Note: This page contains sample records for the topic genomic bac library from Science.gov.
While these samples are representative of the content of Science.gov,
they are not comprehensive nor are they the most current set.
We encourage you to perform a real-time search of Science.gov
to obtain the most current and comprehensive results.
Last update: August 15, 2014.
1

[Construction of genome BAC library for single Branchiostoma belcheri individual].  

PubMed

As one of the closest living invertebrate relatives of vertebrates, amphioxus (subphylum Cephalochordata) occupies a key position in animal evolution and is becoming the best available proxy and model animal for studying the last common ancestor of all chordates, especially vertebrates. As long-term continuous culturing of amphioxus in laboratory became reliable, for pushing this animal to be a more successful model system, whole-genome sequencing of one or more species derived from this branch will be another urgent issue needed to address. In the present study, we described the construction and characterization of a bacterial artificial chromosome (BAC) library, using a single individual of Chinese amphioxus (Branchiostoma belcheri). High quality genomic DNA extracted from the spermary was partially digested with EcoRand EcoRmethylase. Desirable DNA fragments were isolated by pulsed field gel electrophoresis (PFGE), ligated to linearized and phosphorylased carrier pCC1BAC, and then transformed to Escherichia coli EPI300. The constructed library consists of 44 706 clones with the average insert fragment size around 80 kb as estimated by PFGE. The representation of the library is about 9 equivalents to the amhioxus genome. These results indicate that the BAC library will be useful for functional genomic studies and facilitate the whole-genome sequencing of Chinese amphioxus. PMID:20085888

Zhang, Ju-Yong; Huang, Sheng-Feng; Wang, Wei; Xu, An-Long; Wang, Yi-Quan

2010-01-01

2

A sunflower BAC library suitable for PCR screening and physical mapping of targeted genomic regions  

Microsoft Academic Search

A sunflower BAC library consisting of 147,456 clones with an average size of 118 kb has been constructed and characterized. It represents approximately 5× sunflower haploid genome equivalents. The BAC library has been arranged in pools and superpools of DNA allowing screening with various PCR-based markers. Each of the 32 superpools contains 4,608 clones and corresponds to a 36 matrix pools.

Mohamed Fouad Bouzidi; Jérôme Franchel; Quanzhou Tao; Keith Stormo; A. Mraz; Paul Nicolas; Saïd Mouzeyar

2006-01-01

3

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

PubMed Central

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

2011-01-01

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

Tuatara (Sphenodon) genomics: BAC library construction, sequence survey, and application to the DMRT gene family.  

PubMed

The tuatara (Sphenodon punctatus) is of "extraordinary biological interest" as the most distinctive surviving reptilian lineage (Rhyncocephalia) in the world. To provide a genomic resource for an understanding of genome evolution in reptiles, and as part of a larger project to produce genomic resources for various reptiles (evogen.jgi.doe.gov/second_levels/BACs/our_libraries.html), a large-insert bacterial artificial chromosome (BAC) library from a male tuatara was constructed. The library consists of 215 424 individual clones whose average insert size was empirically determined to be 145 kb, yielding a genomic coverage of approximately 6.3x. A BAC-end sequencing analysis of 121 420 bp of sequence revealed a genomic GC content of 46.8%, among the highest observed thus far for vertebrates, and identified several short interspersed repetitive elements (mammalian interspersed repeat-type repeats) and long interspersed repetitive elements, including chicken repeat 1 element. Finally, as a quality control measure the arrayed library was screened with probes corresponding to 2 conserved noncoding regions of the candidate sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig spanning nearly 300 kb was generated, supporting the deep coverage and utility of the library for exploring tuatara genomics. PMID:17135461

Wang, Zhenshan; Miyake, Tsutomu; Edwards, Scott V; Amemiya, Chris T

2006-01-01

6

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.

2011-01-01

7

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.

2013-01-01

8

A pair of new BAC and BIBAC vectors that facilitate BAC/BIBAC library construction and intact large genomic DNA insert exchange  

PubMed Central

Background Large-insert BAC and BIBAC libraries are important tools for structural and functional genomics studies of eukaryotic genomes. To facilitate the construction of BAC and BIBAC libraries and the transfer of complete large BAC inserts into BIBAC vectors, which is desired in positional cloning, we developed a pair of new BAC and BIBAC vectors. Results The new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S have the following features: 1) both contain two 18-bp non-palindromic I-SceI sites in an inverted orientation at positions that flank an identical DNA fragment containing the lacZ selection marker and the cloning site. Large DNA inserts can be excised from the vectors as single fragments by cutting with I-SceI, allowing the inserts to be easily sized. More importantly, because the two vectors contain different antibiotic resistance genes for transformant selection and produce the same non-complementary 3' protruding ATAA ends by I-SceI that suppress self- and inter-ligations, the exchange of intact large genomic DNA inserts between the BAC and BIBAC vectors is straightforward; 2) both were constructed as high-copy composite vectors. Reliable linearized and dephosphorylated original low-copy pIndigoBAC536-S and BIBAC-S vectors that are ready for library construction can be prepared from the high-copy composite vectors pHZAUBAC1 and pHZAUBIBAC1, respectively, without the need for additional preparation steps or special reagents, thus simplifying the construction of BAC and BIBAC libraries. BIBAC clones constructed with the new BIBAC-S vector are stable in both E. coli and Agrobacterium. The vectors can be accessed through our website http://GResource.hzau.edu.cn. Conclusions The two new vectors and their respective high-copy composite vectors can largely facilitate the construction and characterization of BAC and BIBAC libraries. The transfer of complete large genomic DNA inserts from one vector to the other is made straightforward.

2011-01-01

9

Construction of a BAC library for buckwheat genome research - an application to positional cloning of agriculturally valuable traits.  

PubMed

We have constructed a BAC library for common buckwheat Fagopyrum esculentum Moench. The library includes 142,005 clones with an average insert size of approximately 76 kb, equivalent to approximately a 7 to approximately 8-fold coverage of the genome. Polymerase chain reaction based screening of the library with AGAMOUS and FLORICAULA/LEAFY primers, has identified 7 and 9 BACs, respectively, which are consistent with the genome coverage. This library represents the first large insert genomic library for F. esculentum and it can be served as a genetic resource facilitating agricultural, pharmacological, physiological, and evolutionary studies of the species. To demonstrate the utilization of the library for characterizing agriculturally valuable traits, we developed a sequence tagged site marker tightly linked to the dwarf E locus as well as to the self-incompatibility complex locus and screened the library to initiate positional cloning of the causative genes. PMID:19168990

Yasui, Yasuo; Mori, Masashi; Matsumoto, Daiki; Ohnishi, Ohmi; Campbell, Clayton G; Ota, Tatsuya

2008-10-01

10

Human BAC library: construction and rapid screening  

Microsoft Academic Search

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96?000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods.

Shuichi Asakawa; Izumi Abe; Yoshiki Kudoh; Noriyuki Kishi; Yimin Wang; Ryo Kubota; Jun Kudoh; Kazuhiko Kawasaki; Shinsei Minoshima; Nobuyoshi Shimizu

1997-01-01

11

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

PubMed Central

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 crucial for further crop improvements. In this work we report the characterization of three BAC libraries from Clementine (Citrus clementina), one of the most relevant citrus fresh fruit market cultivars, and the analyses of 46.000 BAC end sequences. Clementine is a diploid plant with an estimated haploid genome size of 367 Mb and 2n = 18 chromosomes, which makes feasible the use of genomics tools to boost genetic improvement. Results Three genomic BAC libraries of Citrus clementina were constructed through EcoRI, MboI and HindIII digestions and 56,000 clones, representing an estimated genomic coverage of 19.5 haploid genome-equivalents, were picked. BAC end sequencing (BES) of 28,000 clones produced 28.1 Mb of genomic sequence that allowed the identification of the repetitive fraction (12.5% of the genome) and estimation of gene content (31,000 genes) of this species. BES analyses identified 3,800 SSRs and 6,617 putative SNPs. Comparative genomic studies showed that citrus gene homology and microsyntheny with Populus trichocarpa was rather higher than with Arabidopsis thaliana, a species phylogenetically closer to citrus. Conclusion In this work, we report the characterization of three BAC libraries from C. clementina, and a new set of genomic resources that may be useful for isolation of genes underlying economically important traits, physical mapping and eventually crop improvement in Citrus species. In addition, BAC end sequencing has provided a first insight on the basic structure and organization of the citrus genome and has yielded valuable molecular markers for genetic mapping and cloning of genes of agricultural interest. Paired end sequences also may be very helpful for whole-genome sequencing programs.

Terol, Javier; Naranjo, M Angel; Ollitrault, Patrick; Talon, Manuel

2008-01-01

12

An Oxytricha trifallax micronuclear BAC library  

Microsoft Academic Search

We propose construction of a BAC library for the germline micronuclear genome (MIC) of the ciliate protist Oxytricha trifallax (recently renamed Sterkiella histriomuscorum; alveolate; class Spirotrichia). This library will primarily be used as a tool to characterize the structure and function of the ciliate micronucleus and its relationship to that of the somatic macronucleus, rather than for complete genome sequencing.

Thomas G. Doak; Glenn Herrick; Laura F. Landweber

13

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

Microsoft Academic Search

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 ?14-fold coverage of the presumed ?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

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

2006-01-01

14

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.

Wang, Wei; Bradley, Allan; Huang, Yue

2009-01-01

15

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

16

New genomic resources for the honey bee (Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database  

Microsoft Academic Search

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning en- zyme HindIII in order to develop resources for structural genomics re- search. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and

J. P. Tomkins; M. Luo; G. C. Fang; D. Main; J. L. Goicoechea; M. Atkins; D. A. Frisch; E. Guzmán-Novoa; Y. Yu

17

A novel resource for genomics of Triticeae: BAC library specific for the short arm of rye (Secale cereale L.) chromosome 1R (1RS)  

PubMed Central

Background Genomics of rye (Secale cereale L.) is impeded by its large nuclear genome (1C~7,900 Mbp) with prevalence of DNA repeats (> 90%). An attractive possibility is to dissect the genome to small parts after flow sorting particular chromosomes and chromosome arms. To test this approach, we have chosen 1RS chromosome arm, which represents only 5.6% of the total rye genome. The 1RS arm is an attractive target as it carries many important genes and because it became part of the wheat gene pool as the 1BL.1RS translocation. Results We demonstrate that it is possible to sort 1RS arm from wheat-rye ditelosomic addition line. Using this approach, we isolated over 10 million of 1RS arms using flow sorting and used their DNA to construct a 1RS-specific BAC library, which comprises 103,680 clones with average insert size of 73 kb. The library comprises two sublibraries constructed using HindIII and EcoRI and provides a deep coverage of about 14-fold of the 1RS arm (442 Mbp). We present preliminary results obtained during positional cloning of the stem rust resistance gene SrR, which confirm a potential of the library to speed up isolation of agronomically important genes by map-based cloning. Conclusion We present a strategy that enables sorting short arms of several chromosomes of rye. Using flow-sorted chromosomes, we have constructed a deep coverage BAC library specific for the short arm of chromosome 1R (1RS). This is the first subgenomic BAC library available for rye and we demonstrate its potential for positional gene cloning. We expect that the library will facilitate development of a physical contig map of 1RS and comparative genomics of the homoeologous chromosome group 1 of wheat, barley and rye.

Simkova, Hana; Safar, Jan; Suchankova, Pavla; Kovarova, Pavlina; Bartos, Jan; Kubalakova, Marie; Janda, Jaroslav; Cihalikova, Jarmila; Mago, Rohit; Lelley, Tamas; Dolezel, Jaroslav

2008-01-01

18

Construction of a bacterial artificial chromosome (BAC) library for citrus and identification of BAC contigs containing resistance gene candidates  

Microsoft Academic Search

A BAC library was constructed from the genomic DNA of an intergeneric Citrus and Poncirus hybrid. The library consists of 24,576 clones with an average insert size of 115 kb, representing approximately seven haploid\\u000a genome equivalents and is able to give a greater than 99% probability of isolating single-copy citrus DNA sequences from this\\u000a library. High-density colony hybridization-based library screening

Z. Deng; Q. Tao; Y.-L. Chang; S. Huang; P. Ling; C. Yu; C. Chen; F. G. Gmitter Jr.; H.-B. Zhang

2001-01-01

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

Construction of a bacterial artificial chromosome (BAC) library for potato molecular cytogenetics research.  

PubMed

Lack of reliable techniques for chromosome identification is the major obstacle for cytogenetics research in plant species with large numbers of small chromosomes. To promote molecular cytogenetics research of potato (Solanum tuberosum, 2n = 4x = 48) we developed a bacterial artificial chromosome (BAC) library of a diploid potato species S. bulbocastanum. The library consists of 23,808 clones with an average insert size of 155 kb, and represents approximately 3.7 equivalents to the potato genome. The majority of the clones in the BAC library generated distinct signals on specific potato chromosomes using fluorescence in situ hybridization (FISH). The hybridization signals provide excellent cytological markers to tag individual potato chromosomes. We also demonstrated that the BAC clones can be mapped to specific positions on meiotic pachytene chromosomes. The excellent resolution of pachytene FISH can be used to construct a physical map of potato by mapping molecular marker-targeted BAC clones on pachytene chromosomes. PMID:10701131

Song, J; Dong, F; Jiang, J

2000-02-01

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 bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice  

Microsoft Academic Search

To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic\\u000a DNA was constructed. It consists of 18?432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from\\u000a 37 to 364?kb with an average of 107?kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony

D. Yang; A. Parco; S. Nandi; P. Subudhi; Y. Zhu; G. Wang; N. Huang

1997-01-01

23

Construction of an integrated genetic linkage map for the A genome of Brassica napus using SSR markers derived from sequenced BACs in B. rapa  

Microsoft Academic Search

BACKGROUND: The Multinational Brassica rapa Genome Sequencing Project (BrGSP) has developed valuable genomic resources, including BAC libraries, BAC-end sequences, genetic and physical maps, and seed BAC sequences for Brassica rapa. An integrated linkage map between the amphidiploid B. napus and diploid B. rapa will facilitate the rapid transfer of these valuable resources from B. rapa to B. napus (Oilseed rape,

Jinsong Xu; Xiaoju Qian; Xiaofeng Wang; Ruiyuan Li; Xiaomao Cheng; Yuan Yang; Jie Fu; Shunchang Zhang; Graham J King; Jiangsheng Wu; Kede Liu

2010-01-01

24

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

PubMed

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

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

2013-06-01

25

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

26

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

27

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

SciTech Connect

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

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

2009-04-21

28

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

PubMed Central

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

2010-01-01

29

An integrated BAC and genome sequence physical map of Phytophthora sojae.  

PubMed

Phytophthora spp. are serious pathogens that threaten numerous cultivated crops, trees, and natural vegetation worldwide. The soybean pathogen P. sojae has been developed as a model oomycete. Here, we report a bacterial artificial chromosome (BAC)-based, integrated physical map of the P. sojae genome. We constructed two BAC libraries, digested 8,681 BACs with seven restriction enzymes, end labeled the digested fragments with four dyes, and analyzed them with capillary electrophoresis. Fifteen data sets were constructed from the fingerprints, using individual dyes and all possible combinations, and were evaluated for contig assembly. In all, 257 contigs were assembled from the XhoI data set, collectively spanning approximately 132 Mb in physical length. The BAC contigs were integrated with the draft genome sequence of P. sojae by end sequencing a total of 1,440 BACs that formed a minimal tiling path. This enabled the 257 contigs of the BAC map to be merged with 207 sequence scaffolds to form an integrated map consisting of 79 superscaffolds. The map represents the first genome-wide physical map of a Phytophthora sp. and provides a valuable resource for genomics and molecular biology research in P. sojae and other Phytophthora spp. In one illustration of this value, we have placed the 350 members of a superfamily of putative pathogenicity effector genes onto the map, revealing extensive clustering of these genes. PMID:17153914

Zhang, Xuemin; Scheuring, Chantel; Tripathy, Sucheta; Xu, Zhanyou; Wu, Chengcang; Ko, Angela; Tian, S Ken; Arredondo, Felipe; Lee, Mi-Kyung; Santos, Felipe A; Jiang, Rays H Y; Zhang, Hong-Bin; Tyler, Brett M

2006-12-01

30

Construction of Papaya Male and Female BAC Libraries and Application in Physical Mapping of the Sex Chromosomes  

PubMed Central

Papaya is a major fruit crop in the tropics and has recently evolved sex chromosomes. Towards sequencing the papaya sex chromosomes, two bacterial artificial chromosome (BAC) libraries were constructed from papaya male and female genomic DNA. The female BAC library was constructed using restriction enzyme BstY I and consists of 36,864 clones with an average insert size of 104?kb, providing 10.3x genome equivalents. The male BAC library was constructed using restriction enzyme EcoR I and consists of 55,296 clones with an average insert size of 101?kb, providing 15.0x genome equivalents. The male BAC library was used in constructing the physical map of the male-specific region of the male Y chromosome (MSY) and in filling gaps and extending the physical map of the hermaphrodite-specific region of the Yh chromosome (HSY) and the X chromosome physical map. The female BAC library was used to extend the X physical map gap. The MSY, HSY, and X physical maps offer a unique opportunity to study chromosomal rearrangements, Y chromosome degeneration, and dosage compensation of the papaya nascent sex chromosomes.

Gschwend, Andrea R.; Yu, Qingyi; Moore, Paul; Saski, Christopher; Chen, Cuixia; Wang, Jianping; Na, Jong-Kuk; Ming, Ray

2011-01-01

31

A BAC- and BIBAC-Based Physical Map of the Soybean Genome  

PubMed Central

Genome-wide physical maps are crucial to many aspects of advanced genome research. We report a genome-wide, bacterial artificial chromosome (BAC) and plant-transformation-competent binary large-insert plasmid clone (hereafter BIBAC)-based physical map of the soybean genome. The map was constructed from 78,001 clones from five soybean BAC and BIBAC libraries representing 9.6 haploid genomes and three cultivars, and consisted of 2905 BAC/BIBAC contigs, estimated to span 1408 Mb in physical length. We evaluated the reliability of the map contigs using different contig assembly strategies, independent contig building methods, DNA marker hybridization, and different fingerprinting methods, and the results showed that the contigs were assembled properly. Furthermore, we tested the feasibility of integrating the physical map with the existing soybean composite genetic map using 388 DNA markers. The results further confirmed the nature of the ancient tetraploid origin of soybean and indicated that it is feasible to integrate the physical map with the linkage map even though greater efforts are needed. This map represents the first genome-wide, BAC/BIBAC-based physical map of the soybean genome and would provide a platform for advanced genome research of soybean and other legume species. The inclusion of BIBACs in the map would streamline the utility of the map for positional cloning of genes and QTLs, and functional analysis of soybean genomic sequences.

Wu, Chengcang; Sun, Shuku; Nimmakayala, Padmavathi; Santos, Felipe A.; Meksem, Khalid; Springman, Rachael; Ding, Kejiao; Lightfoot, David A.; Zhang, Hong-Bin

2004-01-01

32

BACs as Tools for the Study of Genomic Imprinting  

PubMed Central

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

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

2011-01-01

33

A BAC-based physical map of the Hessian fly genome anchored to polytene chromosomes  

PubMed Central

Background The Hessian fly (Mayetiola destructor) is an important insect pest of wheat. It has tractable genetics, polytene chromosomes, and a small genome (158 Mb). Investigation of the Hessian fly presents excellent opportunities to study plant-insect interactions and the molecular mechanisms underlying genome imprinting and chromosome elimination. A physical map is needed to improve the ability to perform both positional cloning and comparative genomic analyses with the fully sequenced genomes of other dipteran species. Results An FPC-based genome wide physical map of the Hessian fly was constructed and anchored to the insect's polytene chromosomes. Bacterial artificial chromosome (BAC) clones corresponding to 12-fold coverage of the Hessian fly genome were fingerprinted, using high information content fingerprinting (HIFC) methodology, and end-sequenced. Fluorescence in situ hybridization (FISH) co-localized two BAC clones from each of the 196 longest contigs on the polytene chromosomes. An additional 70 contigs were positioned using a single FISH probe. The 266 FISH mapped contigs were evenly distributed and covered 60% of the genome (95,668 kb). The ends of the fingerprinted BACs were then sequenced to develop the capacity to create sequenced tagged site (STS) markers on the BACs in the map. Only 3.64% of the BAC-end sequence was composed of transposable elements, helicases, ribosomal repeats, simple sequence repeats, and sequences of low complexity. A relatively large fraction (14.27%) of the BES was comprised of multi-copy gene sequences. Nearly 1% of the end sequence was composed of simple sequence repeats (SSRs). Conclusion This physical map provides the foundation for high-resolution genetic mapping, map-based cloning, and assembly of complete genome sequencing data. The results indicate that restriction fragment length heterogeneity in BAC libraries used to construct physical maps lower the length and the depth of the contigs, but is not an absolute barrier to the successful application of the technology. This map will serve as a genomic resource for accelerating gene discovery, genome sequencing, and the assembly of BAC sequences. The Hessian fly BAC-clone assembly, and the names and positions of the BAC clones used in the FISH experiments are publically available at .

Aggarwal, Rajat; Benatti, Thiago R; Gill, Navdeep; Zhao, Chaoyang; Chen, Ming-Shun; Fellers, John P; Schemerhorn, Brandon J; Stuart, Jeff J

2009-01-01

34

Gram negative shuttle BAC vector for heterologous expression of metagenomic libraries  

Microsoft Academic Search

Bacterial artificial chromosome (BAC) vectors enable stable cloning of large DNA fragments from single genomes or microbial assemblages. A novel shuttle BAC vector was constructed that permits replication of BAC clones in diverse Gram-negative species. The “Gram-negative shuttle BAC” vector (pGNS-BAC) uses the F replicon for stable single-copy replication in E. coli and the broad-host-range RK2 mini-replicon for high-copy replication

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

2011-01-01

35

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

Microsoft Academic Search

As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific.

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

2011-01-01

36

Construction and characterization of a half million clone BAC library of durum wheat ( Triticum turgidum ssp. durum )  

Microsoft Academic Search

Durum wheat ( Triticum turgidum ssp. durum, 2 n = 4 x = 28, genomes AB) is an economically important cereal used as the raw material to make pasta and semolina. In this paper we present the construction and characterization of a bacterial artificial chromosome (BAC) library of tetraploid durum wheat cv. Langdon. This variety was selected because of the

A. Cenci; N. Chantret; X. Kong; Y. Gu; O. D. Anderson; T. Fahima; A. Distelfeld; J. Dubcovsky

2003-01-01

37

A BAC end view of the Musa acuminata genome  

PubMed Central

Background Musa species contain the fourth most important crop in developing countries. Here, we report the analysis of 6,252 BAC end-sequences, in order to view the sequence composition of the Musa acuminata genome in a cost effective and efficient manner. Results BAC end sequencing generated 6,252 reads representing 4,420,944 bp, including 2,979 clone pairs with an average read length after cleaning and filtering of 707 bp. All sequences have been submitted to GenBank, with the accession numbers DX451975 – DX458350. The BAC end-sequences, were searched against several databases and significant homology was found to mitochondria and chloroplast (2.6%), transposons and repetitive sequences (36%) and proteins (11%). Functional interpretation of the protein matches was carried out by Gene Ontology assignments from matches to Arabidopsis and was shown to cover a broad range of categories. From protein matching regions of Musa BAC end-sequences, it was determined that the GC content of coding regions was 47%. Where protein matches encompassed a start codon, GC content as a function of position (5' to 3') across 129 bp sliding windows generates a "rice-like" gradient. A total of 352 potential SSR markers were discovered. The most abundant simple sequence repeats in four size categories were AT-rich. After filtering mitochondria and chloroplast matches, thousands of BAC end-sequences had a significant BLASTN match to the Oryza sativa and Arabidopsis genome sequence. Of these, a small number of BAC end-sequence pairs were shown to map to neighboring regions of the Oryza sativa genome representing regions of potential microsynteny. Conclusion Database searches with the BAC end-sequences and ab initio analysis identified those reads likely to contain transposons, repeat sequences, proteins and simple sequence repeats. Approximately 600 BAC end-sequences contained protein sequences that were not found in the existing available Musa expressed sequence tags, repeat or transposon databases. In addition, gene statistics, GC content and profile could also be estimated based on the region matching the top protein hit. A small number of BAC end pair sequences can be mapped to neighboring regions of the Oryza sativa representing regions of potential microsynteny. These results suggest that a large-scale BAC end sequencing strategy has the potential to anchor a small proportion of the genome of Musa acuminata to the genomes of Oryza sativa and possibly Arabidopsis.

Cheung, Foo; Town, Christopher D

2007-01-01

38

Construction of a 7-fold BAC library and cytogenetic mapping of 10 genes in the giant panda (Ailuropoda melanoleuca)  

PubMed Central

Background The giant panda, one of the most primitive carnivores, is an endangered animal. Although it has been the subject of many interesting studies during recent years, little is known about its genome. In order to promote research on this genome, a bacterial artificial chromosome (BAC) library of the giant panda was constructed in this study. Results This BAC library contains 198,844 clones with an average insert size of 108 kb, which represents approximately seven equivalents of the giant panda haploid genome. Screening the library with 15 genes and 8 microsatellite markers demonstrates that it is representative and has good genome coverage. Furthermore, ten BAC clones harbouring AGXT, GHR, FSHR, IRBP, SOX14, TTR, BDNF, NT-4, LH and ZFX1 were mapped to 8 pairs of giant panda chromosomes by fluorescence in situ hybridization (FISH). Conclusion This is the first large-insert genomic DNA library for the giant panda, and will contribute to understanding this endangered species in the areas of genome sequencing, physical mapping, gene cloning and comparative genomic studies. We also identified the physical locations of ten genes on their relative chromosomes by FISH, providing a preliminary framework for further development of a high resolution cytogenetic map of the giant panda.

Liu, Wei; Zhao, Yonghui; Liu, Zhaoliang; Zhang, Ying; Lian, Zhengxing; Li, Ning

2006-01-01

39

Construction and characterization of a BAC-library for a key pollinator, the bumblebee Bombus terrestris L  

Microsoft Academic Search

.  The primitively social bumblebee Bombus terrestris is an ecological model species as well as an important agricultural pollinator. As part of the ongoing development of genomic\\u000a resources for this model organism, we have constructed a publicly available bacterial artificial chromosome (BAC) library\\u000a from males of a field-derived colony. We have shown that this library has a high coverage, which allows

L. Wilfert; M. Muñoz Torres; C. Reber-Funk; R. Schmid-Hempel; J. Tomkins; J. Gadau; P. Schmid-Hempel

2009-01-01

40

Construction of Japanese BAC library Yamato-2 (JY2): a set of 330K clone resources of damage-minimized DNA taken from a genetically established Japanese individual.  

PubMed

A bacterial artificial chromosome (BAC) library referred to as Yamato-2 (JY2), was constructed from a Japanese individual and contained 330,000 clones. Library construction was based on 2 concepts: Japanese pedigree and non-immortalization. Genomic DNA was extracted from white blood cells from umbilical cord blood of a Japanese male individual. Four traits of the sample, (1) amelogenin DNA, (2) short tandem repeat (STR), (3) mitochondrial DNA (mtDNA), and (4) HLA-allele typing, were investigated to verify attribution of the donor. One of the samples with quite good Japanese characteristics was named JY2 and used as a resource for construction of a BAC library. Amelogenin DNA indicated male. STR indicated Mongoloid. MtDNA suggested haplogroup B, which is different from any other diploid whose sequence has been reported. The HLA gene was classified into east-Asian specific haplotype. These results revealed that JY2 was obtained from a Japanese male. We sequenced both ends of 185,012 BAC clones. By using the BLAST search, BAC end sequences (BESs) were mapped on the human reference sequence provided by NCBI. Inserts of individual BAC clones were mapped with both ends properly placed. As a result, 103,647 BAC clones were successfully mapped. The average insert size of BAC calculated from the mapping information was 130 kb. Coverage and redundancy of the reference sequence by successfully mapped BAC clones were 96.4% and 3.9-fold, respectively. This library will be especially suitable as a Japanese standard genome resource. The availability of an accurate library is indispensable for diagnostics or drug-design based on genome information, and JY2 will provide an accurate sequence of the Japanese genome as an important addition to the human genome. PMID:21611881

Terabayashi, Yasunobu; Morita, Keiko; Park, Joon Young; Saito, Soichiro; Shiina, Takashi; Inoko, Hidetoshi; Ishiwata, Isamu; Fujimori, Kazuhiro E; Hirano, Takashi

2011-12-01

41

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

Microsoft Academic Search

To facilitate isolation and characterization of disease and insect resistance genes important to potato, two bacterial artificial chromosome (BAC) libraries were constructed from genomic DNA of the Mexican wild diploid species, Solanum pinnatisectum, which carries high levels of resistance to the most important potato pathogen and pest, the late blight and the Colorado potato beetle (CPB). One of the libraries

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

2004-01-01

42

Construction of an integrated genetic linkage map for the A genome of Brassica napus using SSR markers derived from sequenced BACs in B. rapa  

PubMed Central

Background The Multinational Brassica rapa Genome Sequencing Project (BrGSP) has developed valuable genomic resources, including BAC libraries, BAC-end sequences, genetic and physical maps, and seed BAC sequences for Brassica rapa. An integrated linkage map between the amphidiploid B. napus and diploid B. rapa will facilitate the rapid transfer of these valuable resources from B. rapa to B. napus (Oilseed rape, Canola). Results In this study, we identified over 23,000 simple sequence repeats (SSRs) from 536 sequenced BACs. 890 SSR markers (designated as BrGMS) were developed and used for the construction of an integrated linkage map for the A genome in B. rapa and B. napus. Two hundred and nineteen BrGMS markers were integrated to an existing B. napus linkage map (BnaNZDH). Among these mapped BrGMS markers, 168 were only distributed on the A genome linkage groups (LGs), 18 distrubuted both on the A and C genome LGs, and 33 only distributed on the C genome LGs. Most of the A genome LGs in B. napus were collinear with the homoeologous LGs in B. rapa, although minor inversions or rearrangements occurred on A2 and A9. The mapping of these BAC-specific SSR markers enabled assignment of 161 sequenced B. rapa BACs, as well as the associated BAC contigs to the A genome LGs of B. napus. Conclusion The genetic mapping of SSR markers derived from sequenced BACs in B. rapa enabled direct links to be established between the B. napus linkage map and a B. rapa physical map, and thus the assignment of B. rapa BACs and the associated BAC contigs to the B. napus linkage map. This integrated genetic linkage map will facilitate exploitation of the B. rapa annotated genomic resources for gene tagging and map-based cloning in B. napus, and for comparative analysis of the A genome within Brassica species.

2010-01-01

43

BAC CLONES GENERATED FROM SHEARED DNA  

PubMed Central

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 200 kb. 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 average insert lengths between 50 – 150 kb. The libraries lack chimeric clone problems as determined by mapping paired BAC-end sequences to the assembled fly 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; de Jong, Pieter J.

2007-01-01

44

Strategy to sequence the genome of Corynebacterium glutamicum ATCC 13032: use of a cosmid and a bacterial artificial chromosome library.  

PubMed

The initial strategy of the Corynebacterium glutamicum genome project was to sequence overlapping inserts of an ordered cosmid library. High-density colony grids of approximately 28 genome equivalents were used for the identification of overlapping clones by Southern hybridization. Altogether 18 contiguous genomic segments comprising 95 overlapping cosmids were assembled. Systematic shotgun sequencing of the assembled cosmid set revealed that only 2.84 Mb (86.6%) of the C. glutamicum genome were represented by the cosmid library. To obtain a complete genome coverage, a bacterial artificial chromosome (BAC) library of the C. glutamicum chromosome was constructed in pBeloBAC11 and used for genome mapping. The BAC library consists of 3168 BACs and represents a theoretical 63-fold coverage of the C. glutamicum genome (3.28 Mb). Southern screening of 2304 BAC clones with PCR-amplified chromosomal markers and subsequent insert terminal sequencing allowed the identification of 119 BACs covering the entire chromosome of C. glutamicum. The minimal set representing a 100% genome coverage contains 44 unique BAC clones with an average overlap of 22 kb. A total of 21 BACs represented linking clones between previously sequenced cosmid contigs and provided a valuable tool for completing the genome sequence of C. glutamicum. PMID:11879709

Tauch, Andreas; Homann, Iris; Mormann, Sascha; Rüberg, Silvia; Billault, Alain; Bathe, Brigitte; Brand, Sven; Brockmann-Gretza, Olaf; Rückert, Christian; Schischka, Natalie; Wrenger, Carsten; Hoheisel, Jörg; Möckel, Bettina; Huthmacher, Klaus; Pfefferle, Walter; Pühler, Alfred; Kalinowski, Jörn

2002-04-25

45

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.

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

2014-01-01

46

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.

2011-01-01

47

Genome-Wide Identification of Genes Conferring Energy Related Resistance to a Synthetic Antimicrobial Peptide (Bac8c)  

PubMed Central

A fundamental issue in the design and development of antimicrobials is the lack of understanding of complex modes of action and how this complexity affects potential pathways for resistance evolution. Bac8c (RIWVIWRR-NH2) is an 8 amino acid antimicrobial peptide (AMP) that has been shown to have enhanced activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. We have previously demonstrated that Bac8c appears to interfere with multiple targets, at least in part through the disruption of cytoplasmic membrane related functions, and that resistance to this peptide does not easily develop using standard laboratory methods. Here, we applied a genomics approach, SCalar Analysis of Library Enrichement (SCALEs), to map the effect of gene overexpression onto Bac8c resistance in parallel for all genes and gene combinations (up to ? 10 adjacent genes) in the E. coli genome (a total of ? 500,000 individual clones were mapped). Our efforts identified an elaborate network of genes for which overexpression leads to low-level resistance to Bac8c (including biofilm formation, multi-drug transporters, etc). This data was analyzed to provide insights into the complex relationships between mechanisms of action and potential routes by which resistance to this synthetic AMP can develop.

Spindler, Eileen C.; Boyle, Nanette R.; Hancock, Robert E. W.; Gill, Ryan T.

2013-01-01

48

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

Microsoft Academic Search

Background  Common carp is one of the most important aquaculture teleost fish in the world. Common carp and other closely related Cyprinidae\\u000a species provide over 30% aquaculture production in the world. However, common carp genomic resources are still relatively\\u000a underdeveloped. BAC end sequences (BES) are important resources for genome research on BAC-anchored genetic marker development,\\u000a linkage map and physical map integration,

Peng Xu; Jiongtang Li; Yan Li; Runzi Cui; Jintu Wang; Jian Wang; Yan Zhang; Zixia Zhao; Xiaowen Sun

2011-01-01

49

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

PubMed Central

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

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

2013-01-01

50

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

51

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.

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

52

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.

2012-01-01

53

Mobilization of giant piggyBac transposons in the mouse genome  

PubMed Central

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

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

2011-01-01

54

Mapping of Micro-Tom BAC-End Sequences to the Reference Tomato Genome Reveals Possible Genome Rearrangements and Polymorphisms  

PubMed Central

A total of 93,682 BAC-end sequences (BESs) were generated from a dwarf model tomato, cv. Micro-Tom. After removing repetitive sequences, the BESs were similarity searched against the reference tomato genome of a standard cultivar, “Heinz 1706.” By referring to the “Heinz 1706” physical map and by eliminating redundant or nonsignificant hits, 28,804 “unique pair ends” and 8,263 “unique ends” were selected to construct hypothetical BAC contigs. The total physical length of the BAC contigs was 495, 833, 423?bp, covering 65.3% of the entire genome. The average coverage of euchromatin and heterochromatin was 58.9% and 67.3%, respectively. From this analysis, two possible genome rearrangements were identified: one in chromosome 2 (inversion) and the other in chromosome 3 (inversion and translocation). Polymorphisms (SNPs and Indels) between the two cultivars were identified from the BLAST alignments. As a result, 171,792 polymorphisms were mapped on 12 chromosomes. Among these, 30,930 polymorphisms were found in euchromatin (1 per 3,565?bp) and 140,862 were found in heterochromatin (1 per 2,737?bp). The average polymorphism density in the genome was 1 polymorphism per 2,886?bp. To facilitate the use of these data in Micro-Tom research, the BAC contig and polymorphism information are available in the TOMATOMICS database.

Asamizu, Erika; Shirasawa, Kenta; Hirakawa, Hideki; Sato, Shusei; Tabata, Satoshi; Yano, Kentaro; Ariizumi, Tohru; Shibata, Daisuke; Ezura, Hiroshi

2012-01-01

55

Characterization of porcine endogenous retrovirus clones from the NIH miniature pig BAC library.  

PubMed

Pigs have been considered as donors for xenotransplantation in the replacement of human organs and tissues. However, porcine endogenous retroviruses (PERVs) might transmit new infectious disease to humans during xenotransplantation. To investigate PERV integration sites, 45 PERV-positive BAC clones, including 12 PERV-A, 16 PERV-B, and 17 PERV-C clones, were identified from the NIH miniature pig BAC library. The analysis of 12 selected full-length sequences of PERVs, including the long terminal repeat (LTR) region, identified the expected of open reading frame length, an indicative of active PERV, in all five PERV-C clones and one of the four PERV-B clones. Premature stop codons were observed in only three PERV-A clones. Also, eleven PERV integration sites were mapped using a 5000-rad IMpRH panel. The map locations of PERV-C clones have not been reported before, thus they are novel PERV clones identified in this study. The results could provide basic information for the elimination of site-specific PERVs in selection of pigs for xenotransplantation. PMID:21912484

Yu, Seong-Lan; Jung, Woo-Young; Jung, Kie-Chul; Cho, In-Cheol; Lim, Hyun-Tae; Jin, Dong-Il; Lee, Jun-Heon

2012-01-01

56

VIS-O-BAC: exploratory visualization of functional genome studies from bacteria  

Microsoft Academic Search

Summary: The visualization-aided exploration of complex datasets will allow the research community to formulate novel functional hypotheses leading to a better understanding of biological processes at all levels. Therefore, we have developed a web resource termed VIS-O-BAC designed for the functional investigation of expression data for model systems, such as bacterial pathogens based on a graphical display. Genome-scale datasets derived

Guido Dieterich; Uwe Kärst; Jürgen Wehland; Lothar Jänsch

2006-01-01

57

Construction of a Llama Bacterial Artificial Chromosome Library with Approximately 9-Fold Genome Equivalent Coverage  

PubMed Central

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

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

2012-01-01

58

Comparative genomic hybridisation using a proximal 17p BAC/PAC array detects rearrangements responsible for four genomic disorders  

PubMed Central

Background: Proximal chromosome 17p is a region rich in low copy repeats (LCRs) and prone to chromosomal rearrangements. Four genomic disorders map within the interval 17p11–p12: Charcot–Marie–Tooth disease type 1A, hereditary neuropathy with liability to pressure palsies, Smith–Magenis syndrome, and dup(17)(p11.2p11.2) syndrome. While 80–90% or more of the rearrangements resulting in each disorder are recurrent, several non-recurrent deletions or duplications of varying sizes within proximal 17p also have been characterised using fluorescence in situ hybridisation (FISH). Methods: A BAC/PAC array based comparative genomic hybridisation (array-CGH) method was tested for its ability to detect these genomic dosage differences and map breakpoints in 25 patients with recurrent and non-recurrent rearrangements. Results: Array-CGH detected the dosage imbalances resulting from either deletion or duplication in all the samples examined. The array-CGH approach, in combination with a dependent statistical inference method, mapped 45/46 (97.8%) of the analysed breakpoints to within one overlapping BAC/PAC clone, compared with determinations done independently by FISH. Several clones within the array that contained large LCRs did not have an adverse effect on the interpretation of the array-CGH data. Conclusions: Array-CGH is an accurate and sensitive method for detecting genomic dosage differences and identifying rearrangement breakpoints, even in LCR-rich regions of the genome.

Shaw, C; Shaw, C; Yu, W; Stankiewicz, P; White, L; Beaudet, A; Lupski, J

2004-01-01

59

Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques  

PubMed Central

Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic loxP sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic loxP sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic loxP sites occur frequently and are homogeneously distributed in the genome. Given the mammalian nature of BAC/PAC genomic inserts, we hypothesised that the presence of cryptic loxP sites may affect the ability to grow and modify BAC and PAC clones in E. coli expressing Cre recombinase. We have observed a defect in bacterial growth when some BACs and PACs were transformed into EL350, a DH10B-derived bacterial strain that expresses Cre recombinase under the control of an arabinose-inducible promoter. In this study, we have demonstrated that Cre recombinase expression is leaky in un-induced EL350 cells and that some BAC/PAC sequences contain cryptic loxP sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts.

Semprini, S.; Troup, T.J.; Kotelevtseva, N.; King, K.; Davis, J.R.E.; Mullins, L.J.; Chapman, K.E.; Dunbar, D.R.; Mullins, J.J.

2007-01-01

60

An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction.  

PubMed

Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb. PMID:21221926

Liu, Juan; Li, Jingquan; Feng, Li; Cao, Hui; Cui, Zhongli

2010-12-01

61

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

PubMed

Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses. PMID:12438737

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

2002-01-01

62

Construction of bacterial artificial chromosome libraries and their application in developing PCR-based markers closely linked to a major locus conditioning bruchid resistance in mungbean ( Vigna radiata L. Wilczek)  

Microsoft Academic Search

Bacterial artificial chromosome (BAC) libraries have been widely used in different aspects of genome research. In this paper we report the construction of the first mungbean (Vigna radiata L. Wilczek) BAC libraries. These BAC clones were obtained from two ligations and represent an estimated 3.5 genome equivalents. This correlated well with the screening of nine random single-copy restriction fragment length

M. Miyagi; M. Humphry; Z. Y. Ma; C. J. Lambrides; M. Bateson; C. J. Liu

2004-01-01

63

A first generation bovine BAC-based physical map.  

PubMed

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

64

Generation of a soybean BAC library, and identification of DNA sequences tightly linked to the Rps1-k disease resistance gene  

Microsoft Academic Search

A soybean bacterial artificial chromosome (BAC) library, comprising approximately 45?000 clones, was constructed from high-molecular-weight\\u000a nuclear DNA of cultivar Williams 82, which carries the Rps1-k gene for resistance against Phytophthora sojae. The library is stored in 130 pools with about 350 clones per pool. Completeness of the library was evaluated for 21 random\\u000a sequences including four markers linked to the

S. S. Salimath; M. K. Bhattacharyya

1999-01-01

65

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

66

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

PubMed Central

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

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

2005-01-01

67

Chimeric piggyBac transposases for genomic targeting in human cells.  

PubMed

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

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

2012-08-01

68

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.

2009-01-01

69

High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding  

Microsoft Academic Search

A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used

Mukhlesur Rahman; Zudong Sun; Peter B. E. McVetty; Genyi Li

2008-01-01

70

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.

2014-01-01

71

Technique for Screening and Maintaining Smaller Genomic Libraries.  

National Technical Information Service (NTIS)

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

U. M. X. Sangodkar S. Mavinkurve

1992-01-01

72

Localization and characterization of 170 BAC-derived clones and mapping of 94 microsatellites in the Hessian fly.  

PubMed

Ninety-four microsatellites from enriched genomic libraries of Hessian fly (Hf, Mayetiola destructor [Say]) were localized to 170 cognate clones in an Hf bacterial artificial chromosome (BAC) library. These microsatellite-positive BAC clones were physically mapped to polytene chromosomes by fluorescent in situ hybridization. The mapped microsatellite loci can be used to study the genetic diversity and population structure of Hf. PMID:19592640

Schemerhorn, Brandon J; Crane, Yan M; Morton, Philip K; Aggarwal, Rajat; Benatti, Thiago

2009-01-01

73

A plant-transformation-competent BIBAC/BAC-based map of rice for functional analysis and genetic engineering of its genomic sequence.  

PubMed

Sequencing of the rice genome has provided a platform for functional genomics research of rice and other cereal species. However, multiple approaches are needed to determine the functions of its genes and sequences and to use the genome sequencing results for genetic improvement of cereal crops. Here, we report a plant-transformation-competent, binary bacterial artificial chromosome (BIBAC) and bacterial artificial chromosome (BAC) based map of rice to facilitate these studies. The map was constructed from 20 835 BIBAC and BAC clones, and consisted of 579 overlapping BIBAC/BAC contigs. To facilitate functional analysis of chromosome 8 genomic sequence and cloning of the genes and QTLs mapped to the chromosome, we anchored the chromosomal contigs to the existing rice genetic maps. The chromosomal map consists of 11 contigs, 59 genetic markers, and 36 sequence tagged sites, spanning a total of ca. 38 Mb in physical length. Comparative analysis between the genetic and physical maps of chromosome 8 showed that there are 3 "hot" and 2 "cold" spots of genetic recombination along the chromosomal arms in addition to the "cold spot" in the centromeric region, suggesting that the sequence component contents of a chromosome may affect its local genetic recombination frequencies. Because of its plant transformability, the BIBAC/BAC map could provide a platform for functional analysis of the rice genome sequence and effective use of the sequencing results for gene and QTL cloning and molecular breeding. PMID:17502901

Li, Yaning; Uhm, Taesik; Ren, Chengwei; Wu, Chengcang; Santos, Teofila S; Lee, Mi-Kyung; Yan, Bo; Santos, Felipe; Zhang, Aimin; Scheuring, Chantel; Sanchez, Alma; Millena, Anna C; Nguyen, Henry T; Kou, Hongda; Liu, Daqun; Zhang, Hong-Bin

2007-03-01

74

Complete Genome Sequence of a UL96 Mutant Cytomegalovirus Towne-BAC (Bacterial Artificial Chromosome) Isolate Passaged in Fibroblasts To Allow Accumulation of Compensatory Mutations.  

PubMed

Here, we present the complete genome sequence of a cytomegalovirus Towne-BAC (bacterial artificial chromosome) isolate that we first genetically engineered to mutate the UL96 gene and then serially passaged in human fibroblasts to allow for the accumulation of compensatory mutations. A total of 17 single-base substitutions were discovered in the passaged genome compared to the reference sequence (KF493877). PMID:24158558

Brechtel, Teal M; Tyner, Molly; Tandon, Ritesh

2013-01-01

75

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

Microsoft Academic Search

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

ANNA V. KUKEKOVA; N. V. Vorobieva; V. R. Beklemisheva; J. L. Johnson; S. V. Temnykh; D MITRY V. YUDKIN; L YUDMILA N. TRUT; C. Andre; F. Galibert; G. D. Aguirre; G. M. Acland; A. S. Graphodatsky

2009-01-01

76

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

SciTech Connect

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

Kao, F.T.

1994-04-01

77

BAC CGH-array identified specific small-scale genomic imbalances in diploid DMBA-induced rat mammary tumors  

PubMed Central

Background Development of breast cancer is a multistage process influenced by hormonal and environmental factors as well as by genetic background. The search for genes underlying this malignancy has recently been highly productive, but the etiology behind this complex disease is still not understood. In studies using animal cancer models, heterogeneity of the genetic background and environmental factors is reduced and thus analysis and identification of genetic aberrations in tumors may become easier. To identify chromosomal regions potentially involved in the initiation and progression of mammary cancer, in the present work we subjected a subset of experimental mammary tumors to cytogenetic and molecular genetic analysis. Methods Mammary tumors were induced with DMBA (7,12-dimethylbenz[a]anthrazene) in female rats from the susceptible SPRD-Cu3 strain and from crosses and backcrosses between this strain and the resistant WKY strain. We first produced a general overview of chromosomal aberrations in the tumors using conventional kartyotyping (G-banding) and Comparative Genome Hybridization (CGH) analyses. Particular chromosomal changes were then analyzed in more details using an in-house developed BAC (bacterial artificial chromosome) CGH-array platform. Results Tumors appeared to be diploid by conventional karyotyping, however several sub-microscopic chromosome gains or losses in the tumor material were identified by BAC CGH-array analysis. An oncogenetic tree analysis based on the BAC CGH-array data suggested gain of rat chromosome (RNO) band 12q11, loss of RNO5q32 or RNO6q21 as the earliest events in the development of these mammary tumors. Conclusions Some of the identified changes appear to be more specific for DMBA-induced mammary tumors and some are similar to those previously reported in ACI rat model for estradiol-induced mammary tumors. The later group of changes is more interesting, since they may represent anomalies that involve genes with a critical role in mammary tumor development. Genetic changes identified in this work are at very small scales and thus may provide a more feasible basis for the identification of the target gene(s). Identification of the genes underlying these chromosome changes can provide new insights to the mechanisms of mammary carcinogenesis.

2012-01-01

78

Novel Bacterial Artificial Chromosome Vector pUvBBAC for Use in Studies of the Functional Genomics of Listeria spp  

Microsoft Academic Search

Bacterial artificial chromosome (BAC) vectors are important tools for microbial genome research. We con- structed a novel BAC vector, pUvBBAC, for replication in both gram-negative and gram-positive bacterial hosts. The pUvBBAC vector was used to generate a BAC library for the facultative intracellular pathogen Listeria monocytogenes EGD-e. The library had insert sizes ranging from 68 to 178 kb. We identified

Torsten Hain; Sonja Otten; U. von Both; S. S. Chatterjee; U. Technow; A. Billion; R. Ghai; W. Mohamed; E. Domann; T. Chakraborty

2008-01-01

79

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

80

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

PubMed

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

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

2009-12-01

81

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

PubMed Central

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

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

2009-01-01

82

Chemical Biology\\/ Chemical Genetics\\/ Chemical Genomics: Importance of Chemical Library  

Microsoft Academic Search

A new field of science, chemical biology\\/ chemical genetics\\/ chemical genomics (cb\\/cg\\/cg) has emerged since the late 1990's, especially in the United States. The NIH Roadmap agenda, Molecular Libraries Screening Center Network (MLSCN), became a drive force to push cb\\/cg\\/cg forward. Cb\\/cg\\/cg studies consist of three methodologies, chemical libraries with small molecules, high-throughput screenings, and computational databases. In this review,

Fumihiko Kugawa; Masaru Watanabe; Fuyuhiko Tamanoi

2007-01-01

83

Physical mapping, BAC-end sequence analysis, and marker tagging of the soilborne nematicidal bacterium, Pseudomonas synxantha BG33R.  

PubMed

A bacterial artificial chromosome (BAC) library was constructed for the genome of the rhizosphere-inhabiting fluorescent pseudomonad Pseudomonas synxantha BG33R. Three thousand BAC clones with an average insert size of 140 kbp and representing a 70-fold genomic coverage were generated and arrayed onto nylon membranes. EcoRI fingerprint analysis of 986 BAC clones generated 23 contigs and 75 singletons. Hybridization analysis allowed us to order the 23 contigs and condense them into a single contig, yielding an estimated genome size of 5.1 Mb for P. synxantha BG33R. A minimum-tile path of 47 BACs was generated and end-sequenced. The genetic loci involved in ring nematode egg-kill factor production in BG33R Tn5 mutants, 246 (vgrG homolog), 1122 (sensor kinase homolog), 1233 (UDP-galactose epimerase homolog), 1397 (ferrisiderophore receptor homolog), and 1917 (ribosomal subunit protein homolog), have been mapped onto the minimum-tile BAC library. Two of the genetic regions that flank Tn5 insertions in BG33R egg-kill-negative mutants 1233 and 1397 are separated by a single BAC clone. Fragments isolated by ligation-mediated PCR of the Tn5 mutagenized regions of 29 randomly selected, non-egg-kill-related, insertion mutants have been anchored onto the ordered physical map of P. synxantha. PMID:11881828

Wechter, W P; Begum, D; Presting, G; Kim, J J; Wing, R A; Kluepfel, D A

2002-01-01

84

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

PubMed

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

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

2014-06-01

85

Genomic analysis and marker development for the Tsn1 locus in wheat using bin-mapped ESTs and flanking BAC contigs.  

PubMed

The wheat Tsn1 gene confers sensitivity to the host-selective toxin Ptr ToxA produced by the tan spot fungus (Pyrenophora tritici-repentis). The long-term goal of this research is to isolate Tsn1 using a positional cloning approach. Here, we evaluated 54 ESTs (expressed sequence tags) physically mapped to deletion bin 5BL 0.75-0.76, which is a gene-rich region containing Tsn1. Twenty-three EST loci were mapped as either PCR-based single-stranded conformational polymorphism or RFLP markers in a low-resolution wheat population. The genetic map corresponding to the 5BL 0.75-0.76 deletion bin spans 18.5 cM and contains 37 markers for a density of 2 markers/cM. The EST-based genetic map will be useful for tagging other genes, establishing colinearity with rice, and anchoring sequence ready BAC contigs of the 5BL 0.75-0.76 deletion bin. High-resolution mapping showed that EST-derived markers together with previously developed AFLP-derived markers delineated Tsn1 to a 0.8 cM interval. Flanking markers were used to screen the Langdon durum BAC library and contigs of 205 and 228 kb flanking Tsn1 were assembled, sequenced, and anchored to the genetic map. Recombination frequency averaged 760 kb/cM across the 228 kb contig, but no recombination was observed across the 205 kb contig resulting in an expected recombination frequency of more than 10 Mb/cM. Therefore, chromosome walking within the Tsn1 region may be difficult. However, the sequenced BACs allowed the identification of one microsatellite in each contig for which markers were developed and shown to be highly suitable for marker-assisted selection of Tsn1. PMID:16456656

Lu, H-J; Fellers, J P; Friesen, T L; Meinhardt, S W; Faris, J D

2006-04-01

86

Library Preparation and Data Analysis Packages for Rapid Genome Sequencing  

PubMed Central

High-throughput sequencing (HTS) has quickly become a valuable tool for comparative genetics and genomics and is now regularly carried out in laboratories that are not connected to large sequencing centers. Here we describe an updated version of our protocol for constructing single- and paired-end Illumina sequencing libraries, beginning with purified genomic DNA. The present protocol can also be used for “multiplexing,” i.e. the analysis of several samples in a single flowcell lane by generating “barcoded” or “indexed” Illumina sequencing libraries in a way that is independent from Illumina-supported methods. To analyze sequencing results, we suggest several independent approaches but end users should be aware that this is a quickly evolving field and that currently many alignment (or “mapping”) and counting algorithms are being developed and tested.

Pomraning, Kyle R.; Smith, Kristina M.; Bredeweg, Erin L.; Connolly, Lanelle R.; Phatale, Pallavi A.; Freitag, Michael

2013-01-01

87

Scanning the human genome with combinatorial transcription factor libraries  

Microsoft Academic Search

Despite the critical importance of transcription factors in mediating gene regulation, there exists no general, genome-wide tool that uses transcription factors to induce or silence a target gene or select for a particular phenotype. In the strategy described here, we prepared large combinatorial libraries of artificial transcription factors comprising three or six zinc-finger domains, and selected transcription factor–DNA interactions able

Pilar Blancafort; Laurent Magnenat; Carlos F. Barbas

2003-01-01

88

Sequencing of BAC pools by different next generation sequencing platforms and strategies  

PubMed Central

Background Next generation sequencing of BACs is a viable option for deciphering the sequence of even large and highly repetitive genomes. In order to optimize this strategy, we examined the influence of read length on the quality of Roche/454 sequence assemblies, to what extent Illumina/Solexa mate pairs (MPs) improve the assemblies by scaffolding and whether barcoding of BACs is dispensable. Results Sequencing four BACs with both FLX and Titanium technologies revealed similar sequencing accuracy, but showed that the longer Titanium reads produce considerably less misassemblies and gaps. The 454 assemblies of 96 barcoded BACs were improved by scaffolding 79% of the total contig length with MPs from a non-barcoded library. Assembly of the unmasked 454 sequences without separation by barcodes revealed chimeric contig formation to be a major problem, encompassing 47% of the total contig length. Masking the sequences reduced this fraction to 24%. Conclusion Optimal BAC pool sequencing should be based on the longest available reads, with barcoding essential for a comprehensive assessment of both repetitive and non-repetitive sequence information. When interest is restricted to non-repetitive regions and repeats are masked prior to assembly, barcoding is non-essential. In any case, the assemblies can be improved considerably by scaffolding with non-barcoded BAC pool MPs.

2011-01-01

89

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

PubMed Central

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

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

2006-01-01

90

Chromosome region-specific libraries for human genome analysis  

SciTech Connect

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

Kao, Fa-Ten.

1991-01-01

91

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.

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

2014-01-01

92

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

93

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

Array comparative genomic hybridization (aCGH) is a powerful tool for detecting DNA copy number alterations (CNA). Because diffuse malignant gliomas are often sampled by small biopsies, formalin-fixed paraffin-embedded (FFPE) blocks are often the only tissue available for genetic analysis; FFPE tissues are also needed to study the intratumoral heterogeneity that characterizes these neoplasms. In this paper, we present a combination of evaluations and technical advances that provide strong support for the ready use of oligonucleotide aCGH on FFPE diffuse gliomas. We first compared aCGH using bacterial artificial chromosome (BAC) arrays in 45 paired frozen and FFPE gliomas, and demonstrate a high concordance rate between FFPE and frozen DNA in an individual clone-level analysis of sensitivity and specificity, assuring that under certain array conditions, frozen and FFPE DNA can perform nearly identically. However, because oligonucleotide arrays offer advantages to BAC arrays in genomic coverage and practical availability, we next developed a method of labeling DNA from FFPE tissue that allows efficient hybridization to oligonucleotide arrays. To demonstrate utility in FFPE tissues, we applied this approach to biphasic anaplastic oligoastrocytomas and demonstrate CNA differences between DNA obtained from the two components. Therefore, BAC and oligonucleotide aCGH can be sensitive and specific tools for detecting CNAs in FFPE DNA, and novel labeling techniques enable the routine use of oligonucleotide arrays for FFPE DNA. In combination, these advances should facilitate genome-wide analysis of rare, small and/or histologically heterogeneous gliomas from FFPE tissues. 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

2011-04-01

94

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.

Warden, Charles; Tang, Qiyi; Zhu, Hua

2011-01-01

95

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

PubMed

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

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

2013-01-01

96

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.

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

97

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

PubMed

Spartina species play an important ecological role on salt marshes. Spartina maritima is an Old-World species distributed along the European and North-African Atlantic coasts. This hexaploid species (2n = 6x = 60, 2C = 3,700 Mb) hybridized with different Spartina species introduced from the American coasts, which resulted in the formation of new invasive hybrids and allopolyploids. Thus, S. maritima raises evolutionary and ecological interests. However, genomic information is dramatically lacking in this genus. In an effort to develop genomic resources, we analysed 40,641 high-quality bacterial artificial chromosome-end sequences (BESs), representing 26.7 Mb of the S. maritima genome. BESs were searched for sequence homology against known databases. A fraction of 16.91% of the BESs represents known repeats including a majority of long terminal repeat (LTR) retrotransposons (13.67%). Non-LTR retrotransposons represent 0.75%, DNA transposons 0.99%, whereas small RNA, simple repeats and low-complexity sequences account for 1.38% of the analysed BESs. In addition, 4,285 simple sequence repeats were detected. Using the coding sequence database of Sorghum bicolor, 6,809 BESs found homology accounting for 17.1% of all BESs. Comparative genomics with related genera reveals that the microsynteny is better conserved with S. bicolor compared to other sequenced Poaceae, where 37.6% of the paired matching BESs are correctly orientated on the chromosomes. We did not observe large macrosyntenic rearrangements using the mapping strategy employed. However, some regions appeared to have experienced rearrangements when comparing Spartina to Sorghum and to Oryza. This work represents the first overview of S. maritima genome regarding the respective coding and repetitive components. The syntenic relationships with other grass genomes examined here help clarifying evolution in Poaceae, S. maritima being a part of the poorly-known Chloridoideae sub-family. PMID:23877482

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

2013-12-01

98

Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice ( Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome  

Microsoft Academic Search

The International Rice Genome Sequencing Project has recently announced the high-quality finished sequence that covers nearly\\u000a 95% of the japonica rice genome representing 370 Mbp. Nevertheless, the current physical map of japonica rice contains 62 physical gaps corresponding to approximately 5% of the genome, that have not been identified\\/represented\\u000a in the comprehensive array of publicly available BAC, PAC and other genomic

Jetty S. S. Ammiraju; Yeisoo Yu; Meizhong Luo; Dave Kudrna; HyeRan Kim; Jose L. Goicoechea; Yuichi Katayose; Takashi Matsumoto; Jianzhong Wu; Takuji Sasaki; Rod A. Wing

2005-01-01

99

Postintegration stability of the silkworm piggyBac transposon.  

PubMed

The piggyBac transposon is the most widely used vector for generating transgenic silkworms. The silkworm genome contains multiple piggyBac-like sequences that might influence the genetic stability of transgenic lines. To investigate the postintegration stability of piggyBac in silkworms, we used random insertion of the piggyBac [3 × p3 EGFP afm] vector to generate a W chromosome-linked transgenic silkworm, named W-T. Results of Southern blot and inverse PCR revealed the insertion of a single copy in the W chromosome of W-T at a standard TTAA insertion site. Investigation of 11 successive generations showed that all W-T females were EGFP positive and all males were EGFP negative; PCR revealed that the insertion site was unchanged in W-T offspring. These results suggested that endogenous piggyBac-like elements did not affect the stability of piggyBac inserted into the silkworm genome. PMID:24727025

Jiang, Liang; Sun, Qiang; Liu, Weiqiang; Guo, Huizhen; Peng, Zhengwen; Dang, Yinghui; Huang, Chunlin; Zhao, Ping; Xia, Qingyou

2014-07-01

100

W-derived BAC probes as a new tool for identification of the W chromosome and its aberrations in Bombyx mori.  

PubMed

We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome. PMID:12827381

Sahara, Ken; Yoshido, Atsuo; Kawamura, Naoko; Ohnuma, Akio; Abe, Hiroaki; Mita, Kazuei; Oshiki, Toshikazu; Shimada, Toru; Asano, Shin-Ichiro; Bando, Hisanori; Yasukochi, Yuji

2003-07-01

101

Physical Mapping in a Triplicated Genome: Mapping the Downy Mildew Resistance Locus Pp523 in Brassica oleracea L.  

PubMed Central

We describe the construction of a BAC contig and identification of a minimal tiling path that encompass the dominant and monogenically inherited downy mildew resistance locus Pp523 of Brassica oleracea L. The selection of BAC clones for construction of the physical map was carried out by screening gridded BAC libraries with DNA overgo probes derived from both genetically mapped DNA markers flanking the locus of interest and BAC-end sequences that align to Arabidopsis thaliana sequences within the previously identified syntenic region. The selected BAC clones consistently mapped to three different genomic regions of B. oleracea. Although 83 BAC clones were accurately mapped within a ?4.6 cM region surrounding the downy mildew resistance locus Pp523, a subset of 33 BAC clones mapped to another region on chromosome C8 that was ?60 cM away from the resistance gene, and a subset of 63 BAC clones mapped to chromosome C5. These results reflect the triplication of the Brassica genomes since their divergence from a common ancestor shared with A. thaliana, and they are consonant with recent analyses of the C genome of Brassica napus. The assembly of a minimal tiling path constituted by 13 (BoT01) BAC clones that span the Pp523 locus sets the stage for map-based cloning of this resistance gene.

Carlier, Jorge D.; Alabaca, Claudia S.; Sousa, Nelson H.; Coelho, Paula S.; Monteiro, Antonio A.; Paterson, Andrew H.; Leitao, Jose M.

2011-01-01

102

A small-insert bovine genomic library highly enriched for microsatellite repeat sequences.  

PubMed

A bovine genomic phagemid library was constructed with randomly sheared DNA. Enrichment of this single-stranded DNA library with CA or GT primers resulted in 45% positive clones. The 14% of positive clones with (CA.GT) > 12, and not containing flanking repetitive elements, were sequenced, and the efficiency of marker production was compared with random M13 bacteriophage libraries. Primer sequences and genotyping information are presented for 390 informative bovine microsatellite markers. The genomic frequency for 11 tri- and tetranucleotide repeats was estimated by hybridization to a lambda genomic library. Only GCT, GGT, and GGAT were estimated to have a frequency of > 100 per genome. Enrichment of the phagemid library for these repeats failed to provide a viable source of microsatellite markers in the bovine. Comparison of map interval lengths between 100 markers from the enriched library prepared from randomly sheared DNA and M13 bacteriophage libraries prepared from Mbo1 restriction digests suggested no bias in skeletal genomic coverage based on source of small insert DNA. In conclusion, enrichment of the bovine phagemid library provides a sufficient source of microsatellites so that small repeat lengths and flanking repetitive sequences common in the bovine can be eliminated, resulting in a high percentage of informative markers. PMID:8563170

Stone, R T; Pulido, J C; Duyk, G M; Kappes, S M; Keele, J W; Beattie, C W

1995-10-01

103

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

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

1992-01-01

104

Employing BAC-reporter constructs in the sea anemone Nematostella vectensis.  

PubMed

Changes in the expression and function of genes drive evolutionary change. Comparing how genes are regulated in different species is therefore becoming an important part of evo-devo studies. A key tool for investigating the regulation of genes is represented by bacterial artificial chromosomes (BAC)-reporter constructs. BACs are large insert libraries, often >100 kb, which thus capture the genomic sequences surrounding a gene of interest, including all, or nearly all, of the elements underpinning regulation. Recombinant BACs, containing a reporter gene in place of the endogenous coding sequence of genes, can be utilized to drive the expression of reporter genes under the regulatory control of the gene of interest while still embedded within its genomic context. Systematic deletions within the BAC-reporter construct can be used to identify the minimal reporter in an unbiased way, avoiding the risk of overlooking regulatory elements that may be many kilobases away from the transcription start-site. Nematostella vectensis (Edwardsiidae, Anthozoa, Cnidaria) has become an important model in regenerative biology, ecology, and especially in studies of evo-devo and gene-regulatory networks due to its interesting phylogenetic position and amenability to molecular techniques. The increasing interest in this rising model system also led to a demand for methods that can be used to study the regulation of genes in Nematostella. Here, we present our progress in employing BAC-reporter constructs to visualize gene-expression in Nematostella. Using a new Nematostella-specific recombination cassette, we made nine different BAC-reporter constructs. Although five BAC recombinants gave variable effects, three constructs, namely Nv-bra:eGFP::L10 BAC, Nv-dpp:eGFP::L10 BAC, and Nv-grm:eGFP::L10 BAC delivered promising results. We show that these three constructs express the reporter gene eGFP in 10.4-17.2% of all analyzed larvae, out of which 26.2-41.9% express GFP in a mosaic fashion within the expected domain. In addition to the expression within the known domains, we also observed cases of misexpression of eGFP and examples that could represent actual expression outside the described domain. Furthermore, we deep-sequenced and assembled five different BACs containing Nv-chordin, Nv-foxa, Nv-dpp, Nv-wnta, and Nv-wnt1, to improve assembly around these genes. The use of BAC-reporter constructs will foster cis-regulatory analyses in Nematostella and thus help to improve our understanding of the regulatory network in this cnidarian system. Ultimately, this will advance the comparison of gene-regulation across species and lead to a much better understanding of evolutionary changes and novelties. PMID:23956207

Fischer, Antje H L; Tulin, Sarah; Fredman, David; Smith, Joel

2013-11-01

105

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.

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

2011-01-01

106

Construction and characterisation of a large DNA insert library from the D genome of wheat  

Microsoft Academic Search

A large DNA fragment library consisting of 144?000 clones with an average insert size of 119 kb was constructed from nuclear\\u000a DNA isolated from root and leaf tissue from Triticum tauschii (syn. Aegilops tauschii), the D-genome progenitor of wheat. The library was made in a binary vector that had previously been shown to stably maintain\\u000a large inserts of foreign DNA

O. Moullet; H.-B. Zhang; E. S. Lagudah

1999-01-01

107

RepARK--de novo creation of repeat libraries from whole-genome NGS reads  

PubMed Central

Generation of repeat libraries is a critical step for analysis of complex genomes. In the era of next-generation sequencing (NGS), such libraries are usually produced using a whole-genome shotgun (WGS) derived reference sequence whose completeness greatly influences the quality of derived repeat libraries. We describe here a de novo repeat assembly method—RepARK (Repetitive motif detection by Assembly of Repetitive K-mers)—which avoids potential biases by using abundant k-mers of NGS WGS reads without requiring a reference genome. For validation, repeat consensuses derived from simulated and real Drosophila melanogaster NGS WGS reads were compared to repeat libraries generated by four established methods. RepARK is orders of magnitude faster than the other methods and generates libraries that are: (i) composed almost entirely of repetitive motifs, (ii) more comprehensive and (iii) almost completely annotated by TEclass. Additionally, we show that the RepARK method is applicable to complex genomes like human and can even serve as a diagnostic tool to identify repetitive sequences contaminating NGS datasets.

Koch, Philipp; Platzer, Matthias; Downie, Bryan R.

2014-01-01

108

RepARK-de novo creation of repeat libraries from whole-genome NGS reads.  

PubMed

Generation of repeat libraries is a critical step for analysis of complex genomes. In the era of next-generation sequencing (NGS), such libraries are usually produced using a whole-genome shotgun (WGS) derived reference sequence whose completeness greatly influences the quality of derived repeat libraries. We describe here a de novo repeat assembly method-RepARK (Repetitive motif detection by Assembly of Repetitive K-mers)-which avoids potential biases by using abundant k-mers of NGS WGS reads without requiring a reference genome. For validation, repeat consensuses derived from simulated and real Drosophila melanogaster NGS WGS reads were compared to repeat libraries generated by four established methods. RepARK is orders of magnitude faster than the other methods and generates libraries that are: (i) composed almost entirely of repetitive motifs, (ii) more comprehensive and (iii) almost completely annotated by TEclass. Additionally, we show that the RepARK method is applicable to complex genomes like human and can even serve as a diagnostic tool to identify repetitive sequences contaminating NGS datasets. PMID:24634442

Koch, Philipp; Platzer, Matthias; Downie, Bryan R

2014-05-01

109

A 4Mb BAC\\/PAC contig and complete genomic structure of the GPC5\\/ GPC6 gene cluster on chromosome 13q32  

Microsoft Academic Search

The glypicans compose a family of glycosylphosphatidylinositol-anchored heparan sulfate proteoglycans that may play a role in the control of cell division and growth regulation. So far, six members (GPC1–6) of this family are known in vertebrates. We report the construction of a high-resolution 4 Mb sequence-ready BAC\\/PAC contig of the GPC5\\/GPC6 gene cluster on chromosome region 13q32. The contig indicates

Mark Veugelers; Bart De Cat; Nathalie Delande; Carl Esselens; Ines Bonk; Joris Vermeesch; Peter Marynen; Jean-Pierre Fryns; Guido David

2001-01-01

110

A genome-wide survey of switchgrass genome structure and organization.  

PubMed

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

111

A framework for sequencing the rice genome.  

PubMed

Rice is an important food crop and a model plant for other cereal genomes. The Clemson University Genomics Institute framework project, begun two years ago in anticipation of the now ongoing international effort to sequence the rice genome, is nearing completion. Two bacterial artificial chromosome (BAC) libraries have been constructed from the Oryza sativa cultivar Nipponbare. Over 100,000 BAC end sequences have been generated from these libraries and, at a current total of 28 Mbp, represent 6.5% of the total rice genome sequence. This sequence information has allowed us to draw first conclusions about unique and redundant rice genomic sequences. In addition, more than 60,000 clones (19 genome equivalents) have been successfully fingerprinted and assembled into contigs using FPC software. Many of these contigs have been anchored to the rice chromosomes using a variety of techniques. Hybridization experiments have shown these contigs to be very robust. Contig assembly and hybridization experiments have revealed some surprising insights into the organization of the rice genome, which will have significant repercussions for the sequencing effort. Integration of BAC end sequence data with anchored contig information has provided unexpected revelations on sequence organization at the chromosomal level. PMID:11387975

Presting, G G; Budiman, M A; Wood, T; Yu, Y; Kim, H R; Goicoechea, J L; Fang, E; Blackman, B; Jiang, J; Woo, S S; Dean, R A; Frisch, D; Wing, R A

2001-01-01

112

PCR-Based Ordered Genomic Libraries: a New Approach to Drug Target Identification for Streptococcus pneumoniae  

Microsoft Academic Search

Described here are the development and validation of a novel approach to identify genes encoding drug targets in Streptococcus pneumoniae. The method relies on the use of an ordered genomic library composed of PCR amplicons that were generated under error-prone conditions so as to introduce random mutations into the DNA. Since some of the mutations occur in drug target-encoding genes

Aimee E. Belanger; Angel Lai; Marcia A. Brackman; Donald J. LeBlanc

2002-01-01

113

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

Microsoft Academic Search

An in vitro protein selection method, ribosome display, has been applied to comprehensively identify and map the immunologically relevant proteins of the human pathogen Staphylococcus aureus. A library built up from genomic fragments of the virulent S. aureus COL strain (methicillin-resistant S. aureus) allowed us to screen all possible encoded peptides for immunoreactivity. As selective agents, human sera exhibiting a

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

2003-01-01

114

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

PubMed

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

Summer, Elizabeth J

2009-01-01

115

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.

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

116

Preparation and screening of an arrayed human genomic library generated with the P1 cloning system  

SciTech Connect

The authors describe here the construction and initial characterization of a 3-fold coverage genomic library of the human haploid genome that was prepared using the bacteriophase P1 cloning system. The cloned DNA inserts were produced by size fractionation of a Sau3AI partial digest of high molecular weight genomic DNA isolated from primary cells of human foreskin fibroblasts. The inserts were cloned into the pAd10sacBII vector and packaged in vitro into P1 phage. These were used to generate recombinant bacterial clones, each of which was picked robotically from an agar plate into a well of a 96-well microtiter dish, grown overnight, and stored at -70{degrees}C. The resulting library, designated DMPC-HFF No. 1 series A, consists of {approximately}130,000-140,000 recombinant clones that were stored in 1500 microtiter dishes. To screen the library, clones were combined in a pooling strategy and specific loci were identified by PCR analysis. On average, the library contains two or three different clones for each locus screened. To date the authors have identified a total of 17 clones containing the hypoxanthine-guanine phosphoribosyltransferase, human serum albumin-human {alpha}-fetoprotein, p53, cyclooxygenase I, human apurinic endonuclease, {beta}-polymerase, and DNA ligase I genes. The cloned inserts average 80 kb in size and range from 70 to 95 kb, with one 49-kb insert and one 62-kb insert. 19 refs., 3 figs., 1 tab.

Shepherd, N.S.; Perogner, B.D.; Coulby, J.N.; Ackerman, S.L.; Vaidyanathan, G.; Sauer, R.H.; Balkenhol, T.C.; Sternberg, N. [Dupont Merck Pharmaceutical Co., Wilmington, DE (United States)]|[Central Research and Development Engineering Department and Service Division, Wilmington, DE (United States)

1994-03-29

117

[Construction of the genomic library for cyanobacteria Synechocystis sp. PCC 6803 using a cosmid].  

PubMed

The gene library of the phototrophic cyanobacterium Synechocystis PCC6803 was constructed in the cosmid vector Lorist6. About -3 x 10(3) recombinant clones were obtained, of which 760 were selected for further work. Each clone was kept in an individual well of a microtiter plate. The clone grids obtained were hybridized with two DNA probes, which contained the ndhE and dfr genes of Synechocystis PCC6803. Two groups of probe-overlapping cosmids (11 and 8 clones respectively), containing insertions averaging of 39 kb, were isolated. The capacity of the gene library is nine equivalents of the Synechocystis PCC6803 genome and its representativity exceeds 99%. PMID:8045394

Shestopalov, V I; Nashchokina, O O; Shestakov, S V; Iankovski?, N K

1994-04-01

118

Molecular analysis of DNA and construction of genomic libraries of Mycobacterium leprae.  

PubMed Central

Molecular analysis of DNA from Mycobacterium leprae, "Mycobacterium lufu," and Mycobacterium vaccae has demonstrated that the G + C (guanine plus cytosine) contents of the DNAs are 56, 61, and 65%, respectively, and that the genome sizes are 2.2 X 10(9), 3.1 X 10(9), and 3.1 X 10(9) daltons, respectively. Because of the significant differences in both G + C content and genome size among M. leprae, "M. lufu," and M. vaccae DNAs, these species are not related, although hybridization experiments under nonstringent conditions, with two separate cloned M. leprae DNA inserts as probes, indicate that there are some conserved sequences among the DNAs. The G + C content of Dasypus novemcinctus (armadillo, the animal of choice for cultivating M. leprae) DNA was determined to be 36%. Genomic libraries potentially representing more than 99.99% of each genome were prepared by cloning into the cosmid vector, pHC79, in Escherichia coli K-12. A genomic library representing approximately 95% of the genome of M. vaccae was prepared in pBR322. M. leprae DNA was subcloned from the pHC79::M. leprae library into an expression vector, pYA626. This vector is a 3.8-kilobase derivative of pBR322 in which the promoter region of the asd (aspartate semialdehyde dehydrogenase) gene from Streptococcus mutans has been inserted in place of the EcoRI-to-PstI fragment of pBR322. Several (44% of those tested) pYA626::M. leprae recombinants and one pBR322::M. vaccae recombinant synthesized new polypeptides in minicells of E. coli, indicating that mycobacterial DNA can be expressed in E. coli K-12, although expression is probably dependent upon use of nonmycobacterial promoters recognized by the E. coli transcription-translation apparatus. Images

Clark-Curtiss, J E; Jacobs, W R; Docherty, M A; Ritchie, L R; Curtiss, R

1985-01-01

119

Transposon-mediated BAC transgenesis in human ES cells.  

PubMed

Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs. PMID:22753106

Rostovskaya, Maria; Fu, Jun; Obst, Mandy; Baer, Isabell; Weidlich, Stefanie; Wang, Hailong; Smith, Andrew J H; Anastassiadis, Konstantinos; Stewart, A Francis

2012-10-01

120

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

Microsoft Academic Search

OmniPlex Technology is a new approach to genome amplification and targeted analysis. Initially the entire genome is reformatted into small, amplifiable molecules called Plexisomes, which represent the entire genome as an OmniPlex Library. The whole genome can be amplified en masse using universal primers; using locus-specific primers, regions as large as 50 kb can be amplified. Amplified Plexisomes can be

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

2002-01-01

121

Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.  

PubMed Central

We constructed a partial Sau3A Dictyostelium genomic DNA library in a shuttle vector that replicates extrachromosomally in Dictyostelium cells. This library was used to complement Dictyostelium strain HPS400, which lacks thymidylate synthase activity and requires exogenous thymidine for growth. We have used a modified high-frequency transformation protocol that allows the introduction of the library into a sufficient number of Dictyostelium cells to select complementing plasmids. Using this approach, we have isolated a gene (Thy1) that complements the thymidine growth requirement of HPS400. The gene encodes a 1.2-kilobase RNA and the derived amino acid sequence shows no homology to thymidylate synthase, a protein highly conserved throughout evolution, or any other protein sequence in the data base examined. Thy1 provides an important selectable marker for transforming Dictyostelium cells. In addition, this work suggests that it will be possible to isolate genes that are essential for developmental processes in Dictyostelium by complementation. Images

Dynes, J L; Firtel, R A

1989-01-01

122

Molecular complementation of a genetic marker in Dictyostelium using a genomic DNA library.  

PubMed

We constructed a partial Sau3A Dictyostelium genomic DNA library in a shuttle vector that replicates extrachromosomally in Dictyostelium cells. This library was used to complement Dictyostelium strain HPS400, which lacks thymidylate synthase activity and requires exogenous thymidine for growth. We have used a modified high-frequency transformation protocol that allows the introduction of the library into a sufficient number of Dictyostelium cells to select complementing plasmids. Using this approach, we have isolated a gene (Thy1) that complements the thymidine growth requirement of HPS400. The gene encodes a 1.2-kilobase RNA and the derived amino acid sequence shows no homology to thymidylate synthase, a protein highly conserved throughout evolution, or any other protein sequence in the data base examined. Thy1 provides an important selectable marker for transforming Dictyostelium cells. In addition, this work suggests that it will be possible to isolate genes that are essential for developmental processes in Dictyostelium by complementation. PMID:2813371

Dynes, J L; Firtel, R A

1989-10-01

123

Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements.  

PubMed

We report the heritable germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP. A transformation frequency of 5-10% was obtained. Inheritance of the transgenes has remained stable over more than 15 generations despite the presence of endogenous piggyBac sequences in the B. tryoni genome. The sequence of insertion sites shows the usual canonical pattern of piggyBac integraton into TTAA target sites. An investigation of endogenous piggyBac elements in the B. tryoni genome reveals the presence of sequences almost identical to those reported recently for the B. dorsalis complex of fruit flies and two noctuid moths, suggesting a common origin of piggyBac sequences in these species. The availability of transformation protocols for B. tryoni has the potential to deliver improvements in the performance of the Sterile Insect Technique for this pest species. PMID:20859652

Raphael, K A; Shearman, D C A; Streamer, K; Morrow, J L; Handler, A M; Frommer, M

2011-01-01

124

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

125

A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.  

PubMed

Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan. PMID:17625558

Dietzl, Georg; Chen, Doris; Schnorrer, Frank; Su, Kuan-Chung; Barinova, Yulia; Fellner, Michaela; Gasser, Beate; Kinsey, Kaolin; Oppel, Silvia; Scheiblauer, Susanne; Couto, Africa; Marra, Vincent; Keleman, Krystyna; Dickson, Barry J

2007-07-12

126

Manipulating piggyBac Transposon Chromosomal Integration Site Selection in Human Cells  

PubMed Central

The ability to direct gene delivery to a user-defined chromosomal location would greatly improve gene transfer applications. The piggyBac transposon system is a nonviral gene transfer system proven effective in a variety of cells and tissues, including human cells. We fused a highly site-specific synthetic zinc-finger DNA-binding domain (ZFP) to the N-terminus of the piggyBac transposase and evaluated site-directed genomic integration in human cells. Chimeric ZFP-piggyBac transposase exhibited robust gene transfer activity, targeted binding to a cognate endogenous chromosomal ZFP site in the human genome, and site-directed transposon integration into an episomal plasmid target containing a single ZFP site in human cells. We evaluated the ability of ZFP-piggyBac to direct gene integration into an engineered chromosomal ZFP target site in the human genome and consistently observed a higher degree of ZFP-piggyBac site-directed genomic integration when compared to native piggyBac. Chromatin immunoprecipitation (ChIP) experiments revealed binding of native piggyBac to our engineered TTAA-containing chromosomal target which supported integration, but not a TTAA-deficient chromosomal target which lacked integration. Our results offer insight into the requirements for using a chimeric zinc finger-piggyBac transposase to direct integration into a user-defined chromosomal location.

Kettlun, Claudia; Galvan, Daniel L; George Jr, Alfred L; Kaja, Aparna; Wilson, Matthew H

2011-01-01

127

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

Microsoft Academic Search

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results

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

2008-01-01

128

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

PubMed Central

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

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

2013-01-01

129

A Novel Helper Phage Enabling Construction of Genome-Scale ORF-Enriched Phage Display Libraries  

PubMed Central

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K.

2013-01-01

130

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

131

[Creation of genomic DNA libraries for Pseudomonas mallei and Pseudomonas pseudomallei].  

PubMed

The genomic libraries of P. mallei and P. pseudomallei species were constructed in Escherichia coli. The chromosomal DNA of P. pseudomallei C-141 strain has been cloned into the cosmid vector pHC79 and the broad host range plasmid vector pES154. The chromosomal DNA of P. mallei [symbol: see text]-5 strain has been cloned into the plasmid vector pSUP202. The recombinant clones of the genomic libraries were screened by the enzyme-linked immunoadsorbent assay (ELISA) to detect the production of Pseudomonas antigens: 28 clones were positive. Twelve recombinant strains demonstrated specific antigenic determinants of P. mallei and P. pseudomallei by immunoblotting. Cloned proteins of P. mallei and P. pseudomallei have molecular weights from 30 to 70 kD. A new method for introducing foreign genes into Pseudomonas genomes is offered. P. mallei strains with the chromosomally integrated plasmids pSM are universal recipients for ColEI-based cloning vectors. PMID:9082182

Abaev, I V; Pomerantseva, O M; Astashkin, E I; Pachkunov, D M; Rudnitski?, V Iu; Stagis, I I; Negri?, N V; Bannov, V A; Svetoch, E A

1997-01-01

132

A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions  

PubMed Central

Traditional chromatin analysis methods only test one locus at the time or use different templates for each locus, making a standardized analysis of large genomic regions or many co-regulated genes at different loci a difficult task. On the other hand, genome-wide high-resolution mapping of chromatin accessibility employing massive parallel sequencing platforms generates an extensive data set laborious to analyse and is a cost-intensive method, only applicable to the analysis of a limited set of biological samples. To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis. To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus. Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR.

Basheer, Asjad; Berger, Harald; Reyes-Dominguez, Yazmid; Gorfer, Markus; Strauss, Joseph

2009-01-01

133

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

PubMed

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

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

2013-02-01

134

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

135

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

PubMed Central

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

2011-01-01

136

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.

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

2013-01-01

137

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

138

Screening of cell death genes with a mammalian genome-wide RNAi library.  

PubMed

We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding approximately 200,000 shRNA sequences that targeted 47,400 human transcripts were inserted into a lentivirus vector pFIV-H1-puro, and a pool of pseudovirus particles with a complexity of approximately 200,000 were used to infect target cells. From the cells surviving apoptogenic Fas stimulation, four candidate shRNA sequences were obtained that provided resistance to Fas-induced cell death, including two shRNAs for caspase-8, an shRNA for Bid, and an shRNA for Fas. The reconstructed shRNAs with these sequences were shown to reduce expression of the respective gene products and increase survival after Fas stimulation. When similar selection was performed for tunicamycin-induced apoptosis, no shRNA strongly inhibiting tunicamycin-induced cell death was isolated, although a few reconstructed shRNAs led to a slight increase of survival. Thus, this genome-wide shRNA library proved useful for selection of genes that are involved in cell death, but some limitation was also revealed. PMID:20421362

Tsujii, Hisayo; Eguchi, Yutaka; Chenchik, Alex; Mizutani, Takayuki; Yamada, Kayoko; Tsujimoto, Yoshihide

2010-08-01

139

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

140

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

PubMed

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

141

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.

142

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

143

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.

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

2013-01-01

144

Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries  

PubMed Central

Comprehensive identification of sequences that regulate transcription is one of the major goals of genome biology. Focal alteration in chromatin structure in vivo, detectable through hypersensitivity to DNaseI and other nucleases, is the sine qua non of a diverse cast of transcriptional regulatory elements including enhancers, promoters, insulators, and locus control regions. We developed an approach for genome-scale identification of DNaseI hypersensitive sites (HSs) via isolation and cloning of in vivo DNaseI cleavage sites to create libraries of active chromatin sequences (ACSs). Here, we describe analysis of >61,000 ACSs derived from erythroid cells. We observed peaks in the density of ACSs at the transcriptional start sites of known genes at non-gene-associated CpG islands, and, to a lesser degree, at evolutionarily conserved noncoding sequences. Peaks in ACS density paralleled the distribution of DNaseI HSs. ACSs and DNaseI HSs were distributed between both expressed and nonexpressed genes, suggesting that a large proportion of genes reside within open chromatin domains. The results permit a quantitative approximation of the distribution of HSs and classical cis-regulatory sequences in the human genome.

Sabo, Peter J.; Humbert, Richard; Hawrylycz, Michael; Wallace, James C.; Dorschner, Michael O.; McArthur, Michael; Stamatoyannopoulos, John A.

2004-01-01

145

Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice ( Oryza sativa L.)  

Microsoft Academic Search

The growing number of rice microsatellite markers warrants a comprehensive comparison of allelic variability between the markers\\u000a developed using different methods, with various sequence repeat motifs, and from coding and non-coding portions of the genome.\\u000a We have performed such a comparison over a set of 323 microsatellite markers; 194 were derived from genomic library screening\\u000a and 129 were derived from

Y. G. Cho; T. Ishii; S. Temnykh; X. Chen; L. Lipovich; S. R. McCouch; W. D. Park; N. Ayres; S. Cartinhour

2000-01-01

146

Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library  

Microsoft Academic Search

Background: Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracel- lular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. Results: We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames,

Da-Qiao Ding; Yuki Tomita; Ayumu Yamamoto; Yuji Chikashige; Tokuko Haraguchi; Yasushi Hiraoka

2000-01-01

147

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

Microsoft Academic Search

Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable\\u000a advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library\\u000a construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the

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

2005-01-01

148

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

PubMed Central

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

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

2012-01-01

149

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

PubMed

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

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

2012-01-01

150

Manipulating piggyBac Transposon Chromosomal Integration Site Selection in Human Cells  

Microsoft Academic Search

The ability to direct gene delivery to a user-defined chromosomal location would greatly improve gene transfer applications. The piggyBac transposon system is a nonviral gene transfer system proven effective in a variety of cells and tissues, including human cells. We fused a highly site-specific synthetic zinc-finger DNA-binding domain (ZFP) to the N-terminus of the piggyBac transposase and evaluated site-directed genomic

Claudia Kettlun; Daniel L Galvan; Alfred L George Jr; Aparna Kaja; Matthew H Wilson

2011-01-01

151

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.

152

Mutator System Derivatives Isolated from Sugarcane Genome Sequence.  

PubMed

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

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

2012-09-01

153

A first generation integrated map of the rainbow trout genome  

PubMed Central

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

2011-01-01

154

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

PubMed Central

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.

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

2014-01-01

155

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

PubMed

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

2009-05-15

156

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.

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

2009-01-01

157

An Efficient Method for High-Fidelity BAC/PAC Retrofitting with a Selectable Marker for Mammalian Cell Transfection  

PubMed Central

Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BACs in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination.

Wang, Zunde; Engler, Peter; Longacre, Angelika; Storb, Ursula

2001-01-01

158

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

159

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

PubMed

The Asian corn borer, Ostrinia furnacalis, is a serious pest of corn, sorghum, and cotton in China and other Asian countries. The present study is the first attempt to establish the transgenic line in O. furnacalis using a piggyBac transposon, which will shed light on the future genetic control of O. furnacalis. A piggyBac vector pBac[A3EGFP] was constructed to express enhanced green fluorescence protein (EGFP)under the control of Bombyx mori actin3 promoter. Transient EGFP expression was detected 48 h after preblastodermic microinjection of pBac[A3EGFP] and the excision assay showed the transgenic vector was precisely excised. In G1 animals, PCR (polymerase chain reaction)-based investigations revealed that the exogenous gene had been introduced into O. furnacalis genome and expressed at the transcriptional level. Western blot analysis showed EGFP expression at the protein level, indicating the heritability of the transgene. PMID:22696097

Liu, Dan; Yan, Shanchun; Huang, Yongping; Tan, Anjiang; Stanley, David W; Song, Qisheng

2012-08-01

160

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

PubMed Central

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

Ritschel, Patricia Silva; Lins, Tulio Cesar de Lima; Tristan, Rodrigo Lourenco; Buso, Glaucia Salles Cortopassi; Buso, Jose Amauri; Ferreira, Marcio Elias

2004-01-01

161

Mutagenesis by imprecise excision of the piggyBac transposon in Drosophila melanogaster.  

PubMed

Mutagenesis by transposon-mediated imprecise excision is the most extensively used technique for mutagenesis in Drosophila. Although P-element is the most widely used transposon in Drosophila to generate deletion mutants, it is limited by the insertion coldspots in the genome where P-elements are rarely found. The piggyBac transposon was developed as an alternative mutagenic vector for mutagenesis of non-P-element targeted genes in Drosophila because the piggyBac transposon can more randomly integrate into the genome. Previous studies suggested that the piggyBac transposon always excises precisely from the insertion site without initiating a deletion or leaving behind an additional footprint. This unique characteristic of the piggyBac transposon facilitates reversible gene-transfer in several studies, such as the generation of induced pluripotent stem (iPS) cells from fibroblasts. However, it also raised a potential limitation of its utility in generating deletion mutants in Drosophila. In this study, we report multiple imprecise excisions of the piggyBac transposon at the sepiapterin reductase (SR) locus in Drosophila. Through imprecise excision of the piggyBac transposon inserted in the 5'-UTR of the SR gene, we generated a hypomorphic mutant allele of the SR gene which showed markedly decreased levels of SR expression. Our finding suggests that it is possible to generate deletion mutants by piggyBac transposon-mediated imprecise excision in Drosophila. However, it also suggests a limitation of piggyBac transposon-mediated reversible gene transfer for the generation of induced pluripotent stem (iPS) cells. PMID:22155246

Kim, Heuijong; Kim, Kiyoung; Kim, Jaekwang; Kim, Song-Hee; Yim, Jeongbin

2012-01-01

162

454 sequencing of pooled BAC clones on chromosome 3H of barley  

Microsoft Academic Search

Background  Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp). Among the fast sequencing systems,\\u000a 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report\\u000a the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H.\\u000a \\u000a \\u000a \\u000a \\u000a Results  We sequenced pooled barley

Kazuhiro Sato; Yuka Motoi; Nami Yamaji; Hideya Yoshida

2011-01-01

163

Clone libraries and single cell genome amplification reveal extended diversity of uncultivated magnetotactic bacteria from marine and freshwater environments.  

PubMed

Magnetotactic bacteria (MTB), which orient along the earth's magnetic field using magnetosomes, are ubiquitous and abundant in marine and freshwater environments. Previous phylogenetic analysis of diverse MTB has been limited to few cultured species and the most abundant and conspicuous members of natural populations, which were assigned to various lineages of the Proteobacteria, the Nitrospirae phylum as well as the candidate division OP3. However, their known phylogenetic diversity still not matches the large morphological and ultrastructural variability of uncultured MTB found in environmental communities. Here, we used analysis of 16S rRNA gene clone libraries in combination with microsorting and whole-genome amplification to systematically address the entire diversity of uncultured MTB from two different habitats. This approach revealed extensive and novel diversity of MTB within the freshwater and marine sediment samples. In total, single-cell analysis identified eight different phylotypes, which were only partly represented in the clone libraries, and which could be unambiguously assigned to their respective morphotypes. Identified MTB belonged to the Alphaproteobacteria (seven species) and the Nitrospirae phylum (two species). End-sequencing of a small insert library created from WGA-derived DNA of a novel conspicuous magnetotactic vibrio identified genes with highest similarity to two cultivated MTB as well as to other phylogenetic groups. In conclusion, the combination of metagenomic cloning and single cell sorting represents a powerful approach to recover maximum bacterial diversity including low-abundant magnetotactic phylotypes from environmental samples and also provides access to genomic analysis of uncultivated MTB. PMID:23106823

Kolinko, Sebastian; Wanner, Gerhard; Katzmann, Emanuel; Kiemer, Felizitas; Fuchs, Bernhard M; Schüler, Dirk

2013-05-01

164

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

NASA Astrophysics Data System (ADS)

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

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

2002-06-01

165

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.

2012-01-01

166

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

167

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

PubMed

Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes. PMID:24717434

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

2014-05-22

168

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.

2010-01-01

169

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

170

Identification of Promoter Regions in the Human Genome by Using a Retroviral Plasmid Library-Based Functional Reporter Gene Assay  

PubMed Central

Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a functional selection for promoter regions in the human genome that uses a retroviral plasmid library-based system. This approach enriches for and detects promoter function of isolated DNA fragments in an in vitro cell culture assay. By using this method, we have discovered likely promoters of known and predicted genes, as well as many other putative promoter regions based on the presence of features such as CpG islands. Comparison of sequences of 858 plasmid clones selected by this assay with the human genome draft sequence indicates that a significantly higher percentage of sequences align to the 500-bp segment upstream of the transcription start sites of known genes than would be expected from random genomic sequences. We also observed enrichment for putative promoter regions of genes predicted in at least two annotation databases and for clones overlapping with CpG islands. Functional validation of randomly selected clones enriched by this method showed that a large fraction of these putative promoters can drive the expression of a reporter gene in transient transfection experiments. This method promises to be a useful genome-wide function-based approach that can complement existing methods to look for promoters.

Khambata-Ford, Shirin; Liu, Yueyi; Gleason, Christopher; Dickson, Mark; Altman, Russ B.; Batzoglou, Serafim; Myers, Richard M.

2003-01-01

171

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/.

2014-01-01

172

Contrasting mode of evolution between the MHC class I genomic region and class II region in the three-spined stickleback ( Gasterosteus aculeatus L.; Gasterosteidae: Teleostei)  

Microsoft Academic Search

Major histocompatibility complex (MHC) class I molecules display peptides on cell surfaces for subsequent T-cell recognition\\u000a and are involved in the immune response against intracellular pathogens. In this study, a BAC library was created from a single\\u000a three-spined stickleback and screened for clones containing MHC class I genes. In a 163.2-kb genomic sequence segment of a\\u000a single clone, we identified

Helmut Schaschl; K. Mathias Wegner

2007-01-01

173

CFTR expression from a BAC carrying the complete human gene and associated regulatory elements.  

PubMed

The use of genomic DNA rather than cDNA or mini-gene constructs in gene therapy might be advantageous as these contain intronic and long-range control elements vital for accurate expression. For gene therapy of cystic fibrosis though, no bacterial artificial chromosome (BAC), containing the whole CFTR gene is available. We have used Red homologous recombination to add a to a previously described vector to construct a new BAC vector with a 250.3-kb insert containing the whole coding region of the CFTR gene along with 40.1 kb of DNA 5' to the gene and 25 kb 3' to the gene. This includes all the known control elements of the gene. We evaluated expression by RT-PCR in CMT-93 cells and showed that the gene is expressed both from integrated copies of the BAC and also from episomes carrying the oriP/EBNA-1 element. Sequencing of the human CFTR mRNA from one clone showed that the BAC is functional and can generate correctly spliced mRNA in the mouse background. The BAC described here is the only CFTR genomic construct available on a convenient vector that can be readily used for gene expression studies or in vivo studies to test its potential application in gene therapy for cystic fibrosis. PMID:18657227

Kotzamanis, George; Abdulrazzak, Hassan; Gifford-Garner, Jennifer; Haussecker, Pei Ling; Cheung, Wing; Grillot-Courvalin, Catherine; Harris, Ann; Kittas, Christos; Kotsinas, Athanasios; Gorgoulis, Vassilis G; Huxley, Clare

2009-09-01

174

BAC Modification through Serial or Simultaneous Use of CRE/Lox Technology  

PubMed Central

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

Parrish, Mark; Unruh, Jay; Krumlauf, Robb

2011-01-01

175

Chimeric Mos1 and piggyBac transposases result in site-directed integration  

Microsoft Academic Search

Genetic transformation systems based on Mos1 and piggyBac transposable elements are used to achieve stable chromosomal integration. However, in- tegration sites are randomly distributed in the genome and transgene expression can be influenced by position effects. We developed a novel technology that utilizes chimeric transposases to direct integration into specific sites on a target DNA molecule. The Gal4 DNA binding

K. J. Maragathavally; J. M. Kaminski; C. J. Coates

2006-01-01

176

Highly efficient method for obtaining a subtracted genomic DNA library by the modified in-gel competitive reassociation method.  

PubMed

A highly efficient method to obtain a subtracted genomic DNA library using 1 microgram of target DNA was developed by modification of the previously reported in-gel competitive reassociation procedure. The modified method was based on polymerase chain reaction amplification after selective purification of a target-target reassociated molecule of subtracted DNA fragments to increase cloning efficiency. For a model experimental system, the subtracted DNA library was constructed after two cycles of subtractive reassociation between cervical cancer DNA fragments containing human papilloma virus DNA and the 100-fold excess of dephosphorylated normal tissue DNA fragments which were size-fractionated in agarose gel. Colony hybridization using human papilloma virus DNA as a probe revealed that a more than 500-fold enrichment of human papilloma virus DNA sequences in the subtracted DNA library could easily be obtained. This simple and efficient method will enable us to isolate an unknown foreign DNA fragment and an unknown amplified DNA fragment which might be present in cancer. PMID:7954408

Sasaki, H; Nomura, S; Akiyama, N; Takahashi, A; Sugimura, T; Oishi, M; Terada, M

1994-11-15

177

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

Microsoft Academic Search

BACKGROUND: Currently, two main technologies are used for screening of DNA copy number; the BAC (Bacterial Artificial Chromosome) and the recently developed oligonucleotide-based CGH (Chromosomal Comparative Genomic Hybridization) arrays which are capable of detecting small genomic regions with amplification or deletion. The correlation as well as the discriminative power of these platforms has never been compared statistically on a significant

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

2007-01-01

178

Defining Binge Drinking Quantities through Resulting BACs  

PubMed Central

Binge drinking as a researchable construct has generally been defined as 5 or more drinks on one occasion. However, no study has been conducted to determine if the binge concept that implies “excessive drunkenness” is being optimally captured within that level. Random interviews with breath tests of drinkers returning from visiting bars in Tijuana provide both blood alcohol concentration (BAC) measurements and the self-reported number of drinks consumed. Results indicate that currently used definitions of binge drinking predict relatively low BACs and may not be capturing the “excessive drunkenness” quality of the term. Consumption duration may explain the lower BACs.

Lange, James E.; Voas, Robert B.

2000-01-01

179

Comparative Analysis of Acidobacterial Genomic Fragments from Terrestrial and Aquatic Metagenomic Libraries, with Emphasis on Acidobacteria Subdivision 6? †  

PubMed Central

The bacterial phylum Acidobacteria has a widespread distribution and is one of the most common and diverse phyla in soil habitats. However, members of this phylum have often been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we used a cultivation-independent metagenomic approach to recover genomic information from soilborne members of this phylum. A soil metagenomic fosmid library was screened by PCR targeting acidobacterial 16S rRNA genes, facilitating the recovery of 17 positive clones. Recovered inserts appeared to originate from a range of Acidobacteria subdivisions, with dominance of subdivision 6 (10 clones). Upon full-length insert sequencing, gene annotation identified a total of 350 open reading frames (ORFs), representing a broad range of functions. Remarkably, six inserts from subdivision 6 contained a region of gene synteny, containing genes involved in purine de novo biosynthesis and encoding tRNA synthetase and conserved hypothetical proteins. Similar genomic regions had previously been observed in several environmental clones recovered from soil and marine sediments, facilitating comparisons with respect to gene organization and evolution. Comparative analyses revealed a general dichotomy between marine and terrestrial genes in both phylogeny and G+C content. Although the significance of this homologous gene cluster across subdivision 6 members is not known, it appears to be a common feature within a large percentage of all acidobacterial genomic fragments recovered from both of these environments.

Kielak, Anna M.; van Veen, Johannes A.; Kowalchuk, George A.

2010-01-01

180

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

181

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

182

piggyBac transposon system modification of primary human T cells.  

PubMed

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

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

2012-01-01

183

BacDive--the Bacterial Diversity Metadatabase.  

PubMed

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

184

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.

Sohngen, Carola; Bunk, Boyke; Podstawka, Adam; Gleim, Dorothea; Overmann, Jorg

2014-01-01

185

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

186

Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat.  

PubMed

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

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

2012-12-01

187

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

PubMed

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance "QTL-hotspot" region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement. PMID:24610029

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

2014-03-01

188

Construction of a cloned library of the EcoRI fragments from the human cytomegalovirus genome (strain AD169).  

PubMed Central

The DNA genome of human cytomegalovirus (HCMV) strain AD169 is 158 x 10(6) Mr. Cleavage of the HCMV DNA with the restriction endonuclease EcoRI yields 35 major fragments ranging in size from 0.54 x 10(6) Mr. We have constructed a cloned library of the EcoRI fragments of this strain of HCMV, using the plasmid pACYC184 and the recipient bacterium Escherichia coli strain HB101 RecA-. The viral origin of the cloned inserts was determined by hybridization to viral DNA. The fragments were characterized further by digestion with other restriction enzymes. Several clones were obtained which contained sequences spanning the junction between the long (L) and short (S) components of the viral DNA sequences. These clones differed in molecular weight by multiples of 0.3 x 10(6) to 0.4 x 10(6) Mr. The variability found in the clones was also reflected in the genome. Each clone containing a junction sequence hybridized to a series of bands on Southern filters of EcoRI-digested HCMV DNA. This "ladder effect" provided evidence for a region of heterogeneity within the L-S junction. Images

Tamashiro, J C; Hock, L J; Spector, D H

1982-01-01

189

Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.  

PubMed

Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system. PMID:24535568

Koike-Yusa, Hiroko; Li, Yilong; Tan, E-Pien; Velasco-Herrera, Martin Del Castillo; Yusa, Kosuke

2014-03-01

190

Phenotypic Screening of Escherichia coli K-12 Tn5 Insertion Libraries, Using Whole-Genome Oligonucleotide Microarrays  

PubMed Central

Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Escherichia coli K-12. Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.

Winterberg, Kelly M.; Luecke, John; Bruegl, Amanda S.; Reznikoff, William S.

2005-01-01

191

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

192

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.

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

193

A genomic analysis of Histomonas meleagridis through sequencing of a cDNA library.  

PubMed

Histomonas meleagridis, a flagellated protozoan of the Order Trichomonadida, is the causative agent of blackhead disease in gallinaceous birds. Few genes have been identified in this organism; thus, little is known regarding the molecular basis for its metabolism, virulence, and antigenicity. To identify new genes, a cDNA library derived from a lab strain of H. meleagridis was sequenced and annotated. Data obtained from these experiments identified 3,425 H. meleagridis genes. Analysis of the data allowed the identification of 81 genes coding for putative hydrogenosomal proteins and was used to determine the codon usage frequency. Sequence information also identified bacteria that are cultured with H. meleagridis. Future analysis of these data should provide valuable molecular insights into H. meleagridis and provide the platform for molecular studies aimed at understanding the pathogenesis of blackhead disease. PMID:23075009

Klodnicki, M E; McDougald, L R; Beckstead, R B

2013-04-01

194

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

DOE Data Explorer

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

None

195

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

PubMed Central

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

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

2014-01-01

196

Construction of a library of bovine genomic fragments enriched in CpG islands.  

PubMed

A procedure is described to isolate DNA probes from the bovine genome that are enriched in sites for the so-called rare cutter restriction endonucleases. A collection of SacII (CvCGCGG)-Hin-dIII fragments from bovine sperm was established in the plasmid Bluescript. 180 clones were picked at random and analysed for the presence of inserts with sites for the following rare cutters: EagI, BsshII, NarI, MluI, NruI, NaeI: 70% of the clones contained at least 1 site and 5% contained four different such sites. 22.8% had multiple sites for one or more of the rare cutters tested. Sequence analysis for 16 clones confirmed the cloning of DNA with a G+C content and a proportion of CpG vs GpCs indicative of CpG islands. PMID:8498709

De Rubertis, F; Comincini, S; Leone, P; Rognoni, G; Ferretti, L

1993-02-01

197

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

PubMed Central

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

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

2011-01-01

198

Concomitant reiterative BAC walking and fine genetic mapping enable physical map development for the broad-spectrum late blight resistance region, RB.  

PubMed

The wild potato species Solanum bulbocastanum is a source of genes for potent late blight resistance. We previously mapped resistance to a single region of the S. bulbocastanum chromosome 8 and named the region RB (for "Resistance from S. Bulbocastanum"). We now report physical mapping and contig construction for the RB region via a novel reiterative method of BAC walking and concomitant fine genetic mapping. BAC walking was initiated using RFLP markers previously shown to be associated with late blight resistance. Subcontig extension was accomplished using new probes developed from BAC ends. Significantly, BAC end and partial BAC sequences were also used to develop PCR-based markers to enhance map resolution in the RB region. As they were developed from BAC clones of known position relative to RB, our PCR-based markers are known a priori to be physically closer to the resistance region. These markers allowed the efficient screening of large numbers of segregating progeny at the cotyledon stage, and permitted us to assign the resistance phenotype to a region of approximately 55 kb. Our markers also directed BAC walking efforts away from regions distantly related to RB in favor of the 55-kb region. Because the S. bulbocastanum genotype used in BAC library construction is heterozygous for RB (RB/rb), codominant PCR-based markers, originally developed for fine-scale mapping, were also used to determine homolog origins for individual BAC clones. Ultimately, BAC contigs were constructed for the RB region from both resistant (RB) and susceptible (rb) homologs. PMID:12827499

Bradeen, J M; Naess, S K; Song, J; Haberlach, G T; Wielgus, S M; Buell, C R; Jiang, J; Helgeson, J P

2003-08-01

199

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.

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

200

Organization of phenylalanine ammonia lyase ( PAL ), acidic PR5 and osmotin-like ( OSM ) defence-response gene families in the potato genome  

Microsoft Academic Search

Defence-response (DR) genes are candidates for the genetic functions underlying quantitative resistance to plant pathogens.\\u000a The organization of three DR gene families encoding phenylalanine ammonia lyase (PAL), acidic PR-(pathogenesis-related) protein\\u000a 5, and basic PR-5, or osmotin-like (OSM), proteins was studied in the potato genome. A bacterial artificial chromosome (BAC)\\u000a library containing ~50,000 clones was constructed from high-molecular weight genomic DNA

Rosa A. Castillo Ruiz; Carmen Herrera; Marc Ghislain; Christiane Gebhardt

2005-01-01

201

The effectiveness of reducing illegal blood alcohol concentration (BAC) limits for driving: Evidence for lowering the limit to .05 BAC  

Microsoft Academic Search

PurposeThis scientific review provides a summary of the evidence regarding the benefits of reducing the illegal blood alcohol concentration (BAC) limit for driving and providing a case for enacting a .05 BAC limit.

James C. Fell; Robert B. Voas

2006-01-01

202

Crustins from eyestalk cDNA library of swimming crab Portunus trituberculatus: molecular characterization, genomic organization and expression analysis.  

PubMed

Crustins are cationic, cysteine-rich antimicrobial proteins, containing a single whey acidic protein (WAP) domain in the C-terminal end. Different from the reported Ptcrustin in the hemocytes, two novel crustin genes (PtCrustin2 and PtCrustin3) were cloned completely from the eyestalk cDNA library of Portunus trituberculatus in this study. All PtCrustins share the consensus cysteine motif and are considered as Type I crustins. Four exons and three introns are identified in genomic DNA sequence of PtCrustin3 while three exons and two introns in PtCrustin2. The mRNA transcripts of PtCrustin2 and PtCrustin3 are mainly detected in eyestalk and gills, but not in hemocytes. Although both PtCrutins are up-regulated after challenge of three microorganisms, PtCrustin3 seems to respond more quickly to microbial challenge than Ptcrustin2. Unlike most crustins, both recombinant PtCrustin2 and PtCrustin3 exhibit antibacterial activity against Gram-positive bacteria Micrococcus luteus and Staphyloccocus aureus and Gram-negative bacterium Pseudomonas aeruginosa. In addition, rPtCrustin2 is moderately active against yeast Pichia pastoris and rPtCrustin3 show significant activity against Gram-negative bacterium Vibrio alginolyticus. These results indicate that PtCrustin2 and PtCrustin3 are two novel crustins and play different roles in immune response of P. trituberculatus against microbial challenge. PMID:22971359

Cui, Zhaoxia; Song, Chengwen; Liu, Yuan; Wang, Shuangyan; Li, Qianqian; Li, Xihong

2012-10-01

203

Sequenced BAC anchored reference genetic map that reconciles the ten individual chromosomes of Brassica rapa  

PubMed Central

Background In view of the immense value of Brassica rapa in the fields of agriculture and molecular biology, the multinational Brassica rapa Genome Sequencing Project (BrGSP) was launched in 2003 by five countries. The developing BrGSP has valuable resources for the community, including a reference genetic map and seed BAC sequences. Although the initial B. rapa linkage map served as a reference for the BrGSP, there was ambiguity in reconciling the linkage groups with the ten chromosomes of B. rapa. Consequently, the BrGSP assigned each of the linkage groups to the project members as chromosome substitutes for sequencing. Results We identified simple sequence repeat (SSR) motifs in the B. rapa genome with the sequences of seed BACs used for the BrGSP. By testing 749 amplicons containing SSR motifs, we identified polymorphisms that enabled the anchoring of 188 BACs onto the B. rapa reference linkage map consisting of 719 loci in the 10 linkage groups with an average distance of 1.6 cM between adjacent loci. The anchored BAC sequences enabled the identification of 30 blocks of conserved synteny, totaling 534.9 cM in length, between the genomes of B. rapa and Arabidopsis thaliana. Most of these were consistent with previously reported duplication and rearrangement events that differentiate these genomes. However, we were able to identify the collinear regions for seven additional previously uncharacterized sections of the A genome. Integration of the linkage map with the B. rapa cytogenetic map was accomplished by FISH with probes representing 20 BAC clones, along with probes for rDNA and centromeric repeat sequences. This integration enabled unambiguous alignment and orientation of the maps representing the 10 B. rapa chromosomes. Conclusion We developed a second generation reference linkage map for B. rapa, which was aligned unambiguously to the B. rapa cytogenetic map. Furthermore, using our data, we confirmed and extended the comparative genome analysis between B. rapa and A. thaliana. This work will serve as a basis for integrating the genetic, physical, and chromosome maps of the BrGSP, as well as for studies on polyploidization, speciation, and genome duplication in the genus Brassica.

Kim, HyeRan; Choi, Su Ryun; Bae, Jina; Hong, Chang Pyo; Lee, Seo Yeon; Hossain, Md Jamil; Van Nguyen, Dan; Jin, Mina; Park, Beom-Seok; Bang, Jea-Wook; Bancroft, Ian; Lim, Yong Pyo

2009-01-01

204

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

PubMed

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

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

2007-04-01

205

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

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

206

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.

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

2012-01-01

207

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

PubMed Central

Background Currently, two main technologies are used for screening of DNA copy number; the BAC (Bacterial Artificial Chromosome) and the recently developed oligonucleotide-based CGH (Chromosomal Comparative Genomic Hybridization) arrays which are capable of detecting small genomic regions with amplification or deletion. The correlation as well as the discriminative power of these platforms has never been compared statistically on a significant set of human patient samples. Results In this paper, we present an exhaustive comparison between the two CGH platforms, undertaken at two independent sites using the same batch of DNA from 19 advanced prostate cancers. The comparison was performed directly on the raw data and a significant correlation was found between the two platforms. The correlation was greatly improved when the data were averaged over large chromosomic regions using a segmentation algorithm. In addition, this analysis has enabled the development of a statistical model to discriminate BAC outliers that might indicate microevents. These microevents were validated by the oligo platform results. Conclusion This article presents a genome-wide statistical validation of the oligo array platform on a large set of patient samples and demonstrates statistically its superiority over the BAC platform for the Identification of chromosomic events. Taking advantage of a large set of human samples treated by the two technologies, a statistical model has been developed to show that the BAC platform could also detect microevents.

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

2007-01-01

208

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.

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

209

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.

2011-01-01

210

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

PubMed

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

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

2009-01-01

211

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.

Xu, Zhiyong; Zikos, Dimitrios; Osterrieder, Nikolaus

2014-01-01

212

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

PubMed Central

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

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

2006-01-01

213

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.

Kamenetzky, Laura; Asis, Ramon; Bassi, Sebastian; de Godoy, Fabiana; Bermudez, Luisa; Fernie, Alisdair R.; Van Sluys, Marie-Anne; Vrebalov, Julia; Giovannoni, James J.; Rossi, Magdalena; Carrari, Fernando

2010-01-01

214

Efficient library preparation for next-generation sequencing analysis of genome-wide epigenetic and transcriptional landscapes in embryonic stem cells.  

PubMed

Gene expression in embryonic stem (ES) cells is regulated in part by a network of transcription factors, epigenetic regulators, and histone modifications that influence the underlying chromatin in a way that is conducive or repressive for transcription. Advances in next-generation sequencing technology have allowed for the genome-wide analysis of chromatin constituents and protein-DNA interactions at high resolution in ES cells and other stem cells. While many studies have surveyed genome-wide profiles of a few factors and expression changes at a fixed time point in undifferentiated ES cells, few have utilized an integrative approach to simultaneously survey protein-DNA interactions, histone modifications, and expression programs during ES cell self-renewal and differentiation. To identify transcriptional networks that regulate pluripotency and differentiation, it is important to generate high-quality genome-wide maps of transcription factors, chromatin factors, and histone modifications and to survey global gene expression profiles. Here, to interrogate genome-wide profiles of chromatin features and to survey global gene expression programs in ES cells, we describe protocols for efficient library construction for next-generation sequencing of ChIP-Seq and RNA-Seq samples. PMID:24743988

Kidder, Benjamin L; Zhao, Keji

2014-01-01

215

Generation of an inducible and optimized piggyBac transposon system†  

PubMed Central

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

Cadinanos, Juan; Bradley, Allan

2007-01-01

216

A bacterial artificial chromosome library for 'Jefferson' hazelnut and identification of clones associated with eastern filbert blight resistance and pollen-stigma incompatibility.  

PubMed

European hazelnut (Corylus avellana L.) is the only economically important nut crop in the family Betulaceae. Because of its small genome size (~385 Mb / 1C), relatively short life cycle, availability of a dense linkage map, and amenability to transformation by Agrobacterium, the European hazelnut could serve as a model plant for the Betulaceae. Here we report the construction of a bacterial artificial chromosome (BAC) library for 'Jefferson' hazelnut using the cloning enzyme MboI and the vector pECBAC1 (BamHI site). The library consists of 39,936 clones arrayed in 104,384-well microtitre plates with a mean insert size of 117 kb. The genomic coverage of the library is estimated to be about 12 genome equivalents. This library provides a valuable resource for the map-based cloning of two important genes, the resistance gene from 'Gasaway' that confers resistance to eastern filbert blight caused by the fungus Anisogramma anomala (Peck) E. Müller and the S locus that controls pollen-stigma incompatibility. Fine-resolution mapping near the two loci was carried out using random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers. Fine mapping at the disease resistance locus showed that markers W07-375 and X01-825 flanked the resistance locus at distances of 0.06 and 0.05 cM, respectively. The S locus is flanked by markers 204-950 and KG819-200 at distances of 0.14 and 0.24 cM, respectively. Assuming that 1 cM corresponds to a physical distance of 430 kb, it will take approximately two to three chromosome walks to assemble BAC contigs that span both loci. PMID:21936690

Sathuvalli, Vidyasagar R; Mehlenbacher, Shawn A

2011-10-01

217

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

PubMed Central

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

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

2010-01-01

218

Microsatellite discovery from BAC end sequences and genetic mapping to anchor the soybean physical and genetic maps.  

PubMed

Whole-genome sequencing of the soybean (Glycine max (L.) Merr. 'Williams 82') has made it important to integrate its physical and genetic maps. To facilitate this integration of maps, we screened 3290 microsatellites (SSRs) identified from BAC end sequences of clones comprising the 'Williams 82' physical map. SSRs were screened against 3 mapping populations. We found the AAT and ACT motifs produced the greatest frequency of length polymorphisms, ranging from 17.2% to 32.3% and from 11.8% to 33.3%, respectively. Other useful motifs include the dinucleotide repeats AG, AT, and AG, with frequency of length polymorphisms ranging from 11.2% to 18.4% (AT), 12.4% to 20.6% (AG), and 11.3% to 16.4% (GT). Repeat lengths less than 16 bp were generally less useful than repeat lengths of 40-60 bp. Two hundred and sixty-five SSRs were genetically mapped in at least one population. Of the 265 mapped SSRs, 60 came from BAC singletons not yet placed into contigs of the physical map. One hundred and ten originated in BACs located in contigs for which no genetic map location was previously known. Ninety-five SSRs came from BACs within contigs for which one or more other BACs had already been mapped. For these fingerprinted contigs (FPC) a high percentage of the mapped markers showed inconsistent map locations. A strategy is introduced by which physical and genetic map inconsistencies can be resolved using the preliminary 4x assembly of the whole genome sequence of soybean. PMID:18356965

Shoemaker, Randy C; Grant, David; Olson, Terry; Warren, Wesley C; Wing, Rod; Yu, Yeisoo; Kim, HyeRan; Cregan, Perry; Joseph, Bindu; Futrell-Griggs, Montona; Nelson, Will; Davito, Jon; Walker, Jason; Wallis, John; Kremitski, Colin; Scheer, Debbie; Clifton, Sandra W; Graves, Tina; Nguyen, Henry; Wu, Xiaolei; Luo, Mingcheng; Dvorak, Jan; Nelson, Rex; Cannon, Steven; Tomkins, Jeff; Schmutz, Jeremy; Stacey, Gary; Jackson, Scott

2008-04-01

219

Analysis of the Shotgun Expression Library of the Mycobacterium tuberculosis Genome for Immunodominant Polypeptides: Potential Use in Serodiagnosis  

Microsoft Academic Search

A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens ex- pressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for

Prakash S. Bisen; Sanjay K. Garg; Ram P. Tiwari; P. R. N. Tagore; R. Chandra; R. Karnik; N. Thaker; N. Desai; P. K. Ghosh; M. Fraziano; V. Colizzi

2003-01-01

220

Development of a BAC vector for integration-independent and tight regulation of transgenes in rodents via the Tet system  

Microsoft Academic Search

The establishment of functional transgenic mouse lines is often limited by problems caused by integration site effects on\\u000a the expression construct. Similarly, tetracycline (Tet) controlled transcription units most commonly used for conditional\\u000a transgene expression in mice are strongly influenced by their genomic surrounding. Using bacterial artificial chromosome (BAC)\\u000a technology in constitutive expression systems, it has been shown that integration site

Kai Schönig; David Kentner; Manfred Gossen; Tina Baldinger; Jun Miao; Katrin Welzel; Andreas Vente; Dusan Bartsch; Hermann Bujard

2011-01-01

221

Localization of the mouse kidney disease (kd) gene to a YAC/BAC contig on Chromosome 10.  

PubMed

Mice that are homozygous for the kidney disease (kd) gene on Chromosome (Chr) 10 spontaneously develop a progressive and fatal interstitial nephritis. The disease phenotype is similar to that of the human disease, juvenile nephronophthisis. Using a backcross and intercross breeding strategy and analysis of over 900 resultant progeny, this genetic locus has now been mapped to a minimal co-segregating region of approximately two megabases between D10Mit 193 and D10Mit 38. The location assigned to kd by this study is over 3 cM from the current Mouse Genome Database location. The entire interval has been cloned in yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) clones. Recombinant analysis has permitted assignment of 13 Mit microsatellite markers to positions near or within the region. Two new markers have been identified by using single-strand conformation polymorphism (SSCP) analysis of sequenced BAC ends. Several BAC end sequences align with human BAC clones from Chr 6q2 that contain NR2E1. Snx3, and Ros1. Three murine genes, CD24a, fyn, and ColX reported to map in or near the kd region as defined by this study have been evaluated. Though not definitely excluded, they appear to be unlikely candidates. PMID:11063251

Dell, K M; Li, Y X; Peng, M; Neilson, E G; Gasser, D L

2000-11-01

222

Comparative BAC-based physical mapping of Oryza sativa ssp. indica var. 93-11 and evaluation of the two rice reference sequence assemblies.  

PubMed

Reference sequences are sequences that are used for public consultation, and therefore must be of high quality. Using the whole-genome shotgun/next-generation sequencing approach, many genome sequences of complex higher plants have been generated in recent years, and are generally considered reference sequences. However, none of these sequences has been experimentally evaluated at the whole-genome sequence assembly level. Rice has a relatively simple plant genome, and the genome sequences for its two sub-species obtained using different sequencing approaches were published approximately 10 years ago. This provides a unique system for a case study to evaluate the qualities and utilities of published plant genome sequences. We constructed a robust BAC physical map embedding a large number of BAC end sequences forrice variety 93-11. Through BAC end sequence alignments and tri-assembly comparisons of the 93-11 physical map and the two reference sequences, we found that the Nipponbare reference sequence generated using the clone-by-clone approach has a high quality but still contains small artifact inversions and missing sequences. In contrast, the 93-11 reference sequence generated using the whole-genome shotgun approach contains many large and varied assembly errors, such as inversions, duplications and translocations, as well as missing sequences. The 93-11 physical map provides an invaluable resource for evaluation and improvements toward completion of both Nipponbare and 93-11 reference sequences. PMID:24330235

Pan, Yonglong; Deng, Ying; Lin, Haiyan; Kudrna, David A; Wing, Rod A; Li, Lijia; Zhang, Qifa; Luo, Meizhong

2014-03-01

223

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.

2010-01-01

224

High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding.  

PubMed

A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. PMID:18633592

Rahman, Mukhlesur; Sun, Zudong; McVetty, Peter B E; Li, Genyi

2008-10-01

225

Comparative analysis of acidobacterial genomic fragments from terrestrial and aquatic metagenomic libraries, with emphasis on acidobacteria subdivision 6  

Microsoft Academic Search

The bacterial phylum Acidobacteria has a widespread distribution and is one of the most common and diverse phyla in soil habitats. However, members of this phylum have often been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we used a cultivation-independent metagenomic approach to recover genomic information from soilborne members of this

A. M. Kielak; J. A. Van Veen; G. A. Kowalchuk

2010-01-01

226

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

PubMed

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

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

2011-12-20

227

Genomic markers for malignant progression in pulmonary adenocarcinoma with bronchioloalveolar features  

PubMed Central

Bronchioloalveolar carcinoma (BAC), a subtype of lung adenocarcinoma (ADC) without stromal, vascular, or pleural invasion, is considered an in situ tumor with a 100% survival rate. However, the histological criteria for invasion remain controversial. BAC-like areas may accompany otherwise invasive adenocarcinoma, referred to as mixed type adenocarcinoma with BAC features (AWBF). AWBF are considered to evolve from BAC, representing a paradigm for malignant progression in ADC. However, the supporting molecular evidence remains forthcoming. Here, we have studied the genomic changes of BAC and AWBF by array comparative genomic hybridization (CGH). We used submegabase-resolution tiling set array CGH to compare the genomic profiles of 14 BAC or BAC with focal area suspicious for invasion with those of 15 AWBF. Threshold-filtering and frequency-scoring analysis found that genomic profiles of noninvasive and focally invasive BAC are indistinguishable and show fewer aberrations than tumor cells in BAC-like areas of AWBF. These aberrations occurred mainly at the subtelomeric chromosomal regions. Increased genomic alterations were noted between BAC-like and invasive areas of AWBF. We identified 113 genes that best differentiated BAC from AWBF and were considered candidate marker genes for tumor invasion and progression. Correlative gene expression analyses demonstrated a high percentage of them to be poor prognosis markers in early stage ADC. Quantitative PCR also validated the amplification and overexpression of PDCD6 and TERT on chromosome 5p and the prognostic significance of PDCD6 in early stage ADC patients. We identified candidate genes that may be responsible for and are potential markers for malignant progression in AWBF.

Aviel-Ronen, Sarit; Coe, Bradley P.; Lau, Suzanne K.; da Cunha Santos, Gilda; Zhu, Chang-Qi; Strumpf, Dan; Jurisica, Igor; Lam, Wan L.; Tsao, Ming-Sound

2008-01-01

228

Losing Libraries, Saving Libraries  

ERIC Educational Resources Information Center

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

Miller, Rebecca

2010-01-01

229

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

230

The KoreaBrassica Genome Project: A glimpse of theBrassica genome based on comparative genome analysis withArabidopsis  

Microsoft Academic Search

A complete genome sequence provides unlimited information in the sequenced organ- ism as well as in related taxa. According to the guidance of the Multinational Brassica Genome Project (MBGP), the Korea Brassica Genome Project (KBGP) is sequenc- ing chromosome 1 (cytogenetically oriented chromosome #1) of Brassica rapa .W e have selected 48 seed BACs on chromosome 1 using EST genetic

Tae-Jin Yang; Jung-Sun Kim; Ki-Byung Lim; Soo-Jin Kwon; Jin-A Kim; Mina Jin; Jee Young Park; Myung-Ho Lim; Ho-Il Kim; Seog Hyung Kim; Yong Pyo Lim; Beom-Seok Park

2005-01-01

231

Genomic organization of the Schistosoma mansoni aspartic protease gene, a platyhelminth orthologue of mammalian lysosomal cathepsin D.  

PubMed

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an approximately 8-fold coverage of the schistosome genome. Sequencing of BAC clone 25-J-24 revealed that the cathepsin D gene locus was approximately 13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMalpha. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D. PMID:15302411

Morales, Maria E; Kalinna, Bernd H; Heyers, Oliver; Mann, Victoria H; Schulmeister, Alexandra; Copeland, Claudia S; Loukas, Alex; Brindley, Paul J

2004-08-18

232

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

233

Novel method for selection of antimicrobial peptides from a phage display library by use of bacterial magnetic particles.  

PubMed

Antimicrobial peptides were isolated from a phage display peptide library using bacterial magnetic particles (BacMPs) as a solid support. The BacMPs obtained from "Magnetospirillum magneticum" strain AMB-1 consist of pure magnetite (50 to 100 nm in size) and are covered with a lipid bilayer membrane derived from the invagination of the inner membrane. BacMPs are easily purified from a culture of magnetotactic bacteria by magnetic separation. Approximately 4 x 10(10) PFU of the library phage (complexity, 2.7 x 10(9)) was reacted with BacMPs. The elution of bound phages from BacMPs was performed by disrupting its membrane with phospholipase D treatment. Six candidate peptides, which were highly cationic and could bind onto the BacMP membrane, were obtained. They exhibited antimicrobial activity against Bacillus subtilis but not against Escherichia coli and Saccharomyces cerevisiae. The amino acid substitution of the selected peptide, KPQQHNRPLRHK (peptide 6-7), to enhance the hydrophobicity resulted in obvious antimicrobial activity against all test microorganisms. The present study shows for the first time that a magnetic selection of antimicrobial peptides from the phage display peptide library was successfully achieved by targeting the actual bacterial inner membrane. This BacMP-based method could be a promising approach for a high-throughput screening of antimicrobial peptides targeting a wide range of species. PMID:18952877

Tanaka, Tsuyoshi; Kokuryu, Yoriko; Matsunaga, Tadashi

2008-12-01

234

Flow cytogenetics and plant genome mapping.  

PubMed

The application of flow cytometry and sorting (flow cytogenetics) to plant chromosomes did not begin until the mid-1980s, having been delayed by difficulties in preparation of suspensions of intact chromosomes and discrimination of individual chromosome types. These problems have been overcome during the last ten years. So far, chromosome analysis and sorting has been reported in 17 species, including major legume and cereal crops. While chromosome classification by flow cytometry (flow karyotyping) may be used for quantitative detection of structural and numerical chromosome changes, chromosomes purified by flow sorting were found to be invaluable in a broad range of applications. These included physical mapping using PCR, high-resolution cytogenetic mapping using FISH and PRINS, production of recombinant DNA libraries, targeted isolation of markers, and protein analysis. A great potential is foreseen for the use of sorted chromosomes for construction of chromosome and chromosome-arm-specific BAC libraries, targeted isolation of low-copy (genic) sequences, high-throughput physical mapping of ESTs and other DNA sequences by hybridization to DNA arrays, and global characterization of chromosomal proteins using approaches of proteomics. This paper provides a comprehensive review of the methodology and application of flow cytogenetics, and assesses its potential for plant genome analysis. PMID:14984104

Dolezel, Jaroslav; Kubaláková, Marie; Bartos, Jan; Macas, Jirí

2004-01-01

235

Development of a bacterial artificial chromosome (BAC) recombineering procedure using galK-untranslated region (UTR) for the mutation of diploid genes.  

PubMed

Bacterial artificial chromosome (BAC) recombineering using galK selection allows DNA cloned in Escherichia coli to be modified without introducing an unwanted selectable marker at the modification site. Genomes of some herpesviruses have a pair of inverted repeat sequences that makes it very difficult to introduce mutations into diploid (duplicate) genes using the galK selection method. To mutate diploid genes, we developed a galK-UTR BAC recombineering procedure that blocks one copy of the target diploid gene by insertion of a galK untranslated region (UTR), which enables the simple mutation of the other copy. The blocked copy can then be replaced with an UTR-specific primer pair. The IR2 gene of equine herpesvirus 1 (EHV-1) maps within both the internal (IR) and terminal repeat (TR) of the genomic short region and is expressed at low levels because its promoter is TATA-less. Both IR2 promoters in EHV-1 BAC were replaced with a mutant IR2 promoter containing three Sp1-binding motifs and a consensus TATA box by galK-UTR BAC recombineering. The expression level of the IR2 protein controlled by the modified promoter increased approximately 4-fold as compared to that of wild-type EHV-1. The galK-UTR method will provide a useful tool in studies of herpesviruses. PMID:22407056

Dai, Gan; Kim, Seongman; O'Callaghan, Dennis J; Kim, Seong K

2012-06-01

236

Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli  

PubMed Central

Background Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library. Results Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. Conclusion Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes.

Haney, Steven A; Keeney, David; Chen, Lei; Moghazeh, Soraya; Projan, Steve; Rasmussen, Beth

2003-01-01

237

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

PubMed Central

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

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

2013-01-01

238

PiggyBac Transposon-Mediated Cellular Transgenesis in Mammalian Forebrain by In Utero Electroporation.  

PubMed

In utero electroporation (IUE) is an effective transfection method for delivering plasmid DNA into neural progenitor cells and neurons of mammalian neocortex in vivo. Although IUE is effective at delivering multiple DNA plasmids into populations of cells, unfortunately plasmids delivered into neural progenitor cells remain largely episomal and often get inactivated or lost after cell division. This results in a form of "birthdate" labeling in which only the cell types that do not undergo a second cell division continue to express the transfected plasmids. This limits the application of IUE with standard plasmids and precludes its use in experiments where manipulating or labeling the complete cell lineage of a progenitor is desired. To circumvent this episomal loss of plasmid in IUE, we have used a binary piggyBac transposon system to induce nonviral genomic integration of transgenes. These transgenes do not appear to inactivate after cell division, and this results in stable somatic cellular transgenesis of neurons and glia. Like standard IUE, the system can be used with multiple combinations of plasmids to achieve multicolor labeling and both loss-of-function and gain-of-function manipulations. In this protocol, we describe the method for delivering a binary piggyBac transposon plasmid system by IUE. PMID:24987137

Chen, Fuyi; Maher, Brady J; LoTurco, Joseph J

2014-01-01

239

BacMam: versatile gene delivery technology for GPCR assays.  

PubMed

BacMam viruses are modified baculoviruses that contain mammalian expression cassettes for viral gene delivery and transient expression in mammalian cells. They are easily, inexpensively, and rapidly generated and provide a versatile solution for G protein-coupled receptor (GPCR) cell-based assay development. Using BacMam technology, target gene expression levels are easily controlled and simultaneous delivery of multiple genes is possible, for example, coexpression of a receptor and a G protein or a reporter gene. BacMam viruses are compatible with the GPCR cell-based assay formats typically used in high-throughput screening and provide an unparalleled level of experimental flexibility that is simply not possible when using stable recombinant cell lines. PMID:19513651

Davenport, Elizabeth A; Nuthulaganti, Parvathi; Ames, Robert S

2009-01-01

240

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

MedlinePLUS

... As Blood Alcohol Content (BAC) Increases, So Does Impairment Spring 2014 Table of Contents For purposes of ... define intoxication and provides a rough measure of impairment. Blood Alcohol Content (BAC) 0.0 - 0.05% ...

241

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.

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

242

The Pinus taeda genome is characterized by diverse and highly diverged repetitive sequences  

PubMed Central

Background In today's age of genomic discovery, no attempt has been made to comprehensively sequence a gymnosperm genome. The largest genus in the coniferous family Pinaceae is Pinus, whose 110-120 species have extremely large genomes (c. 20-40 Gb, 2N = 24). The size and complexity of these genomes have prompted much speculation as to the feasibility of completing a conifer genome sequence. Conifer genomes are reputed to be highly repetitive, but there is little information available on the nature and identity of repetitive units in gymnosperms. The pines have extensive genetic resources, with approximately 329000 ESTs from eleven species and genetic maps in eight species, including a dense genetic map of the twelve linkage groups in Pinus taeda. Results We present here the Sanger sequence and annotation of ten P. taeda BAC clones and Genome Analyzer II whole genome shotgun (WGS) sequences representing 7.5% of the genome. Computational annotation of ten BACs predicts three putative protein-coding genes and at least fifteen likely pseudogenes in nearly one megabase of sequence. We found three conifer-specific LTR retroelements in the BACs, and tentatively identified at least 15 others based on evidence from the distantly related angiosperms. Alignment of WGS sequences to the BACs indicates that 80% of BAC sequences have similar copies (? 75% nucleotide identity) elsewhere in the genome, but only 23% have identical copies (99% identity). The three most common repetitive elements in the genome were identified and, when combined, represent less than 5% of the genome. Conclusions This study indicates that the majority of repeats in the P. taeda genome are 'novel' and will therefore require additional BAC or genomic sequencing for accurate characterization. The pine genome contains a very large number of diverged and probably defunct repetitive elements. This study also provides new evidence that sequencing a pine genome using a WGS approach is a feasible goal.

2010-01-01

243

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.

Parker, Jared B.; Walsh, Christopher T.

2013-01-01

244

Suicidal autointegration of sleeping beauty and piggyBac transposons in eukaryotic cells.  

PubMed

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

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

2014-03-01

245

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.

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

2014-01-01

246

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

247

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

248

Highly recombinogenic regions at seed storage protein loci on chromosome 1DS of Aegilops tauschii, the D-genome donor of wheat.  

PubMed Central

A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii, the diploid D-genome donor species of hexaploid wheat. Ae. tauschii was used to overcome some of the limitations commonly associated with molecular studies of wheat such as low levels of DNA polymorphism. Detection of multiple loci by most RFLP probes suggests that gene duplication events have occurred throughout this chromosomal region. Large DNA fragments isolated from a BAC library of Ae. tauschii were used to determine the relationship between physical and genetic distance at seed storage protein loci located at the distal end of chromosome 1DS. Highly recombinogenic regions were identified where the ratio of physical to genetic distance was estimated to be <20 kb/cM. These results are discussed in relation to the genome-wide estimate of the relationship between physical and genetic distance.

Spielmeyer, W; Moullet, O; Laroche, A; Lagudah, E S

2000-01-01

249

SweetBac: a new approach for the production of mammalianised glycoproteins in insect cells.  

PubMed

Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine ?1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showed highly specific binding to the lectin, indicating proper functionality. To confirm these results, PNGase A released N-glycans were analyzed by MALDI-TOF-MS and shown to contain structures with mainly one or two terminal galactose residues. Since the presence of specific N-glycans has an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells. PMID:22485160

Palmberger, Dieter; Wilson, Iain B H; Berger, Imre; Grabherr, Reingard; Rendic, Dubravko

2012-01-01

250

SweetBac: A New Approach for the Production of Mammalianised Glycoproteins in Insect Cells  

PubMed Central

Recombinant production of therapeutically active proteins has become a central focus of contemporary life science research. These proteins are often produced in mammalian cells, in order to obtain products with post-translational modifications similar to their natural counterparts. However, in cases where a fast and flexible system for recombinant production of proteins is needed, the use of mammalian cells is limited. The baculoviral insect cell system has proven to be a powerful alternative for the expression of a wide range of recombinant proteins in short time frames. The major drawback of baculoviral systems lies in the inability to perform mammalian-like glycosylation required for the production of therapeutic glycoproteins. In this study we integrated sequences encoding Caenorhabditis elegans N-acetylglucosaminyltransferase II and bovine ?1,4-galactosyltransferase I into the backbone of a baculovirus genome. The thereby generated SweetBac virus was subsequently used for the production of the human HIV anti-gp41 antibody 3D6 by integrating heavy and light chain open reading frames into the SweetBac genome. The parallel expression of target genes and glycosyltransferases reduced the yield of secreted antibody. However, the overall expression rate, especially in the recently established Tnao38 cell line, was comparable to that of transient expression in mammalian cells. In order to evaluate the ability of SweetBac to generate mammalian-like N-glycan structures on 3D6 antibody, we performed SDS-PAGE and tested for the presence of terminal galactose using Riccinus communis agglutinin I. The mammalianised variants of 3D6 showed highly specific binding to the lectin, indicating proper functionality. To confirm these results, PNGase A released N-glycans were analyzed by MALDI-TOF-MS and shown to contain structures with mainly one or two terminal galactose residues. Since the presence of specific N-glycans has an impact on antibodies ability to exert different effector functions, we tested the binding to human Fc gamma receptor I present on U937 cells.

Palmberger, Dieter; Wilson, Iain B. H.; Berger, Imre; Grabherr, Reingard; Rendic, Dubravko

2012-01-01

251

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.

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

252

Parish Libraries.  

ERIC Educational Resources Information Center

Describes how to develop a cost- and time-efficient parish library. Discusses ongoing promotion of the library; leadership; costs; staffing; shelving and classification systems; location; nonprint media; library policy; and ways to increase visibility. (AEF)

Bingham, Anne

1997-01-01

253

Multimedia Library  

Cancer.gov

Home News and Events Multimedia Library Multimedia Library Learn more about TCGA, its researchers, components and how it works from our multimedia library. Images Image: Glioblastoma 72 DPI | 300 DPIHistopathological image of cerebral glioblastoma.Wikimedia.org

254

[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

255

Digital Libraries  

NSDL National Science Digital Library

This projects introduces digital libraries, digital initiatives, search techniques, and the Instructional Architect Review Rubric. Digital Library Information : The Scope of the Digital Library D-Lib Journal article, 1998 2008 Joint Conference on Digital Libraries (JCDL) Annual meeting devoted to Digital Libraries Initiatives : Digital Libraries Initiative The Initiative's focus is to dramatically advance the means to collect, store, and organize information in digital forms, and make it available for searching, retrieval, and processing via communication networks -- all in ...

Heather

2008-09-29

256

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.

2009-01-01

257

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure  

PubMed Central

Background Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome. Results Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella. Conclusions When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.

2011-01-01

258

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

259

A tiling resolution DNA microarray with complete coverage of the human genome  

Microsoft Academic Search

We constructed a tiling resolution array consisting of 32,433 overlapping BAC clones covering the entire human genome. This increases our ability to identify genetic alterations and their boundaries throughout the genome in a single comparative genomic hybridization (CGH) experiment. At this tiling resolution, we identified minute DNA alterations not previously reported. These alterations include microamplifications and deletions containing oncogenes, tumor-suppressor

Adrian S Ishkanian; Chad A Malloff; Spencer K Watson; Ronald J deLeeuw; Bryan Chi; Bradley P Coe; Antoine Snijders; Donna G Albertson; Daniel Pinkel; Marco A Marra; Victor Ling; Calum MacAulay; Wan L Lam

2004-01-01

260

BacMam technology and its application to drug discovery.  

PubMed

The recombinant baculovirus/insect cell system was firmly established as a leading method for recombinant protein production when a new potential use for these viruses was revealed in 1995. It was reported that engineered recombinant baculoviruses could deliver functional expression cassettes to mammalian cell types; a system which has come to be known as BacMam gene delivery. In the field of high-throughput screening the failure of many common transient gene delivery methods in reproducibility and cell survival has caused investigators to routinely apply stable cell lines in support of cell-based assays. The ease of use, versatility, safety and economics of the BacMam system makes transient gene delivery a viable option in the high-throughput screening setting and in most instances circumvents many of the limitations of stable cell lines. Although a few pharmaceutical companies have embraced the technology, its use is poised to become more widespread with increased familiarity and the emergence of enabling products based on the BacMam system. PMID:23488908

Ames, Robert S; Kost, Thomas A; Condreay, J Patrick

2007-12-01

261

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

PubMed Central

Background Genomics and metagenomics are currently leading research areas, with DNA sequences accumulating at an exponential rate. Although enormous advances in DNA sequencing technologies are taking place, progress is frequently limited by factors such as genomic contig assembly and generation of representative libraries. A number of DNA fragmentation methods, such as hydrodynamic sharing, sonication or DNase I fragmentation, have various drawbacks, including DNA damage, poor fragmentation control, irreproducibility and non-overlapping DNA segment representation. Improvements in these limited DNA scission methods are consequently needed. An alternative method for obtaining higher quality DNA fragments involves partial digestion with restriction endonucleases (REases). We have shown previously that class-IIS/IIC/IIG TspGWI REase, the prototype member of the Thermus sp. enzyme family, can be chemically relaxed by a cofactor analogue, allowing it to recognize very short DNA sequences of 3-bp combined frequency. Such frequently cleaving REases are extremely rare, with CviJI/CviJI*, SetI and FaiI the only other ones found in nature. Their unusual features make them very useful molecular tools for the development of representative DNA libraries. Results We constructed a horse genomic library and a deletion derivative library of the butyrylcholinesterase cDNA coding region using a novel method, based on TaqII, Thermus sp. family bifunctional enzyme exhibiting cofactor analogue specificity relaxation. We used sinefungin (SIN) – an S-adenosylmethionine (SAM) analogue with reversed charge pattern, and dimethylsulfoxide (DMSO), to convert the 6-bp recognition site TaqII (5?-GACCGA-3? [11/9]) into a theoretical 2.9-bp REase, with 70 shortened variants of the canonical recognition sequence detected. Because partial DNA cleavage is an inherent feature of the Thermus sp. enzyme family, this modified TaqII is uniquely suited to quasi-random library generation. Conclusions In the presence of SIN/DMSO, TaqII REase is transformed from cleaving every 4096 bp on average to cleaving every 58 bp. TaqII SIN/DMSO thus extends the palette of available REase prototype specificities. This phenomenon, employed under partial digestion conditions, was applied to quasi-random DNA fragmentation. Further applications include high sensitivity probe generation and metagenomic DNA amplification.

2013-01-01

262

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

PubMed Central

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

2009-01-01

263

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

264

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.

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

2012-01-01

265

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

Microsoft Academic Search

BACKGROUND: Crocodilians (Order Crocodylia) are an ancient vertebrate group of tremendous ecological, social, and evolutionary importance. They are the only extant reptilian members of Archosauria, a monophyletic group that also includes birds, dinosaurs, and pterosaurs. Consequently, crocodilian genomes represent a gateway through which the molecular evolution of avian lineages can be explored. To facilitate comparative genomics within Crocodylia and between

Xueyan Shan; David A Ray; John A Bunge; Daniel G Peterson

2009-01-01

266

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

PubMed Central

Background Physical maps employing libraries of bacterial artificial chromosome (BAC) clones are essential for comparative genomics and sequencing of large and repetitive genomes such as those of the hexaploid bread wheat. The diploid ancestor of the D-genome of hexaploid wheat (Triticum aestivum), Aegilops tauschii, is used as a resource for wheat genomics. The barley diploid genome also provides a good model for the Triticeae and T. aestivum since it is only slightly larger than the ancestor wheat D genome. Gene co-linearity between the grasses can be exploited by extrapolating from rice and Brachypodium distachyon to Ae. tauschii or barley, and then to wheat. Results We report the use of Ae. tauschii for the construction of the physical map of a large distal region of chromosome arm 3DS. A physical map of 25.4 Mb was constructed by anchoring BAC clones of Ae. tauschii with 85 EST on the Ae. tauschii and barley genetic maps. The 24 contigs were aligned to the rice and B. distachyon genomic sequences and a high density SNP genetic map of barley. As expected, the mapped region is highly collinear to the orthologous chromosome 1 in rice, chromosome 2 in B. distachyon and chromosome 3H in barley. However, the chromosome scale of the comparative maps presented provides new insights into grass genome organization. The disruptions of the Ae. tauschii-rice and Ae. tauschii-Brachypodium syntenies were identical. We observed chromosomal rearrangements between Ae. tauschii and barley. The comparison of Ae. tauschii physical and genetic maps showed that the recombination rate across the region dropped from 2.19 cM/Mb in the distal region to 0.09 cM/Mb in the proximal region. The size of the gaps between contigs was evaluated by comparing the recombination rate along the map with the local recombination rates calculated on single contigs. Conclusions The physical map reported here is the first physical map using fingerprinting of a complete Triticeae genome. This study demonstrates that global fingerprinting of the large plant genomes is a viable strategy for generating physical maps. Physical maps allow the description of the co-linearity between wheat and grass genomes and provide a powerful tool for positional cloning of new genes.

2010-01-01

267

Construction of a 1.2Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of Poncirus trifoliata (L.) Raf  

Microsoft Academic Search

The citrus tristeza virus resistance gene ( Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45 696 clones with an average insert size of 80 kb, corresponding to

Zhong-Nan Yang; Xin-Rong Ye; Sandong Choi; Joe Molina; Francis Moonan; Rod A. Wing; Mikeal L. Roose; T. Erik Mirkov

2001-01-01

268

Antibody libraries  

US Patent & Trademark Office Database

The present invention relates to the production of antibody libraries. In particular, the present invention relates to the use of integrating retroviral vectors to generate libraries comprising a plurality of combinations of antibody light chains and heavy chains. The present invention thus provides improved methods of generating and screening antibody libraries comprising large numbers of unique antibodies.

2012-07-17

269

PBmice: an integrated database system of piggyBac (PB) insertional mutations and their characterizations in mice  

PubMed Central

DNA transposon piggyBac (PB) is a newly established mutagen for large-scale mutagenesis in mice. We have designed and implemented an integrated database system called PBmice (PB Mutagenesis Information CEnter) for storing, retrieving and displaying the information derived from PB insertions (INSERTs) in the mouse genome. This system is centered on INSERTs with information including their genomic locations and flanking genomic sequences, the expression levels of the hit genes, and the expression patterns of the trapped genes if a trapping vector was used. It also archives mouse phenotyping data linked to INSERTs, and allows users to conduct quick and advanced searches for genotypic and phenotypic information relevant to a particular or a set of INSERT(s). Sequence-based information can be cross-referenced with other genomic databases such as Ensembl, BLAST and GBrowse tools used in PBmice offer enhanced search and display for additional information relevant to INSERTs. The total number and genomic distribution of PB INSERTs, as well as the availability of each PB insertional LINE can also be viewed with user-friendly interfaces. PBmice is freely available at http://www.idmshanghai.cn/PBmice or http://www.scbit.org/PBmice/.

Sun, Ling V.; Jin, Ke; Liu, Yiming; Yang, Wenwei; Xie, Xing; Ye, Lin; Wang, Li; Zhu, Lin; Ding, Sheng; Su, Yi; Zhou, Jie; Han, Min; Zhuang, Yuan; Xu, Tian; Wu, Xiaohui; Gu, Ning; Zhong, Yang

2008-01-01

270

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

Microsoft Academic Search

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

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

2009-01-01

271

Bac 32, a Novel Bacteriocin Widely Disseminated among Clinical Isolates of Enterococcus faecium  

PubMed Central

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates that had been obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were tested for bacteriocin production. Two hundred seventy-seven (44%) of the strains were bacteriocinogenic; and 193 of these exhibited activity against Enterococcus faecium, Enterococcus hirae, and Enterococcus durans. Strain VRE200 harbors the highly efficient conjugative gentamicin resistance plasmid pG200 (70 kb) and bacteriocin plasmid pTI1 (12.5 kb). The bacteriocin encoded on pTI1 was designated bacteriocin 32 (Bac 32). Bacteriocin 32 was active against E. faecium, E. hirae, and E. durans but showed no activity against Listeria monocytogenes. The Bac 32 genetic locus consists of a bacteriocin gene (bacA) and an immunity gene (bacB). Neither of these genes showed significant homology to any known bacteriocin determinants. The deduced bacA product is 89 amino acids in length, with a putative signal peptide of 19 amino acids at the N terminus. The bacB gene encodes a deduced 55-amino-acid protein without a signal sequence. One hundred eighty-nine strains (97.9%) of the 193 strains with activity against the 3 test enterococcal strains gave rise to the expected specific PCR product with a primer specific for bacA, indicating that there is a high incidence of Bac 32 production among VRE clinical isolates. Data from Southern analyses of plasmid DNA from 189 of the Bac 32-producing strains with a plasmid pTI1-specific probe suggested that 137 (72.5%) of the strains harbored a pTI1-type plasmid. Bac 32 or Bac 32-type bacteriocin activity and the determinant genes were also identified in 22 (39.3%) of a total of 56 vancomycin-sensitive E. faecium clinical isolates, which suggests that this bacteriocin is widely disseminated among E. faecium strains.

Inoue, Takako; Tomita, Haruyoshi; Ike, Yasuyoshi

2006-01-01

272

Genome Mapping and Molecular Breeding of Tomato  

PubMed Central

The cultivated tomato, Lycopersicon esculentum, is the second most consumed vegetable worldwide and a well-studied crop species in terms of genetics, genomics, and breeding. It is one of the earliest crop plants for which a genetic linkage map was constructed, and currently there are several molecular maps based on crosses between the cultivated and various wild species of tomato. The high-density molecular map, developed based on an L. esculentum × L. pennellii cross, includes more than 2200 markers with an average marker distance of less than 1?cM and an average of 750?kbp per cM. Different types of molecular markers such as RFLPs, AFLPs, SSRs, CAPS, RGAs, ESTs, and COSs have been developed and mapped onto the 12 tomato chromosomes. Markers have been used extensively for identification and mapping of genes and QTLs for many biologically and agriculturally important traits and occasionally for germplasm screening, fingerprinting, and marker-assisted breeding. The utility of MAS in tomato breeding has been restricted largely due to limited marker polymorphism within the cultivated species and economical reasons. Also, when used, MAS has been employed mainly for improving simply-inherited traits and not much for improving complex traits. The latter has been due to unavailability of reliable PCR-based markers and problems with linkage drag. Efforts are being made to develop high-throughput markers with greater resolution, including SNPs. The expanding tomato EST database, which currently includes ?214?000 sequences, the new microarray DNA chips, and the ongoing sequencing project are expected to aid development of more practical markers. Several BAC libraries have been developed that facilitate map-based cloning of genes and QTLs. Sequencing of the euchromatic portions of the tomato genome is paving the way for comparative and functional analysis of important genes and QTLs.

Foolad, Majid R.

2007-01-01

273

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

274

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.

Martinez-Conejero, Jose Antonio; Serra, Vicente; Olmo, Ines; Lara, Coral; Simon, Carlos

2014-01-01

275

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

PubMed Central

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

Ting, Jonathan T.; Feng, Guoping

2014-01-01

276

Development of RNAi Libraries for Target Validation and Therapeutics.  

National Technical Information Service (NTIS)

A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require informatio...

T. Giordano

2006-01-01

277

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

278

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

PubMed Central

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

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

2014-01-01

279

Tomorrow's library.  

PubMed

The buildings in which we house libraries are like other special purpose structures; the needs they fill are significantly influenced by technology. A prime function of the library building is to house collections (of people, material, and systems) as well as collections of collections (networks). Electronic formats for library material offer new approaches to information service delivery. An example, the information access station, typifies how traditional functions can be reconfigured with respect to space. Flexible design can help ensure that tomorrow's libraries meet the users' needs, but we need to question all our assumptions about building design including those driven by our understanding of the browsing process. PMID:7842657

Penniman, W D

1994-09-01

280

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: .

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

281

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

282

Abundance and characterization of simple-sequence repeats (SSRs) isolated from a size-fractionated genomic library of Brassica napus L. (rapeseed)  

Microsoft Academic Search

A size-fractionated library of Brassica napus L. (rapeseed), composed of 15000 clones, was screened for the presence of GA-, CA-, and GATA-simple-sequence repeats (SSRs). GA-SSRs were four- and five-fold more abundant than CA- and GATA-SSRs, respectively, and present at a frequency of approximately one SSR for every 100 kb of DNA. Following the sequencing of 124 positive clones, primer pairs

S. Kresovich; A. K. Szewc-McFadden; S. M. Bliek; J. R. McFerson

1995-01-01

283

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

PubMed

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

1996-09-01

284

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.

Amara, R R; Satchidanandam, V

1996-01-01

285

A comprehensive library of DNA-binding site matrices for 55 proteins applied to the complete Escherichia coli K-12 genome1  

Microsoft Academic Search

A major mode of gene regulation occurs via the binding of specific pro- teins to specific DNA sequences. The availability of complete bacterial genome sequences offers an unprecedented opportunity to describe net- works of such interactions by correlating existing experimental data with computational predictions. Of the 240 candidate Escherichia coli DNA- binding proteins, about 55 have DNA-binding sites identified by

Keith Robison; Abigail Manson McGuire; George M. Church

1998-01-01

286

Library Advocacy  

ERIC Educational Resources Information Center

This paper is about the issue of advocacy. Standing at the vanguard of literacy, library media specialists have a unique role. However, it is time for media specialists to advocate their services in a proactive way. If library media specialists cannot, both individually and collectively, put advocacy at the forefront, then students will suffer the…

Plunkett, Kate

2010-01-01

287

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

288

Library Legislation.  

ERIC Educational Resources Information Center

Examination of several library legislation models developed to meet the needs of developed and developing nations reveals that our traditional notion of the library's role in society must be abandoned if we wish to reconcile its benefits to its costs. Four models currently exist: many nations, particularly Asian, have no legislation; most nations,…

Kavass, Igor

289

Library Legislation.  

National Technical Information Service (NTIS)

Examination of several library legislation models developed to meet the needs of developed and developing nations reveals that our traditional notion of the library's role in society must be abandoned if we wish to reconcile its benefits to its costs. Fou...

I. Kavass

1979-01-01

290

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

291

Macintoshed Libraries.  

ERIC Educational Resources Information Center

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

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

292

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

293

BacMam-mediated gene delivery into multipotent mesenchymal stromal cells.  

PubMed

Baculoviruses have been used over the last several decades for high-level protein production in insect cells. Recently, modified baculovirus containing a mammalian promoter, known as BacMam virus, has been shown to give high transduction efficiencies across several cell types with minimal cytopathic effects. Cell types amenable to BacMam transduction include primary and adult stem cells. The shuttle vectors used in the construction of BacMam viruses can hold gene fragments up to 38 kb in size, and multiple BacMam viruses can be used in a single transduction for the delivery of more than one gene. BacMam technology has been used in the delivery and expression of targeted fluorescent protein cellular markers, small interfering RNAi, and extensively in the development of cell-based assays. BacMam offers an ideal method for the delivery and expression of large genes in hard-to-transfect cells such as primary and adult stem cells. In this chapter, we describe methods of generating high titer stocks of BacMam for transducing MSC and their derivatives. PMID:21431539

O'Grady, Michael; Batchelor, Robert H; Scheyhing, Kelly; Kemp, Christopher W; Hanson, George T; Lakshmipathy, Uma

2011-01-01

294

Genetic Analysis of the Sinorhizobium meliloti BacA Protein: Differential Effects of Mutations on Phenotypes  

PubMed Central

Sinorhizobium meliloti strains lacking BacA function are impaired in symbiosis with alfalfa host plants and display altered sensitivities to a number of compounds relative to wild-type strains. With the goal of finding clues to the currently unknown biological function(s) of BacA, we carried out a genetic analysis to determine which amino acids are critical for protein function and to attempt to ascertain whether the multiple phenotypes that result from a bacA-null allele were the result of a common cause or whether BacA has multiple functions. We have created a set of 20 site-directed mutants in which selected individual amino acids in bacA were replaced with glycine residues. The resulting mutants were characterized to determine how the various amino acid changes affected a number of phenotypes associated with loss of BacA function. Mutants H165G, W182G, D198G, and R284G had null phenotypes for all functions assayed, while mutants W57G, S83G, S231G, and K350G were indistinguishable from wild-type strains. The remaining 12 site-directed mutants demonstrate mixed phenotypic characteristics and fall into a number of distinctly different groups. These observations may be consistent with a role for BacA in multiple, nonoverlapping functions.

LeVier, Kristin; Walker, Graham C.

2001-01-01

295

Status and opportunities for genomics research with rainbow trout.  

PubMed

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. PMID:12470823

Thorgaard, Gary H; Bailey, George S; Williams, David; Buhler, Donald R; Kaattari, Stephen L; Ristow, Sandra S; Hansen, John D; Winton, James R; Bartholomew, Jerri L; Nagler, James J; Walsh, Patrick J; Vijayan, Matt M; Devlin, Robert H; Hardy, Ronald W; Overturf, Kenneth E; Young, William P; Robison, Barrie D; Rexroad, Caird; Palti, Yniv

2002-12-01

296

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

297

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.

1998-01-01

298

Gene expression in mammalian cells using BacMam, a modified baculovirus system.  

PubMed

BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. The BacMam system combines the advantages of viral transient expression, ease in generation, and a wide cell tropism. It enables rapid, facile, and flexible gene over-expression experiments to be performed in a variety of mammalian cell lines. Conversion of baculovirus vectors to BacMam vectors involves replacement of the viral specific expression cassette with a mammalian expression cassette or the addition of a mammalian expression cassette. Viruses are produced using standard methods in a few weeks. Mammalian cells transduced with the BacMam viruses have been routinely used as substitutes for stable cell lines. PMID:17951767

Fornwald, James A; Lu, Quinn; Wang, Dayuan; Ames, Robert S

2007-01-01

299

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

300

Development of genomic resources in support of sequencing, assembly, and annotation of the catfish genome.  

PubMed

Major progress has been made in catfish genomics including construction of high-density genetic linkage maps, BAC-based physical maps, and integration of genetic linkage and physical maps. Large numbers of ESTs have been generated from both channel catfish and blue catfish. Microarray platforms have been developed for the analysis of genome expression. Genome repeat structures are studied, laying grounds for whole genome sequencing. USDA recently approved funding of the whole genome sequencing project of catfish using the next generation sequencing technologies. Generation of the whole genome sequence is a historical landmark of catfish research as it opens the real first step of the long march toward genetic enhancement. The research community needs to be focused on aquaculture performance and production traits, take advantage of the unprecedented genome information and technology, and make real progress toward genetic improvements of aquaculture brood stocks. PMID:20430707

Liu, Zhanjiang

2011-03-01

301

Genome-Wide Interrogation of Mammalian Stem Cell Fate Determinants by Nested Chromosome Deletions  

PubMed Central

Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP–based system, we now report the creation and characterization of a collection of ?1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.

MacRae, Tara; Laverdure, Jean-Philippe; Azcoitia, Valeria; Girard, Simon; Chagraoui, Jalila; Ringuette, Nancy; Hebert, Josee; Krosl, Jana; Mayotte, Nadine; Sauvageau, Guy

2010-01-01

302

[The Presidential Libraries.  

ERIC Educational Resources Information Center

There are seven Presidential libraries in various states of existence, from quite active to proposed: (1) Franklin D. Roosevelt Library, (2) Harry S. Truman Library, (3) Herbert Hoover Library, (4) Dwight D. Eisenhower Library, (5) John F. Kennedy Memorial Library (6) Lyndon B. Johnson Library and (7) Rutherford B. Hayes Memorial Library. Each…

Webb, John

303

Bac 32, a novel bacteriocin widely disseminated among clinical isolates of Enterococcus faecium.  

PubMed

A total of 636 vancomycin-resistant Enterococcus faecium (VRE) isolates that had been obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were tested for bacteriocin production. Two hundred seventy-seven (44%) of the strains were bacteriocinogenic; and 193 of these exhibited activity against Enterococcus faecium, Enterococcus hirae, and Enterococcus durans. Strain VRE200 harbors the highly efficient conjugative gentamicin resistance plasmid pG200 (70 kb) and bacteriocin plasmid pTI1 (12.5 kb). The bacteriocin encoded on pTI1 was designated bacteriocin 32 (Bac 32). Bacteriocin 32 was active against E. faecium, E. hirae, and E. durans but showed no activity against Listeria monocytogenes. The Bac 32 genetic locus consists of a bacteriocin gene (bacA) and an immunity gene (bacB). Neither of these genes showed significant homology to any known bacteriocin determinants. The deduced bacA product is 89 amino acids in length, with a putative signal peptide of 19 amino acids at the N terminus. The bacB gene encodes a deduced 55-amino-acid protein without a signal sequence. One hundred eighty-nine strains (97.9%) of the 193 strains with activity against the 3 test enterococcal strains gave rise to the expected specific PCR product with a primer specific for bacA, indicating that there is a high incidence of Bac 32 production among VRE clinical isolates. Data from Southern analyses of plasmid DNA from 189 of the Bac 32-producing strains with a plasmid pTI1-specific probe suggested that 137 (72.5%) of the strains harbored a pTI1-type plasmid. Bac 32 or Bac 32-type bacteriocin activity and the determinant genes were also identified in 22 (39.3%) of a total of 56 vancomycin-sensitive E. faecium clinical isolates, which suggests that this bacteriocin is widely disseminated among E. faecium strains. PMID:16569830

Inoue, Takako; Tomita, Haruyoshi; Ike, Yasuyoshi

2006-04-01

304

BacT\\/Alert: anAutomated Colorimetric Microbial Detection System  

Microsoft Academic Search

BacT\\/Alert (Organon Teknika Corp., Durham, N.C.) isanautomated microbial detection system based on thecolorimetric detection ofC02produced bygrowing microorganisms. Results ofanevaluation ofthemedia, sensor, detection system, anddetection algorithm indicate thatthesystem reliably growsanddetects awide variety ofbacteria andfungi. Results ofalimited pilot clinical trial withaprototype research instrument indicate thatthesystem iscomparable totheradiometric BACTEC460system initsability togrowanddetect microorganisms inblood. Onthebasis ofthese initial findings, large-scale clinical trials comparing BacT\\/Alert withother

THURMAN C. THORPE; MICHAEL L. WILSON; JAMES E. TURNER; L. BARTH RELLER

1990-01-01

305

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

PubMed Central

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

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

2013-01-01

306

Lineage-Specific Expansions of Retroviral Insertions within the Genomes of African Great Apes but Not Humans and Orangutans  

Microsoft Academic Search

Retroviral infections of the germline have the potential to episodically alter gene function and genome structure during the course of evolution. Horizontal transmissions between species have been proposed, but little evidence exists for such events in the human\\/great ape lineage of evolution. Based on analysis of finished BAC chimpanzee genome sequence, we characterize a retroviral element (Pan troglodytes endogenous retrovirus

Chris T. Yohn; Zhaoshi Jiang; Sean D. McGrath; Karen E. Hayden; Philipp Khaitovich; Matthew E. Johnson; Marla Y. Eichler; John D. McPherson; Shaying Zhao; Svante Pääbo; Evan E. Eichler

2005-01-01

307

The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library.  

PubMed

Thirty-four hundred lambda phage clones containing segments of the E. coli chromosome were isolated and used to construct a 4700 kb long integrated restriction map for eight six-base-recognizing enzymes by a rapid mass-analysis method. Our strategy was to measure the sizes of partial restriction enzyme digests by hybridization with a vector probe in a manner analogous to nucleotide sequencing. The data were sorted into groups by a computer program and the boundary clones were further correlated with each other using a mass hybridization method. These clones can be exploited for the isolation of any desired E. coli genes if their map positions are known. Also, the strategy is applicable to analyses of the genomes of other organisms. PMID:3038334

Kohara, Y; Akiyama, K; Isono, K

1987-07-31

308

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

PubMed

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

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

2014-02-01

309

Segmental Duplications and Copy-Number Variation in the Human Genome  

PubMed Central

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders.

Sharp, Andrew J.; Locke, Devin P.; McGrath, Sean D.; Cheng, Ze; Bailey, Jeffrey A.; Vallente, Rhea U.; Pertz, Lisa M.; Clark, Royden A.; Schwartz, Stuart; Segraves, Rick; Oseroff, Vanessa V.; Albertson, Donna G.; Pinkel, Daniel; Eichler, Evan E.

2005-01-01

310

Comparative genome hybridization suggests a role for NRXN1 and APBA2 in schizophrenia  

Microsoft Academic Search

Copy number variations (CNVs) account for a substantial proportion of human genomic variation, and have been shown to cause neurodevelopmental disorders. We sought to determine the relevance of CNVs to the aetiology of schizophrenia (SZ). Whole-genome, high-resolution, tiling path BAC array comparative genomic hybridization (array CGH) was employed to test DNA from 93 individuals with DSM-IV SZ. Common DNA copy

George Kirov; D. Gumus; W. Chen; N. Norton; L. Georgieva; M. Sari; M. C O'Donovan; F. Erdogan; M. J Owen; Hilger Ropers; Reinhard Ullmann

2007-01-01

311

Progress in Understanding and Sequencing the Genome of Brassica rapa  

PubMed Central

Brassica rapa, which is closely related to Arabidopsis thaliana, is an important crop and a model plant for studying genome evolution via polyploidization. We report the current understanding of the genome structure of B. rapa and efforts for the whole-genome sequencing of the species. The tribe Brassicaceae, which comprises ca. 240 species, descended from a common hexaploid ancestor with a basic genome similar to that of Arabidopsis. Chromosome rearrangements, including fusions and/or fissions, resulted in the present-day “diploid” Brassica species with variation in chromosome number and phenotype. Triplicated genomic segments of B. rapa are collinear to those of A. thaliana with InDels. The genome triplication has led to an approximately 1.7-fold increase in the B. rapa gene number compared to that of A. thaliana. Repetitive DNA of B. rapa has also been extensively amplified and has diverged from that of A. thaliana. For its whole-genome sequencing, the Brassica rapa Genome Sequencing Project (BrGSP) consortium has developed suitable genomic resources and constructed genetic and physical maps. Ten chromosomes of B. rapa are being allocated to BrGSP consortium participants, and each chromosome will be sequenced by a BAC-by-BAC approach. Genome sequencing of B. rapa will offer a new perspective for plant biology and evolution in the context of polyploidization.

Hong, Chang Pyo; Kwon, Soo-Jin; Kim, Jung Sun; Yang, Tae-Jin; Park, Beom-Seok; Lim, Yong Pyo

2008-01-01

312

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

PubMed

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

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

2014-01-01

313

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.

Parker, Jared B.; Walsh, Christopher T.

2012-01-01

314

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

Microsoft Academic Search

The BacA protein is essential for the long-term survival of Sinorhizobium meliloti and Brucella abortus within acidic compartments in plant and animal cells, respectively. Since both the S. meliloti and B. abortus bacA mutants have an increased resistance to bleomycin, it was hypothesized that BacA was a transporter of bleomycin and bleomycin-like compounds into the bacterial cell. However, our finding

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

2002-01-01

315

The lepidopteran transposon vector, piggyBac, mediates germ-line transformation in the Mediterranean fruit fly  

Microsoft Academic Search

The piggyBac (IFP2) short inverted terminal repeat transposable element from the cabbage looper Trichop- lusia ni was tested for gene transfer vector function as part of a bipartite vector-helper system in the Mediterranean fruit fly Ceratitis capitata .A piggyBac vector marked with the medf ly white gene was tested with a normally regulated piggyBac transposase helper at two different concentrations

ALFRED M. HANDLER; S USAN D. MCCOMBS; J. FRASER; STEPHEN H. SAUL

1998-01-01

316

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

Microsoft Academic Search

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac

Toshiki Tamura; Chantal Thibert; Corinne Royer; Toshio Kanda; Abraham Eappen; Mari Kamba; Natuo Kômoto; Jean-Luc Thomas; Bernard Mauchamp; Gérard Chavancy; Paul Shirk; Malcolm Fraser; Jean-Claude Prudhomme; Pierre Couble

2000-01-01

317

The effectiveness of a 0.05 blood alcohol concentration (BAC) limit for driving in the United States.  

PubMed

The National Transportation Safety Board recently recommended that states establish a per se blood alcohol concentration (BAC) limit of 0.05 or lower for all drivers who are not already required to adhere to lower BAC limits in a national effort to reduce alcohol-impaired driving. There is strong evidence for adopting this recommendation. A comprehensive review of the literature on BAC limits was conducted. The research indicates that virtually all drivers are impaired regarding at least some driving performance measures at a 0.05 BAC. The risk of being involved in a crash increases significantly at 0.05 BAC and above. The relative risk of being killed in a single-vehicle crash with BACs of 0.05-0.079 is 7-21 times higher than for drivers at 0.00 BAC. Lowering the BAC limit from 0.08 to 0.05 has been a proven effective countermeasure in numerous countries around the world. Most Americans do not believe a person should drive after having two or three drinks in 2 hours. It takes at least four drinks for the average 170-pound male to exceed 0.05 BAC in 2 hours (three drinks for the 137-pound female). Most industrialized nations have established a 0.05 BAC limit or lower for driving. Progress in reducing the proportion of drivers in fatal crashes with illegal BACs has stalled over the past 15 years. Lowering the BAC limit for driving from the current 0.08 to 0.05 has substantial potential to reduce the number of people who drink and drive in the United States and get involved in fatal crashes. PMID:24898061

Fell, James C; Voas, Robert B

2014-06-01

318

Après le bac professionnel ou technologique : la poursuite d'études jusqu'à bac + 2 et sa rentabilité salariale en début de vie active  

Microsoft Academic Search

[fre] Le rendement salarial que les bacheliers professionnels et technologiques peuvent attendre de la poursuite de leurs études dépend de leur réussite, incertaine, au diplôme de niveau bac + 2. Le calcul de ce rendement est fondé sur le supplément de salaire que pourraient escompter ces bacheliers, compte tenu de leurs caractéristiques individuelles, s'ils poursuivaient leurs études en cas de

Stéphanie Moullet

2005-01-01

319

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

320

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

321

Rural Public Libraries in Multitype Library Cooperatives.  

ERIC Educational Resources Information Center

Provides a historical overview of library cooperative efforts, the roles identified for the cooperative library organizations and their members, and associated services. Examines the contributions that rural libraries make toward cooperation among all types of libraries and identifies challenges for rural libraries participating in networks in the…

Ison, Jan

1995-01-01

322

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

PubMed

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

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

2012-02-01

323

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

PubMed Central

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

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

2012-01-01

324

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

PubMed Central

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

2012-01-01

325

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

PubMed

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

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

2014-06-01

326

Expression of Human CAR Splicing Variants in BAC-Transgenic Mice  

PubMed Central

The nuclear receptor constitutive androstane receptor (CAR) is a key regulator for drug metabolism in liver. Human CAR (hCAR) transcripts are subjected to alternative splicing. Some hCAR splicing variants (SVs) have been shown to encode functional proteins by reporter assays. However, in vivo research on the activity of these hCAR SVs has been impeded by the absence of a valid model. This study engineered an hCAR-BAC-transgenic (hCAR-TG) mouse model by integrating the 8.5-kbp hCAR gene as well as 73-kbp upstream and 91-kbp downstream human genomic DNA into the genome of CAR-null mice. A series of experiments demonstrate that (1) the expression of major hCAR mRNA SVs, SV0-4, in livers of hCAR-TG mice is comparable to that in human livers; (2) the hCAR SVs are predominantly expressed in liver, which resembles the tissue distribution of CAR in humans, but diverges from that in mice; and (3) major hCAR mRNA SVs increase markedly in postnatal livers of hCAR-TG mice, which mimics the ontogeny of CAR mRNA in humans. Thus, the transgene likely contains all the functional regulatory elements controlling proper spatial and temporal expression of the hCAR gene. Moreover, hCAR-TG mice respond to the hCAR-specific agonist 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime instead of the mouse CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, as well as the common CAR activator, phenobarbital, suggesting that hCAR is fully functional in livers of transgenic mice. In summary, the hCAR-TG mice developed by this study represent a valid model for studying in vivo function and regulation of hCAR and its splicing variants.

Klaassen, Curtis D.

2013-01-01

327

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

PubMed Central

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

2010-01-01

328

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

329

Construction and characterization of a bacterial artificial chromosome library of peach  

Microsoft Academic Search

A peach [Prunus persica (L.) Batch] bacterial artificial chromosome (BAC) library of var. Jingyu was constructed. Jingyu is a traditional variety,\\u000a that displays many of the important agronomic characters of stone fruits. Since peach leaves are rich in polysaccharides,\\u000a high-molecular-weight (HMW) DNA was extracted from leaf nuclei using a protocol adapted to peach. The HMW DNA embedded in\\u000a agarose plugs

Q. Wang; K. Zhang; X. Qu; J. Jia; J. Shi; D. Jin; B. Wang

2001-01-01

330

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

PubMed

This paper describes the use of advanced fluorescence in situ hybridization (FISH) technologies for genomics and breeding of tomato and related Solanum species. The first part deals with the major determinants of FISH technology: (1) spatial resolution, which depends on the diffraction limit of the microscope and the type of chromosome, chromatin or isolated DNA fibres as target for the hybridisation; (2) the detection sensitivity, which is limited by the sensitivity and dynamic range of the CCD camera and the quality of the microscope, and the amplification system of the weak signals from tiny probe molecules; (3) simultaneous detection of multiple probes labelled directly or indirectly with up to 5 different fluorophores, whether or not in different combinations and/or mixed at different ratios. The power and usability of such multicolour FISH is indispensable when large numbers of bacterial artificial chromosomes (BACs) or other vectors with genomic DNA are available. Mapping of multiple BACs on chromosomes are powerful instruments confirming their assumed genetic position, whereas pooled BACs for a given chromosome arm will reveal the gaps between the BACs or derived contigs of their physical maps. Tandem and dispersed repeats, which are abundant in the genomes of most species, can be analysed in repeat bar coding FISH, showing the major blocks of repeats in heterochromatin and euchromatin areas. Repeat-rich areas of the chromosomes can also be demonstrated by hybridisation of probed Cot fractions of sheared genomic DNA; a powerful method to elucidate the heterochromatin domains for genomic studies. In addition, unlabelled Cot DNA, as blocking agent in BAC-FISH painting, suppresses repetitive sequences from the BACs to hybridise on the chromosomes. Cross-species BAC-FISH painting with labelled probes from tomato and potato BACs and hybridised on the chromosomes of related species, under appropriate conditions, is a powerful instrument to demonstrate chromosomal rearrangements, including inversions and translocations. The technology not only supports phylogenetic studies between the taxa under study but can also be helpful in breeding programs with crops containing introgressed regions from related species when linkage drag or meiotic pairing disturbances between the homoeologues are assumed. In the next steps in comparative genomics, we now can detect smaller chromosomal and DNA rearrangements, diminutions and amplifications of repeats and changes of the epigenetic status of introgressed regions. PMID:20628252

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

2010-07-01

331

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.

332

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.

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

2013-01-01

333

Minnesota Zoological Garden Library.  

ERIC Educational Resources Information Center

Describes the history and functions of the Minnesota Zoological Garden library. Topics covered include the library collections; library services, including online search capabilities; and the various groups of users served by the library. (three references) (CLB)

Norell, Angela

1988-01-01

334

Paraprofessional Library Employees.  

National Technical Information Service (NTIS)

This pamphlet describes library paraprofessionals, answering the questions: (1) What are library paraprofessionals; (2) What do paraprofessionals do; (3) How can you become a library paraprofessional; (4) What is the job market for library paraprofessiona...

C. Evans

1976-01-01

335

Library Services. Miscellaneous Papers.  

National Technical Information Service (NTIS)

Papers on library journal cooperation, interlibrary lending, library services to minorities, and school library media centers, which were presented at the 1983 International Federation of Library Associations (IFLA) conference, include: (1) 'The Co-operat...

1983-01-01

336

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."

337

A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3' untranslated region-mediated gene regulation.  

PubMed

Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3' untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3' UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3' UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome-a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3' UTR of interest. The pBUTR (piggyBac-based 3' UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting. PMID:24753411

Chaudhury, Arindam; Kongchan, Natee; Gengler, Jon P; Mohanty, Vakul; Christiansen, Audrey E; Fachini, Joseph M; Martin, James F; Neilson, Joel R

2014-06-01

338

A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3? untranslated region-mediated gene regulation  

PubMed Central

Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3? untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3? UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3? UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome—a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3? UTR of interest. The pBUTR (piggyBac-based 3? UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.

Chaudhury, Arindam; Kongchan, Natee; Gengler, Jon P.; Mohanty, Vakul; Christiansen, Audrey E.; Fachini, Joseph M.; Martin, James F.; Neilson, Joel R.

2014-01-01

339

High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC.  

PubMed

Transgenesis has been used for expressing human lysozyme (hLZ) in the milk of livestock to improve their disease resistance. Here we describe a human lactoferrin (hLF) BAC as a candidate vector for high-level expression of hLZ in the milk of transgenic mice. Using recombineering, hLF genomic DNA in the hLF BAC was replaced by the hLZ gene (from the ATG start codon to the TAA stop codon), and flanking regions of the hLF gene (a 90-kb 5' and a 30-kb 3') were used as transcriptional control elements for hLZ expression. When this construct was used to generate transgenic mice, rhLZ was highly expressed in the milk of four transgenic mouse lines (1.20-1.76 g/L), was expressed at a lower level in one additional line (0.21 g/L). rhLZ from the milk of these transgenic mice exhibited the same antibacterial activity as native hLZ. Our results suggest a potential approach for producing large amounts of hLZ in the milk of livestock. PMID:21805108

Liu, Shen; Li, Xiangqing; Lu, Dan; Shang, Shengzhe; Wang, Meili; Zheng, Min; Zhang, Ran; Tang, Bo; Li, Qiuyan; Dai, Yunping; Li, Ning

2012-04-01

340

Turkish Libraries: Historical Context.  

ERIC Educational Resources Information Center

Summary of the development of libraries in Turkey highlights the existence of libraries in the ninth century, the Shamssaddin Altunaba Medrese library in Konya, libraries established during the Ottoman era, reports to reform libraries (1869-70, 1909), and reports and library developments attributed to the Republican Era beginning in 1923. (EJS)

Cakin, Irfan

1984-01-01

341

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

342

Public Libraries in Bangladesh.  

ERIC Educational Resources Information Center

Overview of library movement in Bangladesh highlights British (1851-1947) and Pakistan periods (1947-1971), separation of Bangladesh from Pakistan, libraries in development plans (1951-1970), three important public libraries, development of national library, book resources, a library network plan, legislation, finance, leadership, library

Khan, M. H.

1984-01-01

343

Library Buildings and Equipment.  

ERIC Educational Resources Information Center

Six articles discuss library buildings and construction: (1) library buildings and their parts; (2) the North Campus Library of California State University at Long Beach in 1995; (3) new structures for teaching libraries; (4) construction standards for California public libraries; (5) Sick (Library) Building Syndrome; and (6) using focus-group…

Oringdulph, Robert E.; And Others

1990-01-01

344

Functional Identification of the Mouse Circadian Clock Gene by Transgenic BAC Rescue  

PubMed Central

Summary As a complementary approach to positional cloning, we used in vivo complementation with bacterial artificial chromosome (BAC) clones expressed in transgenic mice to identify the circadian Clock gene. A 140 kb BAC transgene completely rescued both the long period and the loss-of-rhythm phenotypes in Clock mutant mice. Analysis with overlapping BAC transgenes demonstrates that a large transcription unit spanning “100,000 base pairs is the Clock gene and encodes a novel basic–helix-loop-helix–PAS domain protein. Overexpression of the Clock transgene can shorten period length beyond the wild-type range, which provides additional evidence that Clock is an integral component of the circadian pacemaking system. Taken together, these results provide a proof of principle that “cloning by rescue” is an efficient and definitive method in mice.

Antoch, Marina P.; Song, Eun-Joo; Chang, Anne-Marie; Vitaterna, Martha Hotz; Zhao, Yaliang; Wilsbacher, Lisa D.; Sangoram, Ashvin M.; King, David P.; Pinto, Lawrence H.; Takahashi, Joseph S.

2013-01-01

345

Chicken genomics charts a path to the genome sequence.  

PubMed

In this paper, the current status of chicken genomics is reviewed. This is timely given the current intense activity centred on sequencing the complete genome of this model species. The genome project is based on a decade of map building by genetic linkage and cytogenetic methods, which are now being replaced by high-resolution radiation hybrid and bacterial artificial chromosome (BAC) contig maps. Markers for map building have generally depended on labour-intensive screening procedures, but in recent years this has changed with the availability of almost 500,000 chicken expressed sequence tags (ESTs). These resources and tools will be critical in the coming months when the chicken genome sequence is being assembled (eg cross-checked with other maps) and annotated (eg gene structures based on ESTs). The future for chicken genome and biological research is an exciting one, through the integration of these resources. For example, through the proposed chicken Ensembl database, it will be possible to solve challenging scientific questions by exploiting the power of a chicken model. One area of interest is the study of developmental mechanisms and the discovery of regulatory networks throughout the genome. Another is the study of the molecular nature of quantitative genetic variation. No other animal species have been phenotyped and selected so intensively as agricultural animals and thus there is much to be learned in basic and medical biology from this research. PMID:15163360

Burt, David W

2004-04-01

346

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.

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

2009-01-01

347

Genomic systematic evolution of ligands by exponential enrichment (Genomic SELEX) for the identification of protein-binding RNAs independent of their expression levels  

Microsoft Academic Search

Genomic systematic evolution of ligands by exponential enrichment (Genomic SELEX) is an experimental procedure for the expression condition-independent identification of protein-binding RNAs. RNA libraries derived from genomic DNA are generated via random priming, PCR amplification and in vitro transcription. Libraries consist of genomic sequences of selected size, and fragments are flanked by constant sequences required for amplification and transcription. This

Christina Lorenz; Frederike von Pelchrzim; Renée Schroeder

2006-01-01

348

Comparative genomics: the key to understanding the human genome project  

Microsoft Academic Search

Summary The sequencing of the human genome is well underway. Technology has ad- vanced, such that the total genomic sequence is possible, along with an extensive catalogue of genes via comprehensive cDNA libraries. With the recent completion of the Saccharomyces cerevisiae sequencing project and the imminent comple- tion of that of Caenorhabditis elegans, the most frequently asked question is how

M. S. Clark

1999-01-01

349

Structure of a KirBac potassium channel with an open bundle crossing indicates a mechanism of channel gating  

Microsoft Academic Search

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying (Kir) potassium channels, and recent crystal structures of both Kir and KirBac channels have provided major insight into their unique structural architecture. However, all of the available structures are closed at the helix bundle crossing, and therefore the structural mechanisms that control opening of their primary activation gate remain unknown. In

Vassiliy N Bavro; Rita De Zorzi; Matthias R Schmidt; João R C Muniz; Lejla Zubcevic; Mark S P Sansom; Catherine Vénien-Bryan; Stephen J Tucker

2012-01-01

350

Genome Dynamics and Evolution of the Mla (Powdery Mildew) Resistance Locus in Barley  

Microsoft Academic Search

Genes that confer defense against pathogens often are clustered in the genome and evolve via diverse mechanisms. To evaluate the organization and content of a major defense gene complex in cereals, we determined the complete se- quence of a 261-kb BAC contig from barley cv Morex that spans the Mla (powdery mildew) resistance locus. Among the 32 predicted genes on

Fusheng Wei; Rod A. Wing; Roger P. Wise

2002-01-01

351

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

352

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.

Fahn?e, Ulrik; Hoper, Dirk; Schirrmeier, Horst; Beer, Martin

2014-01-01

353

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

354

Multiplexed Illumina sequencing libraries from picogram quantities of DNA  

PubMed Central

Background High throughput sequencing is frequently used to discover the location of regulatory interactions on chromatin. However, techniques that enrich DNA where regulatory activity takes place, such as chromatin immunoprecipitation (ChIP), often yield less DNA than optimal for sequencing library preparation. Existing protocols for picogram-scale libraries require concomitant fragmentation of DNA, pre-amplification, or long overnight steps. Results We report a simple and fast library construction method that produces libraries from sub-nanogram quantities of DNA. This protocol yields conventional libraries with barcodes suitable for multiplexed sample analysis on the Illumina platform. We demonstrate the utility of this method by constructing a ChIP-seq library from 100 pg of ChIP DNA that demonstrates equivalent genomic coverage of target regions to a library produced from a larger scale experiment. Conclusions Application of this method allows whole genome studies from samples where material or yields are limiting.

2013-01-01

355

454 sequencing put to the test using the complex genome of barley  

PubMed Central

Background During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising a ~100-fold increase in throughput over Sanger technology – an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley. Results To study its performance in complex genomes, we used 454 technology to sequence four barley Bacterial Artificial Chromosome (BAC) clones and compared the results to those from ABI-Sanger sequencing. All gene containing regions were covered efficiently and at high quality with 454 sequencing whereas repetitive sequences were more problematic with 454 sequencing than with ABI-Sanger sequencing. 454 sequencing provided a much more even coverage of the BAC clones than ABI-Sanger sequencing, resulting in almost complete assembly of all genic sequences even at only 9 to 10-fold coverage. To obtain highly advanced working draft sequences for the BACs, we developed a strategy to assemble large parts of the BAC sequences by combining comparative genomics, detailed repeat analysis and use of low-quality reads from 454 sequencing. Additionally, we describe an approach of including small numbers of ABI-Sanger sequences to produce hybrid assemblies to partly compensate the short read length of 454 sequences. Conclusion Our data indicate that 454 pyrosequencing allows rapid and cost-effective sequencing of the gene-containing portions of large and complex genomes and that its combination with ABI-Sanger sequencing and targeted sequence analysis can result in large regions of high-quality finished genomic sequences.

Wicker, Thomas; Schlagenhauf, Edith; Graner, Andreas; Close, Timothy J; Keller, Beat; Stein, Nils

2006-01-01

356

High frequency of common DNA copy number abnormalities detected by bacterial artificial chromosome array comparative genomic hybridization in 24 breast cancer cell lines.  

PubMed

Breast cancer is a widespread disease in Japan and across the world. Breast cancer cells, as well as most other types of cancer cells, have diverse chromosomal aberrations. Clarifying the character of these chromosomal aberrations should contribute to the development of more suitable therapies, along with the predictions of metastasis and prognosis. Twenty-four breast cancer cell lines were analyzed by bacterial artificial chromosome (BAC) array comparative genomic hybridization (CGH). The array slide contained duplicate spots of 4030 BAC clone DNAs covering the entire human genome with 1 Mbp resolution. In all 24 breast cancer cell lines, frequent and significant amplifications as well as deletions were detected by BAC array CGH. Common DNA copy number gains, detected in 60% (above 15 cell lines) of the 24 breast cancer cell lines were found in 76 BAC clones, located at 1q, 5p, 8q, 9p, 16p, 17q, and 20q. Moreover, common DNA copy number loss was detected in 136 BAC clones, located at 1q, 2q, 3p, 4p, 6q, 8p, 9p, 11p, 13q, 17p, 18q, 19p, Xp, and Xq. The DNA copy number abnormalities found included abnormality of the well-known oncogene cMYC (8q24.21); however, most of them were not reported to relate to breast cancer. BAC array CGH has great potential to detect DNA copy number abnormalities, and has revealed that breast cancer cell lines have substantial heterogeneity. PMID:19222606

Saito, Soichiro; Morita, Keiko; Hirano, Takashi

2009-02-01

357

Assembly of large genomic segments in artificial chromosomes by homologous recombination in Escherichia coli.  

PubMed

We developed a method for the reconstruction of a 100 kb DNA fragment into a bacterial artificial chromosome (BAC). The procedure makes use of iterative rounds of homologous recombination in Escherichia coli. Smaller, overlapping fragments of cloned DNA, such as cosmid clones, are required. They are transferred first into a temperature-sensitive replicon and then into the BAC of choice. We demonstrated the usefulness of this procedure by assembling a 90 kb genomic segment into an E.coli-STREPTOMYCES: artificial chromosome (ESAC). Using this procedure, ESACs are easy to handle and remarkably more stable than the starting cosmids. PMID:11266572

Sosio, M; Bossi, E; Donadio, S

2001-04-01

358

Differential CT features of infectious pneumonia versus bronchioloalveolar carcinoma (BAC) mimicking pneumonia  

Microsoft Academic Search

The purpose of this study was to evaluate retrospectively the differential CT features of bronchioloalveolar carcinoma (BAC) mimicking pneumonia and infectious pneumonia at the lung periphery. CT images were reviewed in 47 patients with focal areas of parenchymal opacification at the lung periphery. We evaluated the presence of ground-glass attenuation, marginal conspicuity of the lesion, CT angiogram sign, air-bronchogram sign,

Tae Hoon Kim; Sang Jin Kim; Young Hoon Ryu; Soo Yoon Chung; Jae Seung Seo; Young Jin Kim; Byoung Wook Choi; Sun Hwa Lee; Sang Ho Cho

2006-01-01

359

Bromate removal during transition from new granular activated carbon (GAC) to biological activated carbon (BAC)  

Microsoft Academic Search

Bromate removal by activated carbon after ozonation is a subject of concern, since bromate is commonly found in the ozonation of bromide-containing water. Though new GAC (granular activated carbon) shows the capacity to reduce bromate to bromide, in the long-term use of GAC following ozonation, its bromate removal rate apparently decreases during transition from new GAC to BAC (biological activated

Mari Asami; Takako Aizawa; Takayuki Morioka; Wataru Nishijima; Akihisa Tabata; Yasumoto Magara

1999-01-01

360

A new positive/negative selection scheme for precise BAC recombineering.  

PubMed

Recombineering technology allows the modification of large DNA constructs without using restriction enzymes, enabling the use of bacterial artificial chromosomes (BACs) in genetic engineering of animals and plants as well as in the studies of structures and functions of chromosomal elements in DNA replication and transcription. Here, we report a new selection scheme of BAC recombineering. A dual kanamycin and streptomycin selection marker was constructed using the kanamycin resistance gene and bacterial rpsL (+) gene. Recombination cassettes generated using this dual marker was used to make precise modifications in BAC constructs in a two-step procedure without leaving behind any unwanted sequences. The dual marker was first inserted into the site of modifications by positive selection of kanamycin resistance. In the second step, the counter-selection of streptomycin sensitivity resulted in the replacement of the dual marker with intended modified sequences. This method of BAC modification worked as efficiently as the previously reported galK method and provided a faster and more cost-effective alternative to the galK method. PMID:19160076

Wang, Shuwen; Zhao, Yuanjun; Leiby, Melanie; Zhu, Jiyue

2009-05-01

361

A meta-analysis of .08 BAC laws in 19 jurisdictions in the United States  

Microsoft Academic Search

More than a dozen studies on the effectiveness of the .08 blood alcohol concentration (BAC) laws have been published; however, those studies have varied both in the statistical methods and the type of outcome measure used, so it is difficult to integrate the findings into an overall estimate of the effectiveness of the law. This study used a consistent outcome

A. Scott Tippetts; Robert B. Voas; James C. Fell; James L. Nichols

2005-01-01

362

Deutsches Museum Library.  

ERIC Educational Resources Information Center

This history of the Library of the Deutsches Museum (Munich, Germany) covers the library's situation at end of the nineteenth century, library collections and buildings, use of the library for research, the rare book collection, and service statistics. Library director, collection and staff size, and main subjects collected are included. (EJS)

Berninger, Ernst H.; Reineke, Eva

1986-01-01

363

Library Research and Statistics.  

ERIC Educational Resources Information Center

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

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

1998-01-01

364

Lithuanian Library History.  

ERIC Educational Resources Information Center

Reviews the history of libraries in Lithuania from 1940 to 1992. Highlights include early book collections, and attitudes toward books and reading; changes after the Soviet invasion, including censorship, lack of timeliness, and information barriers; library networks; library education; research trends; library legislation; library literature; and…

Gudauskas, Renaldas

1994-01-01

365

Marketing the Virtual Library  

ERIC Educational Resources Information Center

Far more people are familiar with their local public or college library facility than their library's website and online resources. In fact, according to a recent survey, 96% of Americans said they had visited a library in person, but less than one-third have visited their online library. Since everyone agrees that online library resources are…

Fagan, Jody Condit

2009-01-01

366

Library Services. Miscellaneous Papers.  

ERIC Educational Resources Information Center

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

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

367

Company profile: Complete Genomics Inc.  

PubMed

Complete Genomics Inc. is a life sciences company that focuses on complete human genome sequencing. It is taking a completely different approach to DNA sequencing than other companies in the industry. Rather than building a general-purpose platform for sequencing all organisms and all applications, it has focused on a single application - complete human genome sequencing. The company's Complete Genomics Analysis Platform (CGA™ Platform) comprises an integrated package of biochemistry, instrumentation and software that sequences human genomes at the highest quality, lowest cost and largest scale available. Complete Genomics offers a turnkey service that enables customers to outsource their human genome sequencing to the company's genome sequencing center in Mountain View, CA, USA. Customers send in their DNA samples, the company does all the library preparation, DNA sequencing, assembly and variant analysis, and customers receive research-ready data that they can use for biological discovery. PMID:21345140

Reid, Clifford

2011-02-01

368

Using array-comparative genomic hybridization to define molecular portraits of primary breast cancers  

Microsoft Academic Search

We analysed 148 primary breast cancers using BAC-arrays containing 287 clones representing cancer-related gene\\/loci to obtain genomic molecular portraits. Gains were detected in 136 tumors (91.9%) and losses in 123 tumors (83.1%). Eight tumors (5.4%) did not have any genomic aberrations in the 281 clones analysed. Common (more than 15% of the samples) gains were observed at 8q11–qtel, 1q21–qtel, 17q11–q12

S-F Chin; Y Wang; N P Thorne; A E Teschendorff; S E Pinder; M Vias; A Naderi; I Roberts; N L Barbosa-Morais; M J Garcia; N G Iyer; T Kranjac; J F R Robertson; S Aparicio; S Tavaré; I Ellis; J D Brenton; C Caldas

2007-01-01

369

Evolution and microsynteny of the apyrase gene family in three legume genomes  

Microsoft Academic Search

Apyrases have been suggested to play important roles in plant nutrition, photomorphogenesis, and nodulation. To help trace the evolution of these genes in the legumes—and possibly, the acquisition of new functions for nodulation—apyrase-containing BACs were sequenced from three legume genomes. Genomic sequences from Medicago truncatula, Glycine max and Lotus japonicus were compared to one another and to corresponding regions in

S. B. Cannon; W. R. McCombie; S. Sato; S. Tabata; R. Denny; L. Palmer; M. Katari; N. D. Young; G. Stacey

2003-01-01

370

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

371

Poultry genome sequences: progress and outstanding challenges.  

PubMed

The first build of the chicken genome sequence appeared in March, 2004 - the first genome sequence of any animal agriculture species. That sequence was done primarily by whole genome shotgun Sanger sequencing, along with the use of an extensive BAC contig-based physical map to assemble the sequence contigs and scaffolds and align them to the known chicken chromosomes and linkage groups. Subsequent sequencing and mapping efforts have improved upon that first build, and efforts continue in search of missing and/or unassembled sequence, primarily on the smaller microchromosomes and the sex chromosomes. In the past year, a draft turkey genome sequence of similar quality has been obtained at a much lower cost primarily due to the development of 'next-generation' sequencing techniques. However, assembly and alignment of the sequence contigs and scaffolds still depended on a detailed BAC contig map of the turkey genome that also utilized comparison to the existing chicken sequence. These 2 land fowl (Galliformes) genomes show a remarkable level of similarity, despite an estimated 30-40 million years of separate evolution since their last common ancestor. Among the advantages offered by these sequences are routine re-sequencing of commercial and research lines to identify the genetic correlates of phenotypic change (for example, selective sweeps), a much improved understanding of poultry diversity and linkage disequilibrium, and access to high-density SNP typing and association analysis, detailed transcriptomic and proteomic studies, and the use of genome-wide marker- assisted selection to enhance genetic gain in commercial stocks. PMID:21335957

Dodgson, J B; Delany, M E; Cheng, H H

2011-01-01

372

Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes.  

PubMed Central

Fluorescence in situ hybridization (FISH) is a powerful tool for physical mapping in human and other mammalian species. However, application of the FISH technique has been limited in plant species, especially for mapping single- or low-copy DNA sequences, due to inconsistent signal production in plant chromosome preparations. Here we demonstrate that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L.) chromosomes by FISH. Repetitive DNA sequences in BAC clones can be suppressed efficiently by using rice genomic DNA as a competitor in the hybridization mixture. BAC clones as small as 40 kb were successfully mapped. To demonstrate the application of the FISH technique in physical mapping of plant genomes, both anonymous BAC clones and clones closely linked to a rice bacterial blight-resistance locus, Xa21, were chosen for analysis. The physical location of Xa21 and the relationships among the linked clones were established, thus demonstrating the utility of FISH in plant genome analysis. Images Fig. 1

Jiang, J; Gill, B S; Wang, G L; Ronald, P C; Ward, D C

1995-01-01

373

Strengthening State Library Administrative Agency (Territoral Library).  

National Technical Information Service (NTIS)

The document describes the Basic State Plan Amendments for the Library Services and Construction Act in Guam and the regulations promulgated thereunder. The major projects described under the plan are: Strengthening State Library Administrative Agency; St...

1974-01-01

374

Library Legislation in India  

ERIC Educational Resources Information Center

The author presents a history of library legislation in India and recommends the establishment of library networks by law to ensure an integrated service which will effectively serve all library patrons. (17 references) (SJ)

Srivastava, Shyan Nath

1972-01-01

375

Supervision in Libraries.  

National Technical Information Service (NTIS)

Although the literature of library administration draws extensively on that of business management, it is difficult to compare library supervision to business or industrial supervision. Library supervisors often do not have managerial training and may con...

M. J. Bailey

1974-01-01

376

Homecoming for Library Symbol.  

ERIC Educational Resources Information Center

Discusses the significance and development of the library symbol and the history of its acceptance by the American Library Association (ALA) and the Canadian Library Association (CLA). Suggestions are made for its use. (CLB)

Egan, Bessie

1987-01-01

377

Sequence analysis of the whole genome of a recombinant Marek's disease virus strain, GX0101, with a reticuloendotheliosis virus LTR insert.  

PubMed

Marek's disease virus Chinese strain GX0101, isolated in 2001, is the first reported recombinant gallid herpesvirus type 2 (GaHV-2) field strain with one reticuloendotheliosis virus (REV) long terminal repeat (LTR) insert. We constructed an infectious bacterial artificial chromosome (BAC) clone of GX0101, which showed characteristics very similar to those of the parental virus in replication and pathogenicity. Using the GX0101 BAC clone, the complete genome of GX0101 was sequenced and analyzed. The length of the GX0101 genome is 178,101 bp, and it contains only one REV-LTR insert at a site 267 bp upstream of the sorf2 gene. PMID:23553452

Su, Shuai; Cui, Ning; Sun, Aijun; Li, Yanpeng; Ding, Jiabo; Chen, Zimeng; Zhao, Peng; Cui, Zhizhong

2013-09-01

378

The Library of Virginia's Digital Library Project.  

ERIC Educational Resources Information Center

Describes The Library of Virginia's Digital Library Project that has made many of its state library collections available via the Internet and World Wide Web. Highlights include digitization decisions; the HTML Web gateway; the online catalog; microfilm digitization; users and use statistics; and future projects. (LRW)

Roderick, Elizabeth; Taylor, Jean Marie; Byrd, Sam; Courson, Glenn

1997-01-01

379

Electronic Library Services: Choosing the Library Future.  

ERIC Educational Resources Information Center

Many futurists envision that the development of information technology will greatly impact the libraries of tomorrow. In response to this prediction, the Montgomery County (Maryland) Department of Public Libraries conducted a study of key trends and issues that the Montgomery County Public Libraries (MCPL) must consider if they are to remain a…

Ecelbarger, Louisa Kay

380

Functional and genetic analysis of haplotypic sequence variation at the nicastrin genomic locus.  

PubMed

Nicastrin (NCSTN) is a component of the ?-secretase complex and therefore potentially a candidate risk gene for Alzheimer's disease. Here, we have developed a novel functional genomics methodology to express common locus haplotypes to assess functional differences. DNA recombination was used to engineer 5 bacterial artificial chromosomes (BACs) to each express a different haplotype of the NCSTN locus. Each NCSTN-BAC was delivered to knockout nicastrin (Ncstn(-/-)) cells and clonal NCSTN-BAC(+)/Ncstn(-/-) cell lines were created for functional analyses. We showed that all NCSTN-BAC haplotypes expressed nicastrin protein and rescued ?-secretase activity and amyloid beta (A?) production in NCSTN-BAC(+)/Ncstn(-/-) lines. We then showed that genetic variation at the NCSTN locus affected alternative splicing in human postmortem brain tissue. However, there was no robust functional difference between clonal cell lines rescued by each of the 5 different haplotypes. Finally, there was no statistically significant association of NCSTN with disease risk in the 4 cohorts. We therefore conclude that it is unlikely that common variation at the NCSTN locus is a risk factor for Alzheimer's disease. PMID:22405046

Hamilton, Gillian; Killick, Richard; Lambert, Jean-Charles; Amouyel, Philippe; Carrasquillo, Minerva M; Pankratz, V Shane; Graff-Radford, Neill R; Dickson, Dennis W; Petersen, Ronald C; Younkin, Steven G; Powell, John F; Wade-Martins, Richard

2012-08-01

381

Transposase mediated construction of RNA-seq libraries  

PubMed Central

RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequenc