniature pig (Sus scrofa), using its blood cells and fibroblasts, respectively. The library contains approximately 153,600 clones ordered in 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 152.3?kb, representing approximately 7.68 genome equivalents of the porcine haploid genome and a 99.93% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. 19 pairs of microsatellite marker primers covering porcine chromosomes were used for screening the BAC library, which showed that each of these markers was positive in the library; the positive clone number was 2 to 9, and the average number was 7.89, which was consistent with 7.68-fold coverage of the porcine genome. And there were no significant differences of genomic BAC library from blood cells and fibroblast cells. Therefore, we identified 19 microsatellite markers that could potentially be used as genetic markers. As a result, this BAC library will serve as a valuable resource for gene identification, physical mapping, and comparative genomics and large-scale genome sequencing in the porcine. PMID:23691508

Liu, Changqing; Guo, Yuo; Lu, Taofeng; Wu, Hongmei; Na, Risu; Li, Xiangchen; Guan, Weijun; Ma, Yuehui

2013-01-01

49

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

50

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...

51

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

PubMed

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

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

2013-10-01

52

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

53

Genomic insight into the common carp (Cyprinus carpio) genome by sequencing analysis of BAC-end sequences  

PubMed Central

Background Common carp is one of the most important aquaculture teleost fish in the world. Common carp and other closely related Cyprinidae species provide over 30% aquaculture production in the world. However, common carp genomic resources are still relatively underdeveloped. BAC end sequences (BES) are important resources for genome research on BAC-anchored genetic marker development, linkage map and physical map integration, and whole genome sequence assembling and scaffolding. Result To develop such valuable resources in common carp (Cyprinus carpio), a total of 40,224 BAC clones were sequenced on both ends, generating 65,720 clean BES with an average read length of 647 bp after sequence processing, representing 42,522,168 bp or 2.5% of common carp genome. The first survey of common carp genome was conducted with various bioinformatics tools. The common carp genome contains over 17.3% of repetitive elements with GC content of 36.8% and 518 transposon ORFs. To identify and develop BAC-anchored microsatellite markers, a total of 13,581 microsatellites were detected from 10,355 BES. The coding region of 7,127 genes were recognized from 9,443 BES on 7,453 BACs, with 1,990 BACs have genes on both ends. To evaluate the similarity to the genome of closely related zebrafish, BES of common carp were aligned against zebrafish genome. A total of 39,335 BES of common carp have conserved homologs on zebrafish genome which demonstrated the high similarity between zebrafish and common carp genomes, indicating the feasibility of comparative mapping between zebrafish and common carp once we have physical map of common carp. Conclusion BAC end sequences are great resources for the first genome wide survey of common carp. The repetitive DNA was estimated to be approximate 28% of common carp genome, indicating the higher complexity of the genome. Comparative analysis had mapped around 40,000 BES to zebrafish genome and established over 3,100 microsyntenies, covering over 50% of the zebrafish genome. BES of common carp are tremendous tools for comparative mapping between the two closely related species, zebrafish and common carp, which should facilitate both structural and functional genome analysis in common carp. PMID:21492448

2011-01-01

54

Construction and Characterization of a Human Bacterial Artificial Chromosome Library  

Microsoft Academic Search

We have constructed an arrayed human genomic BAC library with approximately 4× coverage that is represented by 96,000 BAC clones with average insert size of nearly 140 kb. A new BAC vector that allows color-based positive screening to identify transformants with inserts has increased BAC cloning efficiency. The library was gridded onto hybridization filters at high density for efficient identification

Ung-Jin Kim; Bruce W. Birren; Tatiana Slepak; Valeria Mancino; Cecilie Boysen; Hyung-Lyun Kang; Melvin I. Simon; Hiroaki Shizuya

1996-01-01

55

Use of a Mycobacterium tuberculosis H37Rv Bacterial Artificial Chromosome Library for Genome Mapping, Sequencing, and Comparative Genomics  

PubMed Central

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of ?150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects. PMID:9573111

Brosch, Roland; Gordon, Stephen V.; Billault, Alain; Garnier, Thierry; Eiglmeier, Karin; Soravito, Catherine; Barrell, Bart G.; Cole, Stewart T.

1998-01-01

56

Chromosomal Mapping of Canine-Derived BAC Clones to the Red Fox and American Mink Genomes  

PubMed Central

High-quality sequencing of the dog (Canis lupus familiaris) genome has enabled enormous progress in genetic mapping of canine phenotypic variation. The red fox (Vulpes vulpes), another canid species, also exhibits a wide range of variation in coat color, morphology, and behavior. Although the fox genome has not yet been sequenced, canine genomic resources have been used to construct a meiotic linkage map of the red fox genome and begin genetic mapping in foxes. However, a more detailed gene-specific comparative map between the dog and fox genomes is required to establish gene order within homologous regions of dog and fox chromosomes and to refine breakpoints between homologous chromosomes of the 2 species. In the current study, we tested whether canine-derived gene–containing bacterial artificial chromosome (BAC) clones can be routinely used to build a gene-specific map of the red fox genome. Forty canine BAC clones were mapped to the red fox genome by fluorescence in situ hybridization (FISH). Each clone was uniquely assigned to a single fox chromosome, and the locations of 38 clones agreed with cytogenetic predictions. These results clearly demonstrate the utility of FISH mapping for construction of a whole-genome gene-specific map of the red fox. The further possibility of using canine BAC clones to map genes in the American mink (Mustela vison) genome was also explored. Much lower success was obtained for this more distantly related farm-bred species, although a few BAC clones were mapped to the predicted chromosomal locations. PMID:19546120

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

2009-01-01

57

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

PubMed Central

Background Bread wheat, one of the world’s staple food crops, has the largest, highly repetitive and polyploid genome among the cereal crops. The wheat genome holds the key to crop genetic improvement against challenges such as climate change, environmental degradation, and water scarcity. To unravel the complex wheat genome, the International Wheat Genome Sequencing Consortium (IWGSC) is pursuing a chromosome- and chromosome arm-based approach to physical mapping and sequencing. Here we report on the use of a BAC library made from flow-sorted telosomic chromosome 3A short arm (t3AS) for marker development and analysis of sequence composition and comparative evolution of homoeologous genomes of hexaploid wheat. Results The end-sequencing of 9,984 random BACs from a chromosome arm 3AS-specific library (TaaCsp3AShA) generated 11,014,359?bp of high quality sequence from 17,591 BAC-ends with an average length of 626?bp. The sequence represents 3.2% of t3AS with an average DNA sequence read every 19?kb. Overall, 79% of the sequence consisted of repetitive elements, 1.38% as coding regions (estimated 2,850 genes) and another 19% of unknown origin. Comparative sequence analysis suggested that 70-77% of the genes present in both 3A and 3B were syntenic with model species. Among the transposable elements, gypsy/sabrina (12.4%) was the most abundant repeat and was significantly more frequent in 3A compared to homoeologous chromosome 3B. Twenty novel repetitive sequences were also identified using de novo repeat identification. BESs were screened to identify simple sequence repeats (SSR) and transposable element junctions. A total of 1,057 SSRs were identified with a density of one per 10.4?kb, and 7,928 junctions between transposable elements (TE) and other sequences were identified with a density of one per 1.39?kb. With the objective of enhancing the marker density of chromosome 3AS, oligonucleotide primers were successfully designed from 758 SSRs and 695 Insertion Site Based Polymorphisms (ISBPs). Of the 96 ISBP primer pairs tested, 28 (29%) were 3A-specific and compared to 17 (18%) for 96 SSRs. Conclusion This work reports on the use of wheat chromosome arm 3AS-specific BAC library for the targeted generation of sequence data from a particular region of the huge genome of wheat. A large quantity of sequences were generated from the A genome of hexaploid wheat for comparative genome analysis with homoeologous B and D genomes and other model grass genomes. Hundreds of molecular markers were developed from the 3AS arm-specific sequences; these and other sequences will be useful in gene discovery and physical mapping. PMID:22559868

2012-01-01

58

Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR  

Microsoft Academic Search

Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research. We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes. The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised

Fredrik Sterky; Anders Holmberg; Gunnar Alexandersson; Joakim Lundeberg; Mathias Uhlén

1998-01-01

59

Generation of the first BAC-based physical map of the common carp genome  

PubMed Central

Background Common carp (Cyprinus carpio), a member of Cyprinidae, is the third most important aquaculture species in the world with an annual global production of 3.4 million metric tons, accounting for nearly 14% of the all freshwater aquaculture production in the world. Apparently genomic resources are needed for this species in order to study its performance and production traits. In spite of much progress, no physical maps have been available for common carp. The objective of this project was to generate a BAC-based physical map using fluorescent restriction fingerprinting. Result The first generation of common carp physical map was constructed using four- color High Information Content Fingerprinting (HICF). A total of 72,158 BAC clones were analyzed that generated 67,493 valid fingerprints (5.5 × genome coverage). These BAC clones were assembled into 3,696 contigs with the average length of 476 kb and a N50 length of 688 kb, representing approximately 1.76 Gb of the common carp genome. The largest contig contained 171 BAC clones with the physical length of 3.12 Mb. There are 761 contigs longer than the N50, and these contigs should be the most useful resource for future integrations with linkage map and whole genome sequence assembly. The common carp physical map is available at http://genomics.cafs.ac.cn/fpc/WebAGCoL/Carp/WebFPC/. Conclusion The reported common carp physical map is the first physical map of the common carp genome. It should be a valuable genome resource facilitating whole genome sequence assembly and characterization of position-based genes important for aquaculture traits. PMID:22044723

2011-01-01

60

The wheat D-genome HMW-glutenin locus: BAC sequencing, gene distribution, and retrotransposon clusters.  

PubMed

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 in the same orientation, are 51,893 bp apart, and the separating DNA includes a 31,000-bp cluster of retrotransposons. A second retrotransposon cluster of 32,000 bp follows the x-type HMW-glutenin gene region. Each HMW-glutenin gene is found within a region of mainly unique DNA sequence which includes multiple additional genes including an active endosperm globulin gene not previously reported in the Triticeae family, a leucine-rich-repeat (LRR) type gene truncated at the 5' end of the BAC, a kinase gene of unknown activity, remnants of a paralogous second globulin gene, and genes similar to two hypothetical rice genes. The newly identified globulin genes are assigned to a locus designated Glo-2. Comparison to available orthologous regions of the wheat A and B genomes show rapid sequence divergences flanking the HMW-glutenin genes, and the absence of two hypothetical and unknown genes found 5' to the B-genome x-type ortholog. The region surrounding the Glu-D1 locus is similar to other reported Triticeae BAC sequences; i.e. small gene islands separated by retrotransposon clusters. PMID:12590343

Anderson, O D; Rausch, C; Moullet, O; Lagudah, E S

2003-03-01

61

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...

62

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

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

2009-01-01

63

Comparative analysis of BAC and whole genome shotgun sequences from an Anopheles gambiae region related to Plasmodium encapsulation.  

PubMed

The only natural mechanism of malaria transmission in sub-Saharan Africa is the mosquito, generally Anopheles gambiae. Blocking malaria parasite transmission by stopping the development of Plasmodium in the insect vector would provide a useful alternative to the current methods of malaria control. Toward this end, it is important to understand the molecular basis of the malaria parasite refractory phenotype in An. gambiae mosquito strains. We have selected and sequenced six bacterial artificial chromosome (BAC) clones from the Pen-1 region that is the major quantitative trait locus involved in Plasmodium encapsulation. The sequence and the annotation of five overlapping BAC clones plus one adjacent, but not contiguous clone, totaling 585kb of genomic sequence from the centromeric end of the Pen-1 region of the PEST strain were compared to that of the genome sequence of the same strain produced by the whole genome shotgun technique. This project identified 23 putative mosquito genes plus putative copies of the retrotransposable elements BEL12 and TRANSIBN1_AG in the six BAC clones. Nineteen of the predicted genes are most similar to their Drosophila melanogaster homologs while one is more closely related to vertebrate genes. Comparison of these new BAC sequences plus previously published BAC sequences to the cognate region of the assembled genome sequence identified three retrotransposons present in one sequence version but not the other. One of these elements, Indy, has not been previously described. These observations provide evidence for the recent active transposition of these elements and demonstrate the plasticity of the Anopheles genome. The BAC sequences strongly support the public whole genome shotgun assembly and automatic annotation while also demonstrating the benefit of complementary genome sequences and of human curation. Importantly, the data demonstrate the differences in the genome sequence of an individual mosquito compared to that of a hypothetical, average genome sequence generated by whole genome shotgun assembly. PMID:15944077

Eiglmeier, Karin; Wincker, Patrick; Cattolico, Laurence; Anthouard, Veronique; Holm, Inge; Eckenberg, Ralph; Quesneville, Hadi; Jaillon, Olivier; Collins, Frank H; Weissenbach, Jean; Brey, Paul T; Roth, Charles W

2005-08-01

64

ANALYSIS OF PAPAYA BAC END SEQUENCES REVEALS FIRST INSIGHTS INTO THE ORGANIZATION OF A FRUIT TREE GENOME  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

65

ANALYSIS OF PAPAYA BAC END SEQUENCES REVEALS FIRST INSIGHTS INTO THE ORGANIZATION OF A TREE-FRUIT GENOME  

Technology Transfer Automated Retrieval System (TEKTRAN)

Papaya (Carica papaya L.) is a major tree fruit crop of tropical and subtropical regions with an estimated genome size of 372 Mbp. Here we present the sequence analysis of 4.7% of the papaya genome based on 50,661 BAC end sequences (BESs) representing 17,483,563 high quality bases generated from 26,...

66

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

E-print Network

sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig to the DMRT Gene Family ZHENSHAN WANG, TSUTOMU MIYAKE, SCOTT V. EDWARDS, AND CHRIS T. AMEMIYA From (Hedges and Poling 1999), whereas analysis of small and large mitochondrial ribosomal genes united tuatara

Edwards, Scott

67

Self-excision of the BAC sequences from the recombinant Marek's disease virus genome increases replication and pathogenicity  

PubMed Central

Cloning of full length genomes of herpesviruses as bacterial artificial chromosomes (BAC) has greatly facilitated the manipulation of the genomes of several herpesviruses to identify the pathogenic determinants. We have previously reported the construction of the BAC clone (pRB-1B5) of the highly oncogenic Marek's disease virus (MDV) strain RB-1B, which has proven to be a valuable resource for elucidating several oncogenic determinants. Despite the retention of the BAC replicon within the genome, the reconstituted virus was able to induce tumours in susceptible chickens. Nevertheless, it was unclear whether the presence of the BAC influenced the full oncogenic potential of the reconstituted virus. To maximize the closeness of BAC-derived virus to the parental RB-1B strain, we modified the existing pRB-1B5 clone by restoring the Us2 and by introducing SV40-cre cassette within the loxP sites of the mini-F plasmid, to allow self-excision of the plasmid sequences in chicken cells. The reconstituted virus from the modified clone showed significant improvement in replication in vitro and in vivo. Excision of the BAC sequences also enhanced the pathogenicity to levels similar to that of the parental virus, as the cumulative incidence of Marek's disease in groups infected with the recombinant and the parental viruses showed no significant differences. Thus, we have been able to make significant improvements to the existing BAC clone of this highly oncogenic virus which would certainly increase its usefulness as a valuable tool for studies on identifying the oncogenic determinants of this major avian pathogen. PMID:18230192

Zhao, Yuguang; Petherbridge, Lawrence; Smith, Lorraine P; Baigent, Sue; Nair, Venugopal

2008-01-01

68

Yeast Genomic Library Genomic DNA Sau3AI partial digestion  

E-print Network

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

Odorizzi, Greg

69

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

PubMed

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

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

2013-06-01

70

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

PubMed Central

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

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

2015-01-01

71

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

PubMed

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

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

2015-01-01

72

A first generation bovine BAC-based physical map  

PubMed Central

A first generation clone-based physical map for the bovine genome was constructed combining, fluorescent double digestion fingerprinting and sequence tagged site (STS) marker screening. The BAC clones were selected from an Inra BAC library (105 984 clones) and a part of the CHORI-240 BAC library (26 500 clones). The contigs were anchored using the screening information for a total of 1303 markers (451 microsatellites, 471 genes, 127 EST, and 254 BAC ends). The final map, which consists of 6615 contigs assembled from 100 923 clones, will be a valuable tool for genomic research in ruminants, including targeted marker production, positional cloning or targeted sequencing of regions of specific interest. PMID:14713413

Schibler, Laurent; Roig, Anne; Mahé, Marie-Françoise; Save, Jean-Claude; Gautier, Mathieu; Taourit, Sead; Boichard, Didier; Eggen, André; Cribiu, Edmond P

2004-01-01

73

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome  

PubMed Central

Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes. PMID:19656416

2009-01-01

74

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...

75

Human Genome Anatomy: BACs Integrating the Genetic and Cytogenetic Maps for Bridging Genome and Biomedicine  

Microsoft Academic Search

Human genome sequencing is accelerating rapidly. Multiple genome maps link this sequence to problems in biology and clinical medicine. Because each map represents a different aspect of the structure, content, and behavior of human chromosomes, these fundamental properties must be integrated with the genome to understand disease genes, cancer instability, and human evolution. Cytogenetic maps use 400-850 visible band landmarks

Julie R. Korenberg; Xiao-Ning Chen; Zhiguang Sun; Zheng-Yang Shi; Shaowu Ma; Eddy Vataru; Dean Yimlamai; Jean S. Weissenbach; Hiroaki Shizuya; Melvin I. Simon; Sebastian S. Gerety; Huy Nguyen; Irina S. Zemsteva; Lester Hui; James Silva; Xiaoyun Wu; Bruce W. Birren; Thomas J. Hudson

2006-01-01

76

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH  

PubMed Central

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 ?g usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes. PMID:15284333

Guillaud-Bataille, Marine; Valent, Alexander; Soularue, Pascal; Perot, Christine; Inda, Maria-del-Mar; Receveur, Aline; Smaïli, Sadek; Crollius, Hugues Roest; Bénard, Jean; Bernheim, Alain; Gidrol, Xavier; Danglot, Gisèle

2004-01-01

77

A first generation BAC-based physical map of the half-smooth tongue sole (Cynoglossus semilaevis) genome  

PubMed Central

Background Half-smooth tongue sole (Cynoglossus semilaevis Günther) has been exploited as a commercially important cultured marine flatfish, and female grows 2–3 times faster than male. Genetic studies, especially on the chromosomal sex-determining system of this species, have been carried out in the last decade. Although the genome of half-smooth tongue sole was relatively small (626.9 Mb), there are still some difficulties in the high-quality assembly of the next generation genome sequencing reads without the assistance of a physical map, especially for the W chromosome of this fish due to abundance of repetitive sequences. The objective of this study is to construct a bacterial artificial chromosome (BAC)-based physical map for half-smooth tongue sole with the method of high information content fingerprinting (HICF). Results A physical map of half-smooth tongue sole was constructed with 30, 294 valid fingerprints (7.5?×?genome coverage) with a tolerance of 4 and an initial cutoff of 1e-60. A total of 29,709 clones were assembled into 1,485 contigs with an average length of 539 kb and a N50 length of 664 kb. There were 394 contigs longer than the N50 length, and these contigs will be a useful resource for future integration with linkage map and whole genome sequence assembly. The estimated physical length of the assembled contigs was 797 Mb, representing approximately 1.27 coverage of the half-smooth tongue sole genome. The largest contig contained 410 BAC clones with a physical length of 3.48 Mb. Almost all of the 676 BAC clones (99.9%) in the 21 randomly selected contigs were positively validated by PCR assays, thereby confirming the reliability of the assembly. Conclusions A first generation BAC-based physical map of half-smooth tongue sole was constructed with high reliability. The map will promote genetic improvement programs of this fish, especially integration of physical and genetic maps, fine-mappings of important gene and/or QTL, comparative and evolutionary genomics studies, as well as whole genome sequence assembly. PMID:24650389

2014-01-01

78

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...

79

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis  

Microsoft Academic Search

BACKGROUND: The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects

Giang T. H. Vu; Peter D. S. Caligari; Mike J. Wilkinson

2010-01-01

80

A bacterial artificial chromosome library for the Chinese alligator (Alligator sinensis).  

PubMed

Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90 kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516 kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species. PMID:22759519

He, Ke; Ye, Qing; Zhu, Ying; Chen, Hui; Wan, Qiu-Hong; Fang, Sheng-Guo

2012-10-01

81

Recombineering linear BACs.  

PubMed

Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells. PMID:25239740

Chen, Qingwen; Narayanan, Kumaran

2015-01-01

82

Construction of Two BAC Libraries from Half-Smooth Tongue Sole Cynoglossus semilaevis and Identification of Clones Containing Candidate Sex-Determination Genes  

Microsoft Academic Search

Half-smooth tongue sole (Cynoglossus semilaevis) is an increasingly important aquaculture species in China. It is also a tractable model to study sex chromosome evolution\\u000a and to further elucidate the mechanism of sex determination in teleosts. Two bacterial artificial chromosome (BAC) libraries\\u000a for C. semilaevis, with large, high-quality inserts and deep coverage, were constructed in the BamHI and HindIII sites of

Chang-Wei Shao; Song-Lin Chen; Chantel F. Scheuring; Jian-Yong Xu; Zhen-Xia Sha; Xiao-Li Dong; Hong-Bin Zhang

2010-01-01

83

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...

84

Construction and Characterization of a Schistosoma mansoni Bacterial Artificial Chromosome Library  

Microsoft Academic Search

A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between

Marie-Christine Le Paslier; Raymond J. Pierce; Françoise Merlin; Hirohisa Hirai; Wenjie Wu; David L. Williams; David Johnston; Philip T. LoVerde; Denis Le Paslier

2000-01-01

85

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

PubMed

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

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

2015-01-01

86

Generation of BAC reporter cell lines for drug discovery.  

PubMed

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

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

2015-01-01

87

piggyBac  

PubMed Central

In addition to their natural role in eukaryotic genome evolution, transposons can be powerful tools for functional genomics in diverse taxa. The piggyBac transposon has been applied as such in eukaryotic parasites, both protozoa and helminths, and in several important vector mosquitoes. piggyBac is advantageous for functional genomics because of its ability to transduce a wide range of taxa, its capacity to integrate large DNA ‘cargoes’ relative to other mobile genetic elements, its propensity to target transcriptional units and its ability to re-mobilize without leaving a pattern of non-excised sequences or ‘footprint’ in the genome. We recently demonstrated that piggyBac can integrate transgenes into the genome of the parasitic nematode Strongyloides ratti, an important model for parasitic nematode biology and a close relative of the significant human pathogen S. stercoralis. Unlike transgenes encoded in conventional plasmid vectors, which we assume are assembled into multi-copy episomal arrays as they are in Caenorhabditis elegans, transgenes integrated via piggyBac are not only stably inherited in S. ratti, they are also continuously expressed. This has allowed derivation of the first stable transgene expressing lines in any parasitic nematode, a significant advance in the development of functional genomic tools for these important pathogens. PMID:23914309

Lok, James

2013-01-01

88

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

PubMed

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

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

2014-01-01

89

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries.  

PubMed

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci. PMID:12426598

Rodrigues, N B; Loverde, P T; Romanha, A J; Oliveira, G

2002-01-01

90

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

91

A BAC clone fingerprinting approach to the detection of human genome rearrangements  

PubMed Central

We present a method, called fingerprint profiling (FPP), that uses restriction digest fingerprints of bacterial artificial chromosome clones to detect and classify rearrangements in the human genome. The approach uses alignment of experimental fingerprint patterns to in silico digests of the sequence assembly and is capable of detecting micro-deletions (1-5 kb) and balanced rearrangements. Our method has compelling potential for use as a whole-genome method for the identification and characterization of human genome rearrangements. PMID:17953769

Krzywinski, Martin; Bosdet, Ian; Mathewson, Carrie; Wye, Natasja; Brebner, Jay; Chiu, Readman; Corbett, Richard; Field, Matthew; Lee, Darlene; Pugh, Trevor; Volik, Stas; Siddiqui, Asim; Jones, Steven; Schein, Jacquie; Collins, Collin; Marra, Marco

2007-01-01

92

Gene Transfer Efficiency and Genome-Wide Integration Profiling of Sleeping Beauty, Tol2, and PiggyBac Transposons in Human Primary T Cells  

Microsoft Academic Search

In this study, we compared the genomic integration efficiencies and transposition site preferences of Sleeping Beauty (SB or SB11), Tol2, and piggyBac (PB) transposon systems in primary T cells derived from peripheral blood lymphocytes (PBL) and umbilical cord blood (UCB). We found that PB demonstrated the highest efficiency of stable gene transfer in PBL-derived T cells, whereas SB11 and Tol2

Xin Huang; Hongfeng Guo; Syam Tammana; Yong-Chul Jung; Emil Mellgren; Preetinder Bassi; Qing Cao; Zheng Jin Tu; Yeong C Kim; Stephen C Ekker; Xiaolin Wu; San Ming Wang; Xianzheng Zhou

2010-01-01

93

The Populus Genome and Comparative Genomics  

Microsoft Academic Search

\\u000a \\u000a Populus was the first tree genome, and one of the first plant genomes, to be sequenced. The sequencing project and subsequent annotation\\u000a was a collaborative, international effort, with the bulk of the sequencing carried out by the US Department of Energy Joint\\u000a Genome Institute. Due to the high degree of sequence coverage, the hybrid BAC library-whole genome shotgun approach employed,

Carl J. Douglas; Stephen P. DiFazio

94

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...

95

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 (...

96

Adaptation of a commercial robot for genome library replication  

SciTech Connect

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.

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

1994-01-01

97

BAC sequencing using pooled methods.  

PubMed

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

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

2015-01-01

98

Rainbow smelt (Osmerus mordax) genomic library and EST resources.  

PubMed

Genomic resources in rainbow smelt (Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several complementary deoxyribonucleic acid libraries were constructed, and 36,758 sequences were obtained and combined into 12,159 transcripts. Over half of these transcripts have been identified, several of which have been associated with cold adaptation. These basic resources show high levels of similarity (86%) to salmonid genes and provide initial support for genome duplication in the salmonid ancestor. They also facilitate identification of genes important to fish and direct us toward new technologies for other studies in fish biology. PMID:18386095

von Schalburg, K R; Leong, J; Cooper, G A; Robb, A; Beetz-Sargent, M R; Lieph, R; Holt, R A; Moore, R; Ewart, K V; Driedzic, W R; ten Hallers, B F H; Zhu, B; de Jong, P J; Davidson, W S; Koop, B F

2008-01-01

99

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

PubMed Central

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

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

2011-01-01

100

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

PubMed

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

Chatterjee, Pradeep K

2015-01-01

101

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....

102

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...

103

A physical map of the bovine genome  

Microsoft Academic Search

BACKGROUND: Cattle are important agriculturally and relevant as a model organism. Previously described genetic and radiation hybrid (RH) maps of the bovine genome have been used to identify genomic regions and genes affecting specific traits. Application of these maps to identify influential genetic polymorphisms will be enhanced by integration with each other and with bacterial artificial chromosome (BAC) libraries. The

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

2007-01-01

104

RAPD-based screening of genomic libraries for positional cloning.  

PubMed

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci. PMID:9396827

Dioh, W; Tharreau, D; Lebrun, M H

1997-12-15

105

A First Generation BAC-Based Physical Map of the Asian Seabass (Lates calcarifer)  

Microsoft Academic Search

BackgroundThe Asian seabass (Lates calcarifer) is an important marine foodfish species in Southeast Asia and Australia. Genetic improvement of this species has been achieved to some extent through selective breeding programs since 1990s. Several genomic tools such as DNA markers, a linkage map, cDNA and BAC libraries have been developed to assist selective breeding. A physical map is still lacking,

Jun Hong Xia; Felicia Feng; Grace Lin; Chun Ming Wang; Gen Hua Yue; Bengt Hansson

2010-01-01

106

Python Materials Genomics (pymatgen): A robust, open-source python library for materials analysis  

E-print Network

Python Materials Genomics (pymatgen): A robust, open-source python library for materials analysis-throughput a b s t r a c t We present the Python Materials Genomics (pymatgen) library, a robust, open-source Python library for materials analysis. A key enabler in high-throughput computational materials science

Ceder, Gerbrand

107

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

PubMed

Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ? 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest. PMID:24682816

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

2014-06-01

108

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

PubMed Central

Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ?550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5? cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest. PMID:24682816

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

2014-01-01

109

Using Partial Genomic Fosmid Libraries for Sequencing CompleteOrganellar Genomes  

SciTech Connect

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.

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

2005-08-26

110

Human Cytomegalovirus: Bacterial Artificial Chromosome (BAC) Cloning and Genetic Manipulation  

PubMed Central

Our understanding of human cytomegalovirus (HCMV) biology was long hindered by the inability to perform efficient viral genetic analysis. This hurdle was recently overcome when the genomes of multiple HCMV strains were cloned as infectious bacterial artificial chromosomes (BACs). The BAC system takes advantage of the single-copy F plasmid of E. coli that can stably carry large pieces of foreign DNA. In this system, a recombinant HCMV virus carrying a modified F plasmid is first generated in eukaryotic cells. Recombinant viral genomes are then isolated and recovered in E. coli as BAC clones. BAC-captured viral genomes can be manipulated using prokaryotic genetics, and recombinant virus can be reconstituted from BAC transfection in eukaryotic cells. The BAC reverse genetic system provides a reliable and efficient method to introduce genetic alterations into the viral genome in E.coli and subsequently analyze their effects on virus biology in eukaryotic cells. PMID:22307551

Paredes, Anne M.; Yu, Dong

2011-01-01

111

MultiBac turns sweet.  

PubMed

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

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

2013-01-01

112

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

PubMed Central

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

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

113

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

PubMed

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

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

2014-06-01

114

Herpesvirus BACs: Past, Present, and Future  

PubMed Central

The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determine the function and essentiality of viral genes. In this paper, we discuss the creation of herpesvirus BACs, functional analyses of herpesvirus mutants, and future applications for studies of herpesviruses. We describe commonly used methods to create and mutate herpesvirus BACs (such as site-directed mutagenesis and transposon mutagenesis). We also evaluate the potential future uses of viral BACs, including vaccine development and gene therapy. PMID:21048927

Warden, Charles; Tang, Qiyi; Zhu, Hua

2011-01-01

115

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

PubMed Central

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics. PMID:24037269

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

2013-01-01

116

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

PubMed

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics. PMID:24037269

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

2013-11-01

117

Comparative BAC-based mapping in the white-throated sparrow, a novel behavioral genomics model, using interspecies overgo hybridization  

Microsoft Academic Search

Background  The genomics era has produced an arsenal of resources from sequenced organisms allowing researchers to target species that\\u000a do not have comparable mapping and sequence information. These new \\

Michael N Romanov; Jerry B Dodgson; Rusty A Gonser; Elaina M Tuttle

2011-01-01

118

Genome-wide comparison of paired fresh frozen and formalin-fixed paraffin-embedded gliomas by custom BAC and oligonucleotide array comparative genomic hybridization: facilitating analysis of archival gliomas  

PubMed Central

Molecular genetic analysis of cancer is rapidly evolving as a result of improvement in genomic technologies and the growing applicability of such analyses to clinical oncology. Array based comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA), particularly in solid tumors, and has been applied to the study of malignant gliomas. In the clinical setting, however, gliomas are often sampled by small biopsies and thus formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis, especially for rare types of gliomas. Moreover, the biological basis for the marked intratumoral heterogeneity in gliomas is most readily addressed in FFPE material. Therefore, for gliomas, the ability to use DNA from FFPE tissue is essential for both clinical and research applications. In this study, we have constructed a custom bacterial artificial chromosome (BAC) array and show excellent sensitivity and specificity for detecting CNAs in a panel of paired frozen and FFPE glioma samples. Our study demonstrates a high concordance rate between CNAs detected in FFPE compared to frozen DNA. We have also developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. This labeling technique was applied to a panel of biphasic anaplastic oligoastrocytomas (AOA) to identify genetic changes unique to each component. Together, results from these studies suggest that BAC and oligonucleotide aCGH are sensitive tools for detecting CNAs in FFPE DNA, and can enable genome-wide analysis of rare, small and/or histologically heterogeneous gliomas. PMID:21080181

Mohapatra, Gayatry; Engler, David A.; Starbuck, Kristen D.; Kim, James C.; Bernay, Derek C.; Scangas, George A.; Rousseau, Audrey; Batchelor, Tracy T.; Betensky, Rebecca A.; Louis, David N.

2010-01-01

119

Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays  

PubMed Central

Background Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate® Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. Results A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina’s GoldenGate® Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. Conclusions Illumina’s GoldenGate® Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome. PMID:24010766

2013-01-01

120

A primer on using pooled shRNA libraries for functional genomic screens  

PubMed Central

The discovery of RNA interference (RNAi) has revolutionized genetic analysis in mammalian cells. Loss-of-function RNAi screens enable rapid, functional annotation of the genome. Of the various RNAi approaches, pooled shRNA libraries have received considerable attention because of their versatility. A number of genome-wide shRNA libraries have been constructed against the human and mouse genomes, and these libraries can be readily applied to a variety of screens to interrogate the function of human and mouse genes in an unbiased fashion. We provide an introduction to the technical aspects of using pooled shRNA libraries for genetic screens. PMID:22271906

Hu, Guang; Luo, Ji

2012-01-01

121

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...

122

Bacterial Artificial Chromosome Libraries of Pulse Crops: Characteristics and Applications  

PubMed Central

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

Yu, Kangfu

2012-01-01

123

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

124

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

PubMed

A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen. PMID:10783255

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

2000-04-15

125

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

PubMed

We have designed DOP-PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP-PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome-specific clones spotted on microarrays by use of DNA amplified from flow-sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1-Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet. PMID:12619160

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

2003-04-01

126

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

PubMed Central

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

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

2013-01-01

127

Consequences of normalizing transcriptomic and genomic libraries of plant genomes using a duplex-specific nuclease and tetramethylammonium chloride.  

PubMed

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

Matvienko, Marta; Kozik, Alexander; Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

128

SHORT COMMUNICATION Rainbow Smelt (Osmerus mordax) Genomic Library and EST Resources  

E-print Network

(Osmerus mordax) enable us to examine the genome duplication process in salmonids and test hypotheses relating to the fate of duplicated genes. They further enable us to pursue physiological and ecological studies in smelt. A bacterial artificial chromosome library containing 52,410 clones with an average insert size of 146 kb was constructed. This library represents an 11-fold average coverage of the rainbow smelt (O. mordax) genome. In addition, several

K. V. Ewart; W. R. Driedzic; W. S. Davidson; B. F. Koop

2007-01-01

129

Creation of genome-wide protein expression libraries using random activation of gene expression  

Microsoft Academic Search

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 × 106 individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE

Bruce Sherf; Stephen Rundlett; P. David Jackson; Rob Perry; Scott Cain; Christina Leventhal; Mark Thornton; Rakesh Ramachandran; Jessica Whittington; Laura Lerner; Dana Costanzo; Karen McElligott; Sherry Boozer; Robert Mays; Emery Smith; Neil Veloso; Alison Klika; Jennifer Hess; Kevin Cothren; Kalok Lo; Jason Offenbacher; Joel Danzig; Matt Ducar; John J. Harrington

2001-01-01

130

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

SciTech Connect

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.

Nierman, William C.

2000-02-14

131

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...

132

Genomic analysis using a yeast artificial chromosome library with mouse DNA inserts.  

PubMed Central

A yeast artificial chromosome library with mouse genomic DNA inserts has been constructed. The library encompasses a 2.5-fold coverage of the mouse genome, with an average insert size of 250 kilobases. The screening strategy uses the polymerase chain reaction on pooled DNAs prepared from individually stored clones. The usefulness of the library for chromosome walking was illustrated by constructing a 600-kilobase-long contig of DNA surrounding Hba-ps4, a DNA marker that is tightly linked to the fused (Fu) locus on chromosome 17. Images PMID:1347950

Rossi, J M; Burke, D T; Leung, J C; Koos, D S; Chen, H; Tilghman, S M

1992-01-01

133

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...

134

Pybedtools: a flexible Python library for manipulating genomic datasets and annotations  

PubMed Central

Summary: pybedtools is a flexible Python software library for manipulating and exploring genomic datasets in many common formats. It provides an intuitive Python interface that extends upon the popular BEDTools genome arithmetic tools. The library is well documented and efficient, and allows researchers to quickly develop simple, yet powerful scripts that enable complex genomic analyses. Availability: pybedtools is maintained under the GPL license. Stable versions of pybedtools as well as documentation are available on the Python Package Index at http://pypi.python.org/pypi/pybedtools. Contact: dalerr@niddk.nih.gov; arq5x@virginia.edu Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:21949271

Dale, Ryan K.; Pedersen, Brent S.; Quinlan, Aaron R.

2011-01-01

135

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

PubMed Central

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

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

2010-01-01

136

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

PubMed

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

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

2010-02-01

137

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...

138

Construction and Characterization of Three Wheat Bacterial Artificial Chromosome Libraries  

PubMed Central

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

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

139

A Genome-Wide Survey of Switchgrass Genome Structure and Organization  

PubMed Central

The perennial grass, switchgrass (Panicum virgatum L.), is a promising bioenergy crop and the target of whole genome sequencing. We constructed two bacterial artificial chromosome (BAC) libraries from the AP13 clone of switchgrass to gain insight into the genome structure and organization, initiate functional and comparative genomic studies, and assist with genome assembly. Together representing 16 haploid genome equivalents of switchgrass, each library comprises 101,376 clones with average insert sizes of 144 (HindIII-generated) and 110 kb (BstYI-generated). A total of 330,297 high quality BAC-end sequences (BES) were generated, accounting for 263.2 Mbp (16.4%) of the switchgrass genome. Analysis of the BES identified 279,099 known repetitive elements, >50,000 SSRs, and 2,528 novel repeat elements, named switchgrass repetitive elements (SREs). Comparative mapping of 47 full-length BAC sequences and 330K BES revealed high levels of synteny with the grass genomes sorghum, rice, maize, and Brachypodium. Our data indicate that the sorghum genome has retained larger microsyntenous regions with switchgrass besides high gene order conservation with rice. The resources generated in this effort will be useful for a broad range of applications. PMID:22511929

Sharma, Manoj K.; Sharma, Rita; Cao, Peijian; Jenkins, Jerry; Bartley, Laura E.; Qualls, Morgan; Grimwood, Jane; Schmutz, Jeremy; Rokhsar, Daniel; Ronald, Pamela C.

2012-01-01

140

Chromosome region-specific libraries for human genome analysis  

SciTech Connect

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.

Kao, Fa-Ten.

1992-08-01

141

Phylogenetic Screening of Ribosomal RNA Gene-Containing Clones in Bacterial Artificial Chromosome (BAC) Libraries from Different Depths in Monterey Bay  

Microsoft Academic Search

Marine picoplankton are central mediators of many oceanic biogeochemical processes, but much of their biology and ecology remains ill defined. One approach to better defining these environmentally significant microbes involves the acquisition of genomic data that can provide information about genome content, metabolic capabilities, and population variability in picoplankton assemblages. Previously, we constructed and phylogenetically screened a Bacterial Artificial Chromosome

M. T. Suzuki; C. M. Preston; J. R. de la Torre; G. F. Steward; E. F. DeLong

2004-01-01

142

Alignment of the Genomes of Brachypodium distachyon and Temperate Cereals and Grasses Using Bacterial Artificial Chromosome Landing With Fluorescence in Situ Hybridization  

PubMed Central

As part of an initiative to develop Brachypodium distachyon as a genomic “bridge” species between rice and the temperate cereals and grasses, a BAC library has been constructed for the two diploid (2n = 2x = 10) genotypes, ABR1 and ABR5. The library consists of 9100 clones, with an approximate average insert size of 88 kb, representing 2.22 genome equivalents. To validate the usefulness of this species for comparative genomics and gene discovery in its larger genome relatives, the library was screened by PCR using primers designed on previously mapped rice and Poaceae sequences. Screening indicated a degree of synteny between these species and B. distachyon, which was confirmed by fluorescent in situ hybridization of the marker-selected BACs (BAC landing) to the 10 chromosome arms of the karyotype, with most of the BACs hybridizing as single loci on known chromosomes. Contiguous BACs colocalized on individual chromosomes, thereby confirming the conservation of genome synteny and proving that B. distachyon has utility as a temperate grass model species alternative to rice. PMID:16489232

Hasterok, Robert; Marasek, Agnieszka; Donnison, Iain S.; Armstead, Ian; Thomas, Ann; King, Ian P.; Wolny, Elzbieta; Idziak, Dominika; Draper, John; Jenkins, Glyn

2006-01-01

143

Construction and screening of a genomic library specific for mouse chromosome 16.  

PubMed

We have established a protocol for producing libraries of specific mouse chromosomes. The mouse DNA-containing clones from a genomic library of a hamster-mouse somatic cell hybrid containing only one mouse chromosome are identified by screening with radiolabeled mouse repetitive sequences after specifically blocking hamster repetitive sequences; 95% of the mouse DNA-containing clones are identified. We have applied this protocol in producing a library of mouse chromosome 16, consisting of 14,200 clones or two "chromosome equivalents." Each clone occupies an individual well in a microtiter tray, allowing the entire library to be repeatedly and reproducibly plated and analyzed by hybridization. Further, we have established a protocol for making cDNA probes specifically depleted of highly repetitive sequences for probing libraries of genomic clones. By screening the chromosome 16 library with cDNA probes from mouse liver and brain, we demonstrate the feasibility of identifying expressed sequences and characterizing their patterns of expression. Such chromosome-specific libraries can facilitate the isolation of defined genetic loci as well as form the basis for the production of integrated transcriptional, genetic, and physical maps of entire chromosomes. PMID:2813407

Hochgeschwender, U; Sutcliffe, J G; Brennan, M B

1989-11-01

144

Construction and screening of a genomic library specific for mouse chromosome 16.  

PubMed Central

We have established a protocol for producing libraries of specific mouse chromosomes. The mouse DNA-containing clones from a genomic library of a hamster-mouse somatic cell hybrid containing only one mouse chromosome are identified by screening with radiolabeled mouse repetitive sequences after specifically blocking hamster repetitive sequences; 95% of the mouse DNA-containing clones are identified. We have applied this protocol in producing a library of mouse chromosome 16, consisting of 14,200 clones or two "chromosome equivalents." Each clone occupies an individual well in a microtiter tray, allowing the entire library to be repeatedly and reproducibly plated and analyzed by hybridization. Further, we have established a protocol for making cDNA probes specifically depleted of highly repetitive sequences for probing libraries of genomic clones. By screening the chromosome 16 library with cDNA probes from mouse liver and brain, we demonstrate the feasibility of identifying expressed sequences and characterizing their patterns of expression. Such chromosome-specific libraries can facilitate the isolation of defined genetic loci as well as form the basis for the production of integrated transcriptional, genetic, and physical maps of entire chromosomes. Images PMID:2813407

Hochgeschwender, U; Sutcliffe, J G; Brennan, M B

1989-01-01

145

MOLECULAR STRUCTURE AND ORGANIZATION OF THE WHEAT GENOMIC MANGANESE SUPEROXIDE DISMUTASE GENE  

Technology Transfer Automated Retrieval System (TEKTRAN)

The genomic structure of an MnSOD gene in wheat was elucidated by sequencing a clone from a BAC library of a stripe-rust resistant wheat line. The clone was identified by hybridization with a wheat MnSOD cDNA. The gene consisted of six exons interrupted by five introns with a total length of 4773 nu...

146

A new look towards BAC-based array CGH through a comprehensive comparison with oligo-based array CGH  

E-print Network

flexibility in probe design, greater coverage, and much higher resolution. The latter depends on array design and the cell type homogeneity. Moreover, oligonu- cleotides can more easily be produced for any organism for which the genome has been sequenced... isolated BAC outliers (boxes 1, 2, 3 and 5) and two groups of BAC outliers (boxes 4 and 6) on chromosome 3 of patient sam- ple 817. Isolated BAC outliers point to putative micro- events (microdeletion or small amplification) whereas groups of BAC outliers...

Wicker, Nicolas; Carles, Annaick; Mills, Ian G; Wolf, Maija; Veerakumarasivam, Abhi; Edgren, Henrik; Boileau, Fabrice; Wasylyk, Bohdan; Schalken, Jack A; Neal, David E; Kallioniemi, Olli; Poch, Olivier

2007-03-29

147

Microsatellite-enriched genomic libraries as a source of polymorphic loci for Schistosoma mansoni  

Microsoft Academic Search

Microsatellite markers for Schistosoma mansoni were developed using four genomic microsatellite-enriched libraries. Microsatellites were observed in 65.4% of all sequences. Primer pairs were designed and tested for 23 loci. Eighteen loci produced amplification products, out of which 11 were polymorphic and were further characterized on 100 individ- uals of S. mansoni. Two to 19 alleles per locus were detected. The

N. B. RODRIGUES; M. R. SILVA; M. M. PUCCI; D. J. MINCHELLA; R. SORENSEN; P. T. LOVERDE; A. J. ROMANHA; G. OLIVEIRA

2007-01-01

148

End-sequence profiling: Sequence-based analysis of aberrant genomes  

PubMed Central

Genome rearrangements are important in evolution, cancer, and other diseases. Precise mapping of the rearrangements is essential for identification of the involved genes, and many techniques have been developed for this purpose. We show here that end-sequence profiling (ESP) is particularly well suited to this purpose. ESP is accomplished by constructing a bacterial artificial chromosome (BAC) library from a test genome, measuring BAC end sequences, and mapping end-sequence pairs onto the normal genome sequence. Plots of BAC end-sequences density identify copy number abnormalities at high resolution. BACs spanning structural aberrations have end pairs that map abnormally far apart on the normal genome sequence. These pairs can then be sequenced to determine the involved genes and breakpoint sequences. ESP analysis of the breast cancer cell line MCF-7 demonstrated its utility for analysis of complex genomes. End sequencing of ?8,000 clones (0.37-fold haploid genome clonal coverage) produced a comprehensive genome copy number map of the MCF-7 genome at better than 300-kb resolution and identified 381 genome breakpoints, a subset of which was verified by fluorescence in situ hybridization mapping and sequencing. PMID:12788976

Volik, Stanislav; Zhao, Shaying; Chin, Koei; Brebner, John H.; Herndon, David R.; Tao, Quanzhou; Kowbel, David; Huang, Guiqing; Lapuk, Anna; Kuo, Wen-Lin; Magrane, Gregg; de Jong, Pieter; Gray, Joe W.; Collins, Colin

2003-01-01

149

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

PubMed Central

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

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

1982-01-01

150

Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kbp gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5  

Microsoft Academic Search

We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA\\u000a of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA\\u000a sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome

J. Y. Park; D. H. Koo; C. P. Hong; S. J. Lee; J. W. Jeon; S. H. Lee; P. Y. Yun; B. S. Park; H. R. Kim; J. W. Bang; P. Plaha; I. Bancroft; Y. P. Lim

2005-01-01

151

A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage.  

PubMed Central

The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe. Images PMID:2186372

Anand, R; Riley, J H; Butler, R; Smith, J C; Markham, A F

1990-01-01

152

Reliability analysis of the Ahringer Caenorhabditis elegans RNAi feeding library: a guide for genome-wide screens  

Microsoft Academic Search

Background  The Ahringer C. elegans RNAi feeding library prepared by cloning genomic DNA fragments has been widely used in genome-wide analysis of gene function.\\u000a However, the library has not been thoroughly validated by direct sequencing, and there are potential errors, including: 1)\\u000a mis-annotation (the clone with the retired gene name should be remapped to the actual target gene); 2) nonspecific PCR

Wubin Qu; Changhong Ren; Yuan Li; Jinping Shi; Jiye Zhang; Xiaolei Wang; Xingyi Hang; Yiming Lu; Dongsheng Zhao; Chenggang Zhang

2011-01-01

153

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

PubMed

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

Hao, DaCheng; Yang, Ling; Xiao, PeiGen

2011-03-01

154

Fluorescence in situ hybridization using bacterial artificial chromosome (BAC) clones for the analysis of chromosome rearrangement in Chinese hamster ovary cells  

Microsoft Academic Search

Chromosome identification using Chinese hamster ovary (CHO) genomic bacterial artificial chromosome (BAC) clones has the potential to contribute to the analysis and understanding of chromosomal instability of CHO cell lines and to improve our understanding of chromosome organization during the establishment of recombinant CHO cells. Fluorescence in situ hybridization imaging using BAC clones as probes (BAC-FISH) can provide valuable information

Yihua Cao; Shuichi Kimura; Takayuki Itoi; Kohsuke Honda; Hisao Ohtake; Takeshi Omasa

155

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

PubMed

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

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

2015-03-01

156

BAC transgenic zebrafish for transcriptional promoter and enhancer studies.  

PubMed

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

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

2015-01-01

157

A high utility integrated map of the pig genome  

PubMed Central

Background The domestic pig is being increasingly exploited as a system for modeling human disease. It also has substantial economic importance for meat-based protein production. Physical clone maps have underpinned large-scale genomic sequencing and enabled focused cloning efforts for many genomes. Comparative genetic maps indicate that there is more structural similarity between pig and human than, for example, mouse and human, and we have used this close relationship between human and pig as a way of facilitating map construction. Results Here we report the construction of the most highly continuous bacterial artificial chromosome (BAC) map of any mammalian genome, for the pig (Sus scrofa domestica) genome. The map provides a template for the generation and assembly of high-quality anchored sequence across the genome. The physical map integrates previous landmark maps with restriction fingerprints and BAC end sequences from over 260,000 BACs derived from 4 BAC libraries and takes advantage of alignments to the human genome to improve the continuity and local ordering of the clone contigs. We estimate that over 98% of the euchromatin of the 18 pig autosomes and the X chromosome along with localized coverage on Y is represented in 172 contigs, with chromosome 13 (218 Mb) represented by a single contig. The map is accessible through pre-Ensembl, where links to marker and sequence data can be found. Conclusion The map will enable immediate electronic positional cloning of genes, benefiting the pig research community and further facilitating use of the pig as an alternative animal model for human disease. The clone map and BAC end sequence data can also help to support the assembly of maps and genome sequences of other artiodactyls. PMID:17625002

Humphray, Sean J; Scott, Carol E; Clark, Richard; Marron, Brandy; Bender, Clare; Camm, Nick; Davis, Jayne; Jenks, Andrew; Noon, Angela; Patel, Manish; Sehra, Harminder; Yang, Fengtang; Rogatcheva, Margarita B; Milan, Denis; Chardon, Patrick; Rohrer, Gary; Nonneman, Dan; de Jong, Pieter; Meyers, Stacey N; Archibald, Alan; Beever, Jonathan E; Schook, Lawrence B; Rogers, Jane

2007-01-01

158

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

159

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

SciTech Connect

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.

Korzun, V.N.; Kartel, N.A. [Institute of Genetics and Cytology, Minsk (Belarus); Boerner, A. [Institute of Plant Genetics and Crop Plant Research, Gatersleben (Germany)

1995-06-01

160

Optimization of a genome-wide disordered lentivector-based short hairpin RNA library  

Microsoft Academic Search

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short\\u000a hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations\\u000a by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression\\u000a constructs were

O. A. Guryanova; M. Makhanov; A. A. Chenchik; P. M. Chumakov; E. I. Frolova

2006-01-01

161

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

PubMed

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

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

162

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

PubMed Central

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

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

163

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

Microsoft Academic Search

Background  Rainbow trout (Oncorhynchus mykiss) are cultivated worldwide for aquaculture production and are widely used as a model species to gain knowledge of many aspects\\u000a of fish biology. The common ancestor of the salmonids experienced a whole genome duplication event, making extant salmonids\\u000a such as the rainbow trout an excellent model for studying the evolution of tetraploidization and re-diploidization in vertebrates.

Carine Genet; Patrice Dehais; Yniv Palti; Guangtu Gao; Frederick Gavory; Patrick Wincker; Edwige Quillet; Mekki Boussaha

2011-01-01

164

Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries  

PubMed Central

Background Flax (Linum usitatissimum L.) is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F6-derived recombinant inbred line population provided validation of the SNPs. Results Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents). Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F6 individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%. Conclusions Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from flax. The genotyping-by-sequencing approach proved to be efficient for validation. The SNP resources generated in this work will assist in generating high density maps of flax and facilitate QTL discovery, marker-assisted selection, phylogenetic analyses, association mapping and anchoring of the whole genome shotgun sequence. PMID:23216845

2012-01-01

165

Reasons to build dinoflagellate Symbiodinium BAC libraries  

Microsoft Academic Search

Dinoflagellates are ubiquitous marine and freshwater protists. As free-living photosynthetic plankton, they account for ~50% of the primary productivity of oceans and lakes. As photosynthetic symbionts, they provide essential nutrients to most corals and numerous other marine invertebrates, supporting coral reefs, one of the most diverse ecosystems on earth, a rich food source, and a potential source of future pharmaceuticals.

Thomas G. Doak; Robert B. Moore; Ove Hoegh-Guldberg; Mary Alice Coffroth

166

Library+  

ERIC Educational Resources Information Center

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…

Merrill, Alex

2011-01-01

167

Microcollinearity between autopolyploid sugarcane and diploid sorghum genomes  

PubMed Central

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

2010-01-01

168

High-Throughput Screening of a Corynebacterium glutamicum Mutant Library on Genomic and Metabolic Level  

PubMed Central

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

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

2014-01-01

169

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

PubMed Central

Background Sequencing projects using a clone-by-clone approach require the availability of a robust physical map. The SNaPshot technology, based on pair-wise comparisons of restriction fragments sizes, has been used recently to build the first physical map of a wheat chromosome and to complete the maize physical map. However, restriction fragments sizes shared randomly between two non-overlapping BACs often lead to chimerical contigs and mis-assembled BACs in such large and repetitive genomes. Whole Genome Profiling (WGP™) was developed recently as a new sequence-based physical mapping technology and has the potential to limit this problem. Results A subset of the wheat 3B chromosome BAC library covering 230 Mb was used to establish a WGP physical map and to compare it to a map obtained with the SNaPshot technology. We first adapted the WGP-based assembly methodology to cope with the complexity of the wheat genome. Then, the results showed that the WGP map covers the same length than the SNaPshot map but with 30% less contigs and, more importantly with 3.5 times less mis-assembled BACs. Finally, we evaluated the benefit of integrating WGP tags in different sequence assemblies obtained after Roche/454 sequencing of BAC pools. We showed that while WGP tag integration improves assemblies performed with unpaired reads and with paired-end reads at low coverage, it does not significantly improve sequence assemblies performed at high coverage (25x) with paired-end reads. Conclusions Our results demonstrate that, with a suitable assembly methodology, WGP builds more robust physical maps than the SNaPshot technology in wheat and that WGP can be adapted to any genome. Moreover, WGP tag integration in sequence assemblies improves low quality assembly. However, to achieve a high quality draft sequence assembly, a sequencing depth of 25x paired-end reads is required, at which point WGP tag integration does not provide additional scaffolding value. Finally, we suggest that WGP tags can support the efficient sequencing of BAC pools by enabling reliable assignment of sequence scaffolds to their BAC of origin, a feature that is of great interest when using BAC pooling strategies to reduce the cost of sequencing large genomes. PMID:22289472

2012-01-01

170

NINDS GENSAT BAC Transgenic Project  

NSDL National Science Digital Library

This website from Rockefeller University in New York contains "a gene expression atlas of the central nervous system of the mouse based on bacterial artificial chromosomes (BACs)." GENSAT, or the Gene Expression Nervous System Atlas, contains brain slice images of BAC transgenic mice at the embryonic, postnatal (7 days old), and adult stages, stained to show areas of gene activity. The website comes with a detailed and helpful tutorial that recreates GENSAT's user interface and demonstrates how to manipulate search results.

2008-09-11

171

The Jackson Laboratory: The Mouse Genome Sequence Project  

NSDL National Science Digital Library

Part of the Mouse Genome Informatics program (last reported on in the NSDL Scout Report for the Life Sciences on March 19, 2004) at the Jackson Laboratory, this website presents The Mouse Genome Sequence (MGS) project. MGS is designed "to integrate emerging mouse genomic sequence data with the genetic and biological data available in MGD and GXD." The site links to Eukaryotic Genome Annotation Projects, as well as Sequence Analysis Tools including MouseBlast and Genome Analysis. The site also offers basic background information about the Mouse Genome Sequencing Initiative, and provides site users with access to groups involved in mouse genome sequencing, the BAC clone library, request forms for targeted sequencing, and more.

172

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

PubMed Central

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

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

2013-01-01

173

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

PubMed Central

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

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

2013-01-01

174

Functional screening of metagenome and genome libraries for detection of novel flavonoid-modifying enzymes.  

PubMed

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

Rabausch, U; Juergensen, J; Ilmberger, N; Böhnke, S; Fischer, S; Schubach, B; Schulte, M; Streit, W R

2013-08-01

175

Integrating cytogenetics and genomics in comparative evolutionary studies of cichlid fish  

PubMed Central

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

2012-01-01

176

Bos taurus genome assembly  

PubMed Central

Background We present here the assembly of the bovine genome. The assembly method combines the BAC plus WGS local assembly used for the rat and sea urchin with the whole genome shotgun (WGS) only assembly used for many other animal genomes including the rhesus macaque. Results The assembly process consisted of multiple phases: First, BACs were assembled with BAC generated sequence, then subsequently in combination with the individual overlapping WGS reads. Different assembly parameters were tested to separately optimize the performance for each BAC assembly of the BAC and WGS reads. In parallel, a second assembly was produced using only the WGS sequences and a global whole genome assembly method. The two assemblies were combined to create a more complete genome representation that retained the high quality BAC-based local assembly information, but with gaps between BACs filled in with the WGS-only assembly. Finally, the entire assembly was placed on chromosomes using the available map information. Over 90% of the assembly is now placed on chromosomes. The estimated genome size is 2.87 Gb which represents a high degree of completeness, with 95% of the available EST sequences found in assembled contigs. The quality of the assembly was evaluated by comparison to 73 finished BACs, where the draft assembly covers between 92.5 and 100% (average 98.5%) of the finished BACs. The assembly contigs and scaffolds align linearly to the finished BACs, suggesting that misassemblies are rare. Genotyping and genetic mapping of 17,482 SNPs revealed that more than 99.2% were correctly positioned within the Btau_4.0 assembly, confirming the accuracy of the assembly. Conclusion The biological analysis of this bovine genome assembly is being published, and the sequence data is available to support future bovine research. PMID:19393050

Liu, Yue; Qin, Xiang; Song, Xing-Zhi Henry; Jiang, Huaiyang; Shen, Yufeng; Durbin, K James; Lien, Sigbjørn; Kent, Matthew Peter; Sodeland, Marte; Ren, Yanru; Zhang, Lan; Sodergren, Erica; Havlak, Paul; Worley, Kim C; Weinstock, George M; Gibbs, Richard A

2009-01-01

177

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

PubMed

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

Barakat, Tahsin Stefan; Gribnau, Joost

2015-01-01

178

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

179

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

PubMed

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

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

2014-01-01

180

A Dense Genetic Linkage Map for Common Carp and Its Integration with a BAC-Based Physical Map  

PubMed Central

Background Common carp (Cyprinus carpio) is one of the most important aquaculture species with an annual global production of 3.4 million metric tons. It is also an important ornamental species as well as an important model species for aquaculture research. To improve the economically important traits of this fish, a number of genomic resources and genetic tools have been developed, including several genetic maps and a bacterial artificial chromosome (BAC)-based physical map. However, integrated genetic and physical maps are not available to study quantitative trait loci (QTL) and assist with fine mapping, positional cloning and whole genome sequencing and assembly. The objective of this study was to integrate the currently available BAC-based physical and genetic maps. Results The genetic map was updated with 592 novel markers, including 312 BAC-anchored microsatellites and 130 SNP markers, and contained 1,209 genetic markers on 50 linkage groups, spanning 3,565.9 cM in the common carp genome. An integrated genetic and physical map of the common carp genome was then constructed, which was composed of 463 physical map contigs and 88 single BACs. Combined lengths of the contigs and single BACs covered a physical length of 498.75 Mb, or around 30% of the common carp genome. Comparative analysis between common carp and zebrafish genomes was performed based on the integrated map, providing more insights into the common carp specific whole genome duplication and segmental rearrangements in the genome. Conclusion We integrated a BAC-based physical map to a genetic linkage map of common carp by anchoring BAC-associated genetic markers. The density of the genetic linkage map was significantly increased. The integrated map provides a tool for both genetic and genomic studies of common carp, which will help us to understand the genomic architecture of common carp and facilitate fine mapping and positional cloning of economically important traits for genetic improvement and modification. PMID:23704958

Ji, Peifeng; Zhang, Xiaofeng; Zhao, Zixia; Hou, Guangyuan; Huo, Linhe; Liu, Guiming; Li, Chao; Xu, Peng; Sun, Xiaowen

2013-01-01

181

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

SciTech Connect

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.

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

182

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

PubMed Central

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

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

2014-01-01

183

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

NASA Astrophysics Data System (ADS)

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

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

1998-03-01

184

Rapid identification of gene sequences for transcriptional map assembly by direct cDNA screening of genomic reference libraries.  

PubMed

We have used the direct cDNA screening protocol to identify sequences transcribed in cerebral cortex from a reference library of human Xq28. To derive coding sequences from these genomic clones, we first identified fragments containing transcribed sequences and subjected these to exon trapping or to partial sequencing and analysis by Grail. In a preliminary analysis of three clones, coding sequences from two novel genes expressed in brain were identified. This method allows the rapid identification of coding sequences of genes expressed in specific tissues without recourse to cDNA libraries. The approach is amenable to large scale applications and should be useful for isolating candidate disease genes and in particular for assembling integrated transcriptional maps from large genomic regions. PMID:7874120

Lawrence, B J; Schwabe, W; Kioschis, P; Coy, J F; Poustka, A; Brennan, M B; Hochgeschwender, U

1994-11-01

185

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

PubMed Central

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

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

2009-01-01

186

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

SciTech Connect

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.

Tuskan, Gerald A [ORNL; Gunter, Lee E [ORNL; DiFazio, Stephen P [West Virginia University

2009-01-01

187

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

PubMed

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

Ali, Shawkat; Bakkeren, Guus

2015-01-01

188

Post-Integration Silencing of piggyBac Transposable Elements in Aedes aegypti  

PubMed Central

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites. PMID:23861905

Palavesam, Azhahianambi; Esnault, Caroline; O’Brochta, David A.

2013-01-01

189

Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing.  

PubMed

A Podospora anserina BAC library of 4800 clones has been constructed in the vector pBHYG allowing direct selection in fungi. Screening of the BAC collection for centromeric sequences of chromosome V allowed the recovery of clones localized on either sides of the centromere, but no BAC clone was found to contain the centromere. Seven BAC clones containing 322,195 and 156,244bp from either sides of the centromeric region were sequenced and annotated. One 5S rRNA gene, 5 tRNA genes, and 163 putative coding sequences (CDS) were identified. Among these, only six CDS seem specific to P. anserina. The gene density in the centromeric region is approximately one gene every 2.8kb. Extrapolation of this gene density to the whole genome of P. anserina suggests that the genome contains about 11,000 genes. Synteny analyses between P. anserina and Neurospora crassa show that co-linearity extends at the most to a few genes, suggesting rapid genome rearrangements between these two species. PMID:12892638

Silar, Philippe; Barreau, Christian; Debuchy, Robert; Kicka, Sébastien; Turcq, Béatrice; Sainsard-Chanet, Annie; Sellem, Carole H; Billault, Alain; Cattolico, Laurence; Duprat, Simone; Weissenbach, Jean

2003-08-01

190

Integration of physical and genetic maps of common bean through BAC-derived microsatellite markers  

Microsoft Academic Search

BACKGROUND: Common bean (Phaseolus vulgaris L.) is the most important legume for direct human consumption and the goal of this study was to integrate a recently constructed physical map for the species with a microsatellite based genetic map using a BAC library from the genotype G19833 and the recombinant inbred line population DOR364 × G19833. RESULTS: We searched for simple

Juana M Córdoba; Carolina Chavarro; Jessica A Schlueter; Scott A Jackson; Matthew W Blair

2010-01-01

191

Comparative physical maps derived from BAC end sequences of tilapia (Oreochromis niloticus)  

PubMed Central

Background The Nile tilapia is the second most important fish in aquaculture. It is an excellent laboratory model, and is closely related to the African lake cichlids famous for their rapid rates of speciation. A suite of genomic resources has been developed for this species, including genetic maps and ESTs. Here we analyze BAC end-sequences to develop comparative physical maps, and estimate the number of genome rearrangements, between tilapia and other model fish species. Results We obtained sequence from one or both ends of 106,259 tilapia BACs. BLAST analysis against the genome assemblies of stickleback, medaka and pufferfish allowed identification of homologies for approximately 25,000 BACs for each species. We calculate that rearrangement breakpoints between tilapia and these species occur about every 3 Mb across the genome. Analysis of 35,000 clones previously assembled into contigs by restriction fingerprints allowed identification of longer-range syntenies. Conclusions Our data suggest that chromosomal evolution in recent teleosts is dominated by alternate loss of gene duplicates, and by intra-chromosomal rearrangements (~one per million years). These physical maps are a useful resource for comparative positional cloning of traits in cichlid fishes. The paired BAC end sequences from these clones will be an important resource for scaffolding forthcoming shotgun sequence assemblies of the tilapia genome. PMID:21080946

2010-01-01

192

Identification of novel genes involved in DNA damage response by screening a genome-wide Schizosaccharomyces pombe deletion library  

PubMed Central

Background DNA damage response (DDR) plays pivotal roles in maintaining genome integrity and stability. An effective DDR requires the involvement of hundreds of genes that compose a complicated network. Because DDR is highly conserved in evolution, studies in lower eukaryotes can provide valuable information to elucidate the mechanism in higher organisms. Fission yeast (Schizosaccharomyces pombe) has emerged as an excellent model for DDR research in recent years. To identify novel genes involved in DDR, we screened a genome-wide S. pombe haploid deletion library against six different DNA damage reagents. The library covered 90.5% of the nonessential genes of S. pombe. Results We have identified 52 genes that were actively involved in DDR. Among the 52 genes, 20 genes were linked to DDR for the first time. Flow cytometry analysis of the repair defective mutants revealed that most of them exhibited a defect in cell cycle progression, and some caused genome instability. Microarray analysis and genetic complementation assays were carried out to characterize 6 of the novel DDR genes in more detail. Data suggested that SPBC2A9.02 and SPAC27D7.08c were required for efficient DNA replication initiation because they interacted genetically with DNA replication initiation proteins Abp1 and Abp2. In addition, deletion of sgf73+, meu29+, sec65+ or pab1+ caused improper cytokinesis and DNA re-replication, which contributed to the diploidization in the mutants. Conclusions A genome-wide screen of genes involved in DDR emphasized the key role of cell cycle control in the DDR network. Characterization of novel genes identified in the screen helps to elucidate the mechanism of the DDR network and provides valuable clues for understanding genome stability in higher eukaryotes. PMID:23173672

2012-01-01

193

A rapid method for isolation chromosome band specific cDNAs and genomic cDNAs with microdissection/microcloning libraries  

SciTech Connect

Positional cloning provides a powerful tool for isolation of the gene(s) whose location has been identified while whose biochemical product and function are unknown. Previous techniques for positional cloning require construction of the DNA contig either with YAC, cosmid or bacteriophage clones, which is time consuming. To facilitate this process, we developed a rapid method in which microdissection/microcloning DNA libraries are used as probe pools to directly screen genomic DNA and cDNA libraries. In our initial experiment, we used chromosome band-specific libraries from 5q13 and 2q33 constructed with our microdissection techniques as probe pools to direct screen human genomic library and human brainstem or frontal cortex cDNA libraries using sonicated total human genomic DNA as the competitor. Five genomic DNA clones and 2 cDNA clones have been isolated with the 5q13 library and 11 cDNA clones have been isolated with the 2q33 library. Characterization of 3 genomic and 4 cDNA clones with fluorescence in situ hybridization indicated that these clones are derived from 5q13 and 2q33, respectively. One of our genomic clones had hybridization signals not only at 5q13 but also at 5p13, 5q21 and 5q31, indicating that existence of chromosome-specific repetitive sequences in human chromosome 5. One cDNA clone isolated with 2q33 library contains a polymorphic dinucleotide repeat. Linkage analysis showed that this clone has close linkage with the ALS2 locus previously mapped close to D2S72/D2S116.

He, X.X.; Hentati, A.; Deng, H.X. [Northwestern Univ. Medical School, Chicago, IL (United States)

1994-09-01

194

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

SciTech Connect

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

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

1994-09-01

195

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of spotted maigre ( Nibea albiflora )  

Microsoft Academic Search

In the present study, we reported 13 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library\\u000a of Nibea albiflora. The number of alleles, observed and expected heterozygosity per locus in 44 individuals ranged from 2 to 13, from 0.0909\\u000a to 0.9773 and from 0.0886 to 0.9073, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after\\u000a Bonferroni correction. Analysis of linkage disequilibrium

Shichao Xing; Changwei Shao; Xiaolin Liao; Yongsheng Tian; Songlin Chen

2009-01-01

196

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S  

Microsoft Academic Search

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and\\u000a finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed.\\u000a The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an\\u000a average insert size

Fang Qiu; Demin Jin; Jianmin Fu; Chaoliang Zhang; Weiwu Xie; Rencui Yang; Hongbin Zhang; Bin Wang

1999-01-01

197

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

PubMed Central

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

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

198

A BAC-Based Physical Map of the Major Autosomes of Drosophila melanogaster  

Microsoft Academic Search

We constructed a bacterial artificial chromosome (BAC)-based physical map of chromosomes 2 and 3 of Drosophila melanogaster, which constitute 81% of the genome. Sequence tagged site (STS) content, restriction fingerprinting, and polytene chromosome in situ hybridization approaches were integrated to produce a map spanning the euchromatin. Three of five remaining gaps are in repeat-rich regions near the centromeres. A tiling

Roger A. Hoskins; Catherine R. Nelson; Benjamin P. Berman; Todd R. Laverty; Reed A. George; Lisa Ciesiolka; Mohammed Naeemuddin; Andrew D. Arenson; James Durbin; Robert G. David; Paul E. Tabor; Michael R. Bailey; Denise R. DeShazo; Joseph Catanese; Aaron Mammoser; Kazutoyo Osoegawa; Pieter J. de Jong; Susan E. Celniker; Richard A. Gibbs; Gerald M. Rubin; Steven E. Scherer

2000-01-01

199

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...

200

Highly Efficient Modification of Bacterial Artificial Chromosomes (BACs) Using Novel Shuttle Vectors  

E-print Network

- mosomes (YACs). These include large carrying capacity ( 100­300 kb), high clonal stability, low rate. 2000; McPherson et al. 2001). Indeed, physical contigs are now available for the entire human genome inserts in BACs are sufficiently large in most instances to carry an entire transcription unit and its

Chait, Brian T.

201

SearchSmallRNA: a graphical interface tool for the assemblage of viral genomes using small RNA libraries data  

PubMed Central

Background Next-generation parallel sequencing (NGS) allows the identification of viral pathogens by sequencing the small RNAs of infected hosts. Thus, viral genomes may be assembled from host immune response products without prior virus enrichment, amplification or purification. However, mapping of the vast information obtained presents a bioinformatics challenge. Methods In order to by pass the need of line command and basic bioinformatics knowledge, we develop a mapping software with a graphical interface to the assemblage of viral genomes from small RNA dataset obtained by NGS. SearchSmallRNA was developed in JAVA language version 7 using NetBeans IDE 7.1 software. The program also allows the analysis of the viral small interfering RNAs (vsRNAs) profile; providing an overview of the size distribution and other features of the vsRNAs produced in infected cells. Results The program performs comparisons between each read sequenced present in a library and a chosen reference genome. Reads showing Hamming distances smaller or equal to an allowed mismatched will be selected as positives and used to the assemblage of a long nucleotide genome sequence. In order to validate the software, distinct analysis using NGS dataset obtained from HIV and two plant viruses were used to reconstruct viral whole genomes. Conclusions SearchSmallRNA program was able to reconstructed viral genomes using NGS of small RNA dataset with high degree of reliability so it will be a valuable tool for viruses sequencing and discovery. It is accessible and free to all research communities and has the advantage to have an easy-to-use graphical interface. Availability and implementation SearchSmallRNA was written in Java and is freely available at http://www.microbiologia.ufrj.br/ssrna/. PMID:24607237

2014-01-01

202

Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca sexta shown by five-color BAC-FISH.  

PubMed

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori, together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta, and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15). PMID:18059551

Sahara, Ken; Yoshido, Atsuo; Marec, Frantisek; Fuková, Iva; Zhang, Hong-Bin; Wu, Cheng-Cang; Goldsmith, Marian R; Yasukochi, Yuji

2007-11-01

203

Wheat Genomics: Present Status and Future Prospects  

PubMed Central

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

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

2008-01-01

204

BacDive—the Bacterial Diversity Metadatabase  

PubMed Central

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

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

2014-01-01

205

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

Microsoft Academic Search

Prior to constructing a library of yeast artificial chromosomes (YACs) containing very large human DNA fragments, the authors performed a series of preliminary experiments aimed at developing a suitable protocol. They found an inverse relationship between YAC insert size and transformation efficiency. Evidence of occasional rearrangement within YAC inserts was found resulting in clonally stable internal deletions or clonally unstable

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

1990-01-01

206

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

Microsoft Academic Search

The twehybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cermisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two- hybrid libraries, and

Philip James; John Halladay; Elizabeth A. Craig

1996-01-01

207

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

SciTech Connect

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.

Jean-Michael H. Vos

1999-12-09

208

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

Technology Transfer Automated Retrieval System (TEKTRAN)

To address a general shortage of genomic sequence information in the whitefly Bemisia tabaci Gennadius, we have constructed three cDNA libraries for non-viruliferous whiteflies (eggs, immature instars, and adults)and two from adult insects that fed on tomato plants infected by two geminiviruses: To...

209

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

SciTech Connect

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.

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

2010-05-28

210

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome  

Microsoft Academic Search

BACKGROUND: Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or

Björn Hamberger; Dawn Hall; Mack Yuen; Claire Oddy; Britta Hamberger; Christopher I Keeling; Carol Ritland; Kermit Ritland; Jörg Bohlmann

2009-01-01

211

Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors  

Microsoft Academic Search

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity

David J. Lockhart; Sung-Hou Kim; Laurent Meijer; Sophie LeClerc; Georjana Barnes; David O. Morgan; F. Hernan Espinoza; Soojin Kwon; Andy-Mark W. H. Thunnissen; Lisa Wodicka; Nathanael S. Gray; Peter G. Schultz; Thea C. Norman

1998-01-01

212

PiggyBac Transposon-Mediated, Reversible Gene Transfer in Human Embryonic Stem Cells  

PubMed Central

Permanent and reversible genetic modifications are important approaches to study gene function in different cell types. They are also important for stem cell researchers to explore and test the therapeutic potential of stem cells. The piggyBac transposon from insects is a rising nonviral system that efficiently mutagenizes and mediates gene transfer into the mammalian genome. It is also characterized by its precise excision, leaving no trace sequence behind so that the genomic integrity of the mutated cell can be restored. Here, we use an optimized piggyBac transposon system to mediate gene transfer and expression of a bifunctional fluorescent reporter in human embryonic stem (ES) cells. We provide molecular evidence for transposase-mediated piggyBac integration events and functional evidence for successful expression of a transferred fluorescent protein genes in human ES cells and their in vitro differentiated derivatives. We also demonstrate that the integrated piggyBac transposon can be removed and an undisrupted insertion site can be restored, which implies potential applications for its use in gene therapy and genetics studies. PMID:19740021

Furushima, Kenryo; Hou, Pei-Shan; Ku, Amy T.; Deng, Jian Min; Jang, Chuan-Wei; Fang, Haotian; Adams, Henry P.; Kuo, Min-Liang; Ho, Hong-Nerng

2010-01-01

213

Bac to the future: The use of bac transgenic mice for neuroscience research  

Microsoft Academic Search

The development of methods for engineering bacterial artificial chromosomes (BACs), and for the efficient production of BAC transgenic mice, has allowed the design of in vivo approaches to the analysis of gene expression and function in the brain, which could not be accomplished using traditional methods. These strategies have shed light on the functions of single genes in the nervous

Nathaniel Heintz

2001-01-01

214

Identification of Francisella tularensis genes encoding exported membrane-associated proteins using TnphoA mutagenesis of a genomic library.  

PubMed

Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen. PMID:15458781

Gilmore, Robert D; Bacon, Rendi Murphree; Sviat, Steven L; Petersen, Jeannine M; Bearden, Scott W

2004-10-01

215

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

PubMed Central

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

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

216

The Genome of the Sea Urchin Strongylocentrotus purpuratus  

Microsoft Academic Search

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes,

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

2006-01-01

217

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...

218

Observing copepods through a genomic lens  

PubMed Central

Background Copepods outnumber every other multicellular animal group. They are critical components of the world's freshwater and marine ecosystems, sensitive indicators of local and global climate change, key ecosystem service providers, parasites and predators of economically important aquatic animals and potential vectors of waterborne disease. Copepods sustain the world fisheries that nourish and support human populations. Although genomic tools have transformed many areas of biological and biomedical research, their power to elucidate aspects of the biology, behavior and ecology of copepods has only recently begun to be exploited. Discussion The extraordinary biological and ecological diversity of the subclass Copepoda provides both unique advantages for addressing key problems in aquatic systems and formidable challenges for developing a focused genomics strategy. This article provides an overview of genomic studies of copepods and discusses strategies for using genomics tools to address key questions at levels extending from individuals to ecosystems. Genomics can, for instance, help to decipher patterns of genome evolution such as those that occur during transitions from free living to symbiotic and parasitic lifestyles and can assist in the identification of genetic mechanisms and accompanying physiological changes associated with adaptation to new or physiologically challenging environments. The adaptive significance of the diversity in genome size and unique mechanisms of genome reorganization during development could similarly be explored. Genome-wide and EST studies of parasitic copepods of salmon and large EST studies of selected free-living copepods have demonstrated the potential utility of modern genomics approaches for the study of copepods and have generated resources such as EST libraries, shotgun genome sequences, BAC libraries, genome maps and inbred lines that will be invaluable in assisting further efforts to provide genomics tools for copepods. Summary Genomics research on copepods is needed to extend our exploration and characterization of their fundamental biological traits, so that we can better understand how copepods function and interact in diverse environments. Availability of large scale genomics resources will also open doors to a wide range of systems biology type studies that view the organism as the fundamental system in which to address key questions in ecology and evolution. PMID:21933388

2011-01-01

219

Translating the genetic library: the goals, methods, and applications of the Human Genome Project.  

PubMed Central

The information produced by the Human Genome Project will have profound effects on the medical community, biotechnology companies, research scientists, and others. The ultimate goal of the project is to locate the approximately 100,000 genes that contain the instructions for creating a human being. Once these genes are found, they can be studied to increase understanding of their role in health and disease. Information about the location of a gene, its function, and its connection to disease will be stored in a variety of computer databases. Medical librarians can help assure that this information reaches the people who need it. To aid in this effort, this paper provides an introduction to the Human Genome Project: its goals, examples of methods used to achieve these goals, the types of information produced, and examples of how this information can be used in medicine and basic research. Because rapid research progress makes it difficult to predict the exact course of the project, this paper will focus on the first five years, 1990-1995. PMID:8374581

Keleher, C

1993-01-01

220

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

PubMed Central

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

2010-01-01

221

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...

222

Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)  

Microsoft Academic Search

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned

Swaantje J Roth; Dirk Höper; Martin Beer; Silke Feineis; B Karsten Tischer; Nikolaus Osterrieder

2011-01-01

223

Epigenome analyses using BAC microarrays identify evolutionary conservation of tissue-specific methylation of SHANK3  

Microsoft Academic Search

CpG islands are present in one-half of all human and mouse genes and typically overlap with promoters or exons. We developed a method for high-resolution analysis of the methylation status of CpG islands genome-wide, using arrays of BAC clones and the methylation-sensitive restriction enzyme NotI. Here we demonstrate the accuracy and specificity of the method. By computationally mapping all NotI

Tsui-Ting Ching; Alika K Maunakea; Peter Jun; Chibo Hong; Giuseppe Zardo; Daniel Pinkel; Donna G Albertson; Jane Fridlyand; Jian-Hua Mao; Ksenya Shchors; William A Weiss; Joseph F Costello

2005-01-01

224

Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium Desulfovibrio alaskensis G20  

PubMed Central

ABSTRACT The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5? RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance. PMID:24865553

Kuehl, Jennifer V.; Price, Morgan N.; Ray, Jayashree; Wetmore, Kelly M.; Esquivel, Zuelma; Kazakov, Alexey E.; Nguyen, Michelle; Kuehn, Raquel; Davis, Ronald W.; Hazen, Terry C.; Arkin, Adam P.

2014-01-01

225

Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome  

Microsoft Academic Search

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders(1). We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de

Andrew J Sharp; Sierra Hansen; Rebecca R Selzer; Ze Cheng; Regina Regan; Jane A Hurst; Helen Stewart; Sue M Price; Edward Blair; Raoul C Hennekam; Carrie A Fitzpatrick; Rick Segraves; Todd A Richmond; Cheryl Guiver; Donna G Albertson; Daniel Pinkel; Peggy S Eis; Stuart Schwartz; Samantha J L Knight; Evan E Eichler

2006-01-01

226

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

PubMed

Chromosomes of schistosome parasites among digenetic flukes have a unique evolution because they exhibit the sex chromosomes ZW, which are not found in the other groups of flukes that are hermaphrodites. We conducted molecular cytogenetic analyses for investigating the sex chromosome evolution using chromosome paint analysis and BAC clones mapping. To carry this out, we developed a technique for making paint probes of genomic DNA from a single scraped chromosome segment using a chromosome microdissection system, and a FISH mapping technique for BAC clones. Paint probes clearly identified each of the 8 pairs of chromosomes by a different fluorochrome color. Combination analysis of chromosome paint analysis with Z/W probes and chromosome mapping with 93 BAC clones revealed that the W chromosome of Schistosoma mansoni has evolved by at least four inversion events and heterochromatinization. Nine of 93 BAC clones hybridized with both the Z and W chromosomes, but the locations were different between Z and W chromosomes. The homologous regions were estimated to have moved from the original Z chromosome to the differentiated W chromosome by three inversions events that occurred before W heterohcromatinization. An inversion that was observed in the heterochromatic region of the W chromosome likely occurred after W heterochromatinization. These inversions and heterochromatinization are hypothesized to be the key factors that promoted the evolution of the W chromosome of S. mansoni. PMID:22831897

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

2012-12-01

227

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of obscure puffer ( Takifugu obscurus ) and cross-species amplification  

Microsoft Academic Search

Obscure puffer (Takifugu obscurus) is an anadromous fish species in China. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched\\u000a genomic library of T. obscurus. The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from four to 10, from 0.57\\u000a to 0.86 and from 0.68 to 0.90, respectively. Three loci significantly deviated from

Hongyu Ma; Songlin Chen; Xiaolin Liao; Tianjun Xu; Jiachun Ge

2009-01-01

228

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of starry flounder ( Platichthys stellatus ) and cross-species amplification  

Microsoft Academic Search

Starry flounder (Platichthys stellatus) is a rare fish species in China. Here, we reported 12 polymorphic microsatellite loci isolated from a dinucleotide-enriched\\u000a genomic library of starry flounder (P. stellatus). The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from two to six, from 0.2500\\u000a to 1.0000 and from 0.4512 to 0.7667, respectively. One locus significantly deviated

Guidong Miao; Changwei Shao; Hongyu Ma; Xiaolin Liao; Songlin Chen

2009-01-01

229

Isolation and characterization of polymorphic microsatellite loci from a repeat-enriched genomic library of stone flounder ( Kareius   bicoloratus ) and cross-species amplification  

Microsoft Academic Search

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic\\u000a library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged\\u000a from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci significantly deviated from Hardy–Weinberg

Yong-Sheng Tian; Gui-Dong Miao; Chang-Wei Shao; Xiao-Lin Liao; Song-Lin Chen

2009-01-01

230

Isolation of Brucella abortus ssb and uvrA genes from a genomic library by use of lymphocytes as probes.  

PubMed Central

Brucella abortus proteins from virulent S2308 expressed from a pBluescript II SK- genomic library stimulated peripheral blood mononuclear (PBM) cell proliferation from cattle vaccinated with B. abortus S19. The method described here permits a rapid and directed approach to isolate genes encoding antigens of B. abortus that interact with lymphocytes primed to the living bacterium. The supernatants from the bacterial host JM109 (DE3) were cultured with freshly isolated bovine PBM cells. A total of 300 clones were evaluated. Ten clones were identified that stimulated T-lymphocyte proliferation. Among them, one clone with a 2.5-kb insert stimulated T-lymphocyte proliferation in all three animals, suggesting that the proteins encoded by genes within this fragment may represent immunodominant antigens. DNA sequencing of this clone reveals two large open reading frames (ORFs). ORF II has a high degree of similarity to the Escherichia coli ssb gene, which codes for the single-stranded DNA binding protein. ORF I, in the opposite direction to ORF II, shows similarity to the N terminus of the E. coli uvrA gene, which codes for one of the three subunits of the E. coli ABC excision nuclease. The observation that the PBM cells recognized and proliferated in response to proteins expressed from single clones provides a novel strategy to select bacterial antigens that may prove useful in designing alternative vaccines against brucellosis. PMID:8225607

Zhu, Y; Oliveira, S C; Splitter, G A

1993-01-01

231

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

PubMed

The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs. PMID:24859131

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

2014-10-01

232

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

PubMed

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

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

2008-10-01

233

Genomic Analysis of Wild Tomato Introgressions Determining Metabolism- and Yield-Associated Traits1[C][W  

PubMed Central

With the aim of determining the genetic basis of metabolic regulation in tomato fruit, we constructed a detailed physical map of genomic regions spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 colocated markers from five selected genomic regions, and a total of 614 bacterial artificial chromosome (BAC)/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of Solanum lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analysis of this information resulted in a genome-wide map of a nondomesticated plant species and covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely colinear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated, and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive microsynteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 million years ago. The combined results serve as a reference for comparative studies both at the macrosyntenic and microsyntenic levels. They also provide a valuable tool for fine-mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition. PMID:20118271

Kamenetzky, Laura; Asís, Ramón; Bassi, Sebastián; de Godoy, Fabiana; Bermúdez, Luisa; Fernie, Alisdair R.; Van Sluys, Marie-Anne; Vrebalov, Julia; Giovannoni, James J.; Rossi, Magdalena; Carrari, Fernando

2010-01-01

234

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...

235

Generation of a Genome Scale Lentiviral Vector Library for EF1? Promoter-Driven Expression of Human ORFs and Identification of Human Genes Affecting Viral Titer  

PubMed Central

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

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

2012-01-01

236

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

PubMed

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

Robinson, Karl E; Mahony, Timothy J

2015-01-01

237

Generation of a Complete Single-Gene Knockout Bacterial Artificial Chromosome Library of Cowpox Virus and Identification of Its Essential Genes  

PubMed Central

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

Xu, Zhiyong; Zikos, Dimitrios; Osterrieder, Nikolaus

2014-01-01

238

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

PubMed

Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence. PMID:21969025

Al-Jarbou, Ahmed Nasser

2012-01-01

239

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

PubMed Central

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

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

2014-01-01

240

Fungicidal mechanisms of the antimicrobial peptide Bac8c.  

PubMed

Bac8c (RIWVIWRR-NH2) is an analogue peptide derived through complete substitution analysis of the linear bovine host defense peptide variant Bac2A. In the present study, the antifungal mechanism of Bac8c against pathogenic fungi was investigated, with a particular focus on the effects of Bac8c on the cytoplasmic membrane. We used bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] staining and 3,3'-dipropylthiacarbocyanine iodide [DiSC3(5)] assays to show that Bac8c induced disturbances in the membrane potential of Candida albicans. An increase in membrane permeability and suppression of cell wall regeneration were also observed in Bac8c-treated C. albicans. We studied the effects of Bac8c treatment on model membranes to elucidate its antifungal mechanism. Using calcein and FITC-labeled dextran leakage assays from Bac8c-treated large unilamellar vesicles (LUVs) and giant unilamellar vesicles (GUVs), we found that Bac8c has a pore-forming action on fungal membranes, with an estimated pore radius of between 2.3 and 3.3nm. A membrane-targeted mechanism of action was also supported by the observation of potassium release from the cytosol of Bac8c-treated C. albicans. These results indicate that Bac8c is considered as a potential candidate to develop a novel antimicrobial agent because of its low-cost production characteristics and high antimicrobial activity via its ability to induce membrane perturbations in fungi. PMID:25434926

Lee, Wonyoung; Lee, Dong Gun

2015-02-01

241

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

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

Bearhop, Stuart

242

Matita, a new retroelement from peanut: characterization and evolutionary context in the light of the Arachis A-B genome divergence.  

PubMed

Cultivated peanut is an allotetraploid with an AB-genome. In order to learn more of the genomic structure of peanut, we characterized and studied the evolution of a retrotransposon originally isolated from a resistance gene analog (RGA)-containing bacterial artificial chromosome (BAC) clone. It is a moderate copy number Ty1-copia retrotransposon from the Bianca lineage and we named it Matita. Fluorescent in situ hybridization (FISH) experiments showed that Matita is mainly located on the distal regions of chromosome arms and is of approximately equal frequency on both A- and B-chromosomes. Its chromosome-specific hybridization pattern facilitates the identification of individual chromosomes, a useful cytogenetic tool considering that chromosomes in peanut are mostly metacentric and of similar size. Phylogenetic analysis of Matita elements, molecular dating of transposition events, and an estimation of the evolutionary divergence of the most probable A- and B-donor species suggest that Matita underwent its last major burst of transposition activity at around the same time of the A- and B-genome divergence about 3.5 million years ago. By probing BAC libraries with overgos probes for Matita, resistance gene analogues, and single- or low-copy genes, it was demonstrated that Matita is not randomly distributed in the genome but exhibits a significant tendency of being more abundant near resistance gene homologues than near single-copy genes. The described work is a further step towards broadening the knowledge on genomic and chromosomal structure of peanut and on its evolution. PMID:22120641

Nielen, Stephan; Vidigal, Bruna S; Leal-Bertioli, Soraya C M; Ratnaparkhe, Milind; Paterson, Andrew H; Garsmeur, Olivier; D'Hont, Angélique; Guimarães, Patricia M; Bertioli, David J

2012-01-01

243

Narrowing down the apricot Plum pox virus resistance locus and comparative analysis with the peach genome syntenic region.  

PubMed

Sharka disease, caused by the Plum pox virus (PPV), is one of the main limiting factors for stone fruit crops worldwide. Only a few resistance sources have been found in apricot (Prunus armeniaca L.), and most studies have located a major PPV resistance locus (PPVres) on linkage group 1 (LG1). However, the mapping accuracy was not sufficiently reliable and PPVres was predicted within a low confidence interval. In this study, we have constructed two high-density simple sequence repeat (SSR) improved maps with 0.70 and 0.68 markers/cm, corresponding to LG1 of 'Lito' and 'Goldrich' PPV-resistant cultivars, respectively. Using these maps, and excluding genotype-phenotype incongruent individuals, a new binary trait locus (BTL) analysis for PPV resistance was performed, narrowing down the PPVres support intervals to 7.3 and 5.9 cm in 'Lito' and 'Goldrich', respectively. Subsequently, 71 overlapping oligonucleotides (overgo) probes were hybridized against an apricot bacterial artificial chromosome (BAC) library, identifying 870 single BACs from which 340 were anchored onto a map region of approximately 30-40 cm encompassing PPVres. Partial BAC contigs assigned to the two allelic haplotypes (resistant/susceptible) of the PPVres locus were built by high-information content fingerprinting (HICF). In addition, a total of 300 BAC-derived sequences were obtained, and 257 showed significant homology with the peach genome scaffold_1 corresponding to LG1. According to the peach syntenic genome sequence, PPVres was predicted within a region of 2.16 Mb in which a few candidate resistance genes were identified. PMID:21722293

Vera Ruiz, Elsa María; Soriano, José Miguel; Romero, Carlos; Zhebentyayeva, Tetyana; Terol, Javier; Zuriaga, Elena; Llácer, Gerardo; Abbott, Albert Glenn; Badenes, María Luisa

2011-08-01

244

Partial Complementation of Sinorhizobium meliloti bacA Mutant Phenotypes by the Mycobacterium tuberculosis BacA Protein  

PubMed Central

The Sinorhizobium meliloti BacA ABC transporter protein plays an important role in its nodulating symbiosis with the legume alfalfa (Medicago sativa). The Mycobacterium tuberculosis BacA homolog was found to be important for the maintenance of chronic murine infections, yet its in vivo function is unknown. In the legume plant as well as in the mammalian host, bacteria encounter host antimicrobial peptides (AMPs). We found that the M. tuberculosis BacA protein was able to partially complement the symbiotic defect of an S. meliloti BacA-deficient mutant on alfalfa plants and to protect this mutant in vitro from the antimicrobial activity of a synthetic legume peptide, NCR247, and a recombinant human ?-defensin 2 (HBD2). This finding was also confirmed using an M. tuberculosis insertion mutant. Furthermore, M. tuberculosis BacA-mediated protection of the legume symbiont S. meliloti against legume defensins as well as HBD2 is dependent on its attached ATPase domain. In addition, we show that M. tuberculosis BacA mediates peptide uptake of the truncated bovine AMP, Bac71-16. This process required a functional ATPase domain. We therefore suggest that M. tuberculosis BacA is important for the transport of peptides across the cytoplasmic membrane and is part of a complete ABC transporter. Hence, BacA-mediated protection against host AMPs might be important for the maintenance of latent infections. PMID:23161027

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

2013-01-01

245

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

PubMed Central

Cytomegaloviruses are highly host restricted, resulting in cospeciation 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). Most in vivo experiments performed with RhCMV employed strain 68-1 cloned as a 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 300 bp. To obtain a more precise picture of the actual proteins encoded by the most commonly used molecular clone of RhCMV, we reevaluated the RhCMV 68-1 BAC genome by whole-genome shotgun sequencing and determined the protein content of the resulting RhCMV virions by proteomics. By comparing the RhCMV genome to those of several 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 suggests a high degree of ORF conservation among OWM CMVs, thus decreasing the likelihood that ORFs found only in RhCMV comprise true genes. Moreover, virion proteomics independently validated the revised ORF predictions, since only proteins that were conserved across OWM CMVs 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. PMID:22718821

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

2012-01-01

246

A GAA repeat expansion reporter model of Friedreich's ataxia recapitulates the genomic context and allows rapid screening of therapeutic compounds  

PubMed Central

Friedreich's ataxia (FRDA) is caused by large GAA expansions in intron 1 of the frataxin gene (FXN), which lead to reduced FXN expression through a mechanism not fully understood. Understanding such mechanism is essential for the identification of novel therapies for FRDA and this can be accelerated by the development of cell models which recapitulate the genomic context of the FXN locus and allow direct comparison of normal and expanded FXN loci with rapid detection of frataxin levels. Here we describe the development of the first GAA-expanded FXN genomic DNA reporter model of FRDA. We modified BAC vectors carrying the whole FXN genomic DNA locus by inserting the luciferase gene in exon 5a of the FXN gene (pBAC-FXN-Luc) and replacing the six GAA repeats present in the vector with an ?310 GAA repeat expansion (pBAC-FXN-GAA-Luc). We generated human clonal cell lines carrying the two vectors using site-specific integration to allow direct comparison of normal and expanded FXN loci. We demonstrate that the presence of expanded GAA repeats recapitulates the epigenetic modifications and repression of gene expression seen in FRDA. We applied the GAA-expanded reporter model to the screening of a library of novel small molecules and identified one molecule which up-regulates FXN expression in FRDA patient primary cells and restores normal histone acetylation around the GAA repeats. These results suggest the potential use of genomic reporter cell models for the study of FRDA and the identification of novel therapies, combining physiologically relevant expression with the advantages of quantitative reporter gene expression. PMID:23943791

Lufino, Michele M.P.; Silva, Ana M.; Németh, Andrea H.; Alegre-Abarrategui, Javier; Russell, Angela J.; Wade-Martins, Richard

2013-01-01

247

Library Regulations Library Regulations  

E-print Network

Library 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 of the University by Library Services, and thus there are sections that apply also to non- members of the University

Birmingham, University of

248

LIBRARY SERVICES LIBRARY HOURS  

E-print Network

LIBRARY SERVICES LIBRARY HOURS Up-to-date library hours are posted at http Wesleyan ID that is linked to the library circulation database is needed to charge out library materials to visit the Circulation Office in Olin Library 115 to set up their borrowing privileges. If you have

Royer, Dana

249

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...

250

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

SciTech Connect

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.

Kao, Fa-Ten

1992-08-01

251

The Genome of the Sea Urchin Strongylocentrotus purpuratus  

PubMed Central

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes. PMID:17095691

2011-01-01

252

The genome of the sea urchin Strongylocentrotus purpuratus.  

PubMed

We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes. PMID:17095691

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

2006-11-10

253

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...

254

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

PubMed Central

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

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

255

Whole genome linkage disequilibrium maps in cattle  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

256

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

PubMed Central

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

Parker, Jared B.; Walsh, Christopher T.

2012-01-01

257

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

PubMed Central

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

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

2012-01-01

258

Second-generation genetic linkage map of catfish and its integration with the BAC-based physical map.  

PubMed

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

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

2012-10-01

259

Library System Library System  

E-print Network

Library System #12;Library System 5150 Anthony Wayne Drive David Adamany Undergraduate Library that for the current fiscal year, we've been given an additional $600,000 for our library materials budget. We're very subscriptions. The Wayne State University Libraries are deeply committed to providing our faculty and students

Cinabro, David

260

Isolation and sequence analysis of the wheat B genome subtelomeric DNA  

PubMed Central

Background Telomeric and subtelomeric regions are essential for genome stability and regular chromosome replication. In this work, we have characterized the wheat BAC (bacterial artificial chromosome) clones containing Spelt1 and Spelt52 sequences, which belong to the subtelomeric repeats of the B/G genomes of wheats and Aegilops species from the section Sitopsis. Results The BAC library from Triticum aestivum cv. Renan was screened using Spelt1 and Spelt52 as probes. Nine positive clones were isolated; of them, clone 2050O8 was localized mainly to the distal parts of wheat chromosomes by in situ hybridization. The distribution of the other clones indicated the presence of different types of repetitive sequences in BACs. Use of different approaches allowed us to prove that seven of the nine isolated clones belonged to the subtelomeric chromosomal regions. Clone 2050O8 was sequenced and its sequence of 119 737 bp was annotated. It is composed of 33% transposable elements (TEs), 8.2% Spelt52 (namely, the subfamily Spelt52.2) and five non-TE-related genes. DNA transposons are predominant, making up 24.6% of the entire BAC clone, whereas retroelements account for 8.4% of the clone length. The full-length CACTA transposon Caspar covers 11 666 bp, encoding a transposase and CTG-2 proteins, and this transposon accounts for 40% of the DNA transposons. The in situ hybridization data for 2050O8 derived subclones in combination with the BLAST search against wheat mapped ESTs (expressed sequence tags) suggest that clone 2050O8 is located in the terminal bin 4BL-10 (0.95-1.0). Additionally, four of the predicted 2050O8 genes showed significant homology to four putative orthologous rice genes in the distal part of rice chromosome 3S and confirm the synteny to wheat 4BL. Conclusion Satellite DNA sequences from the subtelomeric regions of diploid wheat progenitor can be used for selecting the BAC clones from the corresponding regions of hexaploid wheat chromosomes. It has been demonstrated for the first time that Spelt52 sequences were involved in the evolution of terminal regions of common wheat chromosomes. Our research provides new insights into the microcollinearity in the terminal regions of wheat chromosomes 4BL and rice chromosome 3S. PMID:19732459

Salina, Elena A; Sergeeva, Ekaterina M; Adonina, Irina G; Shcherban, Andrey B; Afonnikov, Dmitry A; Belcram, Harry; Huneau, Cecile; Chalhoub, Boulos

2009-01-01

261

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

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

2010-01-01

262

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

PubMed Central

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

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

1986-01-01

263

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries  

Microsoft Academic Search

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have

NB Rodrigues; PT LoVerde; AJ Romanha; G Oliveira

2002-01-01

264

Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 – new vectors for in vitro and in vivo delivery  

PubMed Central

Background Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells. Results We describe a series of retrofitting plasmids and a protocol for in vivo loxP/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE carries G418 resistance and oriP/EBNA-1. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice in vivo. We also show that a BAC carrying GFP, oriP and EBNA-1 can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks. Conclusion The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or oriP/EBNA-1 using in vivo expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle in vivo. OriP/EBNA-1 allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks. PMID:12609052

Magin-Lachmann, Christine; Kotzamanis, George; D'Aiuto, Leonardo; Wagner, Ernst; Huxley, Clare

2003-01-01

265

Increased Body Weight of the BAC HD Transgenic Mouse Model of Huntington’s Disease Accounts for Some but Not All of the Observed HD-like Motor Deficits  

PubMed Central

The genome of the Bacterial Artificial Chromosome (BAC) transgenic mouse model of Huntington’s Disease (BAC HD) contains the 170 kb human HTT locus modified by the addition of exon 1 with 97 mixed CAA-CAG repeats. BAC HD mice present robust behavioral deficits in both the open field and the accelerating rotarod tests, two standard behavioral assays of motor function. BAC HD mice, however, also typically present significantly increased body weights relative to wildtype littermate controls (WT) which potentially confounds the interpretation of any motor deficits associated directly with the effects of mutant huntingtin. In order to evaluate this possible confound of body weight, we directly compared the performance of BAC HD and WT female mice under food restricted versus free feeding conditions in both the open field and rotarod tasks to test the hypothesis that some of the motor deficits observed in this HTT-transgenic mouse line results solely from increased body weight. Our results suggest that the rotarod deficit exhibited by BAC HD mice is modulated by both body weight and non-body weight factors resulting from overexpression of full length mutant Htt. When body weights of WT and BAC HD transgenic mice were normalized using restricted feeding, the deficits exhibited by BAC HD mice on the rotarod task were less marked, but were still significant. Since the rotarod deficit between WT and BAC HD mice is attenuated when body weight is normalized by food restriction, utilization of this task in BAC HD mice during pre-clinical evaluation must be powered accordingly and results carefully considered as therapeutic benefit can result from decreased overall body weight and or motoric improvement that may not be related to body mass. Furthermore, after controlling for body weight differences, the hypoactive phenotype displayed by ad libitum fed BAC HD mice in the open field assay was not observed in the BAC HD mice undergoing food restriction. These findings suggest that assessment of spontaneous locomotor activity, as measured in the open field test, may not be the appropriate behavioral endpoint to evaluate the BAC HD mouse during preclinical evaluation since it appears that the apparent hypoactive phenotype in this model is driven primarily by body weight differences. PMID:24042107

Kudwa, Andrea E.; Menalled, Liliana B.; Oakeshott, Stephen; Murphy, Carol; Mushlin, Richard; Fitzpatrick, John; Miller, Sam F.; McConnell, Kristi; Port, Russell; Torello, Justin; Howland, David; Ramboz, Sylvie; Brunner, Dani

2013-01-01

266

BacMam recombinant baculoviruses in G protein-coupled receptor drug discovery.  

PubMed

With completion of the sequencing of the human and mouse genomes, the primary sequences of close to 400 non-olfactory G protein-coupled receptors (GPCRs) have been determined. There are intensive efforts within the pharmaceutical industry to discover and develop new therapeutic agents acting via GPCRs. In addition, there is a concerted effort to identify potential new drug targets from the remaining 150+orphan GPCRs through the identification of their ligands. Access to functionally expressed recombinant receptors underpins both of these key drug discovery activities. Typically, GPCR drug discovery screening activities are carried out using mammalian cell lines stably expressing the target of interest. The influx of new receptor sequences originating from genomic sequencing efforts has caused a shift toward wider applications of transient rather than stable expression systems, especially in support of assays for orphan receptor ligand screening. Recombinant baculoviruses in which the polyhedrin promoter has been replaced with a mammalian promoter, termed BacMam viruses, were originally designed as potential new gene therapy delivery vehicles. This same technology offers numerous advantages as a transient expression system in the assay of membrane-expressed drug targets, including GPCRs. Data presented show that BacMam can be used rapidly to generate robust and pharmacologically authentic GPCR assays in several formats, with the potential to transform drug discovery screening processes for this gene family. PMID:15512844

Ames, Robert; Fornwald, James; Nuthulaganti, Parvathi; Trill, John; Foley, James; Buckley, Peter; Kost, Thomas; Wu, Zining; Romanos, Michael

2004-01-01

267

GENOMIC CHARACTERIZATION AND EXPRESSION ANALYSIS OF THE BACULOVIRAL IAP REPEAT CONTAINING 2 (BIRC2) GENE IN CHANNEL CATFISH, ICTALURUS PUNCTATUS  

Technology Transfer Automated Retrieval System (TEKTRAN)

Clones containing the gene for baculoviral IAP repeat containing 2 (BIRC2) were identified by PCR screening of a gynogenetic channel catfish (Ictalurus punctatus) BAC library. Directed sequencing of ~9.5 kb of the BAC clones revealed eight exons and seven introns in the catfish BIRC2 gene. Express...

268

A conditional piggyBac transposition system for genetic screening in mice identifies oncogenic networks in pancreatic cancer.  

PubMed

Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer. PMID:25485836

Rad, Roland; Rad, Lena; Wang, Wei; Strong, Alexander; Ponstingl, Hannes; Bronner, Iraad F; Mayho, Matthew; Steiger, Katja; Weber, Julia; Hieber, Maren; Veltkamp, Christian; Eser, Stefan; Geumann, Ulf; Öllinger, Rupert; Zukowska, Magdalena; Barenboim, Maxim; Maresch, Roman; Cadiñanos, Juan; Friedrich, Mathias; Varela, Ignacio; Constantino-Casas, Fernando; Sarver, Aaron; Ten Hoeve, Jelle; Prosser, Haydn; Seidler, Barbara; Bauer, Judith; Heikenwälder, Mathias; Metzakopian, Emmanouil; Krug, Anne; Ehmer, Ursula; Schneider, Günter; Knösel, Thomas; Rümmele, Petra; Aust, Daniela; Grützmann, Robert; Pilarsky, Christian; Ning, Zemin; Wessels, Lodewyk; Schmid, Roland M; Quail, Michael A; Vassiliou, George; Esposito, Irene; Liu, Pentao; Saur, Dieter; Bradley, Allan

2015-01-01

269

Genome Improvement at JGI-HAGSC  

SciTech Connect

Since the completion of the sequencing of the human genome, the 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.

Grimwood, Jane: Schmutz, Jeremy, J.: Myers, Richard, M.

2012-03-03

270

Pigeonpea genomics initiative (PGI): an international effort to improve crop productivity of pigeonpea (Cajanus cajan L.)  

PubMed Central

Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an ‘orphan crop legume’. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation’s Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an ‘orphan legume crop’ to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine. PMID:20976284

Penmetsa, R. V.; Dutta, S.; Kulwal, P. L.; Saxena, R. K.; Datta, S.; Sharma, T. R.; Rosen, B.; Carrasquilla-Garcia, N.; Farmer, A. D.; Dubey, A.; Saxena, K. B.; Gao, J.; Fakrudin, B.; Singh, M. N.; Singh, B. P.; Wanjari, K. B.; Yuan, M.; Srivastava, R. K.; Kilian, A.; Upadhyaya, H. D.; Mallikarjuna, N.; Town, C. D.; Bruening, G. E.; He, G.; May, G. D.; McCombie, R.; Jackson, S. A.; Singh, N. K.; Cook, D. R.

2009-01-01

271

Pigeonpea genomics initiative (PGI): an international effort to improve crop productivity of pigeonpea (Cajanus cajan L.).  

PubMed

Pigeonpea (Cajanus cajan), an important food legume crop in the semi-arid regions of the world and the second most important pulse crop in India, has an average crop productivity of 780 kg/ha. The relatively low crop yields may be attributed to non-availability of improved cultivars, poor crop husbandry and exposure to a number of biotic and abiotic stresses in pigeonpea growing regions. Narrow genetic diversity in cultivated germplasm has further hampered the effective utilization of conventional breeding as well as development and utilization of genomic tools, resulting in pigeonpea being often referred to as an 'orphan crop legume'. To enable genomics-assisted breeding in this crop, the pigeonpea genomics initiative (PGI) was initiated in late 2006 with funding from Indian Council of Agricultural Research under the umbrella of Indo-US agricultural knowledge initiative, which was further expanded with financial support from the US National Science Foundation's Plant Genome Research Program and the Generation Challenge Program. As a result of the PGI, the last 3 years have witnessed significant progress in development of both genetic as well as genomic resources in this crop through effective collaborations and coordination of genomics activities across several institutes and countries. For instance, 25 mapping populations segregating for a number of biotic and abiotic stresses have been developed or are under development. An 11X-genome coverage bacterial artificial chromosome (BAC) library comprising of 69,120 clones have been developed of which 50,000 clones were end sequenced to generate 87,590 BAC-end sequences (BESs). About 10,000 expressed sequence tags (ESTs) from Sanger sequencing and ca. 2 million short ESTs by 454/FLX sequencing have been generated. A variety of molecular markers have been developed from BESs, microsatellite or simple sequence repeat (SSR)-enriched libraries and mining of ESTs and genomic amplicon sequencing. Of about 21,000 SSRs identified, 6,698 SSRs are under analysis along with 670 orthologous genes using a GoldenGate SNP (single nucleotide polymorphism) genotyping platform, with large scale SNP discovery using Solexa, a next generation sequencing technology, is in progress. Similarly a diversity array technology array comprising of ca. 15,000 features has been developed. In addition, >600 unique nucleotide binding site (NBS) domain containing members of the NBS-leucine rich repeat disease resistance homologs were cloned in pigeonpea; 960 BACs containing these sequences were identified by filter hybridization, BES physical maps developed using high information content fingerprinting. To enrich the genomic resources further, sequenced soybean genome is being analyzed to establish the anchor points between pigeonpea and soybean genomes. In addition, Solexa sequencing is being used to explore the feasibility of generating whole genome sequence. In summary, the collaborative efforts of several research groups under the umbrella of PGI are making significant progress in improving molecular tools in pigeonpea and should significantly benefit pigeonpea genetics and breeding. As these efforts come to fruition, and expanded (depending on funding), pigeonpea would move from an 'orphan legume crop' to one where genomics-assisted breeding approaches for a sustainable crop improvement are routine. PMID:20976284

Varshney, R K; Penmetsa, R V; Dutta, S; Kulwal, P L; Saxena, R K; Datta, S; Sharma, T R; Rosen, B; Carrasquilla-Garcia, N; Farmer, A D; Dubey, A; Saxena, K B; Gao, J; Fakrudin, B; Singh, M N; Singh, B P; Wanjari, K B; Yuan, M; Srivastava, R K; Kilian, A; Upadhyaya, H D; Mallikarjuna, N; Town, C D; Bruening, G E; He, G; May, G D; McCombie, R; Jackson, S A; Singh, N K; Cook, D R

2010-10-01

272

Prokaryotic Genomes Eurkaryotic Genomes  

E-print Network

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

Qiu, Weigang

273

A physical map of the human genome  

SciTech Connect

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

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

2001-01-01

274

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

PubMed

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

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

2015-01-01

275

The Library The Public Library  

E-print Network

The Library The Public Library The Library Mission The library Function Acquisition Section of the library The library holdings Distribution of the library collections Organzation of Konwledge The library automated system The library services The automated circulation system Recalls Reservation Reference works

276

Efficient Expression of Acetylcholine-Binding Protein from Aplysia californica in Bac-to-Bac System  

PubMed Central

The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP) and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ?5?mg/L, which needs 55?h incubation after infection at the cell density of 2?×?106?cells/mL with an inoculum volume ratio of 1?:?100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies. PMID:25136612

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

2014-01-01

277

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

PubMed

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

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

2012-12-10

278

BAC end sequences corresponding to the B4 resistance gene cluster in common bean: a resource for markers and synteny analyses  

Microsoft Academic Search

In common bean, a complex disease resistance (R) gene cluster, harboring many specific R genes against various pathogens, is located at the end of the linkage group B4. A BAC library of the Meso-american bean genotype\\u000a BAT93 was screened with PRLJ1, a probe previously shown to be specific to the B4 R gene cluster, leading to the identification of 73

Perrine David; Mireille Sévignac; Vincent Thareau; Yann Catillon; Jim Kami; Paul Gepts; Thierry Langin; Valérie Geffroy

2008-01-01

279

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

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.

Vocero-Akbani, A.; Sanjurjo, H.; Fair, K. [Washington Univ. School of Medicine, St. Louis, MO (United States)] [and others

1994-09-01

280

BaC: a thermodynamically stable layered superconductor.  

PubMed

To predict all stable compounds in the Ba-C system, we perform a comprehensive study using first-principles variable-composition evolutionary algorithm USPEX. We find that at 0 K the well-known compound BaC2 is metastable in the whole pressure range 0-40 GPa, while intercalated graphite phase BaC6 is stable at 0-19 GPa. A hitherto unknown layered orthorhombic Pbam phase of BaC has structure consisting of alternating layers of Ba atoms and layers of stoichiometry Ba2C3 containing linear C3 groups and is predicted to be stable in the pressure range 3-32 GPa. From our electron-phonon coupling calculations, the newly found BaC compound is a phonon-mediated superconductor and has a critical superconductivity temperature Tc of 4.32 K at 5 GPa. This compound is dynamically stable at 0 GPa and therefore may be quenchable under normal conditions. PMID:25163859

Wang, Dian-Hui; Zhou, Huai-Ying; Hu, Chao-Hao; Oganov, Artem R; Zhong, Yan; Rao, Guang-Hui

2014-10-14

281

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

PubMed

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

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

2015-03-01

282

The value of avian genomics to the conservation of wildlife  

PubMed Central

Background Genomic studies in non-domestic avian models, such as the California condor and white-throated sparrow, can lead to more comprehensive conservation plans and provide clues for understanding mechanisms affecting genetic variation, adaptation and evolution. Developing genomic tools and resources including genomic libraries and a genetic map of the California condor is a prerequisite for identification of candidate loci for a heritable embryonic lethal condition. The white-throated sparrow exhibits a stable genetic polymorphism (i.e. chromosomal rearrangements) associated with variation in morphology, physiology, and behavior (e.g., aggression, social behavior, sexual behavior, parental care). In this paper we outline the utility of these species as well as report on recent advances in the study of their genomes. Results Genotyping of the condor resource population at 17 microsatellite loci provided a better assessment of the current population's genetic variation. Specific New World vulture repeats were found in the condor genome. Using condor BAC library and clones, chicken-condor comparative maps were generated. A condor fibroblast cell line transcriptome was characterized using the 454 sequencing technology. Our karyotypic analyses of the sparrow in combination with other studies indicate that the rearrangements in both chromosomes 2m and 3a are complex and likely involve multiple inversions, interchromosomal linkage, and pleiotropy. At least a portion of the rearrangement in chromosome 2m existed in the common ancestor of the four North American species of Zonotrichia, but not in the one South American species, and that the 2m form, originally thought to be the derived condition, might actually be the ancestral one. Conclusion Mining and characterization of candidate loci in the California condor using molecular genetic and genomic techniques as well as linkage and comparative genomic mapping will eventually enable the identification of carriers of the chondrodystrophy allele, resulting in improved genetic management of this disease. In the white-throated sparrow, genomic studies, combined with ecological data, will help elucidate the basis of genic selection in a natural population. Morphs of the sparrow provide us with a unique opportunity to study intraspecific genomic differences, which have resulted from two separate yet linked evolutionary trajectories. Such results can transform our understanding of evolutionary and conservation biology. PMID:19607652

2009-01-01

283

Suicidal Autointegration of Sleeping Beauty and piggyBac Transposons in Eukaryotic Cells  

PubMed Central

Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB. PMID:24625543

Devaraj, Anatharam; Singh, Manvendra; Jimenez Orgaz, Ana; Chen, Jia-Xuan; Selbach, Matthias; Ivics, Zoltán; Izsvák, Zsuzsanna

2014-01-01

284

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 ...

285

BacMet: antibacterial biocide and metal resistance genes database.  

PubMed

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

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

2014-01-01

286

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

SciTech Connect

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.

Wei Lin; Weber, A.S. (State Univ. of New York, Buffalo (United States))

1992-05-01

287

A chromosomal genomics approach to assess and validate the desi and kabuli draft chickpea genome assemblies.  

PubMed

With the expansion of next-generation sequencing technology and advanced bioinformatics, there has been a rapid growth of genome sequencing projects. However, while this technology enables the rapid and cost-effective assembly of draft genomes, the quality of these assemblies usually falls short of gold standard genome assemblies produced using the more traditional BAC by BAC and Sanger sequencing approaches. Assembly validation is often performed by the physical anchoring of genetically mapped markers, but this is prone to errors and the resolution is usually low, especially towards centromeric regions where recombination is limited. New approaches are required to validate reference genome assemblies. The ability to isolate individual chromosomes combined with next-generation sequencing permits the validation of genome assemblies at the chromosome level. We demonstrate this approach by the assessment of the recently published chickpea kabuli and desi genomes. While previous genetic analysis suggests that these genomes should be very similar, a comparison of their chromosome sizes and published assemblies highlights significant differences. Our chromosomal genomics analysis highlights short defined regions that appear to have been misassembled in the kabuli genome and identifies large-scale misassembly in the draft desi genome. The integration of chromosomal genomics tools within genome sequencing projects has the potential to significantly improve the construction and validation of genome assemblies. The approach could be applied both for new genome assemblies as well as published assemblies, and complements currently applied genome assembly strategies. PMID:24702794

Ruperao, Pradeep; Chan, Chon-Kit Kenneth; Azam, Sarwar; Karafiátová, Miroslava; Hayashi, Satomi; Cížková, Jana; Saxena, Rachit K; Simková, Hana; Song, Chi; Vrána, Jan; Chitikineni, Annapurna; Visendi, Paul; Gaur, Pooran M; Millán, Teresa; Singh, Karam B; Taran, Bunyamin; Wang, Jun; Batley, Jacqueline; Doležel, Jaroslav; Varshney, Rajeev K; Edwards, David

2014-08-01

288

Frequent Itemsets for Genomic Profiling Jeannette M. de Graaf1  

E-print Network

Profiling 105 (relatively short pieces of DNA) such as bacterial artificial chromosomes (BACsFrequent Itemsets for Genomic Profiling Jeannette M. de Graaf1 , Ren´ee X. de Menezes2,3 , Judith M approach to the study of genomic profiling data. Here a dataset consists of real num- bers describing

Kosters, Walter

289

[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

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.

Simon, M.I.

1994-12-31

290

Action and Timing of BacC and BacD in the Late Stages of Biosynthesis of the Dipeptide Antibiotic Bacilysin  

PubMed Central

Biosynthesis of the dipeptide antibiotic bacilysin, encoded by the seven B. subtilis genes bacA-G, involves diversion of flux from prephenate to the noncognate amino acid anticapsin. The anticapsin warhead is then ligated to the C-terminus of l-alanine to produce mature bacilysin. We have previously noted the formation of two diastereomers of tetrahydrotyrosine (4S- and 4R-H4Tyr) by tandem action of the four purified enzymes BacABGF. BacC (oxidase) and BacD (ligase) have been hypothesized to be remaining late stage enzymes in bacilysin biosynthesis. Using a combination of BacCD in vitro studies, B. subtilis deletion mutants, and isotopic feeding studies, we were able to determine that the H4Tyr diastereomers are actually shunt products that are not on-pathway to bacilysin biosynthesis. Dihydroanticapsin and dihydrobacilysin accumulate in extracts of a ?bacC strain and are processed to anticapsin and then bacilysin on addition of BacC and BacD, respectively. These results suggest the epoxide group in bacilysin is installed in an earlier step of bacilysin biosynthesis, while BacC oxidation of the C7-hydroxyl followed by BacD ligation of anticapsin to l-Ala are the penultimate and ultimate steps of bacilysin biosynthesis. PMID:23317005

Parker, Jared B.; Walsh, Christopher T.

2013-01-01

291

A PiggyBac-Based Recessive Screening Method to Identify Pluripotency Regulators  

PubMed Central

Phenotype driven genetic screens allow unbiased exploration of the genome to discover new biological regulators. Bloom syndrome gene (Blm) deficient embryonic stem (ES) cells provide an opportunity for recessive screening due to frequent loss of heterozygosity. We describe a strategy for isolating regulators of mammalian pluripotency based on conversion to homozygosity of PiggyBac gene trap insertions combined with stringent selection for differentiation resistance. From a screen of 2000 mutants we obtained a disruptive integration in the Tcf3 gene. Homozygous Tcf3 mutants showed impaired differentiation and enhanced self-renewal. This phenotype was reverted in a dosage sensitive manner by excision of one or both copies of the gene trap. These results provide new evidence confirming that Tcf3 is a potent negative regulator of pluripotency and validate a forward screening methodology to identify modulators of pluripotent stem cell biology. PMID:21533166

Guo, Ge; Huang, Yue; Humphreys, Peter; Wang, Xiaozhong; Smith, Austin

2011-01-01

292

Bacterial genomics: the use of DNA microarrays and bacterial artificial chromosomes  

Microsoft Academic Search

Immense amounts of genetic information are contained within microbial genomes. As the number of completely sequenced microbial genomes is increasing, functional and comparative genomic techniques will be employed for sequence analysis and gene characterization. Sequence comparison and expression profiling by DNA microarrays can determine phylogenetic relationships and identify genes while bacterial artificial chromosomes (BACs) allow the study of entire biochemical

Kristen D. Ball; J. T. Trevors

2002-01-01

293

Bacteriocin protein BacL1 of Enterococcus faecalis is a peptidoglycan D-isoglutamyl-L-lysine endopeptidase.  

PubMed

Enterococcus faecalis strains are commensal bacteria in humans and other animals, and they are also the causative agent of opportunistic infectious diseases. Bacteriocin 41 (Bac41) is produced by certain E. faecalis clinical isolates, and it is active against other E. faecalis strains. Our genetic analyses demonstrated that the extracellular products of the bacL1 and bacA genes, which are encoded in the Bac41 operon, coordinately express the bacteriocin activity against E. faecalis. In this study, we investigated the molecular functions of the BacL1 and BacA proteins. Immunoblotting and N-terminal amino acid sequence analysis revealed that BacL1 and BacA are secreted without any processing. The coincidental treatment with the recombinant BacL1 and BacA showed complete bacteriocin activity against E. faecalis, but neither BacL1 nor BacA protein alone showed the bacteriocin activity. Interestingly, BacL1 alone demonstrated substantial degrading activity against the cell wall fraction of E. faecalis in the absence of BacA. Furthermore, MALDI-TOF MS analysis revealed that BacL1 has a peptidoglycan D-isoglutamyl-L-lysine endopeptidase activity via a NlpC/P60 homology domain. These results collectively suggest that BacL1 serves as a peptidoglycan hydrolase and, when BacA is present, results in the lysis of viable E. faecalis cells. PMID:24235140

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

2013-12-27

294

Duplication and differentiation of common carp (Cyprinus carpio) myoglobin genes revealed by BAC analysis.  

PubMed

Two distinct myoglobin (mb) transcripts have been reported in common carp, Cyprinus carpio, which is a hypoxia-tolerant fish living in habitats with greatly fluctuant dissolved oxygen levels. Recombinant protein analysis has shown functional specialization of the two mb transcripts. In this work, analysis for mb-containing bacterial artificial chromosome (BAC) clones indicated different genome loci for common carp myoglobin-1 (mb-1) and myoglobin-2 (mb-2) genes. Fluorescence in situ hybridization (FISH) revealed that mb-1 and mb-2 are located on separate chromosomes. In both of the mb-1 and mb-2 containing BAC clones, gene synteny was well conserved with the homologous region on zebrafish chromosome 1, supporting that the common carp specific mb-2 gene originated from the recent whole genome duplication event in cyprinid lineage. Transcription factor binding sites search indicated that both common carp mb genes lacked specificity Protein 1 (Sp1) and myocyte enhancer factor-2 (MEF2) binding sites, which mediated muscle-specific and calcium-dependent expression in the well-studied mb promoters. Potential hypoxia response elements (HREs) were predicted in the regulatory region of common carp mb genes. These characteristics of common carp mb gene regulatory region well interpreted the hypoxia-inducible, non-muscle expression pattern of mb-1. In the case of mb-2, a 10 bp insertion to the binding site of upstream stimulatory factor (USF), which was a co-factor of hypoxia inducible factor (HIF), might cause the non-response to hypoxia treatment of mb-2. The case of common carp mb gene duplication and subsequent differentiation in expression pattern and protein function provided an example for adaptive evolution toward aquatic hypoxia tolerance. PMID:25026501

Zhao, Zi-Xia; Xu, Peng; Cao, Ding-Chen; Kuang, You-Yi; Deng, Hai-Xia; Zhang, Yan; Xu, Li-Ming; Li, Jiong-Tang; Xu, Jian; Sun, Xiao-Wen

2014-09-15

295

Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.  

PubMed

We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy. PMID:23644548

Chin, Chen-Shan; Alexander, David H; Marks, Patrick; Klammer, Aaron A; Drake, James; Heiner, Cheryl; Clum, Alicia; Copeland, Alex; Huddleston, John; Eichler, Evan E; Turner, Stephen W; Korlach, Jonas

2013-06-01

296

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

PubMed Central

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

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

297

[Microsynteny analysis of tomato and peach genome].  

PubMed

Tomato and peach are two important model species belonging to the families Solanaceae and Rosaceae, respectively. Recently, more and more sequence data generated from their whole genome sequencing projects can be used for bioinformatic analysis. Microsynteny analysis for tomato and peach were conducted to detect conserved syntenic blocks using high quality genetic and physical maps. A large number of microsyntenic regions were detected through three com-parisons: comparison between tomato genetic map and peach physical map, between tomato physical map and peach ge-netic map, and between tomato physical map and peach physical map. Most of the syntenic blocks were short, and each block contained a small number of conserved gene pairs (261 syntenic blocks with only two homolog pairs, and 36 syntenic blocks with more than two homolog pairs). Tomato and peach had noncontinuous fragmentary microsynteny and some syntenic groups composed of complex networks among different chromosomes or BAC contigs. After comparing the ho-mologous proteins with tomato fruit-related EST libraries, a total of 9 proteins were found in different syntenic groups re-lating to fruit development and ripening. Microsynteny identified in this study could facilitate further investigation of fruit development and ripening in these two distantly related species. PMID:20870619

Song, Chi; Wang, Ying

2010-09-01

298

Repetitive sequences, genomic instability and Barrett's esophageal adenocarcinoma  

PubMed Central

Barrett's esophageal adenocarcinoma (BAC) is a cancer associated with heartburn. If gastroesophageal reflux is not treated, the exposure to acid over the years, leads to a premalignant condition known as Barrett's esophagus (BE) which then progresses through low grade and high grade dysplasias to Barrett's adenocarcinoma. Genomic instability, which seems to arise early at BE stage, leads to accrual of mutational changes which underlie the the succession of histological and physiological changes associated with this disease. Genomic instability is therefore an important target for prevention and treatment of cancer and it is important to elucidate the mechanisms associated with this problem. We have shown that elevated/deregulated homologous recombination mediates genomic instability in cancer. Recently we also demonstrated that the mutational rates of individual chromosomes in BAC cells correlate with their ALU frequency. The aims of this article are to briefly discuss different types of repetitive sequences and highlight their importance in physiology of normal and cancer cells, especially BAC. PMID:22479688

2011-01-01

299

Cyclic AMP Effectors in African Trypanosomes Revealed by Genome-Scale RNA Interference Library Screening for Resistance to the Phosphodiesterase Inhibitor CpdA  

PubMed Central

One of the most promising new targets for trypanocidal drugs to emerge in recent years is the cyclic AMP (cAMP) phosphodiesterase (PDE) activity encoded by TbrPDEB1 and TbrPDEB2. These genes were genetically confirmed as essential, and a high-affinity inhibitor, CpdA, displays potent antitrypanosomal activity. To identify effectors of the elevated cAMP levels resulting from CpdA action and, consequently, potential sites for adaptations giving resistance to PDE inhibitors, resistance to the drug was induced. Selection of mutagenized trypanosomes resulted in resistance to CpdA as well as cross-resistance to membrane-permeable cAMP analogues but not to currently used trypanocidal drugs. Resistance was not due to changes in cAMP levels or in PDEB genes. A second approach, a genome-wide RNA interference (RNAi) library screen, returned four genes giving resistance to CpdA upon knockdown. Validation by independent RNAi strategies confirmed resistance to CpdA and suggested a role for the identified cAMP Response Proteins (CARPs) in cAMP action. CARP1 is unique to kinetoplastid parasites and has predicted cyclic nucleotide binding-like domains, and RNAi repression resulted in >100-fold resistance. CARP2 and CARP4 are hypothetical conserved proteins associated with the eukaryotic flagellar proteome or with flagellar function, with an orthologue of CARP4 implicated in human disease. CARP3 is a hypothetical protein, unique to Trypanosoma. CARP1 to CARP4 likely represent components of a novel cAMP signaling pathway in the parasite. As cAMP metabolism is validated as a drug target in Trypanosoma brucei, cAMP effectors highly divergent from the mammalian host, such as CARP1, lend themselves to further pharmacological development. PMID:23877697

Gould, Matthew K.; Bachmaier, Sabine; Ali, Juma A. M.; Alsford, Sam; Tagoe, Daniel N. A.; Munday, Jane C.; Schnaufer, Achim C.; Horn, David

2013-01-01

300

Genomics of hybrid poplar (Populus trichocarpa× deltoides) interacting with forest tent caterpillars (Malacosoma disstria): normalized and full-length cDNA libraries, expressed sequence tags, and a cDNA microarray for the study of insect-induced defences: INSECT-INDUCED DEFENCES IN POPLAR  

Microsoft Academic Search

As part of a genomics strategy to characterize inducible defences against insect herbivory in poplar, we developed a comprehensive suite of functional genomics resources including cDNA libraries, expressed sequence tags (ESTs) and a cDNA microarray platform. These resources are designed to complement the existing poplar genome sequence and poplar ( Populus spp.) ESTs by focusing on herbivore- and elicitor-treated tissues

STEVEN RALPH; CLAIRE ODDY; DAWN COOPER; HESTHER YUEH; SHARON JANCSIK; NATALIA KOLOSOVA; RYAN N. PHILIPPE; DANA AESCHLIMAN; RICK WHITE; DEZENE HUBER; CAROL E. RITLAND; FRANÇOIS BENOIT; TRACEY RIGBY; ANDRÉ NANTEL; YARON S. N. BUTTERFIELD; ROBERT KIRKPATRICK; ELIZABETH CHUN; JERRY LIU; DIANA PALMQUIST; BRIAN WYNHOVEN; JEFFREY STOTT; GEORGE YANG; SARAH BARBER; ROBERT A. HOLT; ASIM SIDDIQUI; STEVEN J. M. JONES; MARCO A. MARRA; BRIAN E. ELLIS; CARL J. DOUGLAS; KERMIT RITLAND; JÖRG BOHLMANN

2006-01-01

301

Discover your Library Medical Library  

E-print Network

Discover your Library Medical Library Welcome to the Gus Fraenkel Medical Library. The Library is a branch of the Flinders University Libraries including: Central (on the Plaza of the north ridge precinct) Law (on level 3 of the Central Library building) Sturt (at the Sturt precinct) as well

302

Genomics Glossary  

NSDL National Science Digital Library

Because genomics is an interdisciplinary science that unites biology, chemistry, physics, and mathematics, its language is diverse and includes terms not always found in dictionaries. This site from Cambridge Healthtech Institute of Massachusetts was designed to help scientists keep on top of this complex language. Loads of terms in categories such as basic genetics, functional and structural genomics, informatics, and genomic-related technology are defined here. Users can access the glossary terms either through a short index of major subject headings or by a longer alphabetically-arranged subject list. The Genomics Glossary deserves bonus points for including links to related resources in the text of its definitions. For example, within the definition of "polymerase chain reaction" are links to sites at Yale Medical School and the National Library of Medicine. In addition, links to pages on nomenclature, a bibliography of Web and print resources, and a FAQ page are available at this fantastic Website.

Chitty, Mary Glen.

303

Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition  

PubMed Central

Background The Solanaceae family contains a number of important crop species including potato (Solanum tuberosum) which is grown for its underground storage organ known as a tuber. Albeit the 4th most important food crop in the world, other than a collection of ~220,000 Expressed Sequence Tags, limited genomic sequence information is currently available for potato and advances in potato yield and nutrition content would be greatly assisted through access to a complete genome sequence. While morphologically diverse, Solanaceae species such as potato, tomato, pepper, and eggplant share not only genes but also gene order thereby permitting highly informative comparative genomic analyses. Results In this study, we report on analysis 89.9 Mb of potato genomic sequence representing 10.2% of the genome generated through end sequencing of a potato bacterial artificial chromosome (BAC) clone library (87 Mb) and sequencing of 22 potato BAC clones (2.9 Mb). The GC content of potato is very similar to Solanum lycopersicon (tomato) and other dicotyledonous species yet distinct from the monocotyledonous grass species, Oryza sativa. Parallel analyses of repetitive sequences in potato and tomato revealed substantial differences in their abundance, 34.2% in potato versus 46.3% in tomato, which is consistent with the increased genome size per haploid genome of these two Solanum species. Specific classes and types of repetitive sequences were also differentially represented between these two species including a telomeric-related repetitive sequence, ribosomal DNA, and a number of unclassified repetitive sequences. Comparative analyses between tomato and potato at the gene level revealed a high level of conservation of gene content, genic feature, and gene order although discordances in synteny were observed. Conclusion Genomic level analyses of potato and tomato confirm that gene sequence and gene order are conserved between these solanaceous species and that this conservation can be leveraged in genomic applications including cross-species annotation and genome sequencing initiatives. While tomato and potato share genic features, they differ in their repetitive sequence content and composition suggesting that repetitive sequences may have a more significant role in shaping speciation than previously reported. PMID:18554403

Zhu, Wei; Ouyang, Shu; Iovene, Marina; O'Brien, Kimberly; Vuong, Hue; Jiang, Jiming; Buell, C Robin

2008-01-01

304

Integration of genetic and physical maps of the chickpea (Cicer arietinum L.) genome using flow-sorted chromosomes.  

PubMed

Cultivated chickpea is the third most important legume after field bean and garden pea worldwide. Despite considerable breeding towards improved yield and resistance to biotic and abiotic stresses, the production of chickpea remained stagnant, but molecular tools are expected to increase the impact of current improvement programs. As a first step towards this goal, various genetic linkage maps have been established and markers linked to resistance genes been identified. However, until now, only one linkage group (LG) has been assigned to a specific chromosome. In the present work, mitotic chromosomes were sorted using flow cytometry and used as template for PCR with primers designed for genomic regions flanking microsatellites. These primers amplify sequence-tagged microsatellite site markers. This approach confirmed the assignment of LG8 to the smallest chromosome H. For the first time, LG5 was linked to the largest chromosome A, LG4 to a medium-sized chromosome E, while LG3 was anchored to the second largest chromosome B. Chromosomes C and D could not be flow-sorted separately and were jointly associated to LG6 and LG7. By the same token, chromosomes F and G were anchored to LG1 and LG2. To establish a set of preferably diagnostic cytogenetic markers, the genomic distribution of various probes was verified using FISH. Moreover, a partial genomic bacterial artificial chromosome (BAC) library was constructed and putative single/low-copy BAC clones were mapped cytogenetically. As a result, two clones were identified localizing specifically to chromosomes E and H, for which no cytogenetic markers were yet available. PMID:21947955

Zatloukalová, Pavlína; H?ibová, Eva; Kubaláková, Marie; Suchánková, Pavla; Simková, Hana; Adoración, Cabrera; Kahl, Günter; Millán, Teresa; Doležel, Jaroslav

2011-08-01

305

BAC-Based Sequencing of Behaviorally-Relevant Genes in the Prairie Vole  

PubMed Central

The prairie vole (Microtus ochrogaster) is an important model organism for the study of social behavior, yet our ability to correlate genes and behavior in this species has been limited due to a lack of genetic and genomic resources. Here we report the BAC-based targeted sequencing of behaviorally-relevant genes and flanking regions in the prairie vole. A total of 6.4 Mb of non-redundant or haplotype-specific sequence assemblies were generated that span the partial or complete sequence of 21 behaviorally-relevant genes as well as an additional 55 flanking genes. Estimates of nucleotide diversity from 13 loci based on alignments of 1.7 Mb of haplotype-specific assemblies revealed an average pair-wise heterozygosity (8.4×10?3). Comparative analyses of the prairie vole proteins encoded by the behaviorally-relevant genes identified >100 substitutions specific to the prairie vole lineage. Finally, our sequencing data indicate that a duplication of the prairie vole AVPR1A locus likely originated from a recent segmental duplication spanning a minimum of 105 kb. In summary, the results of our study provide the genomic resources necessary for the molecular and genetic characterization of a high-priority set of candidate genes for regulating social behavior in the prairie vole. PMID:22238603

McGraw, Lisa A.; Davis, Jamie K.; Thomas, Pamela J.; Young, Larry J.; Thomas, James W.

2012-01-01

306

PiggyBac transposon-mediated gene transfer in Cashmere goat fetal fibroblast cells.  

PubMed

PiggyBac (PB) has recently been found to be functional in various organisms. To verify and exploit its application in the cashmere goat, a PB transposon system including donor and helper vector of was developed, in which the EGFP gene in donor of vector was used as reporter. Cashmere goat fetal fibroblasts cells (GFFs) were transfected with the PB transposon system and the efficiency of gene transfer was determined. Compared with random integration, PB-mediated EGFP expression levels increased 7.78-fold in the GFFs, confirming that the PB transposon system constructed successfully mediated efficient foreign gene integration in the GFFs. To further investigate the characteristics of PB-mediated integration instance, PB integration site distribution in the goat genome was examined. The results showed that PB had a preference for AT rich regions of the goat genome. Thus this study confirms the function of PB transposon in GFFs and provides a potential genetic tool for producing transgenic goats. PMID:22738962

Bai, Ding-Ping; Yang, Ming-Ming; Chen, Yu-Lin

2012-01-01

307

BAC-based sequencing of behaviorally-relevant genes in the prairie vole.  

PubMed

The prairie vole (Microtus ochrogaster) is an important model organism for the study of social behavior, yet our ability to correlate genes and behavior in this species has been limited due to a lack of genetic and genomic resources. Here we report the BAC-based targeted sequencing of behaviorally-relevant genes and flanking regions in the prairie vole. A total of 6.4 Mb of non-redundant or haplotype-specific sequence assemblies were generated that span the partial or complete sequence of 21 behaviorally-relevant genes as well as an additional 55 flanking genes. Estimates of nucleotide diversity from 13 loci based on alignments of 1.7 Mb of haplotype-specific assemblies revealed an average pair-wise heterozygosity (8.4×10(-3)). Comparative analyses of the prairie vole proteins encoded by the behaviorally-relevant genes identified >100 substitutions specific to the prairie vole lineage. Finally, our sequencing data indicate that a duplication of the prairie vole AVPR1A locus likely originated from a recent segmental duplication spanning a minimum of 105 kb. In summary, the results of our study provide the genomic resources necessary for the molecular and genetic characterization of a high-priority set of candidate genes for regulating social behavior in the prairie vole. PMID:22238603

McGraw, Lisa A; Davis, Jamie K; Thomas, Pamela J; Young, Larry J; Thomas, James W

2012-01-01

308

Sex Chromosome Evolution in Amniotes: Applications for Bacterial Artificial Chromosome Libraries  

PubMed Central

Variability among sex chromosome pairs in amniotes denotes a dynamic history. Since amniotes diverged from a common ancestor, their sex chromosome pairs and, more broadly, sex-determining mechanisms have changed reversibly and frequently. These changes have been studied and characterized through the use of many tools and experimental approaches but perhaps most effectively through applications for bacterial artificial chromosome (BAC) libraries. Individual BAC clones carry 100–200?kb of sequence from one individual of a target species that can be isolated by screening, mapped onto karyotypes, and sequenced. With these techniques, researchers have identified differences and similarities in sex chromosome content and organization across amniotes and have addressed hypotheses regarding the frequency and direction of past changes. Here, we review studies of sex chromosome evolution in amniotes and the ways in which the field of research has been affected by the advent of BAC libraries. PMID:20981143

Janes, Daniel E.; Valenzuela, Nicole; Ezaz, Tariq; Amemiya, Chris; Edwards, Scott V.

2011-01-01

309

ChBac3.4: A Novel Proline-Rich Antimicrobial Peptide from Goat Leukocytes  

Microsoft Academic Search

We isolated a new proline-rich peptide, ChBac3.4, from leukocytes of the goat (Capra hirca) and determined its amino acid sequence by Edman degradation and mass spectrometry. ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL–NH2)\\u000a had over 50% sequence identity to the Bac5 peptides found in the leukocytes of goats, sheep and cattle. ChBac3.4 exhibited\\u000a broadspectrum antimicrobial activity, especially under low salt conditions. Since E. coli ML35p treated

Olga Shamova; Dmitriy Orlov; Christin Stegemann; Patricia Czihal; Ralf Hoffmann; Kim Brogden; Nikolay Kolodkin; Galina Sakuta; Alessandro Tossi; Hans-Georg Sahl; Vladimir Kokryakov; Robert I. Lehrer

2009-01-01

310

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...

311

KAIKObase: An integrated silkworm genome database and data mining tool  

PubMed Central

Background The silkworm, Bombyx mori, is one of the most economically important insects in many developing countries owing to its large-scale cultivation for silk production. With the development of genomic and biotechnological tools, B. mori has also become an important bioreactor for production of various recombinant proteins of biomedical interest. In 2004, two genome sequencing projects for B. mori were reported independently by Chinese and Japanese teams; however, the datasets were insufficient for building long genomic scaffolds which are essential for unambiguous annotation of the genome. Now, both the datasets have been merged and assembled through a joint collaboration between the two groups. Description Integration of the two data sets of silkworm whole-genome-shotgun sequencing by the Japanese and Chinese groups together with newly obtained fosmid- and BAC-end sequences produced the best continuity (~3.7 Mb in N50 scaffold size) among the sequenced insect genomes and provided a high degree of nucleotide coverage (88%) of all 28 chromosomes. In addition, a physical map of BAC contigs constructed by fingerprinting BAC clones and a SNP linkage map constructed using BAC-end sequences were available. In parallel, proteomic data from two-dimensional polyacrylamide gel electrophoresis in various tissues and developmental stages were compiled into a silkworm proteome database. Finally, a Bombyx trap database was constructed for documenting insertion positions and expression data of transposon insertion lines. Conclusion For efficient usage of genome information for functional studies, genomic sequences, physical and genetic map information and EST data were compiled into KAIKObase, an integrated silkworm genome database which consists of 4 map viewers, a gene viewer, and sequence, keyword and position search systems to display results and data at the level of nucleotide sequence, gene, scaffold and chromosome. Integration of the silkworm proteome database and the Bombyx trap database with KAIKObase led to a high-grade, user-friendly, and comprehensive silkworm genome database which is now available from URL: . PMID:19843344

Shimomura, Michihiko; Minami, Hiroshi; Suetsugu, Yoshitaka; Ohyanagi, Hajime; Satoh, Chikatada; Antonio, Baltazar; Nagamura, Yoshiaki; Kadono-Okuda, Keiko; Kajiwara, Hideyuki; Sezutsu, Hideki; Nagaraju, Javaregowda; Goldsmith, Marian R; Xia, Qingyou; Yamamoto, Kimiko; Mita, Kazuei

2009-01-01

312

A physical map of the highly heterozygous Populus genome: integration with the genome sequence and genetic map and analysis of haplotype variation  

SciTech Connect

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 first maps of an undomesticated, highly heterozygous plant species. The physical map, consisting of 2,802 contigs, was constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approximately 9.4-fold coverage of the 485+10 Mb Populus genome, as estimated from the genome sequence assembly. BAC ends were sequenced to aid in long-range assembly of whole genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple sequence repeat (SSR)-based markers were derived from the end sequences and used to initiate integration of the BAC and genetic maps. 2,411 physical map contigs, representing 97% of all clones assigned to contigs, were aligned to the sequence assembly (JGI Populus trichocarpa v1.0). 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.

Kelleher, Colin [University of British Columbia, Vancouver; Chiu, Readman [Genome Sciences Centre, Vancouver, BC, Canada; Shin, Heesun [Genome Sciences Centre, Vancouver, BC, Canada; Bosdet, Ian [Genome Sciences Centre, Vancouver, BC, Canada; Krywinski, Martin [Genome Sciences Centre, Vancouver, BC, Canada; Fjell, Chris [Genome Sciences Centre, Vancouver, BC, Canada; Wilkin, Jennifer [University of British Columbia, Vancouver; Yin, Tongming [ORNL; DiFazio, Stephen P [ORNL; Ali, Johar [Genome Sciences Centre, Vancouver, BC, Canada; Asano, Jennifer [Genome Sciences Centre, Vancouver, BC, Canada; Chan, Susanna [Genome Sciences Centre, Vancouver, BC, Canada; Cloutier, Alison [Genome Sciences Centre, Vancouver, BC, Canada; Girn, Noreen [Genome Sciences Centre, Vancouver, BC, Canada; Leach, Stephen [Genome Sciences Centre, Vancouver, BC, Canada; Lee, Darlene [Genome Sciences Centre, Vancouver, BC, Canada; Mathewson, Carrie [Genome Sciences Centre, Vancouver, BC, Canada; Olson, Teika [Genome Sciences Centre, Vancouver, BC, Canada; O'Connor, Katie [Genome Sciences Centre, Vancouver, BC, Canada; Prabhu, Anna-Liisa [Genome Sciences Centre, Vancouver, BC, Canada; Smailus, Duane [Genome Sciences Centre, Vancouver, BC, Canada; Stott, Jeffery [Genome Sciences Centre, Vancouver, BC, Canada; Tsai, Miranda [Genome Sciences Centre, Vancouver, BC, Canada; Wye, Natasaja [Genome Sciences Centre, Vancouver, BC, Canada; Yang, George [Genome Sciences Centre, Vancouver, BC, Canada; Zhuang, Jun [Genome Sciences Centre, Vancouver, BC, Canada; Holt, Robert A. [Genome Sciences Centre, Vancouver, BC, Canada; Putnam, Nicholas [Genome Sciences Centre, Vancouver, BC, Canada; Vrebalov, Julia [Cornell University; Giovannoni, James [Cornell University; Grimwood, Jane [Stanford University; Schmutz, Jeremy [Stanford University; Rokhsar, Daniel [U.S. Department of Energy, Joint Genome Institute; Jones, Steven [Genome Sciences Centre, Vancouver, BC, Canada; Marra, Marco [Genome Sciences Centre, Vancouver, BC, Canada; Tuskan, Gerald A [ORNL; Bohlmann, J. [University of British Columbia, Vancouver; Ellis, Brian [University of British Columbia, Vancouver; Ritland, Kermit [University of British Columbia, Vancouver; Douglas, Carl [University of British Columbia, Vancouver; Schein, Jacqueline [Genome Sciences Centre, Vancouver, BC, Canada

2007-01-01

313

Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition  

PubMed Central

Background Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. Results The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. Conclusion Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking. PMID:24070093

2013-01-01

314

Library Cooperation.  

ERIC Educational Resources Information Center

College and university libraries can work together to enhance library access and share resources, but there are problems inherent in such alliances. Existing library consortia illustrate some important considerations in forging a new library consortium, including complexity of organizational processes, funding issues, technology for sharing both…

Wylie, Neil R.; Yeager, Tamara L.

1999-01-01

315

Library Skills.  

ERIC Educational Resources Information Center

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…

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

316

Library weblogs  

Microsoft Academic Search

A total of 55 weblogs maintained by libraries were identified in late 2003 using Internet search engines and directories. The weblogs were studied using content analysis techniques. Library weblogs were found in just three countries, with the majority being in the USA. Public and academic libraries were more likely to have a weblog than other types of libraries. The most

Laurel A. Clyde

2004-01-01

317

BACs-on-Beads technology: a reliable test for rapid detection of aneuploidies and microdeletions in prenatal diagnosis.  

PubMed

The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples. PMID:24795887

García-Herrero, Sandra; Campos-Galindo, Inmaculada; Martínez-Conejero, José Antonio; Serra, Vicente; Olmo, Inés; Lara, Coral; Simón, Carlos; Rubio, Carmen

2014-01-01

318

BACs-on-Beads Technology: A Reliable Test for Rapid Detection of Aneuploidies and Microdeletions in Prenatal Diagnosis  

PubMed Central

The risk of fetal aneuploidies is usually estimated based on high resolution ultrasound combined with biochemical determination of criterion in maternal blood, with invasive procedures offered to the population at risk. The purpose of this study was to investigate the effectiveness of a new rapid aneuploidy screening test on amniotic fluid (AF) or chorionic villus (CV) samples based on BACs-on-Beads (BoBs) technology and to compare the results with classical karyotyping by Giemsa banding (G-banding) of cultured cells in metaphase as the gold standard technique. The prenatal-BoBs kit was used to study aneuploidies involving chromosomes 13, 18, 21, X, and Y as well as nine microdeletion syndromes in 321 AF and 43 CV samples. G-banding of metaphase cultured cells was performed concomitantly for all prenatal samples. A microarray-based comparative genomic hybridization (aCGH) was also carried out in a subset of samples. Prenatal-BoBs results were widely confirmed by classical karyotyping. Only six karyotype findings were not identified by Prenatal-BoBs, all of them due to the known limitations of the technique. In summary, the BACs-on-Beads technology was an accurate, robust, and efficient method for the rapid diagnosis of common aneuploidies and microdeletion syndromes in prenatal samples. PMID:24795887

Martínez-Conejero, José Antonio; Serra, Vicente; Olmo, Inés; Lara, Coral; Simón, Carlos

2014-01-01

319

Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene  

Microsoft Academic Search

Spermatocytic seminomas are solid tumors found solely in the testis of predominantly elderly individuals. We investigated these tumors using a genome-wide analysis for structural and numerical chromosomal changes through conventional karyotyping, spectral karyotyping, and array comparative genomic hybridization using a 32 K genomic tiling-path resolution BAC platform (confirmed by in situ hybridization). Our panel of five spermatocytic seminomas showed a

L. H. J. Looijenga; R. Hersmus; A. J. M. Gillis; R. Pfundt; H. J. Stoop; R. J. H. L. M. van Gurp; J. Veltman; H. B. Beverloo; E. van Drunen; R. R. Pera; D. T. Schneider; B. Summersgill; J. Shipley; A. McIntyre; P. van der Spek; E. F. P. M. Schoenmakers; J. W. Oosterhuis

2006-01-01

320

The Genome Database Organism-centered listing of available genomic sequence records and projects  

E-print Network

The Genome Database Organism-centered listing of available genomic sequence records and projects http://www.ncbi.nlm.nih.gov/genome National Center for Biotechnology Information · National Library | NCBI Genome | Last Update August 19, 2013 Contact: info@ncbi.nlm.nih.gov Scope Since 2011, the Genome

Levin, Judith G.

321

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...

322

Simulation of a Video-on-Demand Song Bac Toh  

E-print Network

on a video-on-demand system with two levels of cache, RAM and disks, in front of a tape library. Using and libraries. Schools may also be interested in implementing a video-on-demand system to serve classroom materials and lectures on demand. Libraries, which must increasingly archive and provide access to non

323

Library Site Finder MAIN LIBRARY  

E-print Network

Library Site Finder MAIN LIBRARY Burlington Street Tel: 0161 275 3751 THE ALAN GILBERT LEARNING COMMONS Oxford Road Tel: 0161 306 4306 ART & ARCHAEOLOGY LIBRARY Mansfield Cooper Building Tel: 0161 275 3657 BRADDICK LIBRARY School of Physics & Astronomy Brunswick Street Tel: 0161 275 4078 EDDIE DAVIES

Sidorov, Nikita

324

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...

325

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

E-print Network

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 which­Biotechnology Center, University of Wisconsin­Madison, Madison, Wisconsin 53706, USA The accurate mapping of clones

Mishra, Bud

326

Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm  

Microsoft Academic Search

We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses

V. Kokoza; A. Ahmed; E. A. Wimmer; A. S. Raikhel

2001-01-01

327

Status and opportunities for genomics research with rainbow trout  

USGS Publications Warehouse

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.

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

328

Biocenosis of BAC(F)s used for groundwater treatment.  

PubMed

The studies on the biocenosis of biologically active carbon filters (BAC(F)) used in treatment of Miocene water revealed the presence of protozoa of the group of flagellates and ciliates (Glaucoma sp., Opercularia sp.), saprophytic bacteria, phase I and II nitrifying bacteria, Fe(II) and Mn(II) oxidizing bacteria of the family Siderocapsaceae, Leptothrix ochracea and Pedomicrobium sp., as well as microscopic fungi. The stratification of biofilter colonisation by these microorganisms was found to be similar to that occurring in biofilters applied in sewage treatment. A hypothetical model of microbiological transformations in BAC(F)s, brought about by various physiological groups of microorganisms, is presented. It is shown that ozone pre-treatment of water dosed to the biofilter reduces the number of saprophytic bacteria and moulds in its upper layer, as well as Fe(II) oxidizing bacteria of the family Siderocapsaceae across the full section of the biofilter; it does not, however, influence the abundance of nitrifying phase I and II autotrophic bacteria and Mn(II) oxidizing bacteria of the family Siderocapsaceae. The abundance of microorganisms in the biofilter outflow is increased in comparison with that in untreated water; they do not, however, create a health hazard for human beings. PMID:15026224

Grabi?ska-Loniewska, A; Perchu?, M; Korni?owicz-Kowalska, T

2004-04-01

329

piggyBac Transposon-Based Insertional Mutagenesis in Mouse Haploid Embryonic Stem Cells.  

PubMed

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

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

2015-01-01

330

BacA Is Essential for Bacteroid Development in Nodules of Galegoid, but not Phaseoloid, Legumes? †  

PubMed Central

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac71-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae, R. leguminosarum bv. phaseoli, and Rhizobium etli. The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules. PMID:20363949

Karunakaran, Ramakrishnan; Haag, Andreas F.; East, Alison K.; Ramachandran, Vinoy K.; Prell, Jurgen; James, Euan K.; Scocchi, Marco; Ferguson, Gail P.; Poole, Philip S.

2010-01-01

331

Temporal dynamics in the evolution of the sunflower genome as revealed by sequencing and annotation of three large genomic regions  

Microsoft Academic Search

Improved knowledge of genome composition, especially of its repetitive component, generates important informations in both\\u000a theoretical and applied research. In this study, we provide the first insight into the local organization of the sunflower\\u000a genome by sequencing and annotating 349,380 bp from 3 BAC clones, each including one single-copy gene. These analyses resulted\\u000a in the identification of 11 putative gene sequences,

M. Buti; T. Giordani; F. Cattonaro; R. M. Cossu; L. Pistelli; M. Vukich; M. Morgante; A. Cavallini; L. Natali

332

Physical map-assisted whole-genome shotgun sequence assemblies  

PubMed Central

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

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

333

CONSTRUCTION OF A COTTON BAC LIBRARY AND ITS APPLICATION TO GENE ISOLATION  

Technology Transfer Automated Retrieval System (TEKTRAN)

Cotton (Gossypium spp.) is a leading natural fiber crop. This crop is also an excellent source of the oil and protein that are stored in its seed. As a major crop species in the world, cotton has a potentially broad genetic base, reflected in the collections of Gossypium species. Cotton breeders wou...

334

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

PubMed Central

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

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

335

Development and characterization of a pooled Haemophilus influenzae genomic library for the evaluation of gene expression changes associated with mucosal biofilm formation in otitis media  

Microsoft Academic Search

Haemophilus influenzae is one of the most important respiratory pathogens of man. It has been etiologically associated with otitis media, otorrhea, and chronic obstructive pulmonary disease. Identification of new genomic elements will provide novel targets to fight chronic infections caused by this organism. Objective: The new paradigm that chronic infections are caused by bacterial biofilms prompted us to study the

Geza Erdos; Sameera Sayeed; Patricia Antalis; Fen Ze Hu; Jay Hayes; Joseph Goodwin; Richard Dopico; J. Christopher Post; Garth D. Ehrlich

2003-01-01

336

Post-integration stability of piggyBac in Aedes aegypti.  

PubMed

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

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

2007-09-01

337

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

PubMed Central

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

Amara, R R; Satchidanandam, V

1996-01-01

338

Preparation of PAC libraries. Final technical report  

SciTech Connect

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.

Pieter J. de Jong

1997-12-31

339

Dalhousie University Libraries Sexton Design & Technology Library  

E-print Network

1 Dalhousie University Libraries Sexton Design & Technology Library EEXXTTEERRNNAALL RREEVVIIEEWW Marilyn Berger, McGill University Library Jane Philipps, Queen's University Libraries Carolynne Presser, University of Manitoba Libraries RREEPPOORRTT IINNTTRROODDUUCCTTIIOONN Members of the Committee were asked

Brownstone, Rob

340

Qualitative and quantitative resistances to leaf rust finely mapped within two nucleotide-binding site leucine-rich repeat (NBS-LRR)-rich genomic regions of chromosome 19 in poplar.  

PubMed

• R(US) is a major dominant gene controlling quantitative resistance, inherited from Populus trichocarpa, whereas R(1) is a gene governing qualitative resistance, inherited from P. deltoides. • Here, we report a reiterative process of concomitant fine-scale genetic and physical mapping guided by the P. trichocarpa genome sequence. The high-resolution linkage maps were developed using a P. deltoides × P. trichocarpa progeny of 1415 individuals. R(US) and R(1) were mapped in a peritelomeric region of chromosome 19. Markers closely linked to R(US) were used to screen a bacterial artificial chromosome (BAC) library constructed from the P. trichocarpa parent, heterozygous at the locus R(US) . • Two local physical maps were developed, one encompassing the R(US) allele and the other spanning r(US) . The alignment of the two haplophysical maps showed structural differences between haplotypes. The genetic and physical maps were anchored to the genome sequence, revealing genome sequence misassembly. Finally, the R(US) locus was localized within a 0.8-cM interval, whereas R(1) was localized upstream of R(US) within a 1.1-cM interval. • The alignment of the genetic and physical maps with the local reorder of the chromosome 19 sequence indicated that R(US) and R(1) belonged to a genomic region rich in nucleotide-binding site leucine-rich repeat (NBS-LRR) and serine threonine kinase (STK) genes. PMID:21658182

Bresson, Aloïs; Jorge, Véronique; Dowkiw, Arnaud; Guerin, Vanina; Bourgait, Isabelle; Tuskan, Gerald A; Schmutz, Jeremy; Chalhoub, Boulos; Bastien, Catherine; Faivre Rampant, Patricia

2011-10-01

341

Libraries program  

USGS Publications Warehouse

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.

2011-01-01

342

Sequence genomic organization and expression of two channel catfish Ictalurus punctatus Ghrelin receptors  

Technology Transfer Automated Retrieval System (TEKTRAN)

Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration ...

343

Previously uncultured beta-Proteobacteria dominate in biologically active granular activated carbon (BAC) filters.  

PubMed

Bacteria colonizing BAC filters used in drinking water purification from lake water were characterized by morphology, physiological tests, whole cell protein profiles and PLFA (phospholipid fatty acid) composition, and identified by partial 16S rRNA gene sequencing. Epifluorescence revealed prothecate bacteria to dominate in BAC. The majority of the isolates belonged to order Burkholderiales of beta-Proteobacteria, a few to Comamonadaceae but the majority to an undescribed family and the related sequences belonged mainly to uncultured bacteria. Among the less common alpha-Proteobacteria the genus Sphingomonas and the genera Afipia, Bosea or Bradyrhizobium of the Bradyrhizobiaceae family were detected. The majority of cultured bacteria persisting in the BAC biofilter were Burkholderiales, which according to ecological information are efficient in the mineralisation of dissolved organic matter in BAC. The biotechnical potential of the previously uncultured dominant bacteria warrants to be further studied. PMID:19783028

Niemi, R Maarit; Heiskanen, Ilse; Heine, Riitta; Rapala, Jarkko

2009-12-01

344

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

E-print Network

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 ...

Feng, Guoping

345

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

PubMed Central

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

2013-01-01

346

America's Star Libraries: Top-Rated Libraries  

ERIC Educational Resources Information Center

"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,…

Lance, Keith Curry; Lyons, Ray

2009-01-01

347

Motor coordination deficits in Alpk1 mutant mice with the inserted piggyBac transposon  

Microsoft Academic Search

BACKGROUND: ALPK1 (?-kinase 1) is a member of an unconventional alpha-kinase family, and its biological function remains largely unknown. Here we report the phenotypic characterization of one mutant line, in which the piggyBac (PB) transposon is inserted into the Alpk1 gene. RESULTS: The piggyBac(PB) insertion site in mutants was mapped to the first intron of the Alpk1 gene, resulting in

Muyun Chen; Rener Xu

2011-01-01

348

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

PubMed

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

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

2014-11-01

349

Genomic alterations identified by array comparative genomic hybridization as prognostic markers in tamoxifen-treated estrogen receptor-positive breast cancer  

Microsoft Academic Search

BACKGROUND: A considerable proportion of estrogen receptor (ER)-positive breast cancer recurs despite tamoxifen treatment, which is a serious problem commonly encountered in clinical practice. We tried to find novel prognostic markers in this subtype of breast cancer. METHODS: We performed array comparative genomic hybridization (CGH) with 1,440 human bacterial artificial chromosome (BAC) clones to assess copy number changes in 28

Wonshik Han; Mi-Ryung Han; Jason Jongho Kang; Ji-Yeon Bae; Ji Hyun Lee; Young Ju Bae; Jeong Eon Lee; Hyuk-Jae Shin; Ki-Tae Hwang; Sung-Eun Hwang; Sung-Won Kim; Dong-Young Noh

2006-01-01

350

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

PubMed

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

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

2014-03-31

351

Mapping The Icelandic Genome  

NSDL National Science Digital Library

The Berkeley Digital Library SunSITE (see the February 9, 1996 Scout Report) has recently a new digital collection. Mapping The Icelandic Genome is an anthropological forum that examines and debates the "scientific, political, economic, religious, and ethical issues surrounding the deCode Project," a controversial project begun by a biotech company, deCode Genetics, "to produce a comprehensive genomic map of the Icelandic people."

352

Pharmacological Screening Using an FXN-EGFP Cellular Genomic Reporter Assay for the Therapy of Friedreich Ataxia  

PubMed Central

Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol. PMID:23418481

Li, Lingli; Voullaire, Lucille; Sandi, Chiranjeevi; Pook, Mark A.

2013-01-01

353

Library Resources  

Cancer.gov

Library Resources Literature Search via PubMed NOTE: PubMed replaces Entrez (previously limited to Molecular Biology references in Medline). Searches are now free, with no account required, either for Grateful Med or PubMed. National Library of Medicine NIH

354

Privatizing Libraries  

ERIC Educational Resources Information Center

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…

Jerrard, Jane; Bolt, Nancy; Strege, Karen

2012-01-01

355

Engineering large viral DNA genomes using the CRISPR-Cas9 system.  

PubMed

Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus-infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time-consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152?kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat-Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene-ablated HSV but also gene knock-in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein-Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates. PMID:25040500

Suenaga, Tadahiro; Kohyama, Masako; Hirayasu, Kouyuki; Arase, Hisashi

2014-09-01

356

Cancer Genome Anatomy Project  

NSDL National Science Digital Library

The National Cancer Institute has launched the Cancer Genome Anatomy Project to "achieve a comprehensive molecular characterization of normal, precancerous, and malignant cells." Sequenced genes are held as library entries in a database and are available for downloading (fasta format). Each cDNA library entry may include biological source, number of sequences, and library construction detail information. Thousands of gene sequences are available for over 15 cancers, including breast, colon, and prostrate. Contact information for donating or obtaining tissue samples for research purposes is provided.

1997-01-01

357

Dean of Libraries/University Director Library  

E-print Network

Dean of Libraries/University Librarian Director Library Information Systems Director Communications Senior Director Assistant Dean Development Syracuse University Libraries Functional Organizational Chart Librarians and Professionals April 2013 4/25/2013 Syracuse University Libraries Org Chart 1 #12;

McConnell, Terry

358

Stereochemical Outcome at Four Stereogenic Centers During Conversion of Prephenate to Tetrahydrotyrosine by BacABGF in the Bacilysin Pathway†  

PubMed Central

The first four enzymes of the bacilysin antibiotic pathway, BacABGF, convert prephenate to a tetrahydrotyrosine (H4Tyr) diastereomer on the way to the anticapsin warhead of the dipeptide antibiotic. BacB takes the BacA product endocyclic-?4,?8-7R-dihydrohydroxyphenylpyruvate (en-H2HPP) and generates a mixture of 3E- and 3Z-olefins of the exocyclic-?3,?5-dihydrohydroxyphenylpyruvate (ex-H2HPP). The NADH-utilizing BacG then catalyzes a conjugate reduction, adding a pro-S hydride equivalent to C4 to yield tetrahydrohydroxyphenylpyruvate (H4HPP), a transamination away (via BacF) from 2S-H4Tyr. Incubations of the pathway enzymes in D2O yield deuterium incorporation at C8 from BacA and then C9 from BacB action. By 1H-NMR analysis on samples of H4Tyr, the stereochemistry at C4, C8 and C9 can be assigned. BacG (followed by BacF) converts 3E-ex-H2HPP to 2S, 4R, 7R-H4Tyr. The 3Z isomer is instead reduced and transaminated to the opposite diastereomer at C4: 2S, 4S, 7R-H4Tyr. Given that bacilysin has the 2S, 4S stereochemistry in its anticapsin moiety, it is likely that the 2S, 4S-H4Tyr is the diastereomer “on pathway”. NMR determination of the stereochemistry of the CHD samples at C8 and C9 allow assignment of all stereogenic centers (except C3) in this unusual tetrahydro- aromatic amino acid building block, giving insights and constraints on the BacA, BacB, and BacG mechanisms. PMID:22765234

Parker, Jared B.; Walsh, Christopher T.

2012-01-01

359

Microcolinearity in sh2Homologous Regions of the Maize, Rice, and Sorghum Genomes  

Microsoft Academic Search

Large regions of genomic colinearity have been demonstrated among grass species by recombinational mapping, but the degree of chromosomal conservation at the sub-centimorgan level has not been extensively investigated. We cloned the rice and sorghum genes homologous to the sh2 locus of maize on bacterial artificial chromosomes (BACs), and observed that a homologue of the maize a1 gene was also

M. Chen; P. Sanmiguel; A. C. de Oliveira; S.-S. Woo; H. Zhang; R. A. Wing; J. L. Bennetzen

1997-01-01

360

Complete Genome Sequence of Border Disease Virus Genotype 3 Strain Gifhorn  

PubMed Central

The complete genome sequence of the genotype 3 border disease virus strain Gifhorn has been determined; this strain was originally isolated from pigs. This represents the consensus sequence for the virus used to produce the bacterial artificial chromosome (BAC) cDNA clone pBeloGif3, which yields a virus that is severely attenuated in cell culture. PMID:24435861

Fahnøe, Ulrik; Höper, Dirk; Schirrmeier, Horst; Beer, Martin

2014-01-01

361

Tomato Genome Sequencing beyond Heinz 1706 and Update on SOL 100  

E-print Network

Tomato Genome Sequencing beyond Heinz 1706 and Update on SOL 100 #12;Overview ¡Update on Heinz 1706 sequence ¡Other tomato genotypes being (re)sequenced ¡Groups involved in the work ¡Number of different Thompson Institute, University of Oklahoma, and Colorado State University ¡ 1000 BACs sequenced post tomato

Pawlowski, Wojtek

362

Engineering Library Guidance  

E-print Network

Science & Engineering Library Library Guidance April 2014 1 #12;Contents 1 Overview of Waseda University Library 2 Science & Engineering Library and the Student Reading Room 3 WINE: Waseda's Library Catalog3 WINE: Waseda's Library Catalog 4 Library Services 5 Notes 2 #12;How many libraries

Kaji, Hajime

363

Genomic Organization and Evolution of the Vomeronasal Type 2 Receptor-Like (OlfC) Gene Clusters in Atlantic Salmon, Salmo salar  

PubMed Central

There are three major multigene superfamilies of olfactory receptors (OR, V1R, and V2R) in mammals. The ORs are expressed in the main olfactory organ, whereas the V1Rs and V2Rs are located in the vomeronasal organ. Fish only possess one olfactory organ in each nasal cavity, the olfactory rosette; therefore, it has been proposed that their V2R-like genes be classified as olfactory C family G protein-coupled receptors (OlfC). There are large variations in the sizes of OR gene repertoires. Previous studies have shown that fish have between 12 and 46 functional V2R-like genes, whereas humans have lost all functional V2Rs, and frog sp. have more than 240. Pseudogenization of V2R genes is a prevalent event across species. In the mouse and frog genomes, there are approximately double the number of pseudogenes compared with functional genes. An oligonucleotide probe was designed from a conserved sequence from four Atlantic salmon OlfC genes and used to screen the Atlantic salmon bacterial artificial chromosome (BAC) library. Hybridization-positive BACs were matched to fingerprint contigs, and representative BACs were shotgun cloned and sequenced. We identified 55 OlfC genes. Twenty-nine of the OlfC genes are classified as putatively functional genes and 26 as pseudogenes. The OlfC genes are found in two genomic clusters on chromosomes 9 and 20. Phylogenetic analysis revealed that the OlfC genes could be divided into 10 subfamilies, with nine of these subfamilies corresponding to subfamilies found in other teleosts and one being salmon specific. There is also a large expansion in the number of OlfC genes in one subfamily in Atlantic salmon. Subfamily gene expansions have been identified in other teleosts, and these differences in gene number reflect species-specific evolutionary requirements for olfaction. Total RNA was isolated from the olfactory epithelium and other tissues from a presmolt to examine the expression of the odorant genes. Several of the putative OlfC genes that we identified are expressed only in the olfactory epithelium, consistent with these genes encoding odorant receptors. PMID:19221009

Johnstone, Kimberley A.; Ciborowski, Kate L.; Lubieniecki, Krzysztof P.; Chow, William; Phillips, Ruth B.; Koop, Ben F.; Jordan, William C.

2009-01-01

364

INTEGRATED PLANNING: UNIVERSITY LIBRARY your library  

E-print Network

INTEGRATED PLANNING: UNIVERSITY LIBRARY your library engage, enlighten, explore at library.usask.ca Transforming Library Services, Collections and Facilities: The University Library People Plan SUMMARY VERSION OVERVIEW Central themes in the library strategic plan highlight the critical importance which our people

Peak, Derek

365

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

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

Saxena, Swati; Singh, Archana; Archak, Sunil; Behera, Tushar K; John, Joseph K; Meshram, Sudhir U; Gaikwad, Ambika B

2015-01-01

366

Digital Libraries.  

ERIC Educational Resources Information Center

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;…

Fox, Edward A.; Urs, Shalini R.

2002-01-01

367

Telecommunications Library  

NSDL National Science Digital Library

The Telecommunications Library at WilTel, includes resources such as the Long Distance Digest, Telecom Digest, Telecom Glossary, the working papers from The Research Institute for Telecommunications and Information Marketing, among others.

368

Library Juice  

NSDL National Science Digital Library

Library Juice is a current awareness newsletter for librarians and other information professionals, published weekly by editor-librarian Rory Litwin. Each issue of Library Juice compiles recent news, articles, and announcements gleaned from a wide range of mailing lists related to librarianship, information science, intellectual freedom, and social responsibility. The Website posts the current issue of the newsletter and maintains an archive of all past issues.

369

Library of libraries: A novel approach in synthetic combinatorial libraries  

E-print Network

Library of libraries: A novel approach in synthetic combinatorial libraries Nikolai F. Sepetov peptide libraries for pharmacological purposes is not necessarily to find the most active peptide can be used subsequently for developing a drug. Therefore a library of motifs can give as much or more

Lam, Kit S.

370

Controlled Clinical Comparison of BacT/Alert FA Plus and FN Plus Blood Culture Media with BacT/Alert FA and FN Blood Culture Media  

PubMed Central

New blood culture media containing antibiotic-binding polymeric beads have been developed for the BacT/Alert (bioMérieux, Inc., Durham, NC) blood culture system. To assess the performance of these new media, we compared the new BacT/Alert aerobic medium (FA Plus) with resins to BacT/Alert FA medium with activated charcoal and the new BacT/Alert anaerobic medium (FN Plus) to BacT/Alert FN medium at 3 tertiary care medical centers. Study bottle pairs were inoculated with a target volume of 6 to 10 ml of blood from adults and incubated in the BacT/Alert 3D blood culture instrument. In the FA Plus versus FA comparison, there were 1,507 study pairs. Among 170 isolates causing true bloodstream infections (BSIs), significantly more Staphylococcus aureus (P < 0.001) and total microorganisms (P < 0.01) grew in the FA Plus bottle than in the FA bottle. Fewer coagulase-negative staphylococcal (CoNS) contaminants grew in the FA Plus bottle than in the FA bottle (10 versus 22; P = 0.05). In addition, growth was detected earlier in the FA Plus bottle than in the FA bottle (P < 0.001). In the FN Plus versus FN comparison, there were 2,386 study pairs. Among 201 isolates causing true BSIs, significantly more S. aureus (P < 0.001), CoNS (P < 0.005), and total microorganisms (P < 0.001) grew in the FN Plus bottle than in the FN bottle. Also, significantly more CoNS contaminants grew in the FN Plus bottle than in the FN bottle (P < 0.001). Overall, microorganisms were detected earlier in the FN Plus than in the FN bottle (P < 0.001). Medical technologists at all 3 study sites preferred the new media for Gram stain interpretation. We conclude that the FA Plus and FN Plus media provide improved and earlier detection of microorganisms compared with the FA and FN media and are preferable for Gram stain interpretation as well. PMID:24371240

Mirrett, S.; Reller, L. B.; Weinstein, M. P.

2014-01-01

371

Controlled clinical comparison of BacT/alert FA plus and FN plus blood culture media with BacT/alert FA and FN blood culture media.  

PubMed

New blood culture media containing antibiotic-binding polymeric beads have been developed for the BacT/Alert (bioMérieux, Inc., Durham, NC) blood culture system. To assess the performance of these new media, we compared the new BacT/Alert aerobic medium (FA Plus) with resins to BacT/Alert FA medium with activated charcoal and the new BacT/Alert anaerobic medium (FN Plus) to BacT/Alert FN medium at 3 tertiary care medical centers. Study bottle pairs were inoculated with a target volume of 6 to 10 ml of blood from adults and incubated in the BacT/Alert 3D blood culture instrument. In the FA Plus versus FA comparison, there were 1,507 study pairs. Among 170 isolates causing true bloodstream infections (BSIs), significantly more Staphylococcus aureus (P<0.001) and total microorganisms (P<0.01) grew in the FA Plus bottle than in the FA bottle. Fewer coagulase-negative staphylococcal (CoNS) contaminants grew in the FA Plus bottle than in the FA bottle (10 versus 22; P=0.05). In addition, growth was detected earlier in the FA Plus bottle than in the FA bottle (P<0.001). In the FN Plus versus FN comparison, there were 2,386 study pairs. Among 201 isolates causing true BSIs, significantly more S. aureus (P<0.001), CoNS (P<0.005), and total microorganisms (P<0.001) grew in the FN Plus bottle than in the FN bottle. Also, significantly more CoNS contaminants grew in the FN Plus bottle than in the FN bottle (P<0.001). Overall, microorganisms were detected earlier in the FN Plus than in the FN bottle (P<0.001). Medical technologists at all 3 study sites preferred the new media for Gram stain interpretation. We conclude that the FA Plus and FN Plus media provide improved and earlier detection of microorganisms compared with the FA and FN media and are preferable for Gram stain interpretation as well. PMID:24371240

Kirn, T J; Mirrett, S; Reller, L B; Weinstein, M P

2014-03-01

372

BAC to immunology – bacterial artificial chromosome-mediated transgenesis for targeting of immune cells  

PubMed Central

Thirty years after the first transgenic mouse was produced, a plethora of genetic tools has been developed to study immune cells in vivo. A powerful development is the bacterial artificial chromosome (BAC) transgenic approach, combining advantages of both conventional transgenic and knock-in gene-targeting strategies. In immunology the potential of BAC transgenic technology has yet to be fully harvested and, combined with a variety of elegant genetic tools, it will allow the analysis of complex immunological processes in vivo. In this short review, we discuss the applications of BACs in immunology, such as identification of regulatory regions, expression and cell-fate mapping, cell ablation, conditional mutations and the generation of humanized mice. PMID:17437533

Sparwasser, Tim; Eberl, Gérard

2007-01-01

373

Absence of superconductivity down to 80 mK in graphite intercalated BaC 6  

NASA Astrophysics Data System (ADS)

Following recent theoretical and experimental investigations of superconductivity in the graphite intercalated compounds CaC 6 and SrC 6, we report on a very low temperature magnetisation study on BaC 6 down to 80 mK. The data show no trace of superconductivity even at fields as low as 0.7 Oe. Using a McMillan parametrisation of the BCS parameters, we conclude that the Coulomb pseudopotential is expected to be as large as 0.19 in both BaC 6 and SrC 6, i.e. 40% larger than in CaC 6. As an alternative scenario, we argue that extrinsic effects such as intercalant disorder may depress superconductivity in both BaC 6 and SrC 6, as in the case of CaC 6.

Nakamae, S.; Gauzzi, A.; Ladieu, F.; L'Hôte, D.; Eméry, N.; Hérold, C.; Marêché, J. F.; Lagrange, P.; Loupias, G.

2008-03-01

374

America's Star Libraries  

ERIC Educational Resources Information Center

"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…

Lyons, Ray; Lance, Keith Curry

2009-01-01

375

TiO2/UV/O3-BAC processes for removing refractory and hazardous pollutants in raw water.  

PubMed

TiO2/UV/O3-BAC (biological activated carbon) process was employed to treat raw water and compared to UV/O3-BAC process in its optimum parameters (3 mg/L ozone dosage with 15 min oxidation time and 15 min empty bed contact time in BAC). The results showed that the presence of TiO2 improved ozone utilization and biodegradability of the effluent. For the dissolved organic carbon (DOC) removal, TiO2/UV/O3-BAC was more efficient than UV/O3-BAC and its synergetic effect is more than that in UV/O(3)-BAC process. It was showed that small molecules with MW<3000 Da predominated in the raw water accounting for more than 56% DOC, they were increased after oxidation, accounting for more than 64% DOC. GC/MS analysis showed that TiO2/UV/O3-BAC process was effective in removing phthalate esters (PAEs) and persistent organic pollutants (POPs). PAEs' removal ratio reached more than 94% and reduced with the increase of the length of the alkyl side chains and the alkyl branch chains. TiO2/UV/O3-BAC process was also very effective in removing POPs. Polybromobiphenyls' removal rate reached more than 89% and decreased with the increase of substitutional bromines except for 2,2',5,5'-tetrabromobiphenyl, which can be completely removed. PMID:16257116

Li, Laisheng; Zhu, Wanpeng; Zhang, Pengyi; Zhang, Qiuyun; Zhang, Zulin

2006-02-01

376

Estimating Driver Risk Using Alcohol Biomarkers, Interlock BAC Tests and Psychometric Assessments: Initial Descriptives  

PubMed Central

Aim To identify alcohol biomarker and psychometric measures that relate to drivers’ blood alcohol concentration (BAC) patterns from ignition interlock devices (IIDs). Design, Setting, Participants, Measurements In Alberta, Canada, 534 drivers, convicted of driving under the influence of alcohol (DUI), installed IIDs and agreed to participate in a research study. IID BAC tests are an established proxy for predicting future DUI convictions. Three risk groups were defined by rates of failed BAC tests. Program entry and followup blood samples (n=302, 171) were used to measure phosphatidyl ethanol (PETH), carbohydrate deficient transferrin (%CDT), gamma glutamyltransferase (GGT) and other biomarkers. Program entry urine (n=130) was analyzed for ethyl glucuronide (ETG) and ethyl sulfate (ETS). Entry hair samples were tested for fatty acid ethyl esters (FAEE) (n=92) and ETG (n=146). Psychometric measures included the DSM-4 Diagnostic Interview Schedule Alcohol Module, Alcohol Use Disorders Identification Test (AUDIT), the Timeline Followback (TLFB), the Drinker Inventory of Consequences (DRINC), and the Temptation and Restraint Inventory (TRI). Findings Except for FAEE, all alcohol biomarkers were significantly related to the interlock BAC test profiles; higher marker levels predicted higher rates of interlock BAC test failures. PETH, the strongest with an overall ANOVA F ratio of 35.5, had significant correlations with all nine of the other alcohol biomarkers and with 16 of 19 psychometric variables. Urine ETG and ETS were strongly correlated with the IID BAC tests. Conclusions The findings suggest several alcohol biomarkers and assessments could play an important role in the prediction and control of driver alcohol risk when relicensing. PMID:19922520

Marques, Paul; Tippetts, Scott; Allen, John; Javors, Martin; Alling, Christer; Yegles, Michel; Pragst, Fritz; Wurst, Friedrich

2009-01-01

377

LIBRARY REGULATIONS 1. THE LIBRARY POLICY COMMITTEE  

E-print Network

1 LIBRARY REGULATIONS 1. THE LIBRARY POLICY COMMITTEE 1.1 The Library Policy Committee shall shall be: [i] to formulate proposals in regard to policy matters relating to the Library for consideration by the Academic Council and the Governing Body; [ii] to monitor the implementation of Library

Schellekens, Michel P.

378

Library Locations Locations other than Main Library  

E-print Network

Library Locations Locations other than Main Library Example: Feminist Studies HQ1410 .U54 2009 University of California, Santa Barbara Library www.library.ucsb.edu Updated 3-2014 A - B.......................................6 Central M - N..................................................Arts Library (Music Building) P

379

Library Locations Locations other than Main Library  

E-print Network

Library Locations Locations other than Main Library Example: Feminist Studies HQ1410 .U54 2009 Andelson Collection: 2 South, Ethnic & Gender Studies Library (EGSL) Annex: Off campus storage. See www.library.ucsb.edu/depts/access/annex.html Arts Library: 1st Floor, Music Building Asian American Studies: 2 South, Ethnic & Gender Studies

380

UNBC Library External Review 2012 Library Response  

E-print Network

UNBC Library External Review 2012 Library Response November 2, 2012 An external review was conducted of the UNBC Library services in April 2012 by Dr. Vicki Williamson of University of Saskatchewan is available at https://library.unbc.ca/external- review/ . Below is the Library's planned followup

Northern British Columbia, University of

381

Library Annual Report Library Annual Report  

E-print Network

Library Annual Report 2007 Library Annual Report 2007 #12;www.library.uwa.edu.au Our mission: By delivering excellent information resources and services the Library is integral to the University's mission of advancing, transmitting and sustaining knowledge. Our vision: The Library will continue to be at the heart

Tobar, Michael

382

DNA Libraries  

NSDL National Science Digital Library

Teachers' Domain presents this interactive, adapted from the Dolan DNA Learning Center, to help students learn about DNA Libraries, "the tools scientists use to store and reproduce genetic information that they can later access for their research." After an introduction, students can explore more about the different types of DNA libraries by clicking on any of the five DNA vectors: Yeast Artificial Chromosomes, Bacterial Artificial Chromosomes, Cosmids, Bacteriophage Lambda, and Plasmids. On the site, visitors will also find a supplemental background essay, discussion questions, and standards alignment from Teachers' Domain.

383

SFU LIBRARY ANNUAL REPORT  

E-print Network

SFU LIBRARY ANNUAL REPORT 2006/07 #12;22 TABLE OF CONTENTS Message from the University Librarian................................................... ....................................... 7 WAC Bennett Library.................................................................. ....................................... 8 Samuel and Frances Belzberg Library

384

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

PubMed Central

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

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

2013-01-01

385

Public Libraries  

ERIC Educational Resources Information Center

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

Library Journal, 1972

1972-01-01

386

Underground Libraries.  

ERIC Educational Resources Information Center

Discussion of underground buildings constructed primarily during last two decades for various reasons (energy conservation, density of environment, preservation of landscape and historic buildings) notes advantages, disadvantages, and psychological and design considerations. Examples of underground libraries, built mainly in United States, are…

Fuhlrott, Rolf

1986-01-01

387

LOCATIONS OF LIBRARY MATERIALS Syracuse University Libraries include Bird Library, Carnegie Library, and the Geology Library in Heroy Geology  

E-print Network

LOCATIONS OF LIBRARY MATERIALS Syracuse University Libraries include Bird Library, Carnegie Library, and the Geology Library in Heroy Geology Laboratory. Our catalog also includes material housed in the separately administered Law Library in White Hall and the Martin Luther King Jr. Memorial Library in the Department

McConnell, Terry

388

BACs-on-Beads™ (BoBs™) assay for the genetic evaluation of prenatal samples and products of conception.  

PubMed

BACs-on-Beads™ (BoBs™) is a new emerging technology, a modification of comparative genomic hybridization that can be used to detect DNA copy number gains and losses. Here, we describe the application of two different types of BoBs™ assays: (1) Prenatal BoBs (CE-IVD) to detect the most frequent syndromes associated with chromosome microdeletions, as well as the trisomy 13, 18 and 21, and (2) KaryoLite BoBs (RUO) which can detect aneuploidy in all chromosomes by quantifying proximal and terminal regions of each chromosomal arm. The interpretation of the results by BoBsoft™ software is also described. Although BoBs™ may not have the breadth and scope to replace chromosomal microarrays (array comparative genomic hybridization and single nucleotide polymorphism array) in the prenatal setting, particularly when a fetal anomaly has been detected, it is a well suited alternative for FISH or QF-PCR because BoBs™ is comparable, if not superior in terms of cost, turnaround time (TAT) and throughput and accuracy. BoBs™ also has the ability to detect significant fetal mosaicism (?30% with Prenatal BoBs and ?50% with KaryoLite BoBs). However, perhaps the greatest strength of this new technology is the fact that unlike FISH or QF-PCR, it has the ability to detect common microdeletion syndromes or additional aneuploidies, both of which may be easily missed despite excellent prenatal sonography. Thus, when BoBs™ is applied in the correct clinical setting and run and analyzed in appropriate laboratories this technique can improve and augment best practices with a personalization of prenatal care. PMID:25239751

Grati, Francesca Romana; Vialard, François; Gross, Susan

2015-01-01

389

Sequence-Based Analysis of Structural Organization and Composition of the Cultivated Sunflower (Helianthus annuus L.) Genome.  

PubMed

Sunflower is an important oilseed crop, as well as a model system for evolutionary studies, but its 3.6 gigabase genome has proven difficult to assemble, in part because of the high repeat content of its genome. Here we report on the sequencing, assembly, and analyses of 96 randomly chosen BACs from sunflower to provide additional information on the repeat content of the sunflower genome, assess how repetitive elements in the sunflower genome are organized relative to genes, and compare the genomic distribution of these repeats to that found in other food crops and model species. We also examine the expression of transposable element-related transcripts in EST databases for sunflower to determine the representation of repeats in the transcriptome and to measure their transcriptional activity. Our data confirm previous reports in suggesting that the sunflower genome is >78% repetitive. Sunflower repeats share very little similarity to other plant repeats such as those of Arabidopsis, rice, maize and wheat; overall 28% of repeats are "novel" to sunflower. The repetitive sequences appear to be randomly distributed within the sequenced BACs. Assuming the 96 BACs are representative of the genome as a whole, then approximately 5.2% of the sunflower genome comprises non TE-related genic sequence, with an average gene density of 18kbp/gene. Expression levels of these transposable elements indicate tissue specificity and differential expression in vegetative and reproductive tissues, suggesting that expressed TEs might contribute to sunflower development. The assembled BACs will also be useful for assessing the quality of several different draft assemblies of the sunflower genome and for annotating the reference sequence. PMID:24833511

Gill, Navdeep; Buti, Matteo; Kane, Nolan; Bellec, Arnaud; Helmstetter, Nicolas; Berges, Hélène; Rieseberg, Loren H

2014-01-01

390

PiggyBac Mediated Multiplex Gene Transfer in Mouse Embryonic Stem Cell  

PubMed Central

PiggyBac system has been shown to have a high efficiency to mediate gene transfer. However, there are no reports on its efficiency to mediate multiplex transgenes in mouse embryonic stem cells. Here we first established an immortalized feeder cell line by introducing four antibiotic resistance genes simultaneously into the original SNL 76/7 feeder cell line utilizing the PiggyBac system. This is the feeder cell line with the most diverse types of antibiotic resistance genes reported so far, which will enable researchers to perform simultaneous multiplex gene transfer or gene targeting experiments in ES cells. With such feeder cell line, we were able to quantitatively characterize the transposition efficiency of PiggyBac system in mouse ES cells using five transposons carrying different inducible fluorescence proteins and antibiotic resistance genes, and the efficiency ranged from about 2% for one transposon to 0.5% for five transposons. The highly efficient multiplex gene transfer mediated by PiggyBac will no doubt provide researchers with more choices in biomedical research and development. PMID:25517991

Lu, Xibin; Huang, Wei

2014-01-01

391

Functional Complementation and Genetic Deletion Studies of KirBac Channels  

E-print Network

of Medicine, St. Louis, Missouri 63110 The superfamily of prokaryotic inwardly rectifying (KirBac) potassium) potassium channels are regulated by many different cellular factors such as G-proteins, phospha to a wide range of metabolic and physiological stimuli (1, 2). Their importance is illustrated by the fact

Tucker, Stephen J.

392

The Garden of Ulysses: Ferdinand Bac, modernism and the afterlife of myth  

Microsoft Academic Search

Ferdinand Bac's three Mediterranean gardens constructed between 1913 and 1925 are more than a chapter in the history of horticultural design. They are the product of an existential conflict that reflects one of the central cultural dilemmas of the age for designers of all sorts as well as for writers: the problem of epigonism. How was one to create in

Lawrence Joseph

2000-01-01

393

Evaluation of the effects of North Carolina's 0.08% BAC law  

Microsoft Academic Search

This study was conducted to determine whether the lowered BAC limit for drivers in North Carolina resulted in fewer alcohol-related motor vehicle crashes. We used time-series analysis to examine several indicators of alcohol involvement in both injury and fatal crashes between 1991 and 1996. Data from NC crash files as well as the Fatality Analysis Reporting System (FARS) are used.

Robert D. Foss; J. Richard Stewart; Donald W. Reinfurt

2001-01-01

394

BacSim, a simulator for individual-based modelling of bacterial colony growth  

Microsoft Academic Search

The generic, quantitative, spatially explicit, individual-based model BacSim was developed to simulate growth and behaviour of bacteria. The potential of this approach is in relating the properties of microscopic entities - cells - to the properties of macroscopic, complex systems such as biofilms. Here, the growth of a single Escherichia coli cell into a colony was studied. The object-oriented program

Jan-Ulrich Kreft; Ginger Booth; Julian W. T. Wimpenny

1998-01-01

395

Symposium on Presidential Libraries.  

ERIC Educational Resources Information Center

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)

Relyea, Harold C.; And Others

1994-01-01

396

3 Library Regulations Definitions  

E-print Network

3 Library Regulations Definitions In Regulation 3: 'Library' means the University Library as defined in Regulation 3.1; 'Library staff' means the staff of the University Library; 'Librarian' means the University Librarian and Head of Information Resources Directorate or nominee; `Library Committee' means

Mottram, Nigel

397

Sequencing complete mitochondrial and plastid genomes.  

PubMed

Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The "random genome sequencing" protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The "long-PCR-based genome sequencing" protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week. PMID:17406621

Burger, Gertraud; Lavrov, Dennis V; Forget, Lise; Lang, B Franz

2007-01-01

398

Microbe Library  

NSDL National Science Digital Library

The Microbe Library is a searchable microbiology portal provided by the American Society for Microbiology (ASM). The portal contains visual images and animations; curriculum activities for both classroom and laboratory; an articles section containing the tri-annual educational newsletter and annual educational journal, as well as feature articles from ASM News; and educational reviews and resources. The Microbe Library is linked directly to a recommended core curriculum for introductory microbiology education. Visual resources include access to over 300 images, animations, and videos. The posted classroom activities and laboratory exercises were developed by faculty at diverse institutions to improve instruction. They include inquiry-based field-tested materials, student-active learning activities, and ideas for projects or research approaches.

399

Creation and characterization of BAC-transgenic mice with physiological overexpression of epitope-tagged RCAN1 (DSCR1).  

PubMed

The chromosome 21 gene RCAN1, encoding a modulator of the calcineurin (CaN) phosphatase, is a candidate gene for contributing to cognitive disability in people with Down syndrome (DS; trisomy 21). To develop a physiologically relevant model for studying the biochemistry of RCAN1 and its contribution to DS, we generated bacterial artificial chromosome-transgenic (BAC-Tg) mouse lines containing the human RCAN1 gene with a C-terminal HA-FLAG epitope tag incorporated by recombineering. The BAC-Tg was expressed at levels only moderately higher than the native Rcan1 gene: approximately 1.5-fold in RCAN1 (BAC-Tg1) and twofold in RCAN1 (BAC-Tg2). Affinity purification of the RCAN1 protein complex from brains of these mice revealed a core complex of RCAN1 with CaN, glycogen synthase kinase 3-beta (Gsk3b), and calmodulin, with substoichiometric components, including LOC73419. The BAC-Tg mice are fully viable, but long-term synaptic potentiation is impaired in proportion to BAC-Tg dosage in hippocampal brain slices from these mice. RCAN1 can act as a tumor suppressor in some systems, but we found that the RCAN1 BAC-Tg did not reduce mammary cancer growth when present at a low copy number in Tp53;WAP-Cre mice. This work establishes a useful mouse model for investigating the biochemistry and dose-dependent functions of the RCAN1 protein in vivo. PMID:23096997

Xing, Luzhou; Salas, Martha; Zhang, Hong; Gittler, Julia; Ludwig, Thomas; Lin, Chyuan-Sheng; Murty, Vundavalli V; Silverman, Wayne; Arancio, Ottavio; Tycko, Benjamin

2013-02-01

400

Investigation of decolorization of textile wastewater in an anaerobic/aerobic biological activated carbon system (A/A BAC).  

PubMed

The aim of this study is to investigate the decolorization in anaerobic/aerobic biological activated carbon (A/A BAC) system. The experiment was divided into 2 stages; stage I is batch test for preliminary study of dye removal equilibrium time. The preliminary experiment (stage I) provided the optimal data for experimental design of A/A BAC system in SBR (stage II). Stage II is A/A BAC system imitated Sequencing Batch Reactor (SBR) which consist of 5 main periods; fill, react, settle, draw and idle. React period include anaerobic phase followed by aerobic phase. The BAC main media; Granular Activated Carbon (GAC), Mixed Cultures (MC) and Biological Activated Carbon (BAC) were used for dye and organic substances removal in three different solutions; Desizing Agent Solution (DAS), dye Solution (DS) and Synthetic Textile Wastewater (STW). Results indicate that GAC adsorption plays role in dye removal followed by BAC and MC activities, respectively. In the presence desizing agent, decolorization by MC was improved because desizing agent acts as co-substrates for microorganisms. It was found that 50% of dye removal efficiency was achieved in Fill period by MC. GC/MS analysis was used to identify dye intermediate from decolorization. Dye intermediate containing amine group was found in the solution and on BAC surfaces. The results demonstrated that combination of MC and BAC in the system promotes decolorization and dye intermediate removal. In order to improve dye removal efficiency in an A/A BAC system, replacement of virgin GAC, sufficient co-substrates supply and the appropriate anaerobic: aerobic period should be considered. PMID:20836286

Pasukphun, N; Vinitnantharat, S; Gheewala, S

2010-04-01

401

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

SciTech Connect

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.

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

2008-05-22

402

Regulation 28: Library REGULATION 28: LIBRARY  

E-print Network

Regulation 28: Library 180 REGULATION 28: LIBRARY The purpose of this Regulation is to safeguard the common interests of all Library users. All persons are admitted on the understanding that they have read and agreed to observe the Library Regulations. Breach of this Regulation could result in membership being

Sussex, University of

403

The Library UBC LIBRARY CARD APPLICATION FORM  

E-print Network

The Library UBC LIBRARY CARD APPLICATION FORM For Faculty Authorized Users UBC FACULTY MEMBER to the following person so that s/he may borrow Library materials and access services in my name for my UBC September 15th , 2014 Faculty member's statement: I understand that this is a separate library card from my

Michelson, David G.

404

Bacteriocin Protein BacL1 of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan.  

PubMed

Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins, BacL1 and BacA. We previously reported that BacL1 protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan d-isoglutamyl-l-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, the target of BacL1 remains unknown. In this study, we investigated the targeting specificity of BacL1. Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL1 specifically localized at the cell division-associated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells. This specific targeting was dependent on the triple repeat of the SH3 domain located in the region from amino acid 329 to 590 of BacL1. Repression of cell growth due to the stationary state of the growth phase or to treatment with bacteriostatic antibiotics rescued bacteria from the bacteriolytic activity of BacL1 and BacA. The static growth state also abolished the binding and targeting of BacL1 to the cell division-associated site. Furthermore, the targeting of BacL1 was detectable among Gram-positive bacteria with an l-Ala-l-Ala-cross-bridging peptidoglycan, including E. faecalis, Streptococcus pyogenes, or Streptococcus pneumoniae, but not among bacteria with alternate peptidoglycan structures, such as Enterococcus faecium, Enterococcus hirae, Staphylococcus aureus, or Listeria monocytogenes. These data suggest that BacL1 specifically targets the l-Ala-l-Ala-cross-bridged peptidoglycan and potentially lyses the E. faecalis cells during cell division. PMID:25368300

Kurushima, Jun; Nakane, Daisuke; Nishizaka, Takayuki; Tomita, Haruyoshi

2015-01-15

405

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.

406

Diagnosis and Prognostication of Ductal Adenocarcinomas of the Pancreas Based on Genome-Wide DNA Methylation Profiling by Bacterial Artificial Chromosome Array-Based Methylated CpG Island Amplification  

PubMed Central

To establish diagnostic criteria for ductal adenocarcinomas of the pancreas (PCs), bacterial artificial chromosome (BAC) array-based methylated CpG island amplification was performed using 139 tissue samples. Twelve BAC clones, for which DNA methylation status was able to discriminate cancerous tissue (T) from noncancerous pancreatic tissue in the learning cohort with a specificity of 100%, were identified. Using criteria that combined the 12 BAC clones, T-samples were diagnosed as cancers with 100% sensitivity and specificity in both the learning and validation cohorts. DNA methylation status on 11 of the BAC clones, which was able to discriminate patients showing early relapse from those with no relapse in the learning cohort with 100% specificity, was correlated with the recurrence-free and overall survival rates in the validation cohort and was an independent prognostic factor by multivariate analysis. Genome-wide DNA methylation profiling may provide optimal diagnostic markers and prognostic indicators for patients with PCs. PMID:21197409

Gotoh, Masahiro; Arai, Eri; Wakai-Ushijima, Saori; Hiraoka, Nobuyoshi; Kosuge, Tomoo; Hosoda, Fumie; Shibata, Tatsuhiro; Kondo, Tadashi; Yokoi, Sana; Imoto, Issei; Inazawa, Johji; Kanai, Yae

2011-01-01

407

Cell Libraries  

NASA Technical Reports Server (NTRS)

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.

1994-01-01

408

Leabharlann UCD UCD Library  

E-print Network

Leabharlann UCD UCD Library Research Data Management UCD Library is organising a mini at lunchtimes in the Library Link, James Joyce Library (Level 1). Data Caring: Why Manage Your Research Data? Presented by Julia Barrett, Research Services Manager, UCD Library Wednesday, 12 February, 2014 13.00 ­ 14

409

Helpful Websites Ebling Library  

E-print Network

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 Help with NIH Public Access Policy Compliance? · nihpolicy@library.wisc.edu · Trisha Adamus, Ebling

Bohnhoff, David

410

University Libraries Annual Report  

E-print Network

University Libraries 2011­2012 Annual Report #12;University Libraries 2 University of digital collections and services, the physical use of our libraries con- tinues to rise. We increased and Robeson Libraries. We extend- ed library hours. Through donor and departmental gifts we added the De

Hanson, Stephen José

411

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

SciTech Connect

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.

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

2009-03-23

412

Gating of the Bacterial Sodium Channel, NaChBac: Voltage-dependent Charge Movement and Gating Currents  

Microsoft Academic Search

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B.

Alexey Kuzmenkin; Francisco Bezanilla; Ana M. Correa

2004-01-01