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Sample records for aestivum bac genomic

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

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    MacFarlane Amanda J

    2009-07-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

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    Eilertsen Ken

    2001-09-01

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

  4. A complete mitochondrial genome of wheat (Triticum aestivum cv. Chinese Yumai), and fast evolving mitochondrial genes in higher plants

    Indian Academy of Sciences (India)

    Peng Cui; Huitao Liu; Qiang Lin; Feng Ding; Guoyin Zhuo; Songnian Hu; Dongcheng Liu; Wenlong Yang; Kehui Zhan; Aimin Zhang; Jun Yu

    2009-12-01

    Plant mitochondrial genomes, encoding necessary proteins involved in the system of energy production, play an important role in the development and reproduction of the plant. They occupy a specific evolutionary pattern relative to their nuclear counterparts. Here, we determined the winter wheat (Triticum aestivum cv. Chinese Yumai) mitochondrial genome in a length of 452 and 526 bp by shotgun sequencing its BAC library. It contains 202 genes, including 35 known protein-coding genes, three rRNA and 17 tRNA genes, as well as 149 open reading frames (ORFs; greater than 300 bp in length). The sequence is almost identical to the previously reported sequence of the spring wheat (T. aestivum cv. Chinese Spring); we only identified seven SNPs (three transitions and four transversions) and 10 indels (insertions and deletions) between the two independently acquired sequences, and all variations were found in non-coding regions. This result confirmed the accuracy of the previously reported mitochondrial sequence of the Chinese Spring wheat. The nucleotide frequency and codon usage of wheat are common among the lineage of higher plant with a high AT-content of 58%. Molecular evolutionary analysis demonstrated that plant mitochondrial genomes evolved at different rates, which may correlate with substantial variations in metabolic rate and generation time among plant lineages. In addition, through the estimation of the ratio of non-synonymous to synonymous substitution rates between orthologous mitochondrion-encoded genes of higher plants, we found an accelerated evolutionary rate that seems to be the result of relaxed selection.

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

    Science.gov (United States)

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

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

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    Brad S. Coates

    2012-01-01

    Full Text Available 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.

  7. A hybrid BAC physical map of potato: a framework for sequencing a heterozygous genome

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    de Boer Jan M

    2011-12-01

    Full Text Available Abstract Background Potato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH. Results First, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps in silico anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map. Conclusions The AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and

  8. Toward an Integrated BAC Library Resource for Genome Sequencing and Analysis; FINAL

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-02-26

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

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

    Science.gov (United States)

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

    2014-01-01

    Bacterial artificial chromosomes (BACs) are suitable vectors not only to maintain the large genomes of herpesviruses in Escherichia coli but also to enable the traceless introduction of any mutation using modern tools of bacterial genetics. To clone a herpes simplex virus genome, a BAC replication origin is first introduced into the viral genome by homologous recombination in eukaryotic host cells. As part of their nuclear replication cycle, genomes of herpesviruses circularize and these replication intermediates are then used to transform bacteria. After cloning, the integrity of the recombinant viral genomes is confirmed by restriction length polymorphism analysis and sequencing. The BACs may then be used to design virus mutants. Upon transfection into eukaryotic cells new herpesvirus strains harboring the desired mutations can be recovered and used for experiments in cultured cells as well as in animal infection models. PMID:24671676

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

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    Fellers John P

    2009-07-01

    Full Text Available Abstract 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

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

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    Kudrna David

    2011-03-01

    Full Text Available Abstract 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

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

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    Valle Manuel

    2011-07-01

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

  14. Genomic tools development for Aquilegia: construction of a BAC-based physical map

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    Hodges Scott A

    2010-11-01

    Full Text Available Abstract Background The genus Aquilegia, consisting of approximately 70 taxa, is a member of the basal eudicot lineage, Ranuculales, which is evolutionarily intermediate between monocots and core eudicots, and represents a relatively unstudied clade in the angiosperm phylogenetic tree that bridges the gap between these two major plant groups. Aquilegia species are closely related and their distribution covers highly diverse habitats. These provide rich resources to better understand the genetic basis of adaptation to different pollinators and habitats that in turn leads to rapid speciation. To gain insights into the genome structure and facilitate gene identification, comparative genomics and whole-genome shotgun sequencing assembly, BAC-based genomics resources are of crucial importance. Results BAC-based genomic resources, including two BAC libraries, a physical map with anchored markers and BAC end sequences, were established from A. formosa. The physical map was composed of a total of 50,155 BAC clones in 832 contigs and 3939 singletons, covering 21X genome equivalents. These contigs spanned a physical length of 689.8 Mb (~2.3X of the genome suggesting the complex heterozygosity of the genome. A set of 197 markers was developed from ESTs induced by drought-stress, or involved in anthocyanin biosynthesis or floral development, and was integrated into the physical map. Among these were 87 genetically mapped markers that anchored 54 contigs, spanning 76.4 Mb (25.5% across the genome. Analysis of a selection of 12,086 BAC end sequences (BESs from the minimal tiling path (MTP allowed a preview of the Aquilegia genome organization, including identification of transposable elements, simple sequence repeats and gene content. Common repetitive elements previously reported in both monocots and core eudicots were identified in Aquilegia suggesting the value of this genome in connecting the two major plant clades. Comparison with sequenced plant genomes

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

  17. Comparative analysis of catfish BAC end sequences with the zebrafish genome

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    Abernathy Jason

    2009-12-01

    Full Text Available Abstract Background Comparative mapping is a powerful tool to transfer genomic information from sequenced genomes to closely related species for which whole genome sequence data are not yet available. However, such an approach is still very limited in catfish, the most important aquaculture species in the United States. This project was initiated to generate additional BAC end sequences and demonstrate their applications in comparative mapping in catfish. Results We reported the generation of 43,000 BAC end sequences and their applications for comparative genome analysis in catfish. Using these and the additional 20,000 existing BAC end sequences as a resource along with linkage mapping and existing physical map, conserved syntenic regions were identified between the catfish and zebrafish genomes. A total of 10,943 catfish BAC end sequences (17.3% had significant BLAST hits to the zebrafish genome (cutoff value ≤ e-5, of which 3,221 were unique gene hits, providing a platform for comparative mapping based on locations of these genes in catfish and zebrafish. Genetic linkage mapping of microsatellites associated with contigs allowed identification of large conserved genomic segments and construction of super scaffolds. Conclusion BAC end sequences and their associated polymorphic markers are great resources for comparative genome analysis in catfish. Highly conserved chromosomal regions were identified to exist between catfish and zebrafish. However, it appears that the level of conservation at local genomic regions are high while a high level of chromosomal shuffling and rearrangements exist between catfish and zebrafish genomes. Orthologous regions established through comparative analysis should facilitate both structural and functional genome analysis in catfish.

  18. Comparative analysis of catfish BAC end sequences with the zebrafish genome

    OpenAIRE

    Abernathy Jason; Xu Peng; Somridhivej Benjaporn; Ninwichian Parichart; Wang Shaolin; Jiang Yanliang; Liu Hong; Kucuktas Huseyin; Liu Zhanjiang

    2009-01-01

    Abstract Background Comparative mapping is a powerful tool to transfer genomic information from sequenced genomes to closely related species for which whole genome sequence data are not yet available. However, such an approach is still very limited in catfish, the most important aquaculture species in the United States. This project was initiated to generate additional BAC end sequences and demonstrate their applications in comparative mapping in catfish. Results We reported the generation of...

  19. Comparative analysis of catfish BAC end sequences with the zebrafish genome

    OpenAIRE

    Liu, Hong; Jiang, Yanliang; Wang, Shaolin; Ninwichian, Parichart; Somridhivej, Benjaporn; Xu, Peng(Academy of Mathematics and Systems Science, Chinese Academy of Sciences, 100190, Beijing, China); Abernathy, Jason; Kucuktas, Huseyin; Liu, Zhanjiang

    2009-01-01

    Background Comparative mapping is a powerful tool to transfer genomic information from sequenced genomes to closely related species for which whole genome sequence data are not yet available. However, such an approach is still very limited in catfish, the most important aquaculture species in the United States. This project was initiated to generate additional BAC end sequences and demonstrate their applications in comparative mapping in catfish. Results We reported the generation of 43,000 B...

  20. Development of the BAC Physical Maps of Wheat Chromosome 6B for Its Genomic Sequencing

    OpenAIRE

    Kobayashi, A.; Katagiri, S.; Karasawa, W.; Takumi, S.; Doležel, J. (Jaroslav); Ogihara, Y.; Handa, H.

    2015-01-01

    For a purpose of better understanding the genome structure of wheat and accelerating the development of DNA markers for gene isolations and breeding, the Japanese research group, as a member of The International Wheat Genome Sequencing Consortium, is now conducting the physical mapping and genomic sequencing of wheat chromosome 6B of ‘Chinese Spring’ (CS). BAC libraries were constructed respectively using the short and long arm-specific DNAs extracted from the flow-sorted chromosome 6BS and 6...

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

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    Garcia-Mas Jordi

    2010-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-03-18

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

  3. A comprehensive BAC resource

    OpenAIRE

    Zhao, Shaying

    2001-01-01

    The Human Genome Project has generated extensive map and sequence data for a large number of Bacterial Artificial Chromosome (BAC) clones. In order to maximize the efficient use of the data and to minimize the redundant work for the research community, The Institute for Genomic Research (TIGR) comprehensive BAC resource (cBACr) (http://www.tigr.org/tdb/BacResource/BAC_resource_intro.html) was built as an expansion of the TIGR human BAC ends database. This resource coll...

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

    Directory of Open Access Journals (Sweden)

    Talon Manuel

    2008-09-01

    Full Text Available Abstract 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

  5. Study on the Mitochondrial Genome of Sea Island Cotton (Gossypium barbadense) by BAC Library Screening

    Institute of Scientific and Technical Information of China (English)

    SU Ai-guo; LI Shuang-shuang; LIU Guo-zheng; LEI Bin-bin; KANG Ding-ming; LI Zhao-hu; MA Zhi-ying; HUA Jin-ping

    2014-01-01

    The plant mitochondrial genome displays complex features, particularly in terms of cytoplasmic male sterility (CMS). Therefore, research on the cotton mitochondrial genome may provide important information for analyzing genome evolution and exploring the molecular mechanism of CMS. In this paper, we present a preliminary study on the mitochondrial genome of sea island cotton (Gossypium barbadense) based on positive clones from the bacterial artiifcial chromosome (BAC) library. Thirty-ifve primers designed with the conserved sequences of functional genes and exons of mitochondria were used to screen positive clones in the genome library of the sea island cotton variety called Pima 90-53. Ten BAC clones were obtained and veriifed for further study. A contig was obtained based on six overlapping clones and subsequently laid out primarily on the mitochondrial genome. One BAC clone, clone 6 harbored with the inserter of approximate 115 kb mtDNA sequence, in which more than 10 primers fragments could be ampliifed, was sequenced and assembled using the Solexa strategy. Fifteen mitochondrial functional genes were revealed in clone 6 by gene annotation. The characteristics of the syntenic gene/exon of the sequences and RNA editing were preliminarily predicted.

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

    Directory of Open Access Journals (Sweden)

    Saski Christopher A

    2011-09-01

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

  7. Genetic stability of pestivirus genomes cloned into BACs

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Uttenthal, Åse;

    Infectious cDNA clones are a prerequisite for directed genetic manipulations of pestivirus genomes to obtain attenuated pestiviruses designed as new modified live DIVA vaccine candidates against classical swine fever. However, the construction of new infectious pestivirus cDNA clones has been...... hampered due to the large size of the pestivirus genome and due to genetic instability of the cloned cDNA, which in combination with plasmid vectors tend to be unstable and deleterious in the bacterial host. Therefore, new strategies are needed to facilitate construction of stable infectious cDNA clones...... of pestivirus strains. In a collaborative research project, between DTU Vet and FLI, on the establishment of genetically modified pestiviruses engineered specifically for the DIVA principle, we cloned a series of complete pestivirus genomes, obtained by full-length RT-PCR, directly into the bacterial artificial...

  8. The first insight into the salvia (lamiaceae) genome via bac library construction and high-throughput sequencing of target bac clones

    International Nuclear Information System (INIS)

    Salvia is a representative genus of Lamiaceae, a eudicot family with significant species diversity and population adaptibility. One of the key goals of Salvia genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of medicinal plants to increase their health and productivity. Large-insert genomic libraries are a fundamental tool for achieving this purpose. We report herein the construction, characterization and screening of a gridded BAC library for Salvia officinalis (sage). The S. officinalis BAC library consists of 17,764 clones and the average insert size is 107 Kb, corresponding to 3 haploid genome equivalents. Seventeen positive clones (average insert size 115 Kb) containing five terpene synthase (TPS) genes were screened out by PCR and 12 of them were subject to Illumina HiSeq 2000 sequencing, which yielded 28,097,480 90-bp raw reads (2.53 Gb). Scaffolds containing sabinene synthase (Sab), a Sab homolog, TPS3 (kaurene synthase-like 2), copalyl diphosphate synthase 2 and one cytochrome P450 gene were retrieved via de novo assembly and annotation, which also have flanking noncoding sequences, including predicted promoters and repeat sequences. Among 2,638 repeat sequences, there are 330 amplifiable microsatellites. This BAC library provides a new resource for Lamiaceae genomic studies, including microsatellite marker development, physical mapping, comparative genomics and genome sequencing. Characterization of positive clones provided insights into the structure of the Salvia genome. These sequences will be used in the assembly of a future genome sequence for S. officinalis. (author)

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

    Directory of Open Access Journals (Sweden)

    Thorgaard Gary H

    2009-10-01

    Full Text Available Abstract 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. A bacterial artificial chromosome (BAC physical map is needed to facilitate fine mapping of QTL and the selection of positional candidate genes for incorporation in marker-assisted selection (MAS for improving rainbow trout aquaculture production. This resource will also facilitate efforts to obtain and assemble a whole-genome reference sequence for this species. Results The physical map was constructed from DNA fingerprinting of 192,096 BAC clones using the 4-color high-information content fingerprinting (HICF method. The clones were assembled into physical map contigs using the finger-printing contig (FPC program. The map is composed of 4,173 contigs and 9,379 singletons. The total number of unique fingerprinting fragments (consensus bands in contigs is 1,185,157, which corresponds to an estimated physical length of 2.0 Gb. The map assembly was validated by 1 comparison with probe hybridization results and agarose gel fingerprinting contigs; and 2 anchoring large contigs to the microsatellite-based genetic linkage map. Conclusion The production and validation of the first BAC physical map of the rainbow trout genome is described in this paper. We are currently integrating this map with the NCCCWA genetic map using more than 200 microsatellites isolated from BAC end sequences and by identifying BACs that harbor more than 300 previously mapped markers. The availability of an integrated physical and genetic map will enable detailed comparative genome

  10. Construction of a nurse shark (Ginglymostoma cirratum bacterial artificial chromosome (BAC library and a preliminary genome survey

    Directory of Open Access Journals (Sweden)

    Inoko Hidetoshi

    2006-05-01

    Full Text Available Abstract Background Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates. Aims In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC library for the nurse shark, Ginglymostoma cirratum. Results The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible. Conclusion We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

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

    Directory of Open Access Journals (Sweden)

    Hsiao Yu-Yun

    2011-01-01

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

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

    Science.gov (United States)

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

    2008-02-01

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

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

    Science.gov (United States)

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

    2007-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Chaine Christian

    2008-01-01

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

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

    OpenAIRE

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

    2010-01-01

    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 ...

  16. Completely Distinguishing Individual A-genome Chromosomes and Their Karyotyping Analysis by Multiple BAC-FISH

    Institute of Scientific and Technical Information of China (English)

    WANG Kai; GUO Wang-zhen; ZHANG Tian-zhen

    2008-01-01

    @@ Multiple BAC-FISH is a powerful tool for modern cytogenetic researching in both animals and plants.But in cotton,this technique is unavailable due to the high percentage of repetitive sequences.Here,we identified twenty BACs from more than fifty BACs,and successfully demonstrated the use of multiple BAC-FISH for cytogenetie research in a diploid cotton species,G.arboreum.The karyotyping should be a basic application of this technique,but the potential usage such as high-resolution physical mapping construction,assisting BAC-by-BAC sequencing will be invaluable.

  17. A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

    DEFF Research Database (Denmark)

    She, Qunxin; Confalonieri, F.; Zivanovic, Y.;

    2000-01-01

    screening. The PCR approaches included a novel chromosome walking method termed “paired-PCR”. 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were......The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter......-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...

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

    Directory of Open Access Journals (Sweden)

    King Graham J

    2010-10-01

    Full Text Available Abstract 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.

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

    Directory of Open Access Journals (Sweden)

    Waldbieser Geoffrey C

    2007-02-01

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

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

    Science.gov (United States)

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

    2012-08-01

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

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

    Science.gov (United States)

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

  2. Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae)

    OpenAIRE

    Gonthier Lucy; Bellec Arnaud; Blassiau Christelle; Prat Elisa; Helmstetter Nicolas; Rambaud Caroline; Huss Brigitte; Hendriks Theo; Bergès Hélène; Quillet Marie-Christine

    2010-01-01

    Abstract Background The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries ...

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

    Directory of Open Access Journals (Sweden)

    Gonser Rusty A

    2011-06-01

    Full Text Available Abstract 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.

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

    Directory of Open Access Journals (Sweden)

    Blackmon Barbara P

    2011-07-01

    Full Text Available Abstract Background BAC-based physical maps provide for sequencing across an entire genome or a selected sub-genomic region of biological interest. Such a region can be approached with next-generation whole-genome sequencing and assembly as if it were an independent small genome. Using the minimum tiling path as a guide, specific BAC clones representing the prioritized genomic interval are selected, pooled, and used to prepare a sequencing library. Results This pooled BAC approach was taken to sequence and assemble a QTL-rich region, of ~3 Mbp and represented by twenty-seven BACs, on linkage group 5 of the Theobroma cacao cv. Matina 1-6 genome. Using various mixtures of read coverages from paired-end and linear 454 libraries, multiple assemblies of varied quality were generated. Quality was assessed by comparing the assembly of 454 reads with a subset of ten BACs individually sequenced and assembled using Sanger reads. A mixture of reads optimal for assembly was identified. We found, furthermore, that a quality assembly suitable for serving as a reference genome template could be obtained even with a reduced depth of sequencing coverage. Annotation of the resulting assembly revealed several genes potentially responsible for three T. cacao traits: black pod disease resistance, bean shape index, and pod weight. Conclusions Our results, as with other pooled BAC sequencing reports, suggest that pooling portions of a minimum tiling path derived from a BAC-based physical map is an effective method to target sub-genomic regions for sequencing. While we focused on a single QTL region, other QTL regions of importance could be similarly sequenced allowing for biological discovery to take place before a high quality whole-genome assembly is completed.

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

    Directory of Open Access Journals (Sweden)

    Cloutier Sylvie

    2011-05-01

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

  6. Radiation hybrid maps of D-genome of Aegilops tauschii and their application in sequence assembly of large and complex plant genomes

    Science.gov (United States)

    The large and complex genome of bread wheat (Triticum aestivum L., ~17 Gb) requires high-resolution genome maps saturated with ordered markers to assist in anchoring and orienting BAC contigs/ sequence scaffolds for whole genome sequence assembly. Radiation hybrid (RH) mapping has proven to be an e...

  7. Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

    Directory of Open Access Journals (Sweden)

    Emeline Dubois

    2012-01-01

    Full Text Available Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus, ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes.

  8. Analysis of the allohexaploid bread wheat genome (Triticum aestivum) using comparative whole genome shotgun sequencing

    Science.gov (United States)

    The large 17 Gb allopolyploid genome of bread wheat is a major challenge for genome analysis because it is composed of three closely- related and independently maintained genomes, with genes dispersed as small “islands” separated by vast tracts of repetitive DNA. We used a novel comparative genomi...

  9. Development of genomic resources for the prairie vole (Microtus ochrogaster: construction of a BAC library and vole-mouse comparative cytogenetic map

    Directory of Open Access Journals (Sweden)

    Young Larry J

    2010-01-01

    Full Text Available Abstract Background The prairie vole (Microtus ochrogaster is a premier animal model for understanding the genetic and neurological basis of social behaviors. Unlike other biomedical models, prairie voles display a rich repertoire of social behaviors including the formation of long-term pair bonds and biparental care. However, due to a lack of genomic resources for this species, studies have been limited to a handful of candidate genes. To provide a substrate for future development of genomic resources for this unique model organism, we report the construction and characterization of a bacterial artificial chromosome (BAC library from a single male prairie vole and a prairie vole-mouse (Mus musculus comparative cytogenetic map. Results We constructed a prairie vole BAC library (CHORI-232 consisting of 194,267 recombinant clones with an average insert size of 139 kb. Hybridization-based screening of the gridded library at 19 loci established that the library has an average depth of coverage of ~10×. To obtain a small-scale sampling of the prairie vole genome, we generated 3884 BAC end-sequences totaling ~2.8 Mb. One-third of these BAC-end sequences could be mapped to unique locations in the mouse genome, thereby anchoring 1003 prairie vole BAC clones to an orthologous position in the mouse genome. Fluorescence in situ hybridization (FISH mapping of 62 prairie vole clones with BAC-end sequences mapping to orthologous positions in the mouse genome was used to develop a first-generation genome-wide prairie vole-mouse comparative cytogenetic map. While conserved synteny was observed between this pair of rodent genomes, rearrangements between the prairie vole and mouse genomes were detected, including a minimum of five inversions and 16 inter-chromosomal rearrangements. Conclusions The construction of the prairie vole BAC library and the vole-mouse comparative cytogenetic map represent the first genome-wide modern genomic resources developed for this

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

    Directory of Open Access Journals (Sweden)

    Sehgal Sunish K

    2012-05-01

    Full Text Available Abstract 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

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

    OpenAIRE

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

    2010-01-01

    Abstract 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...

  12. [Nuclear and cytoplasmic genome analysis of somatic hybrid of Triticum aestivum L. and Leymus chinensis (Trin.) Tzvel].

    Science.gov (United States)

    Chen, Xiu-Ling; Xia, Guang-Min; Chen, Hui-Min

    2004-08-01

    Intergeneric somatic hybrids were obtained by fusion between protoplasts of Triticum aestivum L. cv. Jinan 177 and Leymus chinensis (Trin.) Tzvel. Protoplasts of L. chinensis were exposed to UV (300 microW/cm(2)) for 30 s, 45 s and 1 min before fusion. The results of morphological and chromosomal observation, isozyme pattern as well as RAPD analysis and the 5S rDNA space sequence analysis showed the hybrid nature of the regenerated colonies of fusion combination T (+) L (UV 30 s). Restriction Fragment Length Po1ymorphism (RFLP) analysis using mitochondrion-specific atp6 and chloroplast-specific rbcL probes showed that most of the organelle genomes of the hybrids originated from L. chinensis and some recombination happened between wheat and L. chinensis. The effects of elimination of the nuclear genome of receptor protoplast on hybrid regeneration and receptor organelle genome elimination are discussed. PMID:15627685

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Gindullis, F; Dechyeva, D; Schmidt, T

    2001-10-01

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

  15. A hybrid BAC physical map of potato: a framework for sequencing a heterozygous genome

    NARCIS (Netherlands)

    Boer, de J.M.; Borm, T.J.A.; Jesse, T.; Brugmans, B.W.; Tang, X.; Bryan, G.J.; Bakker, J.; Eck, van H.J.; Visser, R.G.F.

    2011-01-01

    Background Potato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so

  16. Re-annotation of the physical map of Glycine max for polyploid-like regions by BAC end sequence driven whole genome shotgun read assembly

    Directory of Open Access Journals (Sweden)

    Shultz Jeffry

    2008-07-01

    Full Text Available Abstract Background Many of the world's most important food crops have either polyploid genomes or homeologous regions derived from segmental shuffling following polyploid formation. The soybean (Glycine max genome has been shown to be composed of approximately four thousand short interspersed homeologous regions with 1, 2 or 4 copies per haploid genome by RFLP analysis, microsatellite anchors to BACs and by contigs formed from BAC fingerprints. Despite these similar regions,, the genome has been sequenced by whole genome shotgun sequence (WGS. Here the aim was to use BAC end sequences (BES derived from three minimum tile paths (MTP to examine the extent and homogeneity of polyploid-like regions within contigs and the extent of correlation between the polyploid-like regions inferred from fingerprinting and the polyploid-like sequences inferred from WGS matches. Results Results show that when sequence divergence was 1–10%, the copy number of homeologous regions could be identified from sequence variation in WGS reads overlapping BES. Homeolog sequence variants (HSVs were single nucleotide polymorphisms (SNPs; 89% and single nucleotide indels (SNIs 10%. Larger indels were rare but present (1%. Simulations that had predicted fingerprints of homeologous regions could be separated when divergence exceeded 2% were shown to be false. We show that a 5–10% sequence divergence is necessary to separate homeologs by fingerprinting. BES compared to WGS traces showed polyploid-like regions with less than 1% sequence divergence exist at 2.3% of the locations assayed. Conclusion The use of HSVs like SNPs and SNIs to characterize BACs wil improve contig building methods. The implications for bioinformatic and functional annotation of polyploid and paleopolyploid genomes show that a combined approach of BAC fingerprint based physical maps, WGS sequence and HSV-based partitioning of BAC clones from homeologous regions to separate contigs will allow reliable de

  17. Genome evolution in Reptilia: in silico chicken mapping of 12,000 BAC-end sequences from two reptiles and a basal bird

    Directory of Open Access Journals (Sweden)

    Edwards Scott V

    2009-07-01

    Full Text Available Abstract Background With the publication of the draft chicken genome and the recent production of several BAC clone libraries from non-avian reptiles and birds, it is now possible to undertake more detailed comparative genomic studies in Reptilia. Of interest in particular are the genomic events that transformed the large, repeat-rich genomes of mammals and non-avian reptiles into the minimalist chicken genome. We have used paired BAC end sequences (BESs from the American alligator (Alligator mississippiensis, painted turtle (Chrysemys picta and emu (Dromaius novaehollandiae to investigate patterns of sequence divergence, gene and retroelement content, and microsynteny between these species and chicken. Results From a total of 11,967 curated BESs, we successfully mapped 725, 773 and 2597 sequences in alligator, turtle, and emu, respectively, to sites in the draft chicken genome using a stringent BLAST protocol. Most commonly, sequences mapped to a single site in the chicken genome. Of 1675, 1828 and 2936 paired BESs obtained for alligator, turtle, and emu, respectively, a total of 34 (alligator, 2%, 24 (turtle, 1.3% and 479 (emu, 16.3% pairs were found to map with high confidence and in the correct orientation and with BAC-sized intermarker distances to single chicken chromosomes, including 25 such paired hits in emu mapping to the chicken Z chromosome. By determining the insert sizes of a subset of BAC clones from these three species, we also found a significant correlation between the intermarker distance in alligator and turtle and in chicken, with slopes as expected on the basis of the ratio of the genome sizes. Conclusion Our results suggest that a large number of small-scale chromosomal rearrangements and deletions in the lineage leading to chicken have drastically reduced the number of detected syntenies observed between the chicken and alligator, turtle, and emu genomes and imply that small deletions occurring widely throughout the

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

    Science.gov (United States)

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

    2016-06-01

    Black spot disease, caused by Alternaria alternata Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. In the present study, the locations of black spot disease resistance/susceptibility-related genome regions were studied by fluorescence in situ hybridization using BAC clone (BAC-FISH) on Japanese pear (Pyrus pyrifolia (Burm. f.) Nakai) chromosomes. Root tips of self-pollinated seedlings of 'Osa Gold' were used as materials. Chromosome samples were prepared by the enzymatic maceration and air-drying method. The BAC clone adjacent to the black spot disease-related gene was labeled as a probe for FISH analysis. Black spot disease-related genome regions were detected in telomeric positions of two medium size chromosomes. These two sites and six telomeric 18S-5.8S-25S rDNA sites were located on different chromosomes as determined from the results of multi-color FISH. The effectiveness of the physical mapping of useful genes on pear chromosomes achieved by the BAC-FISH method was unequivocally demonstrated. PMID:27436955

  19. Population- and genome-specific patterns of linkage disequilibrium and SNP variation in spring and winter wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Campbell Kim

    2010-12-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are ideally suited for the construction of high-resolution genetic maps, studying population evolutionary history and performing genome-wide association mapping experiments. Here, we used a genome-wide set of 1536 SNPs to study linkage disequilibrium (LD and population structure in a panel of 478 spring and winter wheat cultivars (Triticum aestivum from 17 populations across the United States and Mexico. Results Most of the wheat oligo pool assay (OPA SNPs that were polymorphic within the complete set of 478 cultivars were also polymorphic in all subpopulations. Higher levels of genetic differentiation were observed among wheat lines within populations than among populations. A total of nine genetically distinct clusters were identified, suggesting that some of the pre-defined populations shared significant proportion of genetic ancestry. Estimates of population structure (FST at individual loci showed a high level of heterogeneity across the genome. In addition, seven genomic regions with elevated FST were detected between the spring and winter wheat populations. Some of these regions overlapped with previously mapped flowering time QTL. Across all populations, the highest extent of significant LD was observed in the wheat D-genome, followed by lower LD in the A- and B-genomes. The differences in the extent of LD among populations and genomes were mostly driven by differences in long-range LD ( > 10 cM. Conclusions Genome- and population-specific patterns of genetic differentiation and LD were discovered in the populations of wheat cultivars from different geographic regions. Our study demonstrated that the estimates of population structure between spring and winter wheat lines can identify genomic regions harboring candidate genes involved in the regulation of growth habit. Variation in LD suggests that breeding and selection had a different impact on each wheat genome both within and

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

  4. Characterization of low-molecular-weight-glutenin subunit genes from the D-genome of Triticum aestivum, Aegilops crassa, Ae. cylindrica and Ae. tauschii

    NARCIS (Netherlands)

    Naghavi, M.R.; Ahmadi, S.; Shanejat-Boushehri, A.A.; Komaei, G.; Struik, P.C.

    2013-01-01

    Twenty low-molecular-weight-glutenin subunit (LMW-GS) gene sequences from the D-genome from Aegilops crassa (2n ¼ 4x ¼ 28), Ae. cylindrica (2n ¼ 4x ¼ 28), Ae. tauschii (2n ¼ 2x ¼ 14) and Triticum aestivum (2n ¼ 6x ¼ 42) were obtained using five sets of specific allele primer pairs. Only the sequence

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

    OpenAIRE

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

    2008-01-01

    Abstract 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 char...

  6. The first insight into the Salvia (Lamiaceae) genome via BAC library construction and high-throughput sequencing of target BAC clones

    OpenAIRE

    Vautrin, Sonia; Song, Chi; Zhu, Ying Jie; Berges, Hélène; Sun, Chao

    2015-01-01

    Salvia is a representative genus of Lamiaceae, a eudicot family with significant species diversity and population adaptibility. One of the key goals of Salvia genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of medicinal plants to increase their health and productivity. Large-insert genomic libraries are a fundamental tool for achieving this purpose. We report herein the cons...

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

    Energy Technology Data Exchange (ETDEWEB)

    Lim, C.S.; Xu, R.X.; Wang, M. [and others

    1996-12-31

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

  8. Genomic Dissection and Expression Profiling Revealed Functional Divergence in Triticum aestivum Leucine Rich Repeat Receptor Like Kinases (TaLRRKs)

    Science.gov (United States)

    Shumayla; Sharma, Shailesh; Kumar, Rohit; Mendu, Venugopal; Singh, Kashmir; Upadhyay, Santosh K.

    2016-01-01

    The leucine rich repeat receptor like kinases (LRRK) constitute the largest subfamily of receptor like kinases (RLK), which play critical roles in plant development and stress responses. Herein, we identified 531 TaLRRK genes in Triticum aestivum (bread wheat), which were distributed throughout the A, B, and D sub-genomes and chromosomes. These were clustered into 233 homologous groups, which were mostly located on either homeologous chromosomes from various sub-genomes or in proximity on the same chromosome. A total of 255 paralogous genes were predicted which depicted the role of duplication events in expansion of this gene family. Majority of TaLRRKs consisted of trans-membrane region and localized on plasma-membrane. The TaLRRKs were further categorized into eight phylogenetic groups with numerous subgroups on the basis of sequence homology. The gene and protein structure in terms of exon/intron ratio, domains, and motifs organization were found to be variably conserved across the different phylogenetic groups/subgroups, which indicated a potential divergence and neofunctionalization during evolution. High-throughput transcriptome data and quantitative real time PCR analyses in various developmental stages, and biotic and abiotic (heat, drought, and salt) stresses provided insight into modus operandi of TaLRRKs during these conditions. Distinct expression of majority of stress responsive TaLRRKs homologous genes suggested their specified role in a particular condition. These results provided a comprehensive analysis of various characteristic features including functional divergence, which may provide the way for future functional characterization of this important gene family in bread wheat.

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

    Science.gov (United States)

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

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

    Directory of Open Access Journals (Sweden)

    Wincker Patrick

    2011-06-01

    Full Text Available Abstract 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 of fish biology. The common ancestor of the salmonids experienced a whole genome duplication event, making extant salmonids such as the rainbow trout an excellent model for studying the evolution of tetraploidization and re-diploidization in vertebrates. However, the lack of a reference genome sequence hampers research progress for both academic and applied purposes. In order to enrich the genomic tools already available in this species and provide further insight on the complexity of its genome, we sequenced a large number of rainbow trout BAC-end sequences (BES and characterized their contents. Results A total of 176,485 high quality BES, were generated, representing approximately 4% of the trout genome. BES analyses identified 6,848 simple sequence repeats (SSRs, of which 3,854 had high quality flanking sequences for PCR primers design. The first rainbow trout repeat elements database (INRA RT rep1.0 containing 735 putative repeat elements was developed, and identified almost 59.5% of the BES database in base-pairs as repetitive sequence. Approximately 55% of the BES reads (97,846 had more than 100 base pairs of contiguous non-repetitive sequences. The fractions of the 97,846 non-repetitive trout BES reads that had significant BLASTN hits against the zebrafish, medaka and stickleback genome databases were 15%, 16.2% and 17.9%, respectively, while the fractions of the non-repetitive BES reads that had significant BLASTX hits against the zebrafish, medaka, and stickleback protein databases were 10.7%, 9.5% and 9.5%, respectively. Comparative genomics using paired BAC-ends revealed several regions of conserved synteny across all the fish species analyzed in this study. Conclusions The characterization of BES provided insights on the rainbow trout genome. The discovery

  11. Construction and characterization of two BAC libraries representing a deep-coverage of the genome of chicory (Cichorium intybus L., Asteraceae

    Directory of Open Access Journals (Sweden)

    Gonthier Lucy

    2010-08-01

    Full Text Available Abstract Background The Asteraceae represents an important plant family with respect to the numbers of species present in the wild and used by man. Nonetheless, genomic resources for Asteraceae species are relatively underdeveloped, hampering within species genetic studies as well as comparative genomics studies at the family level. So far, six BAC libraries have been described for the main crops of the family, i.e. lettuce and sunflower. Here we present the characterization of BAC libraries of chicory (Cichorium intybus L. constructed from two genotypes differing in traits related to sexual and vegetative reproduction. Resolving the molecular mechanisms underlying traits controlling the reproductive system of chicory is a key determinant for hybrid development, and more generally will provide new insights into these traits, which are poorly investigated so far at the molecular level in Asteraceae. Findings Two bacterial artificial chromosome (BAC libraries, CinS2S2 and CinS1S4, were constructed from HindIII-digested high molecular weight DNA of the contrasting genotypes C15 and C30.01, respectively. C15 was hermaphrodite, non-embryogenic, and S2S2 for the S-locus implicated in self-incompatibility, whereas C30.01 was male sterile, embryogenic, and S1S4. The CinS2S2 and CinS1S4 libraries contain 89,088 and 81,408 clones. Mean insert sizes of the CinS2S2 and CinS1S4 clones are 90 and 120 kb, respectively, and provide together a coverage of 12.3 haploid genome equivalents. Contamination with mitochondrial and chloroplast DNA sequences was evaluated with four mitochondrial and four chloroplast specific probes, and was estimated to be 0.024% and 1.00% for the CinS2S2 library, and 0.028% and 2.35% for the CinS1S4 library. Using two single copy genes putatively implicated in somatic embryogenesis, screening of both libraries resulted in detection of 12 and 13 positive clones for each gene, in accordance with expected numbers. Conclusions This

  12. Toward Development of a Whole-genome, BAC/BIBAC-based IntegratedPhysical/Genetic Map of the Cotton Genome Using the Upland Cotton Genetic Standard TM-1: BAC Fingerprinting and Physical Map Contig Construction%利用BAC、BIBAC文库整合构建陆地棉遗传标准系TM 1全基因组的遗传、物理图谱:BAC文库的指纹分析及重叠群构建

    Institute of Scientific and Technical Information of China (English)

    Hong-bin ZHANG; Mi-Kyung LEE; John Z. YU; Russell J. KOHEL; David M. STELLY; Zhan-you XU; Lina COVALEDA; Ke-jiao DING; Liang-tao ZHANG; Cheng-cang WU

    2002-01-01

      We are developing a whole-genome, BAC/BIBAC-based integrated physical/genetic map of the cotton (Gossypium hirsutum L. ) genome using its genetic standard line TM-1 as the reference genotype. Whole-genome physical maps integrated with genetic maps will provide revolutionized tools and platforms for all kinds of genomics research, including large-scale gene mapping, cloning and target DNA marker development.……

  13. Whole genome association mapping of Fusarium head blight resistance in European winter wheat (Triticum aestivum L..

    Directory of Open Access Journals (Sweden)

    Sonja Kollers

    Full Text Available A total of 358 recent European winter wheat varieties plus 14 spring wheat varieties were evaluated for resistance to Fusarium head blight (FHB caused by Fusarium graminearum and Fusarium culmorum in four separate environments. The FHB scores based on FHB incidence (Type I resistance×FHB severity (Type II resistance indicated a wide phenotypic variation of the varieties with BLUE (best linear unbiased estimation values ranging from 0.07 to 33.67. Genotyping with 732 microsatellite markers resulted in 782 loci of which 620 were placed on the ITMI map. The resulting average marker distance of 6.8 cM allowed genome wide association mapping employing a mixed model. Though no clear population structure was discovered, a kinship matrix was used for stratification. A total of 794 significant (-log10(p-value≥3.0 associations between SSR-loci and environment-specific FHB scores or BLUE values were detected, which included 323 SSR alleles. For FHB incidence and FHB severity a total of 861 and 877 individual marker-trait associations (MTA were detected, respectively. Associations for both traits co-located with FHB score in most cases. Consistent associations detected in three or more environments were found on all chromosomes except chromosome 6B, and with the highest number of MTA on chromosome 5B. The dependence of the number of favourable and unfavourable alleles within a variety to the respective FHB scores indicated an additive effect of favourable and unfavourable alleles, i.e. genotypes with more favourable or less unfavourable alleles tended to show greater resistance to FHB. Assessment of a marker specific for the dwarfing gene Rht-D1 resulted in strong effects. The results provide a prerequisite for designing genome wide breeding strategies for FHB resistance.

  14. Integration sites and their characteristic analysis of piggyBac transposon in cattle genome%piggyBac 转座子在牛基因组的整合位点及特征分析

    Institute of Scientific and Technical Information of China (English)

    杜新华; 高雪; 张路培; 高会江; 李俊雅; 许尚忠

    2013-01-01

    piggyBac(PB)转座子作为一种遗传工具被广泛应用于多个物种的转基因及插入突变研究,目前PB 转座子在牛中的相关研究还较少.为了获得PB 转座子在牛基因组中的整合位点,总结其转座特征,文章构建了PB[CMV-EGFP]和pcDNA-PBase 二元转座系统,利用细胞核电转技术共转染牛耳组织成纤维细胞,经G-418 筛选,获得了稳定转染EGFP 的转基因细胞系; 提取细胞基因组DNA,利用基因组步移技术扩增PB 转座子5′ Bac区插入位置的DNA 序列; 通过与牛基因组序列进行BLAST 比对,得到PB 转座子在牛基因组中的插入位点.文章共获得了8 个有效的整合位点,但仅有5 个位点定位到染色体1、2、11 和X 染色体上.序列分析表明:在牛基因组中,PB 转座子可特异性的插入到"TTAA"位置,并整合到基因间的非调控区; 分析整合位点"TTAA"相邻一侧的5 个碱基组成,发现PB 转座子5′端倾向于插入到GC(62.5%)碱基富集区.该研究表明,PB转座子可以在牛基因组中发生转座,获得的整合位点信息为利用PB 转座子在牛上开展遗传学研究提供了理论参考.%As a useful tool for genetic engineering, piggyBac (PB) transposons have been widely used in more than one species of transgenosis or generating mutation studies. At present, the studies about PB transposons in cattle were few. In order to get the PB transposon integration sites and summarize its characteristics in bovine genome, donor plasmid of PB[CMV-EGFP] and helper-dependent plasmid of pcDNA-PBase were constructed and transferred into bovine fibroblasts by Amaxa basic nucleofector kit for primary mammalian fibroblasts. Cell clones stably transfected were obtained after screening by G-418. Genomic DNA of transgenic cells was extracted and the integration sites of PB transposon were de- tected by genome walking technology. Eight integration sites were obtained in bovine genome, although only 5 sites were mapped on

  15. Whole genome association mapping of plant height in winter wheat (Triticum aestivum L..

    Directory of Open Access Journals (Sweden)

    Christine D Zanke

    Full Text Available The genetic architecture of plant height was investigated in a set of 358 recent European winter wheat varieties plus 14 spring wheat varieties based on field data in eight environments. Genotyping of diagnostic markers revealed the Rht-D1b mutant allele in 58% of the investigated varieties, while the Rht-B1b mutant was only present in 7% of the varieties. Rht-D1 was significantly associated with plant height by using a mixed linear model and employing a kinship matrix to correct for population stratification. Further genotyping data included 732 microsatellite markers, resulting in 770 loci, of which 635 markers were placed on the ITMI map plus a set of 7769 mapped SNP markers genotyped with the 90 k iSELECT chip. When Bonferroni correction was applied, a total of 153 significant marker-trait associations (MTAs were observed for plant height and the SSR markers (-log10 (P-value ≥ 4.82 and 280 (-log10 (P-value ≥ 5.89 for the SNPs. Linear regression between the most effective markers and the BLUEs for plant height indicated additive effects for the MTAs of different chromosomal regions. Analysis of syntenic regions in the rice genome revealed closely linked rice genes related to gibberellin acid (GA metabolism and perception, i.e. GA20 and GA2 oxidases orthologous to wheat chromosomes 1A, 2A, 3A, 3B, 5B, 5D and 7B, ent-kaurenoic acid oxidase orthologous to wheat chromosome 7A, ent-kaurene synthase on wheat chromosome 2B, as well as GA-receptors like DELLA genes orthologous to wheat chromosomes 4B, 4D and 7A and genes of the GID family orthologous to chromosomes 2B and 5B. The data indicated that besides the widely used GA-insensitive dwarfing genes Rht-B1 and Rht-D1 there is a wide spectrum of loci available that could be used for modulating plant height in variety development.

  16. Diversity of low-molecular-weight-glutenin subunit genes associated with D-genome in Triticum aestivum, Aegilops crassa, A. cylindrica and A. tauschii

    Directory of Open Access Journals (Sweden)

    Fatemeh Vafadar Shamasbi

    2016-09-01

    Full Text Available The 40% of endosperm protein of common wheat is composed of Low Molecular Weight (LMW Glutenin Subunits. To examine variation in the D genome of wheat, 98 accessions from different areas of Iran were studied using the five Glu-D3-specific pair primers. The amplification percentages of all primer pair sets were 80.61%, 92.86%, 79.59%, 90.82% and 67.35%, respectively. In comparison of the four species, the most observed bands of the first primer pair were found in Ae. tauschii samples. For the second primer pair, the most frequency of the amplified product was found in the T. aestivum samples. For the third primer pair, the Ae. cylindrical samples had the most amplified band. For the fourth primer pair, the most amplified band was found in the T. aestivum samples. The Ae. cylindrica samples had the most frequency band for the fifth primer pair. Based on dendrogram analysis, the accessions were divided in to 18 categories; and also 42 accessions had a bond for any PCR reaction. It is hoped that the result will be effective in molecular analysis and breeding of native landrace plants.

  17. Herpesvirus BACs: Past, Present, and Future

    Directory of Open Access Journals (Sweden)

    Charles Warden

    2011-01-01

    Full Text Available 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.

  18. 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

    Directory of Open Access Journals (Sweden)

    Ritland Carol

    2009-08-01

    Full Text Available Abstract 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 (FLcDNAs. 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

  19. Molecular evolution of Wcor15 gene enhanced our understanding of the origin of A, B and D genomes in Triticum aestivum.

    Science.gov (United States)

    Liu, Fangfang; Si, Hongqi; Wang, Chengcheng; Sun, Genlou; Zhou, Erting; Chen, Can; Ma, Chuanxi

    2016-08-16

    The allohexaploid bread wheat originally derived from three closely related species with A, B and D genome. Although numerous studies were performed to elucidate its origin and phylogeny, no consensus conclusion has reached. In this study, we cloned and sequenced the genes Wcor15-2A, Wcor15-2B and Wcor15-2D in 23 diploid, 10 tetraploid and 106 hexaploid wheat varieties and analyzed their molecular evolution to reveal the origin of the A, B and D genome in Triticum aestivum. Comparative analyses of sequences in diploid, tetraploid and hexaploid wheats suggest that T. urartu, Ae. speltoides and Ae. tauschii subsp. strangulata are most likely the donors of the Wcor15-2A, Wcor15-2B and Wcor15-2D locus in common wheat, respectively. The Wcor15 genes from subgenomes A and D were very conservative without insertion and deletion of bases during evolution of diploid, tetraploid and hexaploid. Non-coding region of Wcor15-2B gene from B genome might mutate during the first polyploidization from Ae. speltoides to tetraploid wheat, however, no change has occurred for this gene during the second allopolyploidization from tetraploid to hexaploid. Comparison of the Wcor15 gene shed light on understanding of the origin of the A, B and D genome of common wheat.

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

    OpenAIRE

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

    2012-01-01

    Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooled Amaranthus hypochondriacus BAC library. Ninety...

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

    Science.gov (United States)

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

  2. 1-Mb resolution array-based comparative genomic hybridization using a BAC clone set optimized for cancer gene analysis

    NARCIS (Netherlands)

    Greshock, J; Naylor, TL; Margolin, A; Diskin, S; Cleaver, SH; Futreal, PA; deJong, PJ; Zhao, SY; Liebman, M; Weber, BL

    2004-01-01

    Array-based comparative genomic hybridization (aCGH) is a recently developed tool for genome-wide determination of DNA copy number alterations. This technology has tremendous potential for disease-gene discovery in cancer and developmental disorders as well as numerous other applications. However, w

  3. Herpesvirus BACs: Past, Present, and Future

    OpenAIRE

    Charles Warden; Qiyi Tang; Hua Zhu

    2011-01-01

    The herpesviridae are a large family of DNA viruses with large and complicated genomes. Genetic manipulation and the generation of recombinant viruses have been extremely difficult. However, herpesvirus bacterial artificial chromosomes (BACs) that were developed approximately 10 years ago have become useful and powerful genetic tools for generating recombinant viruses to study the biology and pathogenesis of herpesviruses. For example, BAC-directed deletion mutants are commonly used to determ...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Lin Grace

    2008-03-01

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

  6. Construction of the Bac-to-Bac System of Bombyx mori Nucleopolyhedroviru

    Institute of Scientific and Technical Information of China (English)

    Jin-shan HUANG; Bi-fang HAO; Xiu-lian SUN; Fei DENG; Hua-lin WANG; Zhi-hong HU

    2007-01-01

    To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae.

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

    Science.gov (United States)

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes. PMID:27511172

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

    OpenAIRE

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

    2002-01-01

    Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidum chromosome was missing in the BAC library, between bp 248727...

  9. Comparative genomic mapping of the bovine Fragile Histidine Triad (FHIT tumour suppressor gene: characterization of a 2 Mb BAC contig covering the locus, complete annotation of the gene, analysis of cDNA and of physiological expression profiles

    Directory of Open Access Journals (Sweden)

    Boussaha Mekki

    2006-05-01

    Full Text Available Abstract Background The Fragile Histidine Triad gene (FHIT is an oncosuppressor implicated in many human cancers, including vesical tumors. FHIT is frequently hit by deletions caused by fragility at FRA3B, the most active of human common fragile sites, where FHIT lays. Vesical tumors affect also cattle, including animals grazing in the wild on bracken fern; compounds released by the fern are known to induce chromosome fragility and may trigger cancer with the interplay of latent Papilloma virus. Results The bovine FHIT was characterized by assembling a contig of 78 BACs. Sequence tags were designed on human exons and introns and used directly to select bovine BACs, or compared with sequence data in the bovine genome database or in the trace archive of the bovine genome sequencing project, and adapted before use. FHIT is split in ten exons like in man, with exons 5 to 9 coding for a 149 amino acids protein. VISTA global alignments between bovine genomic contigs retrieved from the bovine genome database and the human FHIT region were performed. Conservation was extremely high over a 2 Mb region spanning the whole FHIT locus, including the size of introns. Thus, the bovine FHIT covers about 1.6 Mb compared to 1.5 Mb in man. Expression was analyzed by RT-PCR and Northern blot, and was found to be ubiquitous. Four cDNA isoforms were isolated and sequenced, that originate from an alternative usage of three variants of exon 4, revealing a size very close to the major human FHIT cDNAs. Conclusion A comparative genomic approach allowed to assemble a contig of 78 BACs and to completely annotate a 1.6 Mb region spanning the bovine FHIT gene. The findings confirmed the very high level of conservation between human and bovine genomes and the importance of comparative mapping to speed the annotation process of the recently sequenced bovine genome. The detailed knowledge of the genomic FHIT region will allow to study the role of FHIT in bovine cancerogenesis

  10. Genome-wide association mapping of fusarium head blight resistance in wheat (Triticum aestivum L.) using genotyping by sequencing

    Science.gov (United States)

    Fusarium head blight (FHB) is one of the most important wheat diseases worldwide and host resistance displays complex genetic control. A genome-wide association study (GWAS) was performed on 273 winter wheat breeding lines from the mid-western and eastern regions of the US to identify chromosomal re...

  11. Chromosomal location of genomic SSR markers associated with yellow rust resistance in Turkish bread wheat (Triticum aestivum L.)

    Indian Academy of Sciences (India)

    F. Senturk Akfirat; F. Ertugrul; S. Hasancebi; Y. Aydin; K. Akan; Z. Mert; M. Cakir; A. Altinkut Uncuoglu

    2013-08-01

    We have previously reported Xgwm382 as a diagnostic marker for disease resistance against yellow rust in Izgi2001 × ES14 F2 population. Among the same earlier tested 230 primers, one SSR marker (Xgwm311) also amplified a fragment which is present in the resistant parent and in the resistant bulks, but absent in the susceptible parent and in the susceptible bulks. To understand the chromosome group location of these diagnostic markers, Xgwm382 and Xgwm311, in the same population, we selected 16 SSR markers mapped only in one genome of chromosome group 2 around 1–21 cM distance to these diagnostic markers based on the SSR consensus map of wheat. Out of 16 SSRs, Xwmc658 identified resistant F2 individuals as a diagnostic marker for yellow rust disease and provided the location of Xgwm382 and Xgwm311 on chromosome 2AL in our plant material.

  12. Analysis of main effect QTL for thousand grain weight in European winter wheat (Triticum aestivum L. by genome-wide association mapping

    Directory of Open Access Journals (Sweden)

    Christine Désirée Zanke

    2015-09-01

    Full Text Available Grain weight, an essential yield component, is under strong genetic control and at the same time markedly influenced by the environment. Genetic analysis of the thousand grain weight (TGW by genome-wide association study (GWAS was performed with a panel of 358 European winter wheat (Triticum aestivum L. varieties and 14 spring wheat varieties using phenotypic data of field tests in eight environments. Wide phenotypic variations were indicated for the TGW with BLUEs (best linear unbiased estimations values ranging from 35.9 g to 58.2 g with a mean value of 45.4 g and a heritability of H2=0.89. A total of 12 candidate genes for plant height, photoperiodism and grain weight were genotyped on all varieties. Only three candidates, the photoperiodism gene Ppd-D1, dwarfing gene Rht-B1and the TaGW-6A gene were significant explaining up to 14.4%, 2.3% and 3.4% of phenotypic variation, respectively. For a comprehensive genome-wide analysis of TGW-QTL genotyping data from 732 microsatellite markers and a set of 7769 mapped SNP markers genotyped with the 90k iSELECT array were analyzed. In total, 342 significant (-log10 (P-value > 3.0 marker trait associations (MTAs were detected for SSR markers and 1195 MTAs (-log10P-value > 3.0 for SNP markers in all single environments plus the BLUEs. After Bonferroni correction, 28 MTAs remained significant for SSR markers (-log10 (P-value > 4.82 and 58 MTAs for SNP markers (-log10 (P value > 5.89. Apart from chromosomes 4B and 6B for SSR markers and chromosomes 4D and 5D for SNP markers, MTAs were detected on all chromosomes. The highest number of significant SNP markers was found on chromosomes 3B and 1B, while for the SSRs most markers were significant on chromosomes 6D and 3D. Overall, TGW was determined by many markers with small effects. Only three SNP-markers had R2 values above 6%.

  13. BacMet

    DEFF Research Database (Denmark)

    Pal, Chandan; Bengtsson-Palme, Johan; Rensing, Christopher Günther T;

    2014-01-01

    -selection. Information on metal and biocide resistance genes, including their sequences and molecular functions, is, however, scattered. Here, we introduce BacMet (http://bacmet.biomedicine.gu.se) - a manually curated database of antibacterial biocide- and metal-resistance genes based on an in-depth review...

  14. SmartOrBAC

    Directory of Open Access Journals (Sweden)

    Imane BOUIJ-PASQUIER

    2015-11-01

    Full Text Available The emergence of the Internet of Things (IoT paradigm, provides a huge scope for more streamlined living through an increase of smart services but this coincides with an increase in security and privacy concerns, therefore access control has been an important factor in the development of IoT. This work proposes an authorization access model called SmartOrBAC built around a set of security and performance requirements. This model enhances the existing OrBAC (Organization-based Access Control model and adapts it to IoT environments. SmartOrBAC separates the problem into different functional layers and then distributes processing costs between constrained devices and less constrained ones and at the same time addresses the collaborative aspect with a specific solution. This paper also presents the application of SmartOrBAC on a real example of IoT and gives a complexity study demonstrating that even though this model is extensive, it does not add additional complexity regarding traditional access control models.

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

    Directory of Open Access Journals (Sweden)

    Peter J. Maughan

    2012-01-01

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

  16. A BAC clone of MDV strain GX0101 with REV-LTR integration retained its pathogenicity

    Institute of Scientific and Technical Information of China (English)

    SUN AiJun; LAWRENCE Petherbridge; ZHAO YuGuang; LI YanPeng; NAIR Venugopal K; CUI ZhiZhong

    2009-01-01

    The complete genome of Marek's disease virus (MDV) strain GX0101,which was integrated with the LTR sequences of REV,was cloned in Escherichia coli as a bacterial artificial chromosome (BAC).BAC vector sequences were introduced into the US2 locus of the MDV genome by homologous recombination.The viral DNA containing the BAC vector was used to transform Escherichia coli strain of DH10B.Then the recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast (CEF).This BAC viral clone was named bac-GX0101.When the reconstituted virus was inoculated into 1-day-old birds,visceral tumors could be detected as early as 62 d post infection.There was no difference in growth ability and pathogenicity to birds between the BAC derived virus and its parental virus.The BAC derived virus maintained its oncogenicity and immunosuppressive effects.In conclusion,the complete genome of GX0101 strain was successfully cloned into BAC and the infectious clone was rescued.With the powerful BAC manipulation system,the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV.

  17. A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B

    OpenAIRE

    Kobayashi, F; Wu, J. Z.; Kanamori, H; Tanaka, T.; Katagiri, S.; Karasawa, W.; Kaneko, S.; Watanabe, S; Sakaguchi, T; Šafář, J. (Jan); Šimková, H. (Hana); Mukai, Y.; M. Hamada; Saito, M; Hayakawa, K

    2015-01-01

    Background: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with t...

  18. Transposon-mediated BAC transgenesis in human ES cells.

    Science.gov (United States)

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

    2012-10-01

    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

  19. GenMapDB: a database of mapped human BAC clones

    OpenAIRE

    Morley, Michael; Arcaro, Melissa; Burdick, Joshua; Yonescu, Raluca; Reid, Thomas; Kirsch, Ilan R.; Cheung, Vivian G.

    2001-01-01

    GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9–22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequen...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    OpenAIRE

    Cloutier Sylvie; Rathinavelu Rajkumar; Ragupathy Raja

    2011-01-01

    Abstract Background Flax (Linum usitatissimum L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report ...

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

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    Heiner Kuhl

    2011-01-01

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

  3. Technological exploration of BAC-FISH on mitotic chromosomes of maize

    Institute of Scientific and Technical Information of China (English)

    Yongsheng TAO; Zuxin ZHANG; Yonglin CHEN; Lijia LI; Yonglian ZHENG

    2008-01-01

    The rice BAC-DNA was used as probes and fluorescence in situ hybridization (FISH) was applied to the interphase and metaphase mitotic chromosomes of maize. To optimize the BAC-FISH technique, we respect-ively assayed the effect of several factors, including maize or rice genomic Cot DNA used as blocking reagent of DNA, washing temperatures and FAD concentration in the washing buffer and in the hybrid solution. The results show that Cot DNA of maize genome blocked the repet-itive sequence of the rice BAC-DNA when the Cot value was below 50. Meanwhile, it was necessary to adjust the Cot value according to the different probes and their ratios. Decreasing the concentration of FAD in the hybridization mixtures, adjusting the washing rate after hybridization, and most especially, blocking the rice-specific repetitive sequences of BAC-DNA could improve the positive signals of BAC-FISH.

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

    Science.gov (United States)

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

    2016-01-01

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

  5. Estimation of long-terminal repeat element content in the Helicoverpa zea genome from next generation sequencing of reduced representation bacterial artificial chromosome (BAC) pools

    Science.gov (United States)

    The lepidopteran pest insect, Helicoverpa zea, feeds on cultivated corn and cotton crops in North America where control remains challenging due to evolution of resistance to chemical and transgenic insecticidal toxins, yet few genomic resources are available for this species. A bacterial artificial...

  6. Development of genome-specific primers for homoeologous genes in allopolyploid species: the waxy and starch synthase II genes in allohexaploid wheat (Triticum aestivum L. as examples

    Directory of Open Access Journals (Sweden)

    Brûlé-Babel Anita

    2010-05-01

    Full Text Available Abstract Background In allopolypoid crops, homoeologous genes in different genomes exhibit a very high sequence similarity, especially in the coding regions of genes. This makes it difficult to design genome-specific primers to amplify individual genes from different genomes. Development of genome-specific primers for agronomically important genes in allopolypoid crops is very important and useful not only for the study of sequence diversity and association mapping of genes in natural populations, but also for the development of gene-based functional markers for marker-assisted breeding. Here we report on a useful approach for the development of genome-specific primers in allohexaploid wheat. Findings In the present study, three genome-specific primer sets for the waxy (Wx genes and four genome-specific primer sets for the starch synthase II (SSII genes were developed mainly from single nucleotide polymorphisms (SNPs and/or insertions or deletions (Indels in introns and intron-exon junctions. The size of a single PCR product ranged from 750 bp to 1657 bp. The total length of amplified PCR products by these genome-specific primer sets accounted for 72.6%-87.0% of the Wx genes and 59.5%-61.6% of the SSII genes. Five genome-specific primer sets for the Wx genes (one for Wx-7A, three for Wx-4A and one for Wx-7D could distinguish the wild type wheat and partial waxy wheat lines. These genome-specific primer sets for the Wx and SSII genes produced amplifications in hexaploid wheat, cultivated durum wheat, and Aegilops tauschii accessions, but failed to generate amplification in the majority of wild diploid and tetraploid accessions. Conclusions For the first time, we report on the development of genome-specific primers from three homoeologous Wx and SSII genes covering the majority of the genes in allohexaploid wheat. These genome-specific primers are being used for the study of sequence diversity and association mapping of the three homoeologous Wx

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

    Energy Technology Data Exchange (ETDEWEB)

    Pieter J. de Jong

    2000-10-01

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

  8. BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping.

    Science.gov (United States)

    Vu, Giang T H; Dear, Paul H; Caligari, Peter D S; Wilkinson, Mike J

    2010-02-08

    Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical "contig" maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning approximately 10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual "BAC-HAPPY-mapping" to convert BAC landing data into BAC linkage contigs is possible.

  9. Back to BAC: The Use of Infectious Clone Technologies for Viral Mutagenesis

    Directory of Open Access Journals (Sweden)

    Robyn N. Hall

    2012-02-01

    Full Text Available Bacterial artificial chromosome (BAC vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.

  10. New insights into the origin of the B genome of hexaploid wheat: Evolutionary relationships at the SPA genomic region with the S genome of the diploid relative Aegilops speltoides

    Directory of Open Access Journals (Sweden)

    Charmet Gilles

    2008-11-01

    Full Text Available Abstract Background Several studies suggested that the diploid ancestor of the B genome of tetraploid and hexaploid wheat species belongs to the Sitopsis section, having Aegilops speltoides (SS, 2n = 14 as the closest identified relative. However molecular relationships based on genomic sequence comparison, including both coding and non-coding DNA, have never been investigated. In an attempt to clarify these relationships, we compared, in this study, sequences of the Storage Protein Activator (SPA locus region of the S genome of Ae. speltoides (2n = 14 to that of the A, B and D genomes co-resident in the hexaploid wheat species (Triticum aestivum, AABBDD, 2n = 42. Results Four BAC clones, spanning the SPA locus of respectively the A, B, D and S genomes, were isolated and sequenced. Orthologous genomic regions were identified as delimited by shared non-transposable elements and non-coding sequences surrounding the SPA gene and correspond to 35 268, 22 739, 43 397 and 53 919 bp for the A, B, D and S genomes, respectively. Sequence length discrepancies within and outside the SPA orthologous regions are the result of non-shared transposable elements (TE insertions, all of which inserted after the progenitors of the four genomes divergence. Conclusion On the basis of conserved sequence length as well as identity of the shared non-TE regions and the SPA coding sequence, Ae speltoides appears to be more evolutionary related to the B genome of T. aestivum than the A and D genomes. However, the differential insertions of TEs, none of which are conserved between the two genomes led to the conclusion that the S genome of Ae. speltoides has diverged very early from the progenitor of the B genome which remains to be identified.

  11. Genome Wide Single Locus Single Trait, Multi-Locus and Multi-Trait Association Mapping for Some Important Agronomic Traits in Common Wheat (T. aestivum L.).

    Science.gov (United States)

    Jaiswal, Vandana; Gahlaut, Vijay; Meher, Prabina Kumar; Mir, Reyazul Rouf; Jaiswal, Jai Prakash; Rao, Atmakuri Ramakrishna; Balyan, Harindra Singh; Gupta, Pushpendra Kumar

    2016-01-01

    Genome wide association study (GWAS) was conducted for 14 agronomic traits in wheat following widely used single locus single trait (SLST) approach, and two recent approaches viz. multi locus mixed model (MLMM), and multi-trait mixed model (MTMM). Association panel consisted of 230 diverse Indian bread wheat cultivars (released during 1910-2006 for commercial cultivation in different agro-climatic regions in India). Three years phenotypic data for 14 traits and genotyping data for 250 SSR markers (distributed across all the 21 wheat chromosomes) was utilized for GWAS. Using SLST, as many as 213 MTAs (p ≤ 0.05, 129 SSRs) were identified for 14 traits, however, only 10 MTAs (~9%; 10 out of 123 MTAs) qualified FDR criteria; these MTAs did not show any linkage drag. Interestingly, these genomic regions were coincident with the genomic regions that were already known to harbor QTLs for same or related agronomic traits. Using MLMM and MTMM, many more QTLs and markers were identified; 22 MTAs (19 QTLs, 21 markers) using MLMM, and 58 MTAs (29 QTLs, 40 markers) using MTMM were identified. In addition, 63 epistatic QTLs were also identified for 13 of the 14 traits, flag leaf length (FLL) being the only exception. Clearly, the power of association mapping improved due to MLMM and MTMM analyses. The epistatic interactions detected during the present study also provided better insight into genetic architecture of the 14 traits that were examined during the present study. Following eight wheat genotypes carried desirable alleles of QTLs for one or more traits, WH542, NI345, NI170, Sharbati Sonora, A90, HW1085, HYB11, and DWR39 (Pragati). These genotypes and the markers associated with important QTLs for major traits can be used in wheat improvement programs either using marker-assisted recurrent selection (MARS) or pseudo-backcrossing method.

  12. Genome Wide Single Locus Single Trait, Multi-Locus and Multi-Trait Association Mapping for Some Important Agronomic Traits in Common Wheat (T. aestivum L..

    Directory of Open Access Journals (Sweden)

    Vandana Jaiswal

    Full Text Available Genome wide association study (GWAS was conducted for 14 agronomic traits in wheat following widely used single locus single trait (SLST approach, and two recent approaches viz. multi locus mixed model (MLMM, and multi-trait mixed model (MTMM. Association panel consisted of 230 diverse Indian bread wheat cultivars (released during 1910-2006 for commercial cultivation in different agro-climatic regions in India. Three years phenotypic data for 14 traits and genotyping data for 250 SSR markers (distributed across all the 21 wheat chromosomes was utilized for GWAS. Using SLST, as many as 213 MTAs (p ≤ 0.05, 129 SSRs were identified for 14 traits, however, only 10 MTAs (~9%; 10 out of 123 MTAs qualified FDR criteria; these MTAs did not show any linkage drag. Interestingly, these genomic regions were coincident with the genomic regions that were already known to harbor QTLs for same or related agronomic traits. Using MLMM and MTMM, many more QTLs and markers were identified; 22 MTAs (19 QTLs, 21 markers using MLMM, and 58 MTAs (29 QTLs, 40 markers using MTMM were identified. In addition, 63 epistatic QTLs were also identified for 13 of the 14 traits, flag leaf length (FLL being the only exception. Clearly, the power of association mapping improved due to MLMM and MTMM analyses. The epistatic interactions detected during the present study also provided better insight into genetic architecture of the 14 traits that were examined during the present study. Following eight wheat genotypes carried desirable alleles of QTLs for one or more traits, WH542, NI345, NI170, Sharbati Sonora, A90, HW1085, HYB11, and DWR39 (Pragati. These genotypes and the markers associated with important QTLs for major traits can be used in wheat improvement programs either using marker-assisted recurrent selection (MARS or pseudo-backcrossing method.

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

    Science.gov (United States)

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

    2013-04-01

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

  14. Rapid genome mapping in nanochannel arrays for highly complete and accurate de novo sequence assembly of the complex Aegilops tauschii genome.

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    Alex R Hastie

    Full Text Available Next-generation sequencing (NGS technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum. Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.

  15. Is BAC Transgenesis Obsolete? State of the Art in the Era of Designer Nucleases

    Directory of Open Access Journals (Sweden)

    J. Beil

    2012-01-01

    Full Text Available DNA constructs based on bacterial artificial chromosomes (BACs are frequently used to generate transgenic animals as they reduce the influence of position effects and allow predictable expression patterns for genes whose regulatory sequences are not fully identified. Despite these advantages BAC transgenics suffer from drawbacks such as complicated vector construction, low efficiency of transgenesis, and some remaining expression variegation. The recent development of transcription activator-like effector nucleases (TALENs and zinc finger nucleases (ZFNs has resulted in new transgenic techniques which do not have the drawbacks associated with BAC transgenesis. Initial reports indicate that such designer nucleases (DNs allow the targeted insertion of transgenes into endogenous loci by direct injection of the targeting vector and mRNA/DNA encoding the predesigned nucleases into oocytes. This results in the transgene being inserted at a specific locus in the mouse genome, thus circumventing the drawbacks associated with BAC transgenesis.

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

    Science.gov (United States)

    Ali, Shawkat; Bakkeren, Guus

    2015-01-01

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

  17. A dense genetic linkage map for common carp and its integration with a BAC-based physical map.

    Directory of Open Access Journals (Sweden)

    Lan Zhao

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

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

    Science.gov (United States)

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

    2006-12-01

    The Poales (includes the grasses) and Asparagales [includes onion (Allium cepa L.) and asparagus (Asparagus officinalis L.)] are the two most economically important monocot orders. The Poales are a member of the commelinoid monocots, a group of orders sister to the Asparagales. Comparative genomic analyses have revealed a high degree of synteny among the grasses; however, it is not known if this synteny extends to other major monocot groups such as the Asparagales. Although we previously reported no evidence for synteny at the recombinational level between onion and rice, microsynteny may exist across shorter genomic regions in the grasses and Asparagales. We sequenced nine asparagus BACs to reveal physically linked genic-like sequences and determined their most similar positions in the onion and rice genomes. Four of the asparagus BACs were selected using molecular markers tightly linked to the sex-determining M locus on chromosome 5 of asparagus. These BACs possessed only two putative coding regions and had long tracts of degenerated retroviral elements and transposons. Five asparagus BACs were selected after hybridization of three onion cDNAs that mapped to three different onion chromosomes. Genic-like sequences that were physically linked on the cDNA-selected BACs or genetically linked on the M-linked BACs showed significant similarities (e asparagus and rice across these regions. Genic-like sequences that were linked in asparagus were used to identify highly similar (e asparagus and genetic linkages in onion. These results further indicate that synteny among grass genomes does not extend to a sister order in the monocots and that asparagus may not be an appropriate smaller genome model for plants in the Asparagales with enormous nuclear genomes. PMID:17016688

  19. Construction of a BAC library from cucumber (Cucumis sativus L.) and identification of linkage group specific clones

    Institute of Scientific and Technical Information of China (English)

    Yuan Guan; Qi Chen; Junsong Pan; Zheng Li; Huanle He; Aizhong Wu; Rentao Song; Run Cai

    2008-01-01

    A bacterial artificial chromosome (BAC) library consisting of 19,200 clones with an average insert size of 105 kb has been constructed from a cucumber (Cucumis sativus L.) inbred line S94, derived from a cultivar in North China. The entire library was equivalent to approximately 5 haploid cucumber genomes. To facilitate chromosome engineering and anchor the cucumber genetic linkage map to its chromosomes, 15 sequence-characterized amplified regions (SCAR) and seven simple sequence repeats (SSR) markers from each link-age group of cucumber were used to screen an ordered array of pooled BAC DNA with polymerase chain reaction (PCR). Fifteen mark-ers gave at least two positive clones. As a result, 22 BAC clones representing 7 linkage groups of cucumber were identified, which further validated the genome coverage and utility of the library. This BAC library and linkage group specific clones provide essential resources for future research of the cucumber genome.

  20. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Institute of Scientific and Technical Information of China (English)

    Zhang Da; Zhu Houchu; Huang Liuyu

    2013-01-01

    Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC) system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs) have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in ifdelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artiifcial chromosome library (BAC) has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were iflled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93%genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  1. BAC Library Construction and Physical Mapping of Bacillus anthracis A16R

    Directory of Open Access Journals (Sweden)

    Da Zhang

    2013-12-01

    Full Text Available Bacillus anthracis is an endospore-forming bacterium that causes severe inhalational anthrax, and bacillus anthracis A16R is an attenuated strain derived from Bacillus anthracis A16. The development of bacterial artificial chromosome (BAC system has allowed the construction of large insert-size DNA libraries, and the bacterial artificial chromosomes (BACs have become the preferred large insert cloning system for genomic analysis because such libraries are characteristically stable, high in fidelity and easy to handle. To facilitate genome studies of this bacterium, a bacterial artificial chromosome library (BAC has been established from genome DNA of Bacillus anthracis A16R. This library consisted of 9 600 clones randomly selected from more than 15 000 recombinant clones carrying inserts in the plindigoBAC-5 vectors. The mean insert size was 56 kbp, representing an approximate 12-fold genome coverage, while end sequences were obtained from 700 randomly selected clones. Sequences were compared with Bacillus anthracis Ames and Bacillus cereus ATCC 14579 Genome Project databases using the NCBI BLASTN search project. And most BLASTN results showed high identities and that the sequences’ sites could be used as STSs. To construct this physical map, Excel was used for the array of STSs and some gaps of the map were filled up by PCR walking. Artemis-V4 was used in the construction of a genome-wide physical map with 93% genome coverage. The A16R BAC library proved to be a vital tool for the generation of a map that would not only allow the subsequent sequencing of defined areas of genome, but also provide immediate access to clones that were stable and convenient for functional genomic researches.

  2. A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening

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    Deal Karin R

    2010-12-01

    Full Text Available Abstract Background A five-dimensional (5-D clone pooling strategy for screening of bacterial artificial chromosome (BAC clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones. Results A new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87% specific SNP markers on the map with 91% precision. Conclusion A new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.

  3. A BAC-based physical map of Zhikong scallop (Chlamys farreri Jones et Preston.

    Directory of Open Access Journals (Sweden)

    Xiaojun Zhang

    Full Text Available Zhikong scallop (Chlamys farreri is one of the most economically important aquaculture species in China. Physical maps are crucial tools for genome sequencing, gene mapping and cloning, genetic improvement and selective breeding. In this study, we have developed a genome-wide, BAC-based physical map for the species. A total of 81,408 clones from two BAC libraries of the scallop were fingerprinted using an ABI 3130xl Genetic Analyzer and a fingerprinting kit developed in our laboratory. After data processing, 63,641 (∼5.8× genome coverage fingerprints were validated and used in the physical map assembly. A total of 3,696 contigs were assembled for the physical map. Each contig contained an average of 10.0 clones, with an average physical size of 490 kb. The combined total physical size of all contigs was 1.81 Gb, equivalent to approximately 1.5 fold of the scallop haploid genome. A total of 10,587 BAC end sequences (BESs and 167 markers were integrated into the physical map. We evaluated the physical map by overgo hybridization, BAC-FISH (fluorescence in situ hybridization, contig BAC pool screening and source BAC library screening. The results have provided evidence of the high reliability of the contig physical map. This is the first physical map in mollusc; therefore, it provides an important platform for advanced research of genomics and genetics, and mapping of genes and QTL of economical importance, thus facilitating the genetic improvement and selective breeding of the scallop and other marine molluscs.

  4. Suicidal Autointegration of Sleeping Beauty and piggyBac Transposons in Eukaryotic Cells

    OpenAIRE

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

    2014-01-01

    Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from inse...

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Lindblad-Toh Kerstin

    2010-11-01

    Full Text Available Abstract 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.

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

    OpenAIRE

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

    2008-01-01

    Abstract Background The genus Brassica includes the most extensively cultivated vegetable crops worldwide. Investigation of the Brassica genome presents excellent challenges to study plant genome evolution and divergence of gene function associated with polyploidy and genome hybridization. A physical map of the B. rapa genome is a fundamental tool for analysis of Brassica "A" genome structure. Integration of a physical map with an existing genetic map by linking genetic markers and BAC clones...

  8. Construction of an Americn mink Bacterial Artificial Chromosome (BAC) library and sequencing candidate genes important for the fur industry

    DEFF Research Database (Denmark)

    Anistoroaei, Razvan Marian; Hallers, Boudewijn ten; Nefedov, Michael;

    2011-01-01

    contigs (184 kb in average) were assembled. Knowing the complete sequences of these candidate genes will enable confirmation of the association with a phenotype and the finding of causative mutations for the targeted phenotypes.Additionally, 1577 BAC clones were end sequenced; 2505 BAC end sequences (80......BACKGROUND: Bacterial artificial chromosome (BAC) libraries continue to be invaluable tools for the genomic analysis of complex organisms. Complemented by the newly and fast growing deep sequencing technologies, they provide an excellent source of information in genomics projects. RESULTS: Here, we...... consisting of 18,432 clones spotted in duplicate, have been produced for hybridization screening and are publicly available. Overgo probes derived from expressed sequence tags (ESTs), representing 21 candidate genes for traits important for the mink industry, were used to screen the BAC library...

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

    Directory of Open Access Journals (Sweden)

    Lee Soo

    2008-06-01

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

  10. A first generation BAC-based physical map of the Asian seabass (Lates calcarifer.

    Directory of Open Access Journals (Sweden)

    Jun Hong Xia

    Full Text Available BACKGROUND: The Asian seabass (Lates calcarifer is an important marine foodfish species in Southeast Asia and Australia. Genetic improvement of this species has been achieved to some extent through selective breeding programs since 1990s. Several genomic tools such as DNA markers, a linkage map, cDNA and BAC libraries have been developed to assist selective breeding. A physical map is still lacking, although it is essential for positional cloning of genes located in quantitative trait loci (QTL and assembly of whole genome sequences. METHODOLOGY/PRINCIPAL FINDINGS: A genome-wide physical map of the Asian seabass was constructed by restriction fingerprinting of 38,208 BAC clones with SNaPshot HICF FPC technique. A total of 30,454 were assembled into 2,865 contigs. The physical length of the assembled contigs summed up to 665 Mb. Analyses of some contigs using different methods demonstrated the reliability of the assembly. CONCLUSIONS/SIGNIFICANCE: The present physical map is the first physical map for Asian seabass. This physical map will facilitate the fine mapping of QTL for economically important traits and the positional cloning of genes located in QTL. It will also be useful for the whole genome sequencing and assembly. Detailed information about BAC-contigs and BAC clones are available upon request.

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

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    GE Yan; HE GuangChun; WANG ZhiWei; GUO DeDong; QIN Rui; LI RongTian

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Zhao Shaying

    2009-06-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-04-21

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

  15. A Domesticated PiggyBac Transposase Interacts with Heterochromatin and Catalyzes Reproducible DNA Elimination in Tetrahymena

    OpenAIRE

    Alexander Vogt; Kazufumi Mochizuki

    2013-01-01

    The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for ∼30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activ...

  16. Genome Sequencing

    DEFF Research Database (Denmark)

    Sato, Shusei; Andersen, Stig Uggerhøj

    2014-01-01

    The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based on transcr......The current Lotus japonicus reference genome sequence is based on a hybrid assembly of Sanger TAC/BAC, Sanger shotgun and Illumina shotgun sequencing data generated from the Miyakojima-MG20 accession. It covers nearly all expressed L. japonicus genes and has been annotated mainly based...

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

    Directory of Open Access Journals (Sweden)

    Ruvini Ariyadasa

    2012-01-01

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

  18. Extensive conserved synteny of genes between the karyotypes of Manduca sexta and Bombyx mori revealed by BAC-FISH mapping.

    Directory of Open Access Journals (Sweden)

    Yuji Yasukochi

    Full Text Available BACKGROUND: Genome sequencing projects have been completed for several species representing four highly diverged holometabolous insect orders, Diptera, Hymenoptera, Coleoptera, and Lepidoptera. The striking evolutionary diversity of insects argues a need for efficient methods to apply genome information from such models to genetically uncharacterized species. Constructing conserved synteny maps plays a crucial role in this task. Here, we demonstrate the use of fluorescence in situ hybridization with bacterial artificial chromosome probes as a powerful tool for physical mapping of genes and comparative genome analysis in Lepidoptera, which have numerous and morphologically uniform holokinetic chromosomes. METHODOLOGY/PRINCIPAL FINDINGS: We isolated 214 clones containing 159 orthologs of well conserved single-copy genes of a sequenced lepidopteran model, the silkworm, Bombyx mori, from a BAC library of a sphingid with an unexplored genome, the tobacco hornworm, Manduca sexta. We then constructed a BAC-FISH karyotype identifying all 28 chromosomes of M. sexta by mapping 124 loci using the corresponding BAC clones. BAC probes from three M. sexta chromosomes also generated clear signals on the corresponding chromosomes of the convolvulus hawk moth, Agrius convolvuli, which belongs to the same subfamily, Sphinginae, as M. sexta. CONCLUSIONS/SIGNIFICANCE: Comparison of the M. sexta BAC physical map with the linkage map and genome sequence of B. mori pointed to extensive conserved synteny including conserved gene order in most chromosomes. Only a few rearrangements, including three inversions, three translocations, and two fission/fusion events were estimated to have occurred after the divergence of Bombycidae and Sphingidae. These results add to accumulating evidence for the stability of lepidopteran genomes. Generating signals on A. convolvuli chromosomes using heterologous M. sexta probes demonstrated that BAC-FISH with orthologous sequences can be

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

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

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

    Science.gov (United States)

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

    2010-11-19

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

  1. 454 sequencing of pooled BAC clones on chromosome 3H of barley

    Directory of Open Access Journals (Sweden)

    Yamaji Nami

    2011-05-01

    Full Text Available Abstract Background Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp. Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H. Results We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1. Conclusions We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.

  2. A highly redundant BAC library of Atlantic salmon (Salmo salar: an important tool for salmon projects

    Directory of Open Access Journals (Sweden)

    Koop Ben F

    2005-04-01

    Full Text Available Abstract Background As farming of Atlantic salmon is growing as an aquaculture enterprise, the need to identify the genomic mechanisms for specific traits is becoming more important in breeding and management of the animal. Traits of importance might be related to growth, disease resistance, food conversion efficiency, color or taste. To identify genomic regions responsible for specific traits, genomic large insert libraries have previously proven to be of crucial importance. These large insert libraries can be screened using gene or genetic markers in order to identify and map regions of interest. Furthermore, large-scale mapping can utilize highly redundant libraries in genome projects, and hence provide valuable data on the genome structure. Results Here we report the construction and characterization of a highly redundant bacterial artificial chromosome (BAC library constructed from a Norwegian aquaculture strain male of Atlantic salmon (Salmo salar. The library consists of a total number of 305 557 clones, in which approximately 299 000 are recombinants. The average insert size of the library is 188 kbp, representing 18-fold genome coverage. High-density filters each consisting of 18 432 clones spotted in duplicates have been produced for hybridization screening, and are publicly available 1. To characterize the library, 15 expressed sequence tags (ESTs derived overgos and 12 oligo sequences derived from microsatellite markers were used in hybridization screening of the complete BAC library. Secondary hybridizations with individual probes were performed for the clones detected. The BACs positive for the EST probes were fingerprinted and mapped into contigs, yielding an average of 3 contigs for each probe. Clones identified using genomic probes were PCR verified using microsatellite specific primers. Conclusion Identification of genes and genomic regions of interest is greatly aided by the availability of the CHORI-214 Atlantic salmon BAC

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

    Science.gov (United States)

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

    2015-06-01

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

  4. 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

    OpenAIRE

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

    2009-01-01

    Abstract 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 l...

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Déborah Ley

    2014-11-01

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

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

    Directory of Open Access Journals (Sweden)

    You Frank M

    2010-06-01

    Full Text Available Abstract 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

  8. IMPROVING WHEAT TRITICUM AESTIVUM L. BY INTERSPECIFIC AND INTERGENERIC HYBRIDIZATION WITH POACEAE FAMILY SPECIES

    Directory of Open Access Journals (Sweden)

    Czaplicki A.Z.

    2012-08-01

    Full Text Available The related species of the family Poaceae (Triticeae are the source of unprecedented new genes that allow the extension of genetic variation of common wheat Triticum aestivum L. These species have similar homoeologous chromosomes and rDNA sequences very similar to T. aestivum L. [1-3]. This allows the introgression of alien genes and their incorporation into the genomes A, B and D of wheat, where they can function permanently in the wheat genetic systems. Many of them have already been transferred to the varieties of T. aestivum L. [4].The experimental material consisted of 28 lines of winter wheat obtained using the interspecific and intergeneric hybridization of T. aestivum L. with alien species T. durum Desf., T. timopheevii Zhuk., Lolium perenne L. and Aegilops speltoides Taush. Among them, 15 lines were developed from the cross-combination with tetraploid species (AABB T. durum Desf., 4 lines from the combination with other tetraploid species of different genome composition (AAGG T. timopheevii Zhuk., 4 lines from cross with L. perenne L. and 5 lines were the double hybrids (three-generic derived with two related species, T. durum Desf. (AABB and Ae. speltoides Taush (BB.The anther culture method was used for obtaining DH lines from these interspecific and intergeneric hybrids. In in vitro culture 124 green plants were regenerated. The method of cluster analysis grouped hybrids in terms of comprehensive general similarity of the studied traits.

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

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, C

    2005-09-27

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

  10. Immunotoxin BAC5-CT treated nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In this study, immunotoxin (IT) was prepared by conjugating BAC5 and CT with SPDP. The effects of IT on NPC and its mechanisms were explored using double labeled with radioactive nuclides, immunography and electron microscope technique in vivo and in vitro. The specific concentration of BAC5 in the tumor area showed. The radioactivity rate of tumor/nontumor (T/NT) was up to 10.26. IT had cytotoxic effects both on the cultured CNE-2 cell line and tumor multicell spheroides. In vivo, the preliminary result indicated that IT also had a inhibitory action on the nude mice models bearing human NPC (Reported in another article). Under electron microscope, the necrosis and apoptosis of tumor cells were found. The membranes of most tumor cells were found intacted not or corrosined, some of them had the character of apoptosis, including reduce of tumor cells membrane villi, condensation of cytoplasm and pyknosis or cleavage of nuclear. There were many of apoptosis bodies, which were occasionally phagocytosed by tumor cells. The infiltration of immunocytoes in tumor tissue could be seen. The results indicated that BAC5 can specifically combine with NPC cells and BAC5-CT has the inhibitory effect on NPC in vitro and in vivo, mechanism of which may be related to the effects that ‘warhead' CT dissolute the membrane of tumor cells directly, or/and IT promote the infiltration of immunocytoes so as to induce the apoptosis of tumor cell.

  11. Isolation and sequence analysis of the wheat B genome subtelomeric DNA

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    Huneau Cecile

    2009-09-01

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

  12. Annotation and BAC/PAC localization of nonredundant ESTs from drought-stressed seedlings of an indica rice

    Indian Academy of Sciences (India)

    P. Ravindra Babu; A. Chandra Sekhar; Nagabhushana Ithal; G. Markandeya; Arjula R. Reddy

    2002-04-01

    To decipher the genes associated with drought stress response and to identify novel genes in rice, we utilized 1540 high-quality expressed sequence tags (ESTs) for functional annotation and mapping to rice genomic sequences. These ESTs were generated earlier by 3′-end single-pass sequencing of 2000 cDNA clones from normalized cDNA libraries constructed from drought-stressed seedlings of an indica rice. A rice UniGene set of 1025 transcripts was constructed from this collection through the BLASTN algorithm. Putative functions of 559 nonredundant ESTs were identified by BLAST similarity search against public databases. Putative functions were assigned at a stringency E value of 10-6 in BLASTN and BLASTX algorithms. To understand the gene structure and function further, we have utilized the publicly available finished and unfinished rice BAC/PAC (BAC, bacterial artificial chromosome; PAC, P1 artificial chromosome) sequences for similarity search using the BLASTN algorithm. Further, 603 nonredundant ESTs have been mapped to BAC/PAC clones. BAC clones were assigned by a homology of above 95% identity along 90% of EST sequence length in the aligned region. In all, 700 ESTs showed rice EST hits in GenBank. Of the 325 novel ESTs, 128 were localized to BAC clones. In addition, 127 ESTs with identified putative functions but with no homology in IRGSP (International Rice Genome Sequencing Program) BAC/PAC sequences were mapped to the Chinese WGS (whole genome shotgun contigs) draft sequence of the rice genome. Functional annotation uncovered about a hundred candidate ESTs associated with abiotic stress in rice and Arabidopsis that were previously reported based on microarray analysis and other studies. This study is a major effort in identifying genes associated with drought stress response and will serve as a resource to rice geneticists and molecular biologists.

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

    OpenAIRE

    Borenstein, Ronen; Frenkel, Niza

    2009-01-01

    Cloning of large viral genomes into bacterial artificial chromosomes (BACs) facilitates analyses of viral functions and molecular mutagenesis. Previous derivations of viral BACs involved laborious recombinations within infected cells. We describe a single-step production of viral BACs by direct cloning of unit length genomes, derived from circular or head-to-tail concatemeric DNA replication intermediates. The BAC cloning is independent of intracellular recombinations and DNA packaging constr...

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

    Science.gov (United States)

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

    2013-01-01

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

  15. A new active piggyBac-like element in Aphis gossypii

    Institute of Scientific and Technical Information of China (English)

    Guana-Hua Luo; Min Wu; Xiao-Fang Wang; Wei Zhang; Zhao-Jun Han

    2011-01-01

    Nine piggyBac-like elements (PLEs) were identified from the cotton aphid Aphis gossypii Glover.All the PLEs shared high sequence similarity with each other.However,eight of the nine PLEs were unlikely to encode functional transposase due to the existence of disruptive mutations within the coding regions.The other one PLE contained major characteristics of members in the piggyBac family,including TTAA target site duplications,inverted terminal repeats (ITRs),and an open reading frame (ORF) coding for a transposase with a putative DDD domain.This one with an intact transposase ORF was named AgoPLE1.1.The predicted transposase shared 47% similarity with that of Trichoplusia ni piggyBac IFP2.Phylogenetic analyses showed that AgoPLE1.1 was most related to the Heliothis virescens PLEl.1 (HvPLEl.1) element,with 45% and 60% similarity at the nucleotide and amino acid levels,respectively.A functional assay demonstrated that AgoPLEl.1 encoded a functional transposase and was able to cause precise excision in cell cultures.On the other hand,few genomic insertion polymorphisms of AgoPLE1 were observed in the genome of the cotton aphid.These observations suggested that AgoPLEl.1 was a PLE that invaded the cotton aphid genome in recent periods and retained its activity.

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

    Science.gov (United States)

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

    2009-05-01

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

  17. Combining ability for Fusarium head blight resistance in wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Subas Malla

    2010-12-01

    Full Text Available Fusarium head blight (FHB caused by Fusarium graminearum reduces wheat (Triticum aestivum L. em. Thell grain yield and end-use quality worldwide. Three each of FHB susceptible (‘Nekota’, ‘2137’, and ‘Harding’ and resistant (‘Ning 7840’, ‘ND2710’ and ‘BacUp’ parents were included in a partial diallel mating design (Griffing’s Method 4, Model 1. The F4:5 progeny was evaluated for healthy index, undamaged kernels, and deoxynivalenol (DON content following artificial inoculation and mist-irrigation in 2006 and 2007. General combining ability (GCA was highly significant (P < 0.01 for healthy index; whereas specific combining ability (SCA and GCA-by-year interaction were not significant. The combining ability ratio and narrow-sense heritability were 0.95 and 0.83, respectively. The Genotype, Genotype-by-Environment (GGE biplot analysis showed that the ND2710/BacUp combination had the best healthy index and undamaged kernels; whereas Ning 7840 contributed resistance to DON accumulation. The results indicated additive gene effects mainly control that healthy index. Thus, genetic gain in developing resistance in wheat can be achieved through selection.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Ramesh Buyyarapu

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

  20. Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates

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    Yang Ying

    2008-09-01

    Full Text Available Abstract Background αA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12–24 hours, compared to the expression of the endogenous Cryaa gene. Results Here, we used bacterial artificial chromosome (BAC and standard transgenic approaches to examine temporal and spatial regulation of the mouse Cryaa gene. Two BAC transgenes, with EGFP insertions into the third coding exon of Cryaa gene, were created: the intact αA-crystallin 148 kb BAC (αA-BAC and αA-BAC(ΔDCR3, which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the Cryaa gene in lens, but not outside of the lens. The number of cells expressing αA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of αA-crystallin locus derived from αA-BAC(ΔDCR3, 15 kb Cryaa/EGFP. A 15 kb region of Cryaa/EGFP supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of αA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing αA-crystallin in the lens pit. Conclusion We conclude that a 148 kb αA-BAC likely contains all of the regulatory regions required for αA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb Cryaa/EGFP region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic

  1. Generation of BAC transgenic epithelial organoids.

    Directory of Open Access Journals (Sweden)

    Gerald Schwank

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

  2. High Expression of Insulin-like Growth Factor H (IGF-Ⅱ) Using Bac-to-Bac Expression System

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective In order to obtain mature insulin-like growth factor- Ⅱ ( IGF- Ⅱ ), we used Bac-to-Bac baculovirus expression system. Methods Firstly the IGF- Ⅱ cDNA was cloned into a donor plasmid pFastBac1 and the recombinant pFastBac1 was then introduced into competent cells DH 10Bac. Recombinant bacmids were constructed by transposing a mini-Tn7 element from a donor plasmid pFastBac1 to the mini-attTn7 attachment site on the bacmid where the Tn7 transposition functions were provided in trans by a helper plasmid, and then used to transfect Sf9 insect cells to get recombinant baculovirus. The recombinant baculovirus was used to infect insect cells. Results Agarose gel analysis showed that recombinant donor plasmid pFastBac1 was constructed successfully; Agarose gel analysis of PCR products confirmed recombinant bacmid ; SDS-PAGE and Western Blotting showed that a 7KD protein band appeared. Conclusion The mature IGF- Ⅱ with immunogenecity has been expressed and produced by using Bac-to-Bac expression system.

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

    Science.gov (United States)

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

    2003-06-24

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

  4. Molecular Characterization and Global Expression Analysis of Lectin Receptor Kinases in Bread Wheat (Triticum aestivum)

    Science.gov (United States)

    Pandey, Ajay K.; Singh, Kashmir; Upadhyay, Santosh Kumar

    2016-01-01

    Lectin receptor kinases (LRKs) play a critical role in plants during development and stress conditions, but a comprehensive analysis at genome level is still not carried out in Triticum aestivum. Herein, we performed the genome wide identification, characterization and expression analysis of these genes in T. aestivum (TaLRK). In-total 263 TaLRK genes were identified, which were further classified into three groups based on the nature of lectin domain. We identified, two TaLRKs consisted of calcium-dependent lectin (C-LRK), while 84 legume-lectin (L-LRK) and 177 bulb-lectin (B-LRK) domains. The L-LRK and B-LRK genes were distributed throughout the genome of T. aestivum. Most of the TaLRKs were clustered as homologs, which were distributed either in proximity on same chromosome or on homoeologous chromosomes of A, B and D sub-genomes. A total of 9 and 58 duplication events were also predicted in L-LRK and B-LRK, respectively. Phylogenetic analysis indicated conserved evolutionary relationship of homologous and orthologous genes from multiple plant species. Gene ontology analysis indicated TaLRKs role in binding, signaling and receptor activities. Most of the TaLRKs consisted of a trans-membrane domain and predicted to be localized in the plasma-membrane. A diverse expression pattern of TaLRK genes was found in various developmental stages and stress conditions. Some TaLRKs were found to be highly affected during a particular stress, which indicated a specialized role of each LRK gene in a specific stress condition. These results described various characteristic feature and expression pattern of TaLRK genes, which will pave the way for functional characterization in wheat. PMID:27111449

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

    Directory of Open Access Journals (Sweden)

    Jinke Lin

    2011-01-01

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

  6. Comparison of BAC FISH with specific telomeres and centromere probes and chromosome painting on detection of chromosome translocation induced by irradiation%BAC FISH与PAINT法检测辐射诱发染色体易位的比较

    Institute of Scientific and Technical Information of China (English)

    刘青杰; 陆雪; 封江彬; 王晓维; 陈德清

    2008-01-01

    Objective To compare the efficiency of BAC FISH established in our lab and conventional chromosome painting(PAINT)on detection of radiation-induced chromosome translocation.Methods Healthy human peripheral blood samples were irradiated with 0~5.0 Gy 60Co γ-rays.Then chromosome translocations in these samples were detected with BAC FISH and PAINT using chromosomes 1.2 and 4.The genome translocation rates were calculated with observed chromosome translocation rates,and the dose-response curve of two methods were established.Results The genome translocation rates induced by 0~5.0 Gy 60Co γ-rays detected by BAC FISH and PAINT were increased with absorbed doses.The observed translocation rates with BAC FISH were higher than that with PAINT at each dose level.The dose-response curve were Y=0.043 D2+0.0008D+0.0048 for BAC FISH and Y=0.043D2+0.006D+0.0027 for PAINT.Conclusions The translocation rate detected by BAC FISH was higher than that by PAINT,and the parameters β in dose-response curve equation were same by two methods.%目的 比较自行建立的BAC FISH方法和常规染色体涂染(PAINT)方法分析辐射诱发染色体易位有效性的不同.方法 对正常人外周血照射不同剂量(0~5.0 Gy)的60Co γ射线,用1、2和4号染色体特异性端粒和着丝粒探针BAC FISH及PAINT分析染色体易位,将观察到的染色体易位率换算为全基因组易位率,并建立两种方法分析辐射诱发的染色体易位率剂量-效应曲线.结果 用两种方法分析0~5.0 Gy 60Coγ射线诱发的全基因组易位率均随着吸收剂量的增加而增高;在相同的剂量点,BAC FISH染色体易位检出率高于PAINT方法.两种方法分析吸收剂量和全基因组易位率之间的剂量-效应曲线均为二次方程模式,分别为Y=0.043D2+0.0008D+0.0048(BAC FISH)和Y=0.043D2+0.006D+0.0027(PAINT).结论 自行建立的BAC FISH方法分析辐射诱发染色体易位检出率高于常规染色体涂染方法,两种方法建立的

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

    Directory of Open Access Journals (Sweden)

    Jeffrey C. Miecznikowski

    2011-01-01

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

  8. Pig genome sequence - analysis and publication strategy

    DEFF Research Database (Denmark)

    Archibald, Alan L.; Bolund, Lars; Churcher, Carol;

    2010-01-01

    BACKGROUND: The pig genome is being sequenced and characterised under the auspices of the Swine Genome Sequencing Consortium. The sequencing strategy followed a hybrid approach combining hierarchical shotgun sequencing of BAC clones and whole genome shotgun sequencing. RESULTS: Assemblies......) is under construction and will incorporate whole genome shotgun sequence (WGS) data providing > 30x genome coverage. The WGS sequence, most of which comprise short Illumina/Solexa reads, were generated from DNA from the same single Duroc sow as the source of the BAC library from which clones were...

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

    Directory of Open Access Journals (Sweden)

    Jin-Bon Hong

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

  12. Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

    NARCIS (Netherlands)

    Spassova, MI; Prins, M; Stevens, MR; Hille, J; Goldbach, RW; Spassova, Mariana I.; Stevens, Mikel R.; Goldbach, Rob W.

    1999-01-01

    The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens,

  13. [The improvement and application of piggyBac transposon system in mammals].

    Science.gov (United States)

    Qian, Qiujie; Che, Jiaqian; Ye, Lupeng; Zhong, Boxiong

    2014-10-01

    The piggyBac (PB) transposon system is a useful genomic engineering tool due to its high transposition efficiency, precise excision, semi-random insertion and large cargo capacity. But, it still needs to further improve the transgenic efficiency and reduce the risk of endogenous disruption caused by the random insertion of exogenous gene, especially in transgenic experiments of individual mammals. In recent studies, the PB transposase is fused with a DNA binding protein as a chimeric protein, which can guide the transposon to pre-designed loci. Besides, PB transposases obtained by mutagenesis have dramatically enhanced transposition activity and generated a novel function which is excision competent and integration defective. Furthermore, PB transposon system can carry large exogenous DNA fragments up to 207 kb when combining with the bacterial artificial chromosome vector. So far, these modified transposon systems have been widely applied in genome studies, gene therapy and induced pluripotent stem cells (iPS cells). In this study, we review the latest studies on piggyBac transposon system and its application prospect.

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

    Directory of Open Access Journals (Sweden)

    Wilson Melanie

    2003-11-01

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

  15. Pig genome sequence - analysis and publication strategy

    NARCIS (Netherlands)

    Archibald, A.L.; Bolund, L.; Churcher, C.; Fredholm, M.; Groenen, M.A.M.; Harlizius, B.

    2010-01-01

    Background - The pig genome is being sequenced and characterised under the auspices of the Swine Genome Sequencing Consortium. The sequencing strategy followed a hybrid approach combining hierarchical shotgun sequencing of BAC clones and whole genome shotgun sequencing. Results - Assemblies of the B

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

    Science.gov (United States)

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

    2009-03-01

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

  17. L’italiano alla prova del Bac

    Directory of Open Access Journals (Sweden)

    Giulia Massignan

    2013-07-01

    Full Text Available Il lavoro propone alcune attività didattiche di preparazione dell’esame orale di italiano previsto dal Baccalauréat del liceo francese. Tale prova d’esame è stata recentemente oggetto di una riforma ministeriale volta a dare maggior peso alla pratica orale. Dopo uno studio delle nuove modalità della prove d’esame e dei suoi contenuti, ci si concentrerà sull’analisi delle abilità orali necessarie al suo superamento e sugli elementi linguistici e comunicativi su cui porre l’attenzione. Sulla base di queste premesse teoriche e metodologiche, si presentano alcuni interventi didattici di preparazione all’esame: due attività per la prova di comprensione orale e due per la prova di produzione e interazione orale; in entrambi i casi, si insiste sull’importanza di proporre strategie d’ascolto e di parlato insegnabili, che diventino consuetudini attive e parte integrante del bagaglio comunicativo del discente. The italian exam in the FrenchBacThe paper proposes some educational activities to prepare for the oral Italian examination as part of the French Baccalauréat. This exam has recently been the subject of a ministerial reform aimed at giving more weight to the oral practice. After illustrating new examination methods and content, we will focus on the analysis of oral skills necessary for passing the exam and aspects of language and communication on which to focus. Based on these theoretical and methodological premises, some educational exam preparation activities will be presented. Two activities test listening comprehension and two focus on production and oral interaction. The stress is on the importance of proposing teach able listening and speaking strategies, so that they become active habits and integral parts of the communicative learner’s baggage.

  18. A Highly Efficient Protocol of Generating and Analyzing VZV ORF Deletion Mutants Based on a Newly Developed Luciferase VZV BAC System

    OpenAIRE

    Zhang, Zhen; Huang, Ying; Zhu, Hua

    2008-01-01

    Varicella Zoster Virus (VZV) is the causative agent for both varicella (chicken pox) and herpes zoster (shingles). As a member of the human herpesvirus family, VZV contains a large DNA genome, encoding 70 unique open reading frames (ORFs). The functions of the majority of these ORFs remain unknown. Recently, the full-length VZV (P-Oka strain) genome was cloned as a VZV bacteria artificial chromosome (BAC) and additionally a firefly luciferase cassette was inserted to generate a novel lucifera...

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

    Science.gov (United States)

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

    2015-01-01

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

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

    OpenAIRE

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

    2012-01-01

    Nicastrin (NCSTN) is a component of the γ-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+/Nc...

  1. Global transgenerational gene expression dynamics in two newly synthesized allohexaploid wheat (Triticum aestivum lines

    Directory of Open Access Journals (Sweden)

    Qi Bao

    2012-01-01

    Full Text Available Abstract Background Alteration in gene expression resulting from allopolyploidization is a prominent feature in plants, but its spectrum and extent are not fully known. Common wheat (Triticum aestivum was formed via allohexaploidization about 10,000 years ago, and became the most important crop plant. To gain further insights into the genome-wide transcriptional dynamics associated with the onset of common wheat formation, we conducted microarray-based genome-wide gene expression analysis on two newly synthesized allohexaploid wheat lines with chromosomal stability and a genome constitution analogous to that of the present-day common wheat. Results Multi-color GISH (genomic in situ hybridization was used to identify individual plants from two nascent allohexaploid wheat lines between Triticum turgidum (2n = 4x = 28; genome BBAA and Aegilops tauschii (2n = 2x = 14; genome DD, which had a stable chromosomal constitution analogous to that of common wheat (2n = 6x = 42; genome BBAADD. Genome-wide analysis of gene expression was performed for these allohexaploid lines along with their parental plants from T. turgidum and Ae. tauschii, using the Affymetrix Gene Chip Wheat Genome-Array. Comparison with the parental plants coupled with inclusion of empirical mid-parent values (MPVs revealed that whereas the great majority of genes showed the expected parental additivity, two major patterns of alteration in gene expression in the allohexaploid lines were identified: parental dominance expression and non-additive expression. Genes involved in each of the two altered expression patterns could be classified into three distinct groups, stochastic, heritable and persistent, based on their transgenerational heritability and inter-line conservation. Strikingly, whereas both altered patterns of gene expression showed a propensity of inheritance, identity of the involved genes was highly stochastic, consistent with the involvement of diverse Gene Ontology (GO

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

    Science.gov (United States)

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

    2016-01-01

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

  3. [Cytogenetic analysis of alloplasmic recombinant lines (H. vulgare)-T. aestivum with unstable fertility and viability].

    Science.gov (United States)

    Badaeva, E D; Pershina, L A; Bil'danova, L L

    2006-02-01

    Comparative cytogenetic analysis was performed with four alloplasmic recombinant (Hordeum vulgare)-Triticum aestivum lines differing in morphological traits, number of seeds per spike, and seed plumpness. None of the lines displayed introgression of the barley genetic material: the karyotypes included only common wheat chromosomes. Two lines, 79(B) and 79(D), were cytogenetically stable. Plants of lines 79(A) and 79(C) displayed a high frequency of unbalanced chromosome aberrations, including dicentric and polycentric chromosomes, terminal deletions varying in size, acentric fragments, and multiple unidentifiable translocations. Previous studies of the mitochondrial genome showed that the two cytologically unstable lines, which were also unstable in fertility and viability, are characterized by heteroplasmy at the mitochondrial 18S-5S locus (simultaneous presence of barley and wheat fragments). Stable lines 79(B) and 79(D) with normal fertility contained only wheat mitochondrial markers. It was assumed that the substantial instability of the nuclear genome in lines 79(C) and 79(A) was a result of nuclear-cytoplasmic incompatibility and was associated with heteroplasmy, while elimination or considerable reduction of barley material in the mitochondrial genome stabilized the nuclear genome of lines 79(B) and 79(D). In turn, the instability of the nuclear genome was responsible for a decrease in viability and fertility of plants. PMID:16583704

  4. Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice

    Directory of Open Access Journals (Sweden)

    Palm Kaia

    2009-06-01

    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF is a small secreted protein that has important roles in the developing and adult nervous system. Altered expression or changes in the regulation of the BDNF gene have been implicated in a variety of human nervous system disorders. Although regulation of the rodent BDNF gene has been extensively investigated, in vivo studies regarding the human BDNF gene are largely limited to postmortem analysis. Bacterial artificial chromosome (BAC transgenic mice harboring the human BDNF gene and its regulatory flanking sequences constitute a useful tool for studying human BDNF gene regulation and for identification of therapeutic compounds modulating BDNF expression. Results In this study we have generated and analyzed BAC transgenic mice carrying 168 kb of the human BDNF locus modified such that BDNF coding sequence was replaced with the sequence of a fusion protein consisting of N-terminal BDNF and the enhanced green fluorescent protein (EGFP. The human BDNF-BAC construct containing all BDNF 5' exons preceded by different promoters recapitulated the expression of endogenous BDNF mRNA in the brain and several non-neural tissues of transgenic mice. All different 5' exon-specific BDNF-EGFP alternative transcripts were expressed from the transgenic human BDNF-BAC construct, resembling the expression of endogenous BDNF. Furthermore, BDNF-EGFP mRNA was induced upon treatment with kainic acid in a promotor-specific manner, similarly to that of the endogenous mouse BDNF mRNA. Conclusion Genomic region covering 67 kb of human BDNF gene, 84 kb of upstream and 17 kb of downstream sequences is sufficient to drive tissue-specific and kainic acid-induced expression of the reporter gene in transgenic mice. The pattern of expression of the transgene is highly similar to BDNF gene expression in mouse and human. This is the first study to show that human BDNF gene is regulated by neural activity.

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

    Science.gov (United States)

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

    2014-11-01

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

  6. A PiggyBac-based recessive screening method to identify pluripotency regulators.

    Directory of Open Access Journals (Sweden)

    Ge Guo

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

  7. Whole genome linkage disequilibrium maps in cattle

    Science.gov (United States)

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

  8. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

    Directory of Open Access Journals (Sweden)

    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

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

    Directory of Open Access Journals (Sweden)

    Dieter Palmberger

    Full Text Available 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 β1,4-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

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

    OpenAIRE

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

    2011-01-01

    Abstract Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows mainte...

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

    OpenAIRE

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

    2009-01-01

    Abstract 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 chromos...

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

    OpenAIRE

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

    2009-01-01

    Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via...

  13. 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

    Directory of Open Access Journals (Sweden)

    Ma Wujun

    2010-05-01

    Full Text Available Abstract 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

  14. A Domesticated piggyBac Transposase Plays Key Roles in Heterochromatin Dynamics and DNA Cleavage during Programmed DNA Deletion in Tetrahymena thermophila

    OpenAIRE

    Cheng CY; Vogt A; Mochizuki K.; Yao MC

    2010-01-01

    Transposons comprise large fractions of eukaryotic genomes and provide genetic reservoirs for the evolution of new cellular functions. We identified TPB2, a homolog of the piggyBac transposase gene that is required for programmed DNA deletion in Tetrahymena. TPB2 was expressed exclusively during the time of DNA excision, and its encoded protein Tpb2p was localized in DNA elimination heterochromatin structures. Notably, silencing of TPB2 by RNAi disrupts the final assembly of these heterochrom...

  15. Microbial Genomics Research in China

    Institute of Scientific and Technical Information of China (English)

    ZHAOGuo-ping

    2004-01-01

    Microorganisms, including phage/virus, were initial targets and tools for developing DNA sequencing technology. Microbial genomic study was started as a model system for the Human Genome Project (HGP) and it did successfully supported the HGP, particularly with respect to BAC contig construction and large-scale shotgun sequencing and assembly. Microbial genomics study has become the fastest developed genomics discipline along with HGP, taking the advantage of the organisms' highly diversified physiology, extremely long history of evolution, close relationship with human/environment,as well as relatively small genome sizes and simple systems for functional analysis.

  16. Microbial Genomics Research in China

    Institute of Scientific and Technical Information of China (English)

    ZHAO Guo-ping

    2004-01-01

    @@ Microorganisms, including phage/virus, were initial targets and tools for developing DNA sequencing technology. Microbial genomic study was started as a model system for the Human Genome Project (HGP) and it did successfully supported the HGP, particularly with respect to BAC contig construction and large-scale shotgun sequencing and assembly. Microbial genomics study has become the fastest developed genomics discipline along with HGP, taking the advantage of the organisms' highly diversified physiology, extremely long history of evolution, close relationship with human/environment,as well as relatively small genome sizes and simple systems for functional analysis.

  17. Intermediate fertile Triticum aestivum (+) Agropyron elongatum somatic hybrids are generated by low doses of UV irradiation

    Institute of Scientific and Technical Information of China (English)

    Ai Xia CHENG; Guang Min XIA; Da Ying ZHI; Hui Min CHEN

    2004-01-01

    We report the production and characterization of somatic hybrids between Triticum aestivum L. and Agropyron elongatum (Host) Nevishi (the synonym is Thinopyrum ponticum). Asymmetric protoplast fusion was performed between Agropyron elongatum protoplasts irradiated with a low UV dose and protoplasts of wheat taken from nonregenerable suspension cultures. More than 40 green plantlets were obtained from 15 regenerated clones and one of them produced seeds. The phenotypes of the hybrid plants and seeds were intermediate between wheat and Agropyron elongatum. All of the regenerated calli and plants were verified as intergeneric hybrids on the basis of morphological observation and analysis of isozyme,cytological,5SrDNA spacer sequences and random amplified polymorphic DNA (RAPD). RFLP analysis of the mitochondrial genome revealed evidence of random segregation and recombination of mtDNA.

  18. An active piggyBac-like element in Macdunnoughia crassisigna

    Institute of Scientific and Technical Information of China (English)

    Min Wu; Zhi-Chan Sun; Chun-Lin Hu; Gu-Feng Zhang; Zhao-Jun Han

    2008-01-01

    In this paper,a highly conserved piggyBac-like sequence.designated as McrPLE was cloned from a lepidopteran insect,Macdunnoughia crassisigna.It is 2 472 bp long in full length with a single open reading frame and encodes a 595 amino acid transPOsase.It shares identical terminal and sub-terminal repeats with T. ni IFP2 and iS flanked by the typical TTAA target-site duplications.Alignment and phylogeneric analysis revealed that McrPLE had greater than 99.5%identity and appeared to be the closest one in phylogeny to IFP2 among the PLEs so far found in various species.Plasmid.based excision and transposition assay proved it Was mobile in cell culture.Otherwise.McPLE element and all other highly conserved IFP2 sequences reported previously were found to share three corrution nucleotide substitutions.This suggests that the original IFP2 may be a related variant of a predecessor element that became widespread.The existence of nearly identical piggyBac sequence in reproductively isolated species Was thought also a strong indication of horizontal transmission,which raises important considerations for the stability and practical use of piggyBac transformation vectors.

  19. Genome Improvement at JGI-HAGSC

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-03-03

    Since the completion of the sequencing of the human genome, the Joint Genome Institute (JGI) has rapidly expanded its scientific goals in several DOE mission-relevant areas. At the JGI-HAGSC, we have kept pace with this rapid expansion of projects with our focus on assessing, assembling, improving and finishing eukaryotic whole genome shotgun (WGS) projects for which the shotgun sequence is generated at the Production Genomic Facility (JGI-PGF). We follow this by combining the draft WGS with genomic resources generated at JGI-HAGSC or in collaborator laboratories (including BAC end sequences, genetic maps and FLcDNA sequences) to produce an improved draft sequence. For eukaryotic genomes important to the DOE mission, we then add further information from directed experiments to produce reference genomic sequences that are publicly available for any scientific researcher. Also, we have continued our program for producing BAC-based finished sequence, both for adding information to JGI genome projects and for small BAC-based sequencing projects proposed through any of the JGI sequencing programs. We have now built our computational expertise in WGS assembly and analysis and have moved eukaryotic genome assembly from the JGI-PGF to JGI-HAGSC. We have concentrated our assembly development work on large plant genomes and complex fungal and algal genomes.

  20. Genotyping of somatic hybrids between Festuca arundinacea Schreb. and Triticum aestivum L.

    Science.gov (United States)

    Cai, Yunfei; Xiang, Fengning; Zhi, Daying; Liu, Heng; Xia, Guangmin

    2007-10-01

    In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration. PMID:17594098

  1. Performance of BAC process for treatment of micro-polluted water

    Institute of Scientific and Technical Information of China (English)

    WANG Chen; MA Fang; SHAN Dan; YANG Ji-xian; Chang Chein-chi

    2008-01-01

    Biological activated carbon (BAC) has been developed on the granular activated carbon by immobi-lization of selected and acclimated species of bacteria to treat the micro-polluted water. The BAC removal effi-ciencies for nitrobenzene, permanganate index, turbidity and ammonia were investigated. Effects of shock load-ing and SEM (Scanning Electron Microscope) observation on BAC were studied. Baekwashing and its intensity of BAC were also discussed. The results showed that BAC took short time to start up and recover to the normal condition after shock loading. The shock loading studies showed that the removal efficiency of BAC was not completely inhibited even at high concentration of nitrobenzene. Backwashing performed once every 10- 20 d,or an average of 15 d. Backwashing intensity was 12-14 L/( s·m2) with air and 3-4 L/( s·m2) with water.

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

    Directory of Open Access Journals (Sweden)

    Yandell Mark

    2010-07-01

    Full Text Available Abstract 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

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

    OpenAIRE

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

    2006-01-01

    We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the...

  4. Widespread distribution of the piggyBac transposon in various bactrocera species

    International Nuclear Information System (INIS)

    Full text: The piggyBac transposable element from the Lepidopteran species Trichoplusia ni is currently the most widely used vector for insect transgenesis. Consequently, the presence of piggyBac-like sequences has been investigated, by PCR and Southern analysis, in different species of target genera such as Ceratitis, Bactrocera and Anastrepha, along with Tirhitromina and Rhagoletis. PiggyBac-like sequences were detected in several Bactrocera species. The evolution of the piggyBac-like sequences is discussed with respect to the phylogenies of the hosts. (author)

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

    Directory of Open Access Journals (Sweden)

    Yongming Wang

    2014-03-01

    Full Text Available 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.

  6. A physical map of the human genome

    Energy Technology Data Exchange (ETDEWEB)

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

    2001-01-01

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

  7. The Physical Location of Gm-2 and Gm-6 in O.officinalis with BAC-FISH Based on Comparative RFLP Map of Wild Rice and Cultivated Rice

    Institute of Scientific and Technical Information of China (English)

    QIN Rui; WEI Wen-hui; NING Shun-bin; JIN Wei-wei; HE Guang-cun; SONG Yun-chun

    2002-01-01

    Rice BAC library is used widely in rice genome research due to its distinctive advantages over other library systems. In this study, two rice BAC clones closely linked to rice gall midge resistance, Gm-2 and Gm-6, were in situ hybridized to Oryza officinalis chromosomes. They were located on the long arm of chromosome 4 with FL 72.33% and 77.10% respectively and their FL was consistent with the selective marker of rice, RG214 and RZ569. The frequency of signal detection was 61.2% and 59.5% respectively.Our study was based on comparative RFLP map of wild rice, O. officinalis, and cultivated rice, O. sativa.

  8. Breeding of T. aestivum-Ag. intermedium translocation lines with T. timopheevii cytoplasm and characterization by GISH

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A male-sterile T. aestivum-Ag. intermedium partial amphiploid with cytoplasm of T. timopheevii as a female parent was crossed to common wheat. The hybrid was backcrossed to the male parent several times continually and self-crossed at last. Two stable lines with common wheat phenotype, H96269-2 and H96278, have been obtained. The chromosome numbers of the two lines are all 2n = 42 in somatic cells. By inoculation test, the two lines show a high level of resistance to yellow rust. Through genomic in situ hybridization (GISH) with Ag. intermedium total genomic DNA as a probe, it is demonstrated that the two stable lines are all small segmental translocation lines, and the translocated chromosome segments from Ag. intermedium are located on the short arm terminals of wheat chromosomes. Genetics analysis suggests that the yellow rust resistance gene(s) are probably located on the translocated chromosome segments of Ag. intermedium.

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

    Science.gov (United States)

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

    2008-01-01

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

  10. High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides.

    NARCIS (Netherlands)

    Carvalho, B; Ouwerkerk, E; Meijer, G.A.; Ylstra, B.

    2004-01-01

    BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as

  11. Toward Elucidating the Structure of Tetraploid Cotton Genome

    Institute of Scientific and Technical Information of China (English)

    GUO Wang-zhen

    2008-01-01

    @@ Upland cotton has the highest yield,and accounts for >95% of world cotton production.Decoding upland cotton genomes will undoubtedly provide the ultimate reference and resource for structural,functional,and evolutionary studies of the species.Here,we employed GeneTrek and BAC tagging information approaches to predict the general composition and structure of the allotetraploid cotton genome.

  12. A non-autonomous insect piggyBac trasposable element is mobile in tobacco

    Science.gov (United States)

    The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid onl...

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

    Science.gov (United States)

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

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

    Directory of Open Access Journals (Sweden)

    Jonathan T Ting

    2014-04-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Roth Swaantje J

    2011-01-01

    Full Text Available Abstract Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV. We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR as a bacterial artificial chromosome (BAC in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool.

  16. Potentially toxic elements in foodcrops: Triticum aestivum L., Zea mays L.

    Science.gov (United States)

    Bini, Claudio; Fontana, Silvia; Squizzato, Stefania; Minello, Fabiola; Fornasier, Flavio; Wahsha, Mohammad

    2013-04-01

    Soil is the basis of the ecosystems and of our system of food production. Crops can uptake heavy metals and potentially toxic elements from the soil and store them in the roots or translocate them to the aerial parts. Excessive content of these elements in edible parts can produce toxic effects and, through the food chain and food consumption, result in a potential hazard for human health. In this study soils and plants (spring wheat, Triticum aestivum L. and maize, Zea mays L.) from a tannery district in North-East Italy were analyzed to determine pedological characters, soil microbial indicators and the content of some major and micro-nutrients and potentially toxic elements (Al, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Ni, P, Pb, S, Zn, V). The soils of the area are moderately polluted; Cr is the most important inorganic contaminant, followed by Ni, Cu and V. Factor analysis evidenced that the contaminants are in part anthropogenic and in part geogenic. Major anthropogenic origin was detected for Cr, Ni (from industrial activities), Zn, Cu, Cd (from agriculture practices). Biological Absorption Coefficient (BAC) from soil to plant roots and Translocation factor (TF) within the plant were calculated; major nutrients (K, P, S) and some micronutrients (Cu, Zn, Mg, Mn) are easily absorbed and translocated, whilst other nutrients (Ca, Fe) and potentially toxic elements or micronutrients (Al, Cd, Cr, Ni, Pb, V) are not accumulated in the seeds of the two considered species. However, the two edible species proved differently able to absorb and translocate elements, and this suggests to consider separately every species as potential PHEs transporter to the food chain and to humans. Cr concentrations in seeds and other aerial parts (stem and leaves) of the examined plants are higher than the values found for the same species and for other cereals grown on unpolluted soils. Comparing the Cr levels in edible parts with recommended dietary intake, besides other possible Cr sources

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

    Directory of Open Access Journals (Sweden)

    Sandra García-Herrero

    2014-01-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Changqing Liu

    2014-03-01

    Full Text Available 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.

  20. Cloning and expression of human UDP-glucuronosyltransferase 1A4 in Bac-to-Bac system

    International Nuclear Information System (INIS)

    UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from uridine diphosphate-glucuronic acid (UDP-GA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGT1A4, the Bac-to-Bac expression system was used to express the recombinant UGT1A4 with His-tag on the C-terminal. The His-tagged recombinant UGT1A4 expressed in Spodoptera frugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55 kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14 ± 15 pmol/min/mg protein (n=3) with 0.5 mM imipramine by HPLC, but was not detectable in blank Sf9 cells. It paved the way for the further studies for drug glucuronidation by UGT1A4. The purification of the UGT1A4 can be done by Ni-resin. This is helpful to do research on the structure of the UFT1A4

  1. Analysis and location of a rice BAC clone containing telomeric DNA sequences

    Institute of Scientific and Technical Information of China (English)

    翟文学; 陈浩; 颜辉煌; 严长杰; 王国梁; 朱立煌

    1999-01-01

    BAC2, a rice BAC clone containing (TTTAGGG)n homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG) n-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescence in situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.

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

    Science.gov (United States)

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

  3. piggyBac转座子及其在转基因昆虫中的应用%Advances on piggyBac transposon and its applications in insect transgenesis

    Institute of Scientific and Technical Information of China (English)

    王建军; 王常春; 韩召军

    2009-01-01

    piggyBac是一种从粉纹夜蛾Trichoplusia ni.中分离到的、具有TTAA插入位点特异性的DNA转座子.piggyBac可在昆虫基因组中准确切离,转化频率较高,并且不受宿主因子的限制,是目前转基因昆虫研究中应用最广的转座子载体.近年来的研究发现,piggyBac类转座子广泛分布于昆虫和其他生物基因组中.文章从piggyBac的结构、转座特性、在转基因昆虫中的应用以及piggyBac类转座子的分布等几个方面综述了piggyBac的研究进展.%The piggyBac element is a TTAA-insertion site specific DNA transposon and was originally discovered in Trichoplusia ni. The piggyBac element is capable of precise excision and shows high frequency of transformation in insect genome, and its activity is less restricted by host factors. These characteristics make piggyBac the most widely used gene vector in insect transgenesis. Recent studies revealed that piggyBac-like element is distributed in a wide variety of insect and other organisms. In this review, we introduced the structure and transposition characteristics of piggyBac and its application in insect transgenesis, and the distribution of piggyBac-like elements was also described.

  4. L’iconostase de Rădeana-Bacău

    Directory of Open Access Journals (Sweden)

    Marina Sabados

    2010-01-01

    Full Text Available L’analyse complexe (peinture et sculpture décorative, iconographie et style de l’iconostase de Rădeana, dans le département de Bacău, un ensemble presque inconnu, donation du voïvode Gheorghe Ştefan de 1658, relève sa place singulière dans l’histoire moldave du genre. Au sujet de la peinture, les conclusions mènent vers un atelier galicien, tandis que la sculpture décorative à la manière baroque s’avère précoce par rapport aux ensembles contemporains (ceux ruthènes y compris et pourrait représenter le modèle, en tant qu’architecture et ornementation, des iconostases moldaves du XVIIIe siècle.

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

    Science.gov (United States)

    Li, Tianda; Shuai, Ling; Mao, Junjie; Wang, Xuepeng; Wang, Mei; Zhang, Xinxin; Wang, Leyun; Li, Yanni; Li, Wei; Zhou, Qi

    2016-01-01

    Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids. We showed that the transgenic rats expressed the RFP or eGFP gene in many organs and had the capability to transmit the marker gene to the next generation through germline integration. In addition, rat embryonic stem cells (ESCs) carrying an RFP reporter gene can be derived from the blastocysts of the transgenic rats. Moreover, the RFP gene can be detected in chimeras derived from RFP ESCs via blastocyst injection. This work suggests that PB-mediated transgenesis is a powerful tool to generate transgenic rats expressing fluorescent proteins with high efficiency, and this technique can be used to derive rat ESCs expressing a reporter protein. PMID:27624004

  6. Evaluation of chronic lymphocytic leukemia by BAC-based microarray analysis

    Directory of Open Access Journals (Sweden)

    McDaniel Lisa D

    2011-02-01

    Full Text Available Abstract Background Chronic lymphocytic leukemia (CLL is a highly variable disease with life expectancies ranging from months to decades. Cytogenetic findings play an integral role in defining the prognostic significance and treatment for individual patients. Results We have evaluated 25 clinical cases from a tertiary cancer center that have an established diagnosis of CLL and for which there was prior cytogenetic and/or fluorescence in situ hybridization (FISH data. We performed microarray-based comparative genomic hybridization (aCGH using a bacterial artificial chromosome (BAC-based microarray designed for the detection of known constitutional genetic syndromes. In 15 of the 25 cases, aCGH detected all copy number imbalances identified by prior cytogenetic and/or FISH studies. For the majority of those not detected, the aberrations were present at low levels of mosaicism. Furthermore, for 15 of the 25 cases, additional abnormalities were detected. Four of those cases had deletions that mapped to intervals implicated in inherited predisposition to CLL. For most cases, aCGH was able to detect abnormalities present in as few as 10% of cells. Although changes in ploidy are not easily discernable by aCGH, results for two cases illustrate the detection of additional copy gains and losses present within a mosaic tetraploid cell population. Conclusions Our results illustrate the successful evaluation of CLL using a microarray optimized for the interrogation of inherited disorders and the identification of alterations with possible relevance to CLL susceptibility.

  7. Direct Sensitivity Test of the MB/BacT System

    Directory of Open Access Journals (Sweden)

    Barreto Angela Maria Werneck

    2002-01-01

    Full Text Available In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23% trisodium phosphate solution, incubated for 24 h at 35°C, and neutralized v/v with 20% monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 µg/ml, Isoniazid - 0.2 µg/ml, Pyrazinamide - 100 µg/ml, Ethambutol - 2.5 µg/ml, Ethionamide - 1.25 µg/ml, and Streptomycin - 2 µg/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100% sensitivity at 95% Confidence Interval (82.2-100 and 100% specificity at 95% Confidence Interval (84.5-100, followed by Isoniazid and Pyrazinamide. In this experiment, 92% of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram.

  8. Screening of tissues pooled cDNA library using probes by restricted fragments of BAC positive clones of ovine MHC%以绵羊MHC 区段BAC 克隆酶切片段为探针杂交筛选绵羊混合组织cDNA 文库

    Institute of Scientific and Technical Information of China (English)

    杨小亮; 白大章; 邱巍; 董慧芹; 李大全; 陈芳; 马润林; Hugh T Blair; 高剑峰

    2012-01-01

    Under the premise what we have known bacterial artificial chromosome(BAC) clone sequence information and gene annotation predicted in the Chinese Merino sheep major histocompatibility complex (MHC) region, the digested fragments from 6 BAC clones that were located in the MHC region of the Chinese Merino sheep genome BAC library, which were used to screen the cDNA library using plaque in situ hybridization as probes. The full length of positive cDNA clones (sequences) isolated were completely sequenced, and the sequences obtained were aligned with the corresponding known sequence information and the BAC clones with gene annotation. Meanwhile, the sequence similarity was searched in NCBI Blastn database. This work aimed at verification of accuracy of the gene annotation results and initial analysis of gene (sequence) function. At last, 27 positive cDNA clones (sequences) in total were screened through two runs of hybridization. It was also found that these sequences could be positioned in the corresponding BAC clones, and 25 sequences were located in exon area of the annotated gene. It was verified that 23 sequences had the highest sequence similarity with those in the Bos taurus by searching against the NCBI Blastn database; moreover, the function of these sequences were closely relate to immunology.%在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC 文库MHC 区段的6 个BAC 克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA 文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC 克隆比对以及在NCBI Blastn 数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析.实验中,经过两轮杂交共筛选出27 个cDNA 阳性克隆(序

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

    Directory of Open Access Journals (Sweden)

    Manoj K Sharma

    Full Text Available 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.

  10. Exploiting BAC-end sequences for the mining, characterization and utility of new short sequences repeat (SSR) markers in Citrus.

    Science.gov (United States)

    Biswas, Manosh Kumar; Chai, Lijun; Mayer, Christoph; Xu, Qiang; Guo, Wenwu; Deng, Xiuxin

    2012-05-01

    The aim of this study was to develop a large set of microsatellite markers based on publicly available BAC-end sequences (BESs), and to evaluate their transferability, discriminating capacity of genotypes and mapping ability in Citrus. A set of 1,281 simple sequence repeat (SSR) markers were developed from the 46,339 Citrus clementina BAC-end sequences (BES), of them 20.67% contained SSR longer than 20 bp, corresponding to roughly one perfect SSR per 2.04 kb. The most abundant motifs were di-nucleotide (16.82%) repeats. Among all repeat motifs (TA/AT)n is the most abundant (8.38%), followed by (AG/CT)n (4.51%). Most of the BES-SSR are located in the non-coding region, but 1.3% of BES-SSRs were found to be associated with transposable element (TE). A total of 400 novel SSR primer pairs were synthesized and their transferability and polymorphism tested on a set of 16 Citrus and Citrus relative's species. Among these 333 (83.25%) were successfully amplified and 260 (65.00%) showed cross-species transferability with Poncirus trifoliata and Fortunella sp. These cross-species transferable markers could be useful for cultivar identification, for genomic study of Citrus, Poncirus and Fortunella sp. Utility of the developed SSR marker was demonstrated by identifying a set of 118 markers each for construction of linkage map of Citrus reticulata and Poncirus trifoliata. Genetic diversity and phylogenetic relationship among 40 Citrus and its related species were conducted with the aid of 25 randomly selected SSR primer pairs and results revealed that citrus genomic SSRs are superior to genic SSR for genetic diversity and germplasm characterization of Citrus spp.

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

    Directory of Open Access Journals (Sweden)

    Thomas Ann

    2009-07-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  13. Comparative Analysis of Codon Usage Patterns Among Mitochondrion, Chloroplast and Nuclear Genes in Triticum aestivum L.

    Institute of Scientific and Technical Information of China (English)

    Wen-Juan Zhang; Jie Zhou; Zuo-Feng Li; Li Wang; Xun Gu; Yang Zhong

    2007-01-01

    In many organisms, the difference in codon usage patterns among genes reflects variation in local base compositional biases and the intensity of natural selection. In this study, a comparative analysis was performed to investigate the characteristics of codon bias and factors in shaping the codon usage patterns among mitochondrion,chloroplast and nuclear genes in common wheat (Triticum aestivum L.). GC contents in nuclear genes were higher than that in mitochondrion and chloroplast genes. The neutrality and correspondence analyses indicated that the codon usage in nuclear genes would be a result of relative strong mutational bias, while the codon usage patterns of rnitochondrion and chloroplast genes were more conserved in GC content and influenced by translation level.The Parity Rule 2 (PR2) plot analysis showed that pyrimidines were used more frequently than purines at the third codon position in the three genomes. In addition, using a new alterative strategy, 11, 12, and 24 triplets were defined as preferred codons in the mitochondrion, chloroplast and nuclear genes, respectively. These findings suggested that the mitochondrion, chloroplast and nuclear genes shared particularly different features of codon usage and evolutionary constraints.

  14. A novel system for Agrobacterium-mediated transformation of wheat( Triticum aestivum L.) cells

    Institute of Scientific and Technical Information of China (English)

    XUYAO; BAOJIANLI; JINGFENJIA

    1993-01-01

    A new approach for transforming the cultured cells of wheat (Triticum aestivum L.cv.Ganmai 8)was developed vsing Agrobacterium tumefaciens. The features of the optimum procedure were:(a)both combined synthetic signal molecules and multiple natural extracts from susceptible plants were used to pretreat the primary vigorous Agrobacterium(PVA)cells for approximately 16h:(b)the gyratory magnetic field condition was used during cocultivation;(c)the cocultivating period and selecting condition were modified;(d)the recipient cells were at exuberant metabolism and active division while infected with Agrobacterium.Both neomycin phosphotransferase and nopaline synthase assays demonstrated the expression of NPT Ⅱ and NOS genes.located on the T-DNA segment of chimaeric plasmid pGV3850::1103neo.in transformed wheat cell colonies by adopting the techniques of dot blot ndPAGE or high voltage paper electrophoresis,Integration of the foreign genes into wheat genome was confirmed by Southerm blot hybridization.Moreover.a relatively rational method was described for the estimation of transformation frequencies from cultured cell levels.

  15. Development and Identification of Triticum aestivum L.-Thinopyrum bessarabicum L(o)ve Chromosome Translocations

    Institute of Scientific and Technical Information of China (English)

    ZHUANG Li-fang; QI Zeng-jun; CHEN Pei-du; FENG Yi-gao; LIU Da-jun

    2004-01-01

    With ass7istance of chromosome C-banding and genomic in situ hybridization(GISH)combined with meiotic analysis,five germplasms with homozygous wheat-Th. Bessarabicum chromosome translocations were developed and identified among BC1F5 progenies of the cross between T. Aestivum cv. Chinese Spring and Chinese Spring-Th. Bessarabicum amphiploid. These lines included Tj01 and Tj02(2n=44)containing a pair of wheat-Th. Bessarabicum translocation chromosomes besides a pair of added Th. Bessarabicum chromosomes,Tj03(2n=44)with a pair of added interspecific translocation chromosomes,Tj04(2n=44)containing a pair of interspecific translocation chromosomes besides an added pair of Th. Bessarabicum chromosome arms and Tj05(2n=46)containing a pair of interspecific translocation chromosomes besides two pairs of added intact alien chromosomes. The breakpoints of all the translocations were found to be not around centromere. Meanwhile,all the lines showed normal plant growth,development and fertility,while the translocation chromosomes transmitted regularly. The obtained translocations might be of use for transferring elite genes from Th. Bessarabicum into wheat.

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

  17. Red-mediated transposition and final release of the mini-F vector of a cloned infectious herpesvirus genome.

    Directory of Open Access Journals (Sweden)

    Felix Wussow

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

  18. PiggyBac Transposon Mutagenesis: A Tool for Cancer Gene Discovery in Mice

    OpenAIRE

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

    2010-01-01

    Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon/transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed the unique qualities o...

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

    Science.gov (United States)

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

    2016-06-01

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

  20. Uncertainty of Blood Alcohol Concentration (BAC Results as Related to Instrumental Conditions: Optimization and Robustness of BAC Analysis Headspace Parameters

    Directory of Open Access Journals (Sweden)

    Haleigh A. Boswell

    2015-12-01

    Full Text Available Analysis of blood alcohol concentration is a routine analysis performed in many forensic laboratories. This analysis commonly utilizes static headspace sampling, followed by gas chromatography combined with flame ionization detection (GC-FID. Studies have shown several “optimal” methods for instrumental operating conditions, which are intended to yield accurate and precise data. Given that different instruments, sampling methods, application specific columns and parameters are often utilized, it is much less common to find information on the robustness of these reported conditions. A major problem can arise when these “optimal” conditions may not also be robust, thus producing data with higher than desired uncertainty or potentially inaccurate results. The goal of this research was to incorporate the principles of quality by design (QBD in the adjustment and determination of BAC (blood alcohol concentration instrumental headspace parameters, thereby ensuring that minor instrumental variations, which occur as a matter of normal work, do not appreciably affect the final results of this analysis. This study discusses both the QBD principles as well as the results of the experiments, which allow for determination of more favorable instrumental headspace conditions. Additionally, method detection limits will also be reported in order to determine a reporting threshold and the degree of uncertainty at the common threshold value of 0.08 g/dL. Furthermore, the comparison of two internal standards, n-propanol and t-butanol, will be investigated. The study showed that an altered parameter of 85 °C headspace oven temperature and 15 psi headspace vial pressurization produces the lowest percent relative standard deviation of 1.3% when t-butanol is implemented as an internal standard, at least for one very common platform. The study also showed that an altered parameter of 100 °C headspace oven temperature and 15-psi headspace vial pressurization

  1. Genetic Relationships Among Five Basic Genomes St, E, A, B and D in Triticeae Revealed by Genomic Southern and in situ Hybridization

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The St and E are two important basic genomes in the perennial tribe Triticeae (Poaceae). They exist in many perennial species and are very closely related to the A, B and D genomes of bread wheat (Triticum aestivum L.). Genomic Southern hybridization and genomic in situ hybridization (GISH) were used to analyze the genomic relationships between the two genomes (St and E) and the three basic genomes (A, B and D) of T. aestivum. The semi-quantitative analysis of the Southern hybridization suggested that both St and E genomes are most closely related to the D genome, then the A genome, and relatively distant to the B genome. GISH analysis using St and E genomic DNA as probes further confirmed the conclusion.St and E are the two basic genomes of Thinopyrum ponticum (StStEeEbEx) and Th. intermedium (StEeEb), two perennial species successfully used in wheat improvement. Therefore, this paper provides a possible answer as to why most of the spontaneous wheat- Thinopyrum translocations and substitutions usually happen in the D genome, some in the A genome and rarely in the B genome. This would develop further use of alien species for wheat improvement, especially those containing St or E in their genome components.

  2. Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

    Science.gov (United States)

    Kamboj, Aman; Saini, Mohini; Rajan, Lekshmi S; Patel, Chhabi Lal; Chaturvedi, V K; Gupta, Praveen K

    2015-12-15

    To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector. PMID:26478540

  3. Ectomycorrhizal communities in a productive Tuber aestivum Vittad. orchard: composition, host influence and species replacement

    OpenAIRE

    Benucci, Gian Maria Niccolò; Raggi, Lorenzo; Albertini, Emidio; Grebenc, Tine; Bencivenga, Mattia; Falcinelli, Mario; Di Massimo, Gabriella

    2011-01-01

    Truffles (Tuber spp.) and other ectomycorrhizal species form species-rich assemblages in the wild as well as in cultivated ecosystems. We aimed to investigate the ectomycorrhizal communities of hazels and hornbeams that are growing in a 24-year-old Tuber aestivum orchard. We demonstrated that the ectomycorrhizal communities included numerous species and were phylogenetically diverse. Twenty-nine ectomycorrhizal taxa were identified. Tuber aestivum ectomycorrhizae were abundant (9.3%), only th...

  4. Cloning of a novel phosphateserine aminotransferase gene from a Triticum aestivum-Elytrigia elongatum alien substitution line with resistance to powdery mildew

    Institute of Scientific and Technical Information of China (English)

    HE Daoyi; WANG Honggang

    2005-01-01

    Shannong 551, a T. aestivum-E. elongatum alien substitution line with resistance to powdery mildew, was inoculated with pathogenic spores of powdery mildew. The leaf samples were prepared 48 h after inoculation for scanning electron microscopy. The result showed that germination of spores and growth of young mycelia on leaves of Shannong 551 were suppressed at the early stage of infection. At the same time, RNAs were prepared from the leaves for the cloning of WRP1 and RPW2 by cDNA RDA and RACE technology. BLAST analysis of the sequences indicated that both WRP1 and RPW2 were novel genes. WRP1 contains no complete ORF. RPW2 contains the conserved structure domain of aminotransferase, and its DNA sequence shares high homology with genes of phosphateserine aminotransferase in many organisms. Therefore, it is speculated as a novel phosphateserine aminotransferase gene. The results of Northern blot suggested that expression of RPW2 occurred at the early stage of infection by powdery mildew. Southern blot using the probe of RPW2, in which there was strong hybridizing signals in both genome of Shannong 551 and E. elongatum, but not in those of Jinan 13 and Lumai No.5, indicated that RPW2 derived from the genome of E. elongatum.

  5. Competition increases sensitivity of wheat (Triticum aestivum to biotic plant-soil feedback.

    Directory of Open Access Journals (Sweden)

    W H Gera Hol

    Full Text Available Plant-soil feedback (PSF and plant competition play an important role in structuring vegetation composition, but their interaction remains unclear. Recent studies suggest that competing plants could dilute pathogenic effects, whereas the standing view is that competition may increase the sensitivity of the focal plant to PSF. In agro-ecosystems each of these two options would yield contrasting outcomes: reduced versus enhanced effects of weeds on crop biomass production. To test the effect of competition on sensitivity to PSF, we grew Triticum aestivum (Common wheat with and without competition from a weed community composed of Vicia villosa, Chenopodium album and Myosotis arvensis. Plants were grown in sterilized soil, with or without living field inoculum from 4 farms in the UK. In the conditioning phase, field inocula had both positive and negative effects on T. aestivum shoot biomass, depending on farm. In the feedback phase the differences between shoot biomass in T. aestivum monoculture on non-inoculated and inoculated soils had mostly disappeared. However, T. aestivum plants growing in mixtures in the feedback phase were larger on non-inoculated soil than on inoculated soil. Hence, T. aestivum was more sensitive to competition when the field soil biota was present. This was supported by the statistically significant negative correlation between shoot biomass of weeds and T. aestivum, which was absent on sterilized soil. In conclusion, competition in cereal crop-weed systems appears to increase cereal crop sensitivity to soil biota.

  6. Highly Efficient Modification of Bacterial Artificial Chromosomes (BACs) Using Novel Shuttle Vectors Containing the R6Kγ Origin of Replication

    OpenAIRE

    Gong, Shiaoching; Yang, Xiangdong William; Li, Chenjian; Heintz, Nathaniel

    2002-01-01

    Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the ...

  7. A comparative physical map reveals the pattern of chromosomal evolution between the turkey (Meleagris gallopavo and chicken (Gallus gallus genomes

    Directory of Open Access Journals (Sweden)

    Delany Mary E

    2011-09-01

    Full Text Available Abstract Background A robust bacterial artificial chromosome (BAC-based physical map is essential for many aspects of genomics research, including an understanding of chromosome evolution, high-resolution genome mapping, marker-assisted breeding, positional cloning of genes, and quantitative trait analysis. To facilitate turkey genetics research and better understand avian genome evolution, a BAC-based integrated physical, genetic, and comparative map was developed for this important agricultural species. Results The turkey genome physical map was constructed based on 74,013 BAC fingerprints (11.9 × coverage from two independent libraries, and it was integrated with the turkey genetic map and chicken genome sequence using over 41,400 BAC assignments identified by 3,499 overgo hybridization probes along with > 43,000 BAC end sequences. The physical-comparative map consists of 74 BAC contigs, with an average contig size of 13.6 Mb. All but four of the turkey chromosomes were spanned on this map by three or fewer contigs, with 14 chromosomes spanned by a single contig and nine chromosomes spanned by two contigs. This map predicts 20 to 27 major rearrangements distinguishing turkey and chicken chromosomes, despite up to 40 million years of separate evolution between the two species. These data elucidate the chromosomal evolutionary pattern within the Phasianidae that led to the modern turkey and chicken karyotypes. The predominant rearrangement mode involves intra-chromosomal inversions, and there is a clear bias for these to result in centromere locations at or near telomeres in turkey chromosomes, in comparison to interstitial centromeres in the orthologous chicken chromosomes. Conclusion The BAC-based turkey-chicken comparative map provides novel insights into the evolution of avian genomes, a framework for assembly of turkey whole genome shotgun sequencing data, and tools for enhanced genetic improvement of these important agricultural and

  8. Chicken genome mapping - Constructing part of a road map for mining this bird's DNA

    NARCIS (Netherlands)

    Aerts, J.

    2005-01-01

    The aim of the research presented in this thesis was to aid in the international chicken genome mapping effort. To this purpose, a significant contribution was made to the construction of the chicken whole-genome BAC-based physical map (presented in Chapter A). An important aspect of this constructi

  9. Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids.

    Science.gov (United States)

    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

    2014-05-01

    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.

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

    OpenAIRE

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

    2011-01-01

    Bactofilins are fibre-forming bacterial cytoskeletal proteins. Here, we report the structural and biochemical characterization of MXAN_7475 (BacM), one of the four bactofilins of Myxococcus xanthus. Absence of BacM leads to a characteristic ‘crooked’ cell morphology and an increased sensitivity to antibiotics targeting cell wall biosynthesis. The absence of the other three bactofilins MXAN_4637–4635 (BacN-P) has no obvious phenotype. In M. xanthus, BacM exists as a 150-amino-acid full-length ...

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

    Science.gov (United States)

    Sato, Masahiro; Maeda, Kosuke; Koriyama, Miyu; Inada, Emi; Saitoh, Issei; Miura, Hiromi; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi; Miyoshi, Kazuchika

    2016-01-01

    The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes. PMID:27589724

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

    Science.gov (United States)

    Sato, Masahiro; Maeda, Kosuke; Koriyama, Miyu; Inada, Emi; Saitoh, Issei; Miura, Hiromi; Ohtsuka, Masato; Nakamura, Shingo; Sakurai, Takayuki; Watanabe, Satoshi; Miyoshi, Kazuchika

    2016-01-01

    The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes. PMID:27589724

  13. Toward Elucidating the Structure of Tetraploid Cotton Genome

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Upland cotton has the highest yield,and accounts for >95% of world cotton production.Decoding upland cotton genomes will undoubtedly provide the ultimate reference and resource for structural,functional,and evolutionary studies of the species.Here,we employed GeneTrek and BAC

  14. MRS培养基组分对Lactobacillus J23合成抗菌肽Bac-J23的影响%Effect of MRS medium components on bacteriocin Bac-J23 production from Lactobacillus J23

    Institute of Scientific and Technical Information of China (English)

    易华西; 韩雪; 杜明; 张兰威

    2012-01-01

    The effect of MRS medium ingredient on the bacteriocin Bac-J23 produced by Lactobacillus J23 were studied. The results showed that 5g/L acetic acid sodium, sucrose and Yeast extraction could stimulate the growth of strain Lactobacillus J23 and induce Bac-J23 production. Higher concentration KH2PO4 (15g/L~20g/L) and 0.2g/L MgSO4 were propitious to Bac-J23 production, while MnSO4 and Tween-80 had no influence to the growth of Lactobacillus J23 and the production of Bac-J23.%研究了培养基MRS组分对乳酸菌Lactobacillus J23产生广谱抗菌肽Bac-J23的影响,并对其组成进行了优化.结果表明,当培养基中浓度为5g/L的乙酸钠对Bac-J23的合成具有刺激诱导作用,蔗糖对Bac-J23合成的最有利,酵母提取物对Lactobacillus J23的生长和Bac-J23的合成均具有明显的促进作用,KH2PO4(15g/L~20g/L)有利于Bac-J23的合成,MgSO4 (0.2g/L)对合成Bac-J23最有利,MnSO4和Tween-80对Bac-J23合成及Lactobacillus J23生长均没有明显影响.

  15. 蛇毒cystatin在Bac to Bac杆状病毒表达系统的表达与鉴定%Expression and Identification of Cystatin from Snake Venom in Bac-to-Bac Baculovirus Expression System

    Institute of Scientific and Technical Information of China (English)

    张晓艳; 林旭; 林建银

    2006-01-01

    目的 探讨蛇毒cystatin在Bac to Bac杆状病毒表达系统中的表达.方法 PCR扩增蛇毒cystatin基因,将其克隆到pFastBacHTc中,通过转化E.Coli DH10Bac筛选克隆,抽提重组Bacmid/cystatin,后者经Cellfectin介导转染Sf9细胞,获取重组病毒,扩增病毒并感染Sf9细胞进行表达,SDS-PAGE、Western-blot分析鉴定表达蛋白66.结果 获得重组cystatin的杆状病毒,Sf9细胞能表达出与蛇毒cystatin单抗、5×His单抗结合的蛋白,相对分子质量约15 kD.结论 蛇毒cystatin在Bac to Bac杆状病毒表达系统中成功表达.

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

    Science.gov (United States)

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

    2009-01-01

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

  17. NetPhosBac - A predictor for Ser/Thr phosphorylation sites in bacterial proteins

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Soufi, Boumediene; Jers, Carsten;

    2009-01-01

    sites in two bacterial model organisms Bacillus subtilis and Escherichia coli. Interestingly, the analysis of these phosphorylation sites revealed that most of them are not characteristic for eukaryotic-type protein kinases, which explains the poor performance of eukaryotic data-trained phosphorylation....... Moreover, NetPhosBac predictions of phosphorylation sites in E. coli proteins were experimentally verified on protein and site-specific levels. In conclusion, NetPhosBac clearly illustrates the advantage of taxa-specific predictors and we hope it will provide a useful asset to the microbiological community....

  18. An abbreviated version of the brief assessment of cognition in schizophrenia (BACS

    Directory of Open Access Journals (Sweden)

    MD Yasuhiro Kaneda

    2015-06-01

    Full Text Available Background and Objectives: A short version of the Brief Assessment of Cognition in Schizophrenia (BACS was derived. Methods: We calculated the corrected item-total correlation (CITC for each test score relative to the composite score, and then computed the proportion of variance that each test shares with the global score excluding that test (Rt² = CITCt² and the variance explained per minute of administration time for each test (Rt²/mint. Results and Conclusions: The 3 tests with the highest Rt²/mint, Symbol Coding, Digit Sequencing, and Token Motor, were selected for the Abbreviated BACS.

  19. Antidiabetic and Antioxidant Properties of Triticum aestivum in Streptozotocin-Induced Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Yogesha Mohan

    2013-01-01

    Full Text Available The antidiabetic and antioxidant potential of Triticum aestivum were evaluated by using in vivo methods in normal and streptozotocin-induced diabetic rats. Diabetes was induced in the Wistar strain albino rats by injecting streptozotocin at a dose of 55 mg/kg body weight. Ethanolic extracts of Triticum aestivum at doses of 100 mg/kg body weight were administered orally for 30 days. Various parameters were studied and the treatment group with the extract showed a significant increase in the liver glycogen and a significant decrease in fasting blood glucose, glycosylated hemoglobin levels, and serum marker enzyme levels. The total cholesterol and serum triglycerides levels, low density lipoprotein, and very low density lipoprotein were also significantly reduced and the high density lipoprotein level was significantly increased upon treatment with the Triticum aestivum ethanol extract. A significant decrease in the levels of lipid peroxides, superoxide dismutase, and glutathione peroxidise and increase in the levels of vitamin E, catalase, and reduced glutathione were observed in Triticum aestivum treated diabetic rats. Thus, from this study we conclude that ethanolic extract of Triticum aestivum exhibited significant antihyperglycemic, hypolipidemic, and antioxidant activities in streptozotocin-induced diabetic rats.

  20. Characterization of Aegilops kotschyi Boiss. x Triticum aestivum L. hybrid lines

    Directory of Open Access Journals (Sweden)

    Roman Prażak

    2014-01-01

    Full Text Available A study of four F5 and one BC1F1 Aegilops kotschyi Boiss. x Triticum aestivum L. hybrid lines was conducted to determine their quantitative morphological and qualitative features as well as a molecular investigation was carried out. Observations of ten quantitative traits showed that the F5 hybrid lines exhibited intermediate values between Ae. kotschyi Boiss. and T. aestivum L., or had similar traits to one of the parents. These hybrid lines had a significantly lower number and weight of grains per main spike, main spike fertility and 1000-grain weight than T. aestivum L. cv. ‘Rusałka’. The BC1F1 hybrid line was characterized by wheat-like fertility and phenotype. The F5 hybrid lines were characterized by much higher variability of the analysed morphological traits than T. aestivum L. cv. ‘Rusałka’. Grains of the hybrid lines had higher protein and micronutrient (iron and zinc content than wheat grains. The presence of DNA fragments specific to Ae. kotschyi Boiss. in the genotypes of the hybrid lines was confirmed by seven ISSR (Inter Simple Sequence Repeats molecular markers. Two ISSR markers – ISSR23690 and ISSR33650 – were the most effective for germplasm analysis of the hybrid lines. The analysed lines can become a source material for improvement of common wheat T. aestivum L. in crossing programs.

  1. The Chimp Genome Reveals a Retroviral Invasion in Primate Evolution

    OpenAIRE

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

    2005-01-01

    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 1 [PTERV1]) that has become integrated in the germline of Africa...

  2. Characterization of a PDR type ABC transporter gene from wheat (Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    SHANG Yi; XIAO Jin; MA LuLin; WANG HaiYan; QI ZengJun; CHEN PeiDu; LIU DaJun; WANG XiuE

    2009-01-01

    DON,as a virulence factor,plays an important role in the infection of Fusarium graminearum in wheat.The infection ability of F. graminearum depends on its capacity of producing DON. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. In this study,GeneChip analysis indicated that an EST encoding an ATP-binding cassette (ABC) transporter was up-regulated by 45 times in a wheat landrace Wangshuibai,which is resistant to DON accumulation.A pair of EST-derived primers were designed based on the EST sequence,and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp,consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kD. BLAST analysis indicated that the gene may belong to pleiotropic drug resistance (PDR) sub-family,and hence designated as TaPDR1 (Triticum aestivum pleiotropic drug resistance). TaPDR1 was located on chromosome 5A of wheat using nulliaomic-tetrasomic lines of Chinese Spring. TaPDR1 was up-regulated by induction of both DON and F. graminearum. Expression patterns of TaPDR1 were different in wild-type Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1,a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON accumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress,such as JA and SA,nor by abiotic stresses,such as heat,cold,wounding and NaCI. However,TaPDR1 expression was

  3. Multi-platform next-generation sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis

    NARCIS (Netherlands)

    Dalloul, R.A.; Long, J.A.; Zimin, A.V.; Aslam, M.L.; Crooijmans, R.P.M.A.; Megens, H.J.W.C.; Groenen, M.

    2010-01-01

    A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more th

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

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

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

  5. Draft Genome Sequence of the Phenazine-Producing Pseudomonas fluorescens Strain 2-79.

    Science.gov (United States)

    Nesemann, Kai; Braus-Stromeyer, Susanna A; Thuermer, Andrea; Daniel, Rolf; Mavrodi, Dmitri V; Thomashow, Linda S; Weller, David M; Braus, Gerhard H

    2015-03-26

    Pseudomonas fluorescens strain 2-79, a natural isolate of the rhizosphere of wheat (Triticum aestivum L.), possesses antagonistic potential toward several fungal pathogens. We report the draft genome sequence of strain 2-79, which comprises 5,674 protein-coding sequences.

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

    Science.gov (United States)

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

  7. Comparison of Transformation Efficiency of piggyBac Transposon among Three Different Silkworm Bombyx mori Strains

    Institute of Scientific and Technical Information of China (English)

    Boxiong ZHONG; Jianying LI; Jin'e CHEN; Jian YE; Songdong YU

    2007-01-01

    The transformation rate of three different strains of silkworm Bombyx mori was compared after the introduction of enhanced green fluorescence protein (EGFP)-encoding genes into the silkworm eggs by microinjection of a mixture of piggyBac vector and helper plasmid containing a transposase-encoding sequence. Although there were no significant differences among the three strains in the percentages of fertile moths in microinjected eggs (P=0.1258), the percentages of Go transformed moths in fertile moths and injected eggs were both significantly different (P=0.01368 and P=0.02398, respectively). The transformation rate of the Nistari strain (Indian strain) was significantly higher than that of the other two strains, Golden-yellow-cocoon (Vietnamese strain) and Jiaqiu (Chinese strain), which had similar rate. These results indicate that the transformation efficiency of the piggyBac-based system might vary with silkworm strains with different genetic backgrounds. The presence of endogenous piggyBac-like elements might be an important factor influencing the transformation efficiency of introduced piggyBac-derived vectors, and the diverse amount and activation in different silkworm strains might account for the significant differences.

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

    Science.gov (United States)

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

    2016-06-01

    The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision-making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co-opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov-Chain Monte-Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac.

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

    International Nuclear Information System (INIS)

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

  10. Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology

    NARCIS (Netherlands)

    Tang, X.; Boer, de J.M.; Eck, van H.J.; Bachem, C.W.B.; Visser, R.G.F.; Jong, de J.H.

    2009-01-01

    A cytogenetic map has been developed for diploid potato (Solanum tuberosum), in which the arms of the 12 potato bivalents can be identified in pachytene complements using multicolor fluorescence in situ hybridization (FISH) with a set of 60 genetically anchored bacterial artificial chromosome (BAC)

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

    Science.gov (United States)

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

    2016-06-01

    The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision-making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co-opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov-Chain Monte-Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac. PMID:27027400

  12. Construction of a full bacterial artificial chromosome (BAC) library of Oryza sativa genome

    Institute of Scientific and Technical Information of China (English)

    TAOQUANZHOU; HAIYINGZHAO; 等

    1994-01-01

    We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 106 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.

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

    Science.gov (United States)

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

    2013-01-01

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

  14. Characterization of Aegilops kotschyi Boiss. x Triticum aestivum L. hybrid lines

    OpenAIRE

    Roman Prażak; Edyta Paczos-Grzęda

    2014-01-01

    A study of four F5 and one BC1F1 Aegilops kotschyi Boiss. x Triticum aestivum L. hybrid lines was conducted to determine their quantitative morphological and qualitative features as well as a molecular investigation was carried out. Observations of ten quantitative traits showed that the F5 hybrid lines exhibited intermediate values between Ae. kotschyi Boiss. and T. aestivum L., or had similar traits to one of the parents. These hybrid lines had a significantly lower number and weight of gra...

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

    Directory of Open Access Journals (Sweden)

    Tabet-Canale Kamila

    2011-04-01

    Full Text Available Abstract 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

  16. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

    Directory of Open Access Journals (Sweden)

    Guillaume Manat

    Full Text Available Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2 catalyze the dephosphorylation of C55-PP molecules on the same (outer side of the plasma membrane.

  17. Whole genome comparative analysis of channel catfish (Ictalurus punctatus) with four model fish species

    OpenAIRE

    Jiang, Yanliang; Gao, Xiaoyu; Liu, Shikai; Zhang, Yu; Liu, Hong; Sun, Fanyue; Bao, Lisui; Waldbieser, Geoff; Liu, Zhanjiang

    2013-01-01

    Background Comparative mapping is a powerful tool to study evolution of genomes. It allows transfer of genome information from the well-studied model species to non-model species. Catfish is an economically important aquaculture species in United States. A large amount of genome resources have been developed from catfish including genetic linkage maps, physical maps, BAC end sequences (BES), integrated linkage and physical maps using BES-derived markers, physical map contig-specific sequences...

  18. Complete Genomes of Classical Swine Fever Virus Cloned into Bacterial Artificial Chromosomes

    OpenAIRE

    Rasmussen, Thomas Bruun; Reimann, I; Uttenthal, Åse; De Beer, M.

    2011-01-01

    Complete genome amplification of viral RNA provides a new tool for the generation of modified pestiviruses. We have used our full-genome amplification strategy for generation of amplicons representing complete genomes of classical swine fever virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome (BAC) generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived. Our strategy allows construction of stable infec...

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

    Science.gov (United States)

    Pasukphun, N; Vinitnantharat, S; Gheewala, S

    2010-04-01

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

  20. Introduction of temperature-sensitive helper and donor plasmids into Bac-to-Bac baculovirus expression systems

    Institute of Scientific and Technical Information of China (English)

    Zhihong; Huang; Ao; Li; Mengjia; Pan; Wenbi; Wu; Meijin; Yuan; Kai; Yang

    2015-01-01

    In the baculovirus shuttle vector(bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome,and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive(ts) plasmid, p SIM6. Using the resulting ts helper plasmid p MON7124 ts and the ts donor plasmid p FB1ts-PH-GFP, a recombinant bacmid,b Ac WT-PG(-), was constructed, and the transposition efficiency was found to be 33.1%. The plasmids were then removed by culturing at 37 °C. For b Ac WT-PG(-), the infectious progeny virus titer and the protein expression level under the control of the polyhedrin promoter were similar to those of a bacmid constructed with unmodified helper and donor plasmids. These ts plasmids will be useful for obtaining plasmid-free bacmids for both heterologous protein production and fundamental studies of baculovirus biology.

  1. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

    Directory of Open Access Journals (Sweden)

    Zhou Yi

    2007-06-01

    Full Text Available Abstract Background The zebrafish (Danio rerio is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data. Results Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed. Conclusion The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.

  2. Evaluation of assembly strategies using RNA-seq data associated with grain development of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Li, Huai-Zhu; Gao, Xiang; Li, Xiao-Yan; Chen, Qi-Jiao; Dong, Jian; Zhao, Wan-Chun

    2013-01-01

    Wheat (Triticum aestivum L.) is one of the most important crops cultivated worldwide. Identifying the complete transcriptome of wheat grain could serve as foundation for further study of wheat seed development. However, the relatively large size and the polyploid complexity of the genome have been substantial barriers to molecular genetics and transcriptome analysis of wheat. Alternatively, RNA sequencing has provided some useful information about wheat genes. However, because of the large number of short reads generated by RNA sequencing, factors that are crucial to transcriptome assembly, including software, candidate parameters and assembly strategies, need to be optimized and evaluated for wheat data. In the present study, four cDNA libraries associated with wheat grain development were constructed and sequenced. A total of 14.17 Gb of high-quality reads were obtained and used to assess different assembly strategies. The most successful approach was to filter the reads with Q30 prior to de novo assembly using Trinity, merge the assembled contigs with genes available in wheat cDNA reference data sets, and combine the resulting assembly with an assembly from a reference-based strategy. Using this approach, a relatively accurate and nearly complete transcriptome associated with wheat grain development was obtained, suggesting that this is an effective strategy for generation of a high-quality transcriptome from RNA sequencing data. PMID:24349528

  3. Association analysis of grain traits with SSR markers between Aegilops tauschii and hexaploid wheat (Triticum aestivumL.)

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jing-lan; WANG Hong-wei; ZHANG Xiao-cun; DU Xu-ye; LI An-fei; KONG Ling-rang

    2015-01-01

    Seven important grain traits, including grain length (GL), grain width (GW), grain perimeter (GP), grain area (GA), grain length/width ratio (GLW), roundness (GR), and thousand-grain weight (TGW), were analyzed using a set of 139 simple sequence repeat (SSR) markers in 130 hexaploid wheat varieties and 193Aegilops tauschiaccessions worldwide. In total, 1612 aleles inAe. tauschiand 1360 aleles in hexaploid wheat (Triticum aestivumL.) were detected throughout the D genome. 197 marker-trait associations inAe. tauschi were identiifed with 58 different SSR loci in 3 environments, and the average phenotypic variation value (R2) ranged from 0.68 to 15.12%. In contrast, 208 marker-trait associations were identiifed in wheat with 66 different SSR markers in 4 environments and the average phenotypicR2ranged from 0.90 to 19.92%. Further analysis indicated that there are 6 common SSR loci present in bothAe. tauschi and hexaploid wheat, which are signiifcantly associated with the 5 investigated grain traits (i.e., GA, GP, GR, GL, and TGW) and in total, 16 aleles derived from the 6 aforementioned SSR loci were shared byAe. tauschi and hexaploid wheat. These preliminary data suggest the existence of common aleles may explain the evolutionary process and the selection betweenAe. tauschi and hexaploid wheat. Furthermore, the genetic differentiation of grain shape and thousand-grain weight were observed in the evolutionary developmental process fromAe. tauschi to hexaploid wheat.

  4. Heat stress in wheat (Triticum aestivum L.) : Effects on grain growth and quality traits

    NARCIS (Netherlands)

    Spiertz, J.H.J.; Hamer, R.J.; Xu, H.; Primo Martin, C.; Don, C.; Putten, van der P.E.L.

    2006-01-01

    Heat stress effects on grain dry mass and quality were studied in spring wheat genotypes (Triticum aestivum L.). Three cultivars were chosen with respect to heat tolerance: Lavett (genotype 1), selected for temperate growing conditions and two CIMMYT cultivars, Ciano-79 (genotype 2) and Attila (geno

  5. Heat stress in wheat (Triticum aestivum L.): Effects on grain growth and quality traits

    NARCIS (Netherlands)

    Spiertz, J.H.J.; Hamer, R.J.; Xu, H.; Primo-Martin, C.; Don, C.; Putten, P.E.L. van der

    2006-01-01

    Heat stress effects on grain dry mass and quality were studied in spring wheat genotypes (Triticum aestivum L.). Three cultivars were chosen with respect to heat tolerance: Lavett (genotype 1), selected for temperate growing conditions and two CIMMYT cultivars, Ciano-79 (genotype 2) and Attila (geno

  6. In vitro effect of wheat bran (Triticum aestivum) extract on calcium oxalate urolithiasis crystallization.

    Science.gov (United States)

    Sekkoum, Khaled; Cheriti, Abdelkrim; Taleb, Safia

    2011-10-01

    Urolithiasis can lead to the loss of renal function in some cases. In this study, we tested the inhibiting effect of wheat bran (Triticum aestivum L) extract on calcium oxalate crystallization in a turbidimetric model, by FTIR spectroscopy, and polarized microscopy. The results show that this plant extract has a major inhibitory effect on calcium oxalate crystallization. PMID:22164778

  7. Metabolic Pathways of the Wheat (Triticum aestivum)Endosperm Amyloplast Revealed by Proteomics

    Science.gov (United States)

    By definition, amyloplasts are plastids specialized for starch production. However, amyloplasts have a broader range of functions that are not widely recognized. A recent proteomic study of amyloplasts isolated from wheat (Triticum aestivum Butte 86) endosperm suggested that they share many of the ...

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

    Science.gov (United States)

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

    2010-07-01

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

  9. Genetic transformation of Drosophila willistoni using piggyBac transposon and GFP

    Directory of Open Access Journals (Sweden)

    Manuela Finokiet

    2007-01-01

    Full Text Available Studies were carried out on the use of piggyBac transposable element as vector and the green fluorescent protein (EGFP from the jellyfish, Aquorea victoria, as a genetic marker for the transformation of Drosophila willistoni. Preblastoderm embryos of D. willistoni white mutant were microinjected with a plasmid containing the EGFP marker and the piggyBac ITRs, together with a helper plasmid containing the piggyBac transposase placed under the control of the D. melanogaster hsp70 promoter. G0 adults transformants were recovered at a frequency of approximately 67%. Expression of EGFP in larvae, pupae and adults was observed up to the third generation, suggesting that this transposon was not stable in D. willistoni. Transformed individuals displayed high levels of EGFP expression during larvae and adult stages in the eye, abdomen, thorax and legs, suggesting a wide expression pattern in this species than reported to other species of Drosophilidae.Descrevemos neste trabalho a transformação genética de Drosophila willistoni empregando o elemento transponível piggyBac como vetor e o gene EGFP (green fluorescent protein retirado da água-viva Aquorea victoria, como marcador de transformação. Embriões de D. willistoni em estágio pré-blastoderme, mutantes para o gene white, foram microinjetados com plasmídio contendo o marcador EGFP e as regiões ITRs do transposon piggyBac concomitantemente com um plasmídio auxiliar possuindo o gene da transposase de piggyBac sobre o controle do promotor do gene hsp70 de Drosophila melanogaster. Adultos transformantes Go foram gerados em uma taxa de 67%. A expressão de GFP em larvas, pupas e adultos foi observada somente até a terceira geração, sugerindo que este transposon não é estável em D. willistoni. Os indivíduos transformados exigem um alto nível de expressão de EGFP durante os estágios de larva e, também em adultos o gene marcador é expresso nos olhos, abdome, tórax e patas, mostrando um

  10. Dynamics of rhizosphere properties and antioxidative responses in wheat (Triticum aestivum L.) under cadmium stress.

    Science.gov (United States)

    Li, Yonghua; Wang, Li; Yang, Linsheng; Li, Hairong

    2014-04-01

    In this study, we performed a rhizobox experiment to examine the dynamic changes in the rhizosphere properties and antioxidant enzyme responses of Triticum aestivum L. under three levels of cadmium stress. A set of micro-techniques (i.e., Rhizobox and Rhizon SMS) were applied for the dynamically non-destructive collection of the rhizosphere soil solution to enable the observation at a high temporal resolution. The dynamics of soluble cadmium and dissolved organic carbon (DOC) in the rhizosphere soil solutions of the Triticum aestivum L. were characterised by the sequence week 0 after sowing (WAS0)soil solution pH was found to follow an opposite distribution pattern. Systematically, both superoxide dismutase (SOD) and catalase (CAT) activities in the leaves of the Triticum aestivum L. increased concomitantly with increasing cadmium levels (p>0.05) and growth duration (pstress with a decrease at high cadmium stress (p>0.05). These results suggested the enhancement of DOC production and the greater antioxidant enzyme activities were two important protective mechanisms of Triticum aestivum L. under cadmium stress, whereas rhizosphere acidification might be an important mechanism for the mobilisation of soil cadmium. The results also revealed that plant-soil interactions strongly influence the soil solution chemistry in the rhizosphere of Triticum aestivum L., that, in turn, can stimulate chemical and biochemical responses in the plants. In most cases, these responses to cadmium stress were sensitive and might allow us to develop strategies for reducing the risks of the cadmium contamination to crop production. PMID:24580822

  11. SSR-based identification of genetic groups within European populations of Tuber aestivum Vittad.

    Science.gov (United States)

    Molinier, Virginie; Murat, Claude; Peter, Martina; Gollotte, Armelle; De la Varga, Herminia; Meier, Barbara; Egli, Simon; Belfiori, Beatrice; Paolocci, Francesco; Wipf, Daniel

    2016-02-01

    Tuber species are ectomycorrhizal ascomycetes establishing relationships with different host trees and forming hypogeous fruiting bodies known as truffles. Among Tuber species, Tuber aestivum Vittad. has a wide distributional range being found naturally all over Europe. Here, we performed large-scale population genetic analyses in T. aestivum to (i) investigate its genetic diversity at the European scale, (ii) characterize its genetic structure and test for the presence of ecotypes and (iii) shed light into its demographic history. To reach these goals, 230 ascocarps from different populations were genotyped using 15 polymorphic simple sequence repeat markers. We identified 181 multilocus genotypes and four genetic groups which did not show a clear geographical separation; although, one of them was present exclusively in Southeast France, Italy and Spain. Fixation index values between pairs of genetic groups were generally high and ranged from 0.29 to 0.45. A significant deficit of heterozygosity indicated a population expansion instead of a recent population bottleneck, suggesting that T. aestivum is not endangered in Europe, not even in Mediterranean regions. Our study based on a large-scale population genetic analysis suggests that genetically distinct populations and likely ecotypes within T. aestivum are present. In turn, this study paves the way to future investigations aimed at addressing the biological and/or ecological factors that have concurred in shaping the population genetic structure of this species. Present results should also have implications for the truffle market since defining genetic markers are now possible at least for some specific T. aestivum genetic groups. PMID:26070448

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

    Science.gov (United States)

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

    2013-12-01

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

  13. Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis.

    NARCIS (Netherlands)

    Hehir-Kwa, J.Y.; Egmont-Peterson, M.; Janssen, I.M.; Smeets, D.F.C.M.; Geurts van Kessel, A.H.M.; Veltman, J.A.

    2007-01-01

    Recently, comparative genomic hybridization onto bacterial artificial chromosome (BAC) arrays (array-based comparative genomic hybridization) has proved to be successful for the detection of submicroscopic DNA copy-number variations in health and disease. Technological improvements to achieve a high

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

    Science.gov (United States)

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

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Mazzuchelli Juliana

    2012-09-01

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

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

    OpenAIRE

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

    1983-01-01

    A brief overview of the Bibliographic Access and Control System developed by the Washington University School of Medicine Library is presented. Because the system has been described in two previous reports, this paper focuses on its relationship to other automated programs (i.e., PHILSOM and OCTANET), education of users, evaluation of the system, and outreach to the medical center. In operation for more than two years, BACS represents the computerization of much of the managerial and operatio...

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

    OpenAIRE

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

    2014-01-01

    An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable ge...

  19. BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus.

    Science.gov (United States)

    Argilaguet, Jordi M; Pérez-Martín, Eva; López, Sergio; Goethe, Martin; Escribano, J M; Giesow, Katrin; Keil, Günther M; Rodríguez, Fernando

    2013-04-01

    Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17 days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development.

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

    OpenAIRE

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

    2013-01-01

    Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivate...

  1. GENSAT BAC Cre-recombinase driver lines to study the functional organization of cerebral cortical and basal ganglia circuits

    OpenAIRE

    Gerfen, Charles R.; Paletzki, Ronald; Heintz, Nathaniel

    2013-01-01

    Recent development of molecular genetic techniques are rapidly advancing understanding of the functional role of brain circuits in behavior. Critical to this approach is the ability to target specific neuron populations and circuits. The collection of over 250 BAC Cre-recombinase driver lines produced by the GENSAT project provides a resource for such studies. Here we provide characterization of GENSAT BAC-Cre driver lines with expression in specific neuroanatomical pathways within the cerebr...

  2. Neutralization of endotoxin in vitro and in vivo by Bac7(1-35), a proline-rich antibacterial peptide.

    Science.gov (United States)

    Ghiselli, Roberto; Giacometti, Andrea; Cirioni, Oscar; Circo, Raffaella; Mocchegiani, Federico; Skerlavaj, Barbara; D'Amato, Giuseppina; Scalise, Giorgio; Zanetti, Margherita; Saba, Vittorio

    2003-06-01

    Lipopolysaccharides (LPS), or endotoxins, are structural components of gram-negative bacteria implicated in the pathogenesis of septic shock. In this study the antiendotoxin activity of Bac7(1-35), a synthetic peptide based on the sequence of a proline-rich antibacterial peptide from bovine neutrophils, was investigated in vitro and in an experimental rat model of gram-negative septic shock. The ability of Bac7(1-35) to bind LPS from Escherichia coli O111:B4 was determined using a sensitive Limulus chromogenic assay. In the in vivo study, adult male Wistar rats were given an intraperitoneal injection of 1 x 10(9) colony-forming units of E. coli ATCC 25922. All animals were randomized to receive intraperitoneally 1 mg/kg Bac7(1-35), or isotonic sodium chloride solution (control group C1), 60 mg/kg of piperacillin and 1 mg/kg polymyxin B, 1 mg/kg of polymyxin B plus 60 mg/kg of piperacillin, and 1 mg/kg of Bac7(1-35) plus 60 mg/kg of piperacillin. Each group included 15 animals. Bac7(1-35) was found to completely inhibit the LPS procoagulant activity at approximately 10 microM peptide concentration, as determined by in vitro LAL chromogenic assay. Treatment with Bac7(1-35) resulted in significant decrease in plasma endotoxin levels and lethality rates compared with saline injected control animals. No statistically significant differences were noted between Bac7(1-35) and polymyxin B in reducing all variables measured. These results provide evidence for the ability of Bac7(1-35) to effectively bind LPS and protect animals from lethal effects of this molecule, and point to its potential use for the treatment of endotoxin-induced septic shock. PMID:12785015

  3. Assessment of acrylamide degradation potential of Pseudomonas aeruginosa BAC-6 isolated from industrial effluent.

    Science.gov (United States)

    Chandrashekar, Vijayashree; Chandrashekar, Chandrika; Shivakumar, Rajath; Bhattacharya, Sourav; Das, Arijit; Gouda, Bhaskar; Rajan, Subbaramiah Sundara

    2014-07-01

    Acrylamide finds diverse industrial applications but is considered an environmental threat because of its neurotoxic, carcinogenic, and teratogenic effects. Certain bacteria enzymatically degrade acrylamide to acrylic acid and ammonia. The present investigation was carried out to isolate and identify an acrylamide-degrading bacterium from industrial effluent. Bacterial growth and extent of acrylamide degradation in the presence of different acrylamide concentrations, nutrients, varied range of pH, and temperature were analyzed. Among the eight acrylamide-degrading isolates, isolate BAC-6 demonstrated the highest degradation, and based upon the partial 16S rDNA sequencing, it was identified as Pseudomonas aeruginosa. P. aeruginosa BAC-6 grew over a wide range of acrylamide concentrations, but the highest degradation was recorded at 500 mg/L concentration with concomitant cell growth. Among the carbon supplements, mannitol supported the highest growth and degradation. Maximum degradation was reported at neutral pH. A mesophilic temperature range (25-40 °C) facilitated conducive bacterial growth followed by degradation. The highest degradation and bacterial growth were observed at 30 and 35 °C, respectively. Thus, it could be inferred from the present investigation that cultural conditions strongly affected the degradation potential of P. aeruginosa BAC-6 and advocated the utilization of the isolate in bioremediation of sites polluted with acrylamide.

  4. A BAC-based transgenic mouse specifically expresses an inducible Cre in the urothelium.

    Directory of Open Access Journals (Sweden)

    Tian Huai Shen

    Full Text Available Cre-loxp mediated conditional knockout strategy has played critical roles for revealing functions of many genes essential for development, as well as the causal relationships between gene mutations and diseases in the postnatal adult mice. One key factor of this strategy is the availability of mice with tissue- or cell type-specific Cre expression. However, the success of the traditional molecular cloning approach to generate mice with tissue specific Cre expression often depends on luck. Here we provide a better alternative by using bacterial artificial chromosome (BAC-based recombineering to insert iCreERT2 cDNA at the ATG start of the Upk2 gene. The BAC-based transgenic mice express the inducible Cre specifically in the urothelium as demonstrated by mRNA expression and staining for LacZ expression after crossing with a Rosa26 reporter mouse. Taking into consideration the size of the gene of interest and neighboring genes included in a BAC, this method should be widely applicable for generation of mice with tissue specific gene expression or deletions in a more specific manner than previously reported.

  5. The complete chloroplast genome sequence of Brachypodium distachyon: sequence comparison and phylogenetic analysis of eight grass plastomes

    Directory of Open Access Journals (Sweden)

    Anderson Olin D

    2008-07-01

    Full Text Available Abstract Background Wheat, barley, and rye, of tribe Triticeae in the Poaceae, are among the most important crops worldwide but they present many challenges to genomics-aided crop improvement. Brachypodium distachyon, a close relative of those cereals has recently emerged as a model for grass functional genomics. Sequencing of the nuclear and organelle genomes of Brachypodium is one of the first steps towards making this species available as a tool for researchers interested in cereals biology. Findings The chloroplast genome of Brachypodium distachyon was sequenced by a combinational approach using BAC end and shotgun sequences derived from a selected BAC containing the entire chloroplast genome. Comparative analysis indicated that the chloroplast genome is conserved in gene number and organization with respect to those of other cereals. However, several Brachypodium genes evolve at a faster rate than those in other grasses. Sequence analysis reveals that rice and wheat have a ~2.1 kb deletion in their plastid genomes and this deletion must have occurred independently in both species. Conclusion We demonstrate that BAC libraries can be used to sequence plastid, and likely other organellar, genomes. As expected, the Brachypodium chloroplast genome is very similar to those of other sequenced grasses. The phylogenetic analyses and the pattern of insertions and deletions in the chloroplast genome confirmed that Brachypodium is a close relative of the tribe Triticeae. Nevertheless, we show that some large indels can arise multiple times and may confound phylogenetic reconstruction.

  6. Controlling of crustacean zooplankton reproduction in biological activated carbon (BAC) filters by strengthen operation and management of conventional process

    Institute of Scientific and Technical Information of China (English)

    LIU Li-jun; ZHANG Jin-song; LI Xiao-wei; HE Jun-guo

    2010-01-01

    To counter the mass reproduction and penetration of crustacean zooplankton in Biological Activated Carbon (BAC) filters which may result in the presence of organisms in potable water and water pollution,this paper analyzed the factors affecting organisms' reproduction in BAC filters.A comparative study was performed on the density and composition of crustacean zooplankton of the concerned water treatment units of two advanced water plants (Plant A and B) which with the same raw water and the same treatment technique in southern China.The results obtained show that the crustaceans' density and composition was very different between the sand filtered water of Plant A and Plant B.which Harpacticoida bred sharply in the sediment tanks and penetrated sand filter into BAC falters was the primary reason of crustaceans reproduce in BAC filters of Plant A.For prevention of the organisms reproduction in BAC,some strengthen measures was taken including pre-chlorination,cleaning coagulation tanks and sediment tanks completely,increasing sludge disposal frequency to stop organisms enter BAC filters,and the finished water quality was improved and enhanced.

  7. Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes.

    Science.gov (United States)

    Beier, Sebastian; Himmelbach, Axel; Schmutzer, Thomas; Felder, Marius; Taudien, Stefan; Mayer, Klaus F X; Platzer, Matthias; Stein, Nils; Scholz, Uwe; Mascher, Martin

    2016-07-01

    Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path. PMID:26801048

  8. Complete Genome Sequence of Border Disease Virus Genotype 3 Strain Gifhorn

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

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

    Science.gov (United States)

    Ostermann, Eleonore; Spohn, Michael; Indenbirken, Daniela

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Aline Crucello

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

  12. Heavy metal uptake, translocation, and bioaccumulation studies of Triticum aestivum cultivated in contaminated dredged materials.

    Science.gov (United States)

    Shumaker, Ketia L; Begonia, Gregorio

    2005-08-01

    Phytoremediation is a technology that uses vegetation to remediate contaminants from water, soil, and sediments. Unlike traditional remediation techniques such as soil washing or vitrification, phytoremediation offers a technology that is solar-driven, aesthetically pleasing, and cost effective. Recent studies indicate that winter wheat (Triticum aestivum L.) is a potential accumulator for heavy metals such as lead (Pb) and cadmium (Cd) in hydroponic systems. Based on these findings, a laboratory study was conducted with the primary objective of determining the phytoaccumulation capability of this plant species for heavy metals from contaminated dredged materials (DMs) originating from two confined disposal facilities (CDF). The United States Army Corps of Engineers (USACE) manages several hundred million cubic meters of DMs each year, and 5 to 10 % of these DMs require special handling because they are contaminated with hazardous substances that can move from the substrates into food webs causing unacceptable risk outside CDFs. Phytoremediation may offer an alternative to decrease this risk. Chemical analyses by USACE personnel identified 17 metals in various DMs, but in this present study, only zinc (Zn) and Cd were investigated. Pre-germinated seeds of the test plants were planted under laboratory conditions in pots containing the various DMs and reference soil. Four weeks after planting, plants were harvested and separated into roots and shoots for biomass production and tissue metal concentrations analyses. Results showed that T. aestivum plants have the capacity to tolerate and grow in multiple-metal contaminated DMs with the potential of accumulating various amounts of Zn and Cd. Root and shoot biomass of T. aestivum were not significantly affected by the DMs on which the plants were grown suggesting that this plant species can grow just as well on DMs contaminated by various metals as in the reference soil. No significant differences in the Zn tissue

  13. Heavy Metal Uptake, Translocation, and Bioaccumulation Studies of Triticum aestivum Cultivated in Contaminated Dredged Materials

    Directory of Open Access Journals (Sweden)

    Gregorio Begonia

    2005-08-01

    Full Text Available Phytoremediation is a technology that uses vegetation to remediate contaminants from water, soil, and sediments. Unlike traditional remediation techniques such as soil washing or vitrification, phytoremediation offers a technology that is solar-driven, aesthetically pleasing, and cost effective. Recent studies indicate that winter wheat (Triticum aestivum L. is a potential accumulator for heavy metals such as lead (Pb and cadmium (Cd in hydroponic systems. Based on these findings, a laboratory study was conducted with the primary objective of determining the phytoaccumulation capability of this plant species for heavy metals from contaminated dredged materials (DMs originating from two confined disposal facilities (CDF. The United States Army Corps of Engineers (USACE manages several hundred million cubic meters of DMs each year, and 5 to 10 % of these DMs require special handling because they are contaminated with hazardous substances that can move from the substrates into food webs causing unacceptable risk outside CDFs. Phytoremediation may offer an alternative to decrease this risk. Chemical analyses by USACE personnel identified 17 metals in various DMs, but in this present study, only zinc (Zn and Cd were investigated. Pre-germinated seeds of the test plants were planted under laboratory conditions in pots containing the various DMs and reference soil. Four weeks after planting, plants were harvested and separated into roots and shoots for biomass production and tissue metal concentrations analyses. Results showed that T. aestivum plants have the capacity to tolerate and grow in multiple-metal contaminated DMs with the potential of accumulating various amounts of Zn and Cd. Root and shoot biomass of T. aestivum were not significantly affected by the DMs on which the plants were grown suggesting that this plant species can grow just as well on DMs contaminated by various metals as in the reference soil. No significant differences in the Zn

  14. An Efficient Approach for the Development of Locus Specific Primers in Bread Wheat (Triticum aestivum L. and Its Application to Re-Sequencing of Genes Involved in Frost Tolerance.

    Directory of Open Access Journals (Sweden)

    Steve Babben

    Full Text Available Recent declines in costs accelerated sequencing of many species with large genomes, including hexaploid wheat (Triticum aestivum L.. Although the draft sequence of bread wheat is known, it is still one of the major challenges to developlocus specific primers suitable to be used in marker assisted selection procedures, due to the high homology of the three genomes. In this study we describe an efficient approach for the development of locus specific primers comprising four steps, i.e. (i identification of genomic and coding sequences (CDS of candidate genes, (ii intron- and exon-structure reconstruction, (iii identification of wheat A, B and D sub-genome sequences and primer development based on sequence differences between the three sub-genomes, and (iv; testing of primers for functionality, correct size and localisation. This approach was applied to single, low and high copy genes involved in frost tolerance in wheat. In summary for 27 of these genes for which sequences were derived from Triticum aestivum, Triticum monococcum and Hordeum vulgare, a set of 119 primer pairs was developed and after testing on Nulli-tetrasomic (NT lines, a set of 65 primer pairs (54.6%, corresponding to 19 candidate genes, turned out to be specific. Out of these a set of 35 fragments was selected for validation via Sanger's amplicon re-sequencing. All fragments, with the exception of one, could be assigned to the original reference sequence. The approach presented here showed a much higher specificity in primer development in comparison to techniques used so far in bread wheat and can be applied to other polyploid species with a known draft sequence.

  15. Antarctic notothenioid fishes: genomic resources and strategies for analyzing an adaptive radiation.

    Science.gov (United States)

    Detrich, H W; Amemiya, Chris T

    2010-12-01

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

  16. Pituitary-specific overexpression of porcine follicle-stimulating hormone leads to improvement of female fecundity in BAC transgenic mice.

    Directory of Open Access Journals (Sweden)

    Mingjun Bi

    Full Text Available Follicle-stimulating hormone (FSH is a pituitary glycoprotein that, together with luteinizing hormone, plays a crucial role in ovarian folliculogenesis and female fertility. We previously found that FSH beta is a major gene controlling high prolificacy of Chinese Erhualian pigs. To directly study the biological effects on reproductive function of porcine FSH (pFSH for polyovulatory species, we generated a novel gain-of-function mouse model using a bacterial artificial chromosome (BAC system to jointly introduce 92 kb and 165 kb genomic fragments comprising the pFSH α- and β-subunit genes. These directed the physiological expression of pFSH with the same temporal and spatial pattern as endogenous FSH in female transgenic (TG mice. Serum levels of biologically active pFSH heterodimers in independent TG lines ranged from 6.36 to 19.83 IU/L. High basal pFSH activity led to a significant reduction of serum LH and testosterone levels in TG females compared to wild-type (WT littermates, yet endogenous FSH and estradiol levels were significantly elevated. Interestingly, ovarian histology showed that the number of corpora lutea was significantly higher at 14 and 28 weeks of age in TG females and breeding curves revealed that mean litter sizes of TG females were obviously larger than for WT littermates before 52 weeks of age. These findings indicate that pituitary-specific overexpression of pFSH within physiological boundaries can increase ovulation rate and litter size, but it does not cause reproductive defects. Therefore, our TG mouse model provides exciting insights for investigating the actions of pFSH in vivo.

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

    Science.gov (United States)

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

    2005-05-01

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

  18. Cloning the simian varicella virus genome in E. coli as an infectious bacterial artificial chromosome

    OpenAIRE

    Gray, Wayne L.; Zhou, Fuchun; Noffke, Juliane; Tischer, B Karsten

    2011-01-01

    Simian varicella virus (SVV) is closely related to human varicella-zoster virus and causes varicella and zoster-like disease in nonhuman primates. In this study, a mini-F replicon was inserted into a SVV cosmid and infectious SVV was generated by co-transfection of Vero cells with overlapping SVV cosmids. The entire SVV genome, cloned as a bacterial artificial chromosome (BAC), was stably propagated upon serial passage in E. coli. Transfection of pSVV-BAC DNA into Vero cells yielded infectiou...

  19. Heavy Metal Uptake, Translocation, and Bioaccumulation Studies of Triticum aestivum Cultivated in Contaminated Dredged Materials

    OpenAIRE

    Gregorio Begonia; Shumaker, Ketia L.

    2005-01-01

    Phytoremediation is a technology that uses vegetation to remediate contaminants from water, soil, and sediments. Unlike traditional remediation techniques such as soil washing or vitrification, phytoremediation offers a technology that is solar-driven, aesthetically pleasing, and cost effective. Recent studies indicate that winter wheat (Triticum aestivum L.) is a potential accumulator for heavy metals such as lead (Pb) and cadmium (Cd) in hydroponic systems. Based on these findings, a labora...

  20. Contaminants in organically and conventionally produced winter wheat (Triticum aestivum) in Belgium

    OpenAIRE

    Pussemier, Luc; Harcz, Peter; De Temmerman, Ludwig; De Voghel, Simon; Waegeneers, Nadia; Wilmart, Olivier; Vromman, Valérie; Schmit, Jean-François; Moons, Emmanuelle; Van Peteghem, Carlos; De Saeger, Sarah; Schneider, Yves Jacques; Larondelle, Yvan

    2007-01-01

    Abstract A database has been compiled with the levels of important contaminants (mycotoxins, heavy metals and pesticides) measured from 2002 to 2005 in winter wheat (Triticum aestivum) grown in Belgium according to the organic and conventional farming systems. Assuming no further change in contaminant levels during cereal processing and during the preparation of foodstuffs, conservative intakes are estimated for the consumers of cereal based products such as flour, bread, breakfast...

  1. Genetic and ecological aspects of gene flow from wheat (Triticum aestivum L.) to Aegilops L. species

    OpenAIRE

    Schoenenberger, Nicola; Küpfer, Philippe

    2006-01-01

    Genetic and ecological aspects of gene flow from wheat (Triticum aestivum L.) to Aegilops L. species Hybridisation and introgression from crops to wild relatives is a key issue in risk assessment. In the present study, hybridisation and introgression dynamics from hexaploid wheat (2n=42) to tetraploid Aegilops species (2n=28) were investigated by experiments in natural conditions or in the greenhouse, and by genetic analyses. In order to study crop-weed hybridisation as a function of distance...

  2. Distribution of cadmium, copper, and zinc in the caryopsis of wheat (Triticum aestivum L. )

    Energy Technology Data Exchange (ETDEWEB)

    Pieczonka, K.; Rosopulo, A.

    1985-12-01

    Cadmium, copper, and zinc were quantitatively determined in the whole grain, the germ, the aleurone layer, the outer pericarp, and the endosperm from the caryopsis of wheat (Triticum aestivum L.) by the methods of direct solid microsampling and flame-AAS, respectively. Metal concentrations markedly differed among the tissues investigated. Both methods used in this study produced almost identical heavy metal concentrations. However, the techniques dramatically differed in the amounts of grain material required for analysis.

  3. BacT/ALERT 3 D系统在结核性脑膜炎诊断中的应用%Value of BacT/ALERT 3 D microbial detection system in rapid diagnosis of tuberculosis men-ingitis

    Institute of Scientific and Technical Information of China (English)

    张继萍; 任丽娟; 李永利; 叶迎宾; 黄秀香; 郑立恒

    2015-01-01

    目的:评价BacT/ALERT 3D系统在诊断结核性脑膜炎中的应用价值。方法:收集2009年1月~2014年6月间200例结核性脑膜炎患者早期脑脊液标本480份,每份标本分别用离心涂片法、罗氏培养法和BacT/ALERT 3D系统培养法检测,对其结果进行分析,总结3种方法对确诊结核性脑膜炎之间的差异。结果:480份脑脊液标本中,离心涂片法、罗氏培养法、BacT/ALERT 3D系统培养法的结核杆菌阳性检出率分别为3.13%、5.42%、10.63%,BacT/ALERT 3D系统培养法结核杆菌初代分离时间短于罗氏培养法。结论:BacT/ALERT 3D系统有较高的检出率、较快的检出时间,有助于提高结核性脑膜炎的发现率,值得临床推广应用。%Objective:To evaluate the application value of BacT/ALERT 3D microbial detection system in rapid diagnosis of tuberculosis meningitis . Methods:480 specimens in cerebrospinsl fluid ( CSF) of 200 patients diagnosed with tuberculosis meningitis were collected from January 2009 to June 2014.These specimens were processed for detection of the mycobacterium tuberculosis by centrifugal smear acid-fast stain ( Ziehl-Neelsen staining) and Lowenstein-Jensen cultivation,as well as the BacT/ALERT 3D system.The above results were analyzed and compared regarding the positive rates in order to evaluate the value in diagnosing tuberculosis meningitis .Results:The positive rates of the centrifugal smear acid-fast smear,Lowenstein-Jensen cultiva-tion and BacT/ALERT 3D system cultivation were 3.13%,5.42% and 10.63% respectively.Conclusion: BacT/Alert 3D microbial detection system may lead to higher sensitivity and shortened detection time ,which is worthy of wider clinical recommendation .

  4. Genome engineering and gene drive in the mosquito aedes aegypti

    OpenAIRE

    St John, Oliver Tudor Lockhart; Sinkins, Steven; Alphey, Luke

    2012-01-01

    Genetic control strategies are a novel method for reducing populations of pest insects such as the yellow fever mosquito Aedes aegypti, a major vector of several important arboviral diseases. This thesis describes efforts to develop new tools to engineer the Ae. aegypti genome and to better understand existing tools, and furthermore to use these to engineer a gene drive system in Ae. aegypti. The piggyBac transposon was found to be extremely stable in the germline of Ae. aegypti, and transpos...

  5. Expression of SETD4 in Bac-to-Bac baculovirus expression system%SETD4蛋白在Bac-to-Bac杆状病毒系统的表达

    Institute of Scientific and Technical Information of China (English)

    雷烨铭; 崔航; 钟玙沄; 王义乾; 黄穗; 赵舒祺; 蔡军伟; 姜勇; 刘靖华

    2015-01-01

    目的:通过昆虫杆状病毒表达系统表达SETD4(SET domain-containing 4)蛋白,并纯化SETD4蛋白,为深入探讨SETD4的功能奠定基础。方法提取小鼠正常肝组织的RNA,通过RT-PCR扩增SETD4基因,并克隆至pFastBac-HTB构建重组载体,再转座获得重组杆粒;通过脂质体介导将重组杆粒转染SF9细胞产生重组病毒,扩增病毒感染细胞并获得重组蛋白;利用Ni2+亲和柱来纯化蛋白,并通过Western Blot及考马斯亮蓝染色鉴定SETD4蛋白。结果经双酶切鉴定及测序证实SETD4基因插入了供体质粒;经PCR鉴定证实SETD4基因插入了穿梭载体;经考马斯亮蓝染色证实纯化得到重组蛋白,用His-Tag抗体和SETD4特异性抗体在50 kD处可检测到目的条带。结论成功利用昆虫杆状病毒表达系统够表达了SETD4,并纯化了SETD4。%Objective To express SET domain- containing 4 (SETD4) protein through using baculovirus expression system and purify the expressed product to explore the functions of SETD4 protein and further understand the biological roles of SET family proteins. Methods The SETD4 gene was amplified by RT-PCR from mouse normal liver tissue. The gene was then inserted into pFastBac-HTB vector to form the recombinant donor plasmid which was further transformed into DH10Bac to construct the recombined bacmid. Next the bacmid was transfected to sf9 cells for package of the recombinant baculovirus particles. The recombinant SETD4 protein was expressed from the cells transduced by the recombinant baculovirus and was purified by NI-NTA resin. Purified protein was examined by coomassie brilliant blue staining and Western Blotting. Results The donor plasmid and recombined bacmid were successfully prepared and the recombinant baculovirus particles were produced from sf9 cells. The SETD4 protein was obtained and confirmed by brilliant blue staining and western blotting with a His-tag antibody and a specific SETD4 antibody

  6. Ectomycorrhizal communities above and below ground and truffle productivity in a Tuber aestivum orchard

    Directory of Open Access Journals (Sweden)

    Elena Salerni

    2014-08-01

    Full Text Available Aim of study: The diversity of ectomycorrhizal fungal communities (EM above (EMFb and below (EMMt ground associated with Quercus cerris L., Q. pubescens Willd., and Pinus nigra J.F.Arnold was analyzed.Area of study: A 20 year-old orchard that produces Tuber aestivum truffles, located a few kilometers from Chiusi della Verna (latitude 43° 41’ 53’’; longitude 11° 56’ 9’’ in Tuscany (central Italy was observed.Material and Methods: This investigation combined analyses of EMFb, EMMt, T. aestivum productivity, different host trees, and statistical data on community ecology.Main results: The EM communities showed high species richness and differed slightly in relation to both the host tree and their location above or below ground, providing frequent findings of Tricholoma and Tomentella, respectively. Positive correlations were found between the number of truffles and host trees, and between the weight and number of truffles and EMFb.Research highlights: Mycorrhizal fungi and truffle production are not in competition.Key words: Fungal communities; fruiting bodies; morphotypes; Tuber aestivum; competition; Italy.

  7. The role of pH in Tuber aestivum syn. uncinatum mycorrhiza development within commercial orchards

    Directory of Open Access Journals (Sweden)

    Paul W. Thomas

    2013-12-01

    Full Text Available The accepted advice when establishing a plantation of Tuber aestivum syn. uncinatum is that young inoculated trees should be planted on calcareous soils with a naturally high pH level. When a site is employed that has a naturally low pH level, lime is often applied to raise the pH to a considered ideal level of c.7.5. However, this may not be the correct approach. Here we present data from 33 data points taken from commercial truffle orchards in England, UK. Soil pH is correlated to Tuber aestivum syn. uncinatum mycorrhiza survivorship and development. The optimal observed pH was 7.51 but the actual optimal pH for cultivation may be higher. Sub optimal pH levels lead to a reduction of Tuber aestivum syn. uncinatum mycorrhiza. This reduction is not permanent and mycorrhization levels may be improved within a 12 month period by amending the soil pH. The importance of understanding the interaction of pH with other variables and the results in relation truffle cultivation are discussed.

  8. Ecological characteristics of a Hungarian summer truffle (Tuber aestivum Vittad. producing area

    Directory of Open Access Journals (Sweden)

    Csorbai A. Gógán

    2013-12-01

    Full Text Available Hungary has outstanding environment for natural truffle production in some regions including plain and hilly areas. The most famous of all the natural summer truffle (Tuber aestivum Vittad. habitats is the commonly called Jászság region. This area is situated in the middle of Hungary, between river Danube and Tisza. The flatland area is basically covered by river alluviums with main soils of chernozems, fluvisols, solonchaks and arenosols. Climate of the region is typically continental: warm and dry summers and cold winters vary. The area is traditionally of agricultural use, although strong afforestation was made in the late 1950’s. The English oak (Quercus robur L. populations planted at that time gave a basis for current excellent truffle production. Nowadays the region has proved to be the best natural summer truffle (T. aestivum producing area of Hungary with early season opening (June and high quality truffles as early as August. In the research the best truffle producing forest blocks were selected for ecological investigation. Results of the detailed site description showed uniform climate characteristics and dominance of English oak (Q. robur or mixed English oak-Turkey oak (Quercus cerris L. forests. Soil types revealed differences from earlier findings: dominance of gleysols and water affected chernozems was declared. Soil chemical parameters are in accordance with literature data: pH, organic matter and active carbonate content of the examined soils fall within the range indicated as the requirement of T. aestivum.

  9. Optimum condition of Bac-J23 production from Lactobacillus J23%Lactobacillus J23发酵合成抗茵肽Bac-J23培养条件的优化研究

    Institute of Scientific and Technical Information of China (English)

    易华西; 韩雪; 杜明; 张兰威

    2012-01-01

    pH, temperature and inoculation size dependent property were observed when Lactobacillus paracasei-J23 produced Bac-J23. The optimum condition to Bac-J23 synthesize were 36.8 ℃, pHS.2 and 2.13×10^4 cfu/mL inoculum size. Under the optimum condition, the production of Bac-J23 was improved 1.68 folds and reached 5400 U/mL.%研究了培养条件(接种量、发酵温度、初始pH)对Lactobacillus J23发酵合成广谱抗茵肽Bac-J23的影响,并采用响应面法进行了优化。结果表明,Lactobacillus-J23生产Bac-J23体现出温度、pH梯度和接种剂量依赖性,响应面优化结果表明最适温度和pH分别为36.8℃和5.2,接种量阈值为2.13×10^4cfu/mL。

  10. KAIKObase: An integrated silkworm genome database and data mining tool

    Directory of Open Access Journals (Sweden)

    Nagaraju Javaregowda

    2009-10-01

    Full Text Available Abstract 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

  11. Genomic organization and evolution of the Atlantic salmon hemoglobin repertoire

    Directory of Open Access Journals (Sweden)

    Phillips Ruth B

    2010-10-01

    Full Text Available Abstract Background The genomes of salmonids are considered pseudo-tetraploid undergoing reversion to a stable diploid state. Given the genome duplication and extensive biological data available for salmonids, they are excellent model organisms for studying comparative genomics, evolutionary processes, fates of duplicated genes and the genetic and physiological processes associated with complex behavioral phenotypes. The evolution of the tetrapod hemoglobin genes is well studied; however, little is known about the genomic organization and evolution of teleost hemoglobin genes, particularly those of salmonids. The Atlantic salmon serves as a representative salmonid species for genomics studies. Given the well documented role of hemoglobin in adaptation to varied environmental conditions as well as its use as a model protein for evolutionary analyses, an understanding of the genomic structure and organization of the Atlantic salmon α and β hemoglobin genes is of great interest. Results We identified four bacterial artificial chromosomes (BACs comprising two hemoglobin gene clusters spanning the entire α and β hemoglobin gene repertoire of the Atlantic salmon genome. Their chromosomal locations were established using fluorescence in situ hybridization (FISH analysis and linkage mapping, demonstrating that the two clusters are located on separate chromosomes. The BACs were sequenced and assembled into scaffolds, which were annotated for putatively functional and pseudogenized hemoglobin-like genes. This revealed that the tail-to-tail organization and alternating pattern of the α and β hemoglobin genes are well conserved in both clusters, as well as that the Atlantic salmon genome houses substantially more hemoglobin genes, including non-Bohr β globin genes, than the genomes of other teleosts that have been sequenced. Conclusions We suggest that the most parsimonious evolutionary path leading to the present organization of the Atlantic salmon

  12. Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues

    Directory of Open Access Journals (Sweden)

    Fraser Tresa S

    2008-08-01

    Full Text Available Abstract Background The piggyBac transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase (TPase itself. A thorough understanding of just how piggyBac works may lead to more effective use of this important mobile element. A PSORTII analysis of the TPase amino acid sequence predicts a bipartite nuclear localization signal (NLS near the c-terminus, just upstream of a putative ZnF (ZnF. Results We fused the piggyBac TPase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP in the Drosophila melanogaster inducible metallothionein protein. Using Drosophila Schneider 2 (S2 cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of piggyBac localization with a scanning confocal microscope 48 hours after induction with copper sulphate. Conclusion Through n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the piggyBac TPase open reading frame, we found that not only is the PSORTII-predicted NLS required for the TPase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this region for nuclear localization.

  13. Physical location of rice Gm-6,Pi-5(t) genes in O. officinalis with BAC-FISH

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A fluorescence in situ hybridization (FISH) procedure was adoptedto physically map two rice BAC clones 24E21 and 4F22 linked to Gm-6 and Pi-5(t) in O. officinalis. FISH results showed that the two BAC clones were located at 4L. The percentage distance from the centromere to the hybridization sites was 72±2.62 for 24E21 and 54±5.43 for 4F22, the detection rates were 52.70% and 61.2%. The results obtained from the BAC and plasmid clones, RG214 and RZ565 of cultivated rice and O. officinalis were the same. This suggested that the markers, RG214 and RZ565 of cultivated rice and O. officinalis were in the same BAC clones. The homologous sequences of Gm-6 and Pi-5(t) in O. officinalis were positions that signals existed on the 4L. Many signals were observed when no Cot-1 DNA blocked. This also showed that repetitive sequences were some homolgous between cultivated rice and O. officinalis. The identification of chromosome 4 of O. officinalis is based on Jena et al. (1994). In our study, we discussed the possibility of physical map in O. officinalis with rice BAC clones.

  14. Expression of Human CAR Splicing Variants in BAC-Transgenic Mice

    OpenAIRE

    Zhang, Yu-Kun Jennifer; Lu, Hong; Klaassen, Curtis D.

    2012-01-01

    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 wel...

  15. Reframing Nationality through Local and Regional Social Practices – Europe Direct Bacău Relay

    Directory of Open Access Journals (Sweden)

    Camelia Cmeciu

    2009-06-01

    Full Text Available Paraphrasing the famous European syntagm “unity in diversity”, we will introduce anothersyntagm, namely “nationality in diversity”, thus laying an emphasis on the strategies (social practicesadopted by each of the thirty one Europe Direct relays in Romania in order to achieve their goals.Having as theoretical background the four semiotic systems (represented participants, interactiveparticipants, composition, modality of social semiotics (van Leeuwen, Kress, 2001, 2005, we willinterpret the 2009 campaign promoted by Europe Direct Bacău as an alternative interweaving ofcognitive, affective and behavioural effects.

  16. Germline transformation of Aedes fluviatilis (Diptera:Culicidae) with the piggyBac transposable element.

    Science.gov (United States)

    Rodrigues, Flávia Guimarães; Oliveira, Sabrina Barbosa; Rocha, Bruno Coelho; Moreira, Luciano Andrade

    2006-11-01

    The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz), which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors. PMID:17160283

  17. Germline transformation of Aedes fluviatilis (Diptera:Culicidae with the piggyBac transposable element

    Directory of Open Access Journals (Sweden)

    Flávia Guimarães Rodrigues

    2006-11-01

    Full Text Available The technique to generate transgenic mosquitoes requires adaptation for each target species because of aspects related to species biology, sensitivity to manipulation and rearing conditions. Here we tested different parameters on the microinjection procedure in order to obtain a transgenic Neotropical mosquito species. By using a transposon-based strategy we were able to successfully transform Aedes fluviatilis (Lutz, which can be used as an avian malaria model. These results demonstrate the usefulness of the piggyBac transposable element as a transformation vector for Neotropical mosquito species and opens up new research frontiers for South American mosquito vectors.

  18. Reprogramming of mouse amniotic fluid cells using a PiggyBac transposon system

    Directory of Open Access Journals (Sweden)

    E. Bertin

    2015-11-01

    Full Text Available Induced pluripotent stem (iPS cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Amniotic fluid (AF cells are easy to obtain from routinely scheduled procedures for prenatal diagnosis and iPS cells have been generated from human AF. Here, we generated iPS cells from mouse AF cells, using a non-viral-based approach constituted by the PiggyBac (PB transposon system. All iPS cell lines obtained exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo.

  19. Genetic and physiological architecture of early vigor in Aegilops tauschii, the D-genome of hexaploid wheat. A quantitative trait loci analysis

    NARCIS (Netherlands)

    Steege, ter M.W.; Ouden, den F.M.; Lambers, H.; Stam, P.; Peeters, A.J.M.

    2005-01-01

    Plant growth can be studied at different organizational levels, varying from cell, leaf, and shoot to the whole plant. The early growth of seedlings is important for the plant's establishment and its eventual success. Wheat (Triticum aestivum, genome AABBDD) seedlings exhibit a low early growth rate

  20. Effect of 2,4-D on Seedling Physiology and Cytogenetical Studies in Triticum aestivum and Phalaris minor ( Gramineae )%Effect of 2,4-D on Seedling Physiology and Cytogenetical Studies in Triticum aestivum and Phalaris minor (Gramineae)

    Institute of Scientific and Technical Information of China (English)

    Muhammad Rashid Khan; Khawaja Muhammad Aslam

    2006-01-01

    Effects of 2,4-D on seedling growth and chromosomal abnormalities were studied in Triticum aestivum and Phalaris minor. Seeds were soaked at different concentrations of 2,4 -D (0.01%, 0.1%, 1.0% ) for 4, 8, 12 and 16 hours. 2,4-D suppressed the germination more severely in P. minor than in T. aestivum. Shoot and root length was retarded with the increase of concentration and time of treatment in both species. Generally radical was more negatively affected than coleoptile and emergence of radical was not observed at 1.0% concentration at 8, 12, and 16 hours of treatment in T. aestivum while in P. minor there was a total lack of radical emergence at 1.0% concentration for all durations of treatment. Stiff and curled roots and undifferentiated callus like scutellar tissues were observed in T. aestivum, while in P. minor the coleoptile obtained was lean, pale green in colour and was lying flat on filter paper. Mitotic index decreased, while chromosomal abnormalities, bridges and laggards were increased with the increase of concentration and soaking time however, laggards were not observed in T. aestivum. Clumping and chain formation of chromosomes at metaphase was also noticed in P. minor.

  1. Quantitative Traits of Ion Beam Induced Mutagenesis in Triticum aestivum

    Institute of Scientific and Technical Information of China (English)

    Huan FANG; Zhen JIAO

    2012-01-01

    [Objective] The aim of this study was to elucidate the quantitative traits of plants mutagenized by ion beam. [Method] The particular variation phenotypes, a- gronomic traits, and protein and wet gluten contents of progenies derived from the same ion beam induced mutant were investigated. [Result] Morphological polymor- phism existed in some individuals. Plant height, spike length and protein content were significantly influenced by ion beam, and effective tiller number and wet gluten content were moderately influenced. Multiple comparisons of all the indices within groups indicated genomic instability among these groups. Coefficient of variation im- plied the differences within group were very low. [Conclusion] Ion beam irradiation displayed characteristics of multi-directivity and non-directiveness. It aroused multiple variations in the same mutant. Instability among progeny indicates cells had different fate even in the same irradiated tissue. It may take several generations for mutants to stabilize particular phenotypes. The effects of ion beam irradiation may be the in- terrelated direct irradiation damage, indirect irradiation damage and late effect, such as bystander effect and adaptive response.

  2. The Varicella-Zoster Virus Genome

    OpenAIRE

    Cohen, Jeffrey I.

    2010-01-01

    The varicella-zoster virus (VZV) genome contains at least 70 genes, and all but 6 have homologs in herpes simplex virus. Cosmids and BACs corresponding to the VZV parental Oka and vaccine Oka viruses have been used to “knock-out” 34 VZV genes. Seven VZV genes (ORF4, 5, 9, 21, 29, 62, and 68) have been shown to be required for growth in vitro. Recombinant viruses expressing several markers (e.g. beta-galactosidase, green fluorescence protein, luciferase) and several foreign viral genes (from h...

  3. An innovative process to improve turbidity and Organics Removal by BAC filters

    Science.gov (United States)

    Miao, Jia; Zhao, Qingliang; Wang, Baozhen; Li, Ji; Zhang, Jinsong

    2006-10-01

    The turbidity criterion for the product water of a WTP according to the State Project ‘863’ on the safeguard technology of drinking water in the southern areas of China is 0.1 NTU. The turbidity removal in the activated carbon filter was analyzed in a pilot-scale test and an innovative technology to improve the turbidity removal in a biologically activated carbon (BAC) filter was put forward in order to meet the criterion. Experimental results showed that the enhanced filtration by adding polymerized aluminium chloride (PAC) into the BAC filter was quite effective in turbidity control. The effluent turbidity was kept at a stable level (mean) of 0.033 NTU with a high removal of about 80% for influent turbidity of 0.110 0240 NTU with an addition of PAC at 0.05 mg L-1, meeting the requirement for filtrate turbidity equal to or less than 0.1 NTUC totally. In addition, the larger the PAC dosage was, the lower the effluent turbidity was. However, further improvement of turbidity removal was not obvious for PAC dosages beyond 0.l0 mg L-, and an optimal PAC dosage in the range of 0.05 0.10 mg L- was proposed.

  4. An Innovative Process to Improve Turbidity and Organics Removal by BAC Filters

    Institute of Scientific and Technical Information of China (English)

    MIAO Jia; ZHAO Qingliang; WANG Baozhen; LI Ji; ZHANG Jinsong

    2006-01-01

    The turbidity criterion for the product water of a WTP according to the State Project ‘863' on the safeguard technology of drinking water in the southern areas of China is 0.1 NTU. The turbidity removal in the activated carbon filter was analyzed in a pilot-scale test and an innovative technology to improve the turbidity removal in a biologically activated carbon (BAC) filter was put forward in order to meet the criterion. Experimental results showed that the enhanced filtration by adding polymerized aluminium chloride (PAC) into the BAC filter was quite effective in turbidity control. The effluent turbidity was kept at a stable level (mean) of 0.033 NTU with a high removal of about 80% for influent turbidity of 0.110 -0240 NTU with an addition of PAC at 0.05 mg L-1, meeting the requirement for filtrate turbidity equal to or less than 0.1NTUC totally. In addition, the larger the PAC dosage was, the lower the effluent turbidity was. However, further improvement of turbidity removal was not obvious for PAC dosages beyond 0.10 mg L- 1, and an optimal PAC dosage in the range of 0.05 - 0.10 mg L- 1 was proposed.

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

    Directory of Open Access Journals (Sweden)

    Xu Rener

    2011-01-01

    Full Text Available Abstract Background ALPK1 (α-kinase 1 is a member of an unconventional alpha-kinase family, and its biological function remains largely unknown. Here we report the phenotypic characterization of one mutant line, in which the piggyBac (PB transposon is inserted into the Alpk1 gene. Results The piggyBac(PB insertion site in mutants was mapped to the first intron of the Alpk1 gene, resulting in the effective disruption of the intact Alpk1 transcript expression. The transposon-inserted Alpk1 homozygous mutants (Alpk1PB/PB displayed severe defects in motor coordination in a series of behavioral analysis, including dowel test, hanging wire test, rotarod analysis and footprint analysis. However, the cerebellar architecture, Purkinje cell morphology and electrophysiology of the Purkinje cells appeared normal in mutants. The motor coordination deficits in the Alpk1PB/PB mice were rescued by transgenic mice expressing the full-length Alpk1-coding sequence under the control of the ubiquitous expression promoter. Conclusions Our results indicate that ALPK1 plays an important role in the regulation of motor coordination. Alpk1PB/PB mice would be a useful model to provide a clue to the better understanding of the cellular and molecular mechanisms of ALPK1 in the control of fine motor activities.

  6. Characterization of Porcine Endogenous Retrovirus Clones from the NIH Miniature Pig BAC Library

    Directory of Open Access Journals (Sweden)

    Seong-Lan Yu

    2012-01-01

    Full Text Available 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.

  7. Aerobic biodegradation of a sulfonated phenylazonaphthol dye by a bacterial community immobilized in a multistage packed-bed BAC reactor.

    Science.gov (United States)

    Ruiz-Arias, Alfredo; Juárez-Ramírez, Cleotilde; de los Cobos-Vasconcelos, Daniel; Ruiz-Ordaz, Nora; Salmerón-Alcocer, Angélica; Ahuatzi-Chacón, Deifilia; Galíndez-Mayer, Juvencio

    2010-11-01

    A microbial community able to aerobically degrade the azo dye Acid Orange 7 was selected from riparian or lacustrine sediments collected at sites receiving textile wastewaters. Three bacterial strains, pertaining to the genera Pseudomonas, Arthrobacter, and Rhizobium, constitute the selected community. The biodegradation of AO7 was carried out in batch-suspended cell culture and in a continuously operated multistage packed-bed BAC reactor. The rapid decolorization observed in batch culture, joined to a delay of about 24 h in COD removal and cell growth, suggests that enzymes involved in biodegradation of the aromatic amines generated after AO7 azo-bond cleavage (1-amino-2-naphthol [1-A2N] and 4-aminobenzenesulfonic acid [4-ABS]), are inducible in this microbial consortium. After this presumptive induction period, the accumulated byproducts, measured through COD, were partially metabolized and transformed in cell mass. At all azo dye loading rates used, complete removal of AO7 and 1-A2N was obtained in the multistage packed-bed BAC reactor (PBR).; however, the overall COD (eta ( COD )) and 4-ABS (eta ( ABS )) removal efficiencies obtained in steady state continuous culture were about 90%. Considering the toxicity of 1-A2N, its complete removal has particular relevance. In the first stages of the packed-bed BAC reactor (Fig. 4a-c), major removal was observed. In the last stage, only a slight removal of COD and 4-ABS was obtained. Comparing to several reported studies, the continuously operated multistage packed-bed BAC reactor showed similar or superior results. In addition, the operation of large-packed-bed BAC reactors could be improved by using several shallow BAC bed stages, because the pressure drop caused by bed compaction of a support material constituted by small and fragile particles can be reduced.

  8. Endosperm Tolerance of Paternal Aneuploidy Allows Radiation Hybrid Mapping of the Wheat D-Genome and a Measure of γ Ray-Induced Chromosome Breaks

    OpenAIRE

    Tiwari, Vijay K; Oscar Riera-Lizarazu; Hilary L Gunn; Kasandra Lopez; M Javed Iqbal; Kianian, Shahryar F; Leonard, Jeffrey M.

    2012-01-01

    Physical mapping and genome sequencing are underway for the ≈17 Gb wheat genome. Physical mapping methods independent of meiotic recombination, such as radiation hybrid (RH) mapping, will aid precise anchoring of BAC contigs in the large regions of suppressed recombination in Triticeae genomes. Reports of endosperm development following pollination with irradiated pollen at dosages that cause embryo abortion prompted us to investigate endosperm as a potential source of RH mapping germplasm. H...

  9. Fractionation of Rare Earth Elements in Plants Ⅰ. Fractionation Patterns and Their Forming Mechanisms in Different Organs of Triticum Aestivum

    Institute of Scientific and Technical Information of China (English)

    Liang Tao; Ding Shiming; Zhang Chaosheng; Zhang Zili; Yan Juncai; Li Haitao

    2005-01-01

    Fractionations of rare earth elements (REEs) and the forming mechanisms in plants were studied using Triticum aestivum as plant material with application of exogenous REEs and hydroponic culture. REEs were significantly fractionated in different parts of Triticum aestivum. M-type tetrad effect could be observed in both root and shoot of Triticum aestivum, which might result from the different abilities of REEs to form phosphate precipitation. Middle REEs (MREEs), light REEs (LREEs) and heavy REEs (HREEs) were enriched in root, stem and leaf of Triticum aestivum, respectively. REE speciation calculations using VMINTEQ program show REEs in simulated xylem solution mainly exist as REE-EDTA- and RE3+, but only HREEs are enriched in REE-EDTA-, while LREEs are enriched in the other REE species. It is suggested that the fractionation between LREEs and HREEs might be caused by the uptake of REE-EDTA- in Triticum aestivum leaves, but might result from the uptake of the other REE species in their stems.

  10. Construction of Oryza Sativa genome contigs by fingerprint strategy

    Institute of Scientific and Technical Information of China (English)

    TAOQUAZHOU; GUOFANHONG; 等

    1995-01-01

    We described the construction of BAC contigs of the genome of a indica variety of Oryza sativa.Guang Lu Ai 4. An entire representative(Sixfold coverage of rice chromosomes)and genetically stable BAC library of rice genome constructed in this lab has been systematically analysed by restriction enzyme fragmentation and polyacrylamide gel electrophoresis.And all the images thus obtained were subject to image-processing,which consisted of preliminary location of bands,cooperative tracking of lanes by correlation of adjacent bads.a precise densitometric pass,alignment at the marker bands with the standard,optional interactive editing,and normalization of the accepted bands.The contigs were generated based on the Computer Software specially designed for genome mapping.The number of contigs with 600 kb in length on average was 464.of contigs with 1000kb in length on average was 107; of contigs with 1500 kb in length on average was Construction of Oryza Sativa genome contigs.23.Therefor,all the contigs we have obtained ampunted up to 420 megabases in length.Considering the size of rice genome(430 megabased),the contigs generated in this lab have covered nearly 98% of the rice genome.We are now in the process of mapping the contigs to chromosomes.

  11. Structural and enzymatic characterization of BacD, an l-amino acid dipeptide ligase from Bacillus subtilis

    OpenAIRE

    Shomura, Yasuhito; Hinokuchi, Emi; Ikeda, Hajime; Senoo, Akihiro; Takahashi, Yuichi; Saito, Jun-ichi; Komori, Hirofumi; Shibata,Naoki; Yonetani, Yoshiyuki; Higuchi, Yoshiki

    2012-01-01

    BacD is an ATP-dependent dipeptide ligase responsible for the biosynthesis of l-alanyl-l-anticapsin, a precursor of an antibiotic produced by Bacillus spp. In contrast to the well-studied and phylogenetically related d-alanine: d-alanine ligase (Ddl), BacD synthesizes dipeptides using l-amino acids as substrates and has a low substrate specificity in vitro. The enzyme is of great interest because of its potential application in industrial protein engineering for the environmentally friendly b...

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

    Directory of Open Access Journals (Sweden)

    Navdeep Gill

    2014-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Masahiro Gotoh

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-06-01

    Cowpea (Vigna unguiculata) is an annual legume grown in tropical and subtropical regions, which is economically relevant due to high protein content in dried beans, green pods, and leaves. In this work, a comparative cytogenetic study between V. unguiculata and Phaseolus vulgaris (common bean) was conducted using BAC-FISH. Sequences previously mapped in P. vulgaris chromosomes (Pv) were used as probes in V. unguiculata chromosomes (Vu), contributing to the analysis of macrosynteny between both legumes. Thirty-seven clones from P. vulgaris 'BAT93' BAC library, corresponding to its 11 linkage groups, were hybridized in situ. Several chromosomal rearrangements were identified, such as translocations (between BACs from Pv1 and Pv8; Pv2 and Pv3; as well as Pv2 and Pv11), duplications (BAC from Pv3), as well as paracentric and pericentric inversions (BACs from Pv3, and Pv4, respectively). Two BACs (from Pv2 and Pv7), which hybridized at terminal regions in almost all P. vulgaris chromosomes, showed single-copy signal in Vu. Additionally, 17 BACs showed no signal in V. unguiculata chromosomes. The present results demonstrate the feasibility of using BAC libraries in comparative chromosomal mapping and karyotype evolution studies between Phaseolus and Vigna species, and revealed several macrosynteny and collinearity breaks among both legumes. PMID:25634499

  15. Responses of alloplasmic (cytoplasm=Triticum timopheevii) and euplasmic wheats (Triticum aestivum) to photoperiod and vernalization.

    Science.gov (United States)

    Ward, R W; Heyne, E G; Paulsen, G M

    1983-07-01

    Studies were conducted to determine the influence of the male sterility-inducing cytoplasm of Triticum timopheevii (Zhuk.) Zhuk. on response of several common winter wheat (T. aestivum L.) nuclear genotypes to photoperiod and vernalization. Comparative studies of cytoplasmic substitution lines provide information on the role of the cytoplasmic genetic mechanism in growth and development. In the case of cytoplasmic male sterility-based hybrid production systems, ubiquity of sterility-inducing cytoplasm in derived hybrids warrants thorough characterization of its influence on plant phenotype. Factorial combinations of cytoplasm (T. timopheevii and T. aestivum), nuclear genotype, and photoperiod or vernalization treatments were evaluated under hydroponic conditions in controlled environment chambers. Interaction of cytoplasm, photoperiod, and nuclear genotype was significant in one or more experiments for days to anthesis and potential spikelet number, and interaction of cytoplasm, vernalization, and nuclear genotype was significant for days to spike emergence. Long day length was associated with increased percentage seed set in one study, but interactions of photoperiod and cytoplasm were not detected for percentage seed set. Interactions involving cytoplasm and photoperiod or vernalization were interpreted as evidence of the existence of genetic factors in cytoplsam of T. timopheevii which alter photoperiod or vernalization responses of alloplasmic plants relative to responses exhibited by euplasmic plants. Since photoperiod and vernalization responses are critical to adaptation, T. timopheevii cytoplasm can alter adaptability of T. aestivum. The specific effect would be nuclear genotype dependent, and does not appear to be of a magnitude greater than that induced by nuclear genetic variability at loci conditioning photoperiod or vernalization responses or other adaptation-determining characteristics. Normal multilocation/year testing of alloplasmic hybrids should

  16. DIVERSIDAD GENÉTICA DE GLUTENINAS Y GLIADINAS EN TRIGOS HARINEROS (Triticum aestivum L.) MEXICANOS

    OpenAIRE

    Eliel Martínez-Cruz; Eduardo Espitia-Rangel; Héctor E. Villaseñor-Mir; José D. Molina-Galán; Ignacio Benítez-Riquelme; Amalio Santacruz-Varela; Roberto J. Peña-Bautista

    2010-01-01

    Las gluteninas y las gliadinas del trigo harinero (Triticum aestivum L.) tienen una función fundamental en la definición de la calidad de panificación. Con el objetivo de caracterizar la composición de las subunidades de gluteninas de alto (G-APM) y bajo (G-BPM) peso molecular, y de las omega;-gliadinas, en 72 progenitores usados por el programa de fitomejoramiento de trigo harinero para temporal del Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (CEVAMEX-INIFAP) y en...

  17. The effect of foliar fungicides on the mycoflora of seeds of Triticum aestivum

    Directory of Open Access Journals (Sweden)

    Janusz Błaszkowski

    2014-08-01

    Full Text Available The effect of three foliar fungicides. i.e., Bayloton 25 WP, Dithane M-45, and Funaben K. on the mycoflora associated with the seeds of spring Triticum aestivum cv. Kolibri cultivated in the field was investigated. The fungicide which highly reduced the number of both fungal colonies and species was Funaben K. Of the fungi most frequently occurring. Only Funaben K applied on the seeds reduced the proportion of seeds with Alternaria alternata, Cladosporium spp.. and Septoria nodorum. In contrast. seeds from plants traeted with Funaben K harboured significantly more colonies of non-sporulating fungi.

  18. Effects of high temperature after anthesis on starch granules in grains of wheat (Triticum aestivum L.)

    OpenAIRE

    Liu, P.; Guo, W; Jiang, Z.; Pu, H.; Feng, C.; Zhu, X.; Peng, Y.; KUANG, A.; LITTLE, C. R.

    2010-01-01

    SUMMARY The effect of high temperatures (above 25°C) on starch concentration and the morphology of starch granules in the grains of wheat (Triticum aestivum L.) were studied. Wheat plants of cultivars Yangmai 9 (weak-gluten) and Yangmai 12 (medium-gluten) were treated with high temperatures for 3 days at different times after anthesis. The results showed that the starch concentration of grains given a heat-shock treatment above 30°C were lower than those developing at normal temperature in bo...

  19. Ectomycorrhizal communities above and below ground and truffle productivity in a Tuber aestivum orchard

    OpenAIRE

    Elena Salerni; Maria D'Aguanno; Pamela Leonardi; Claudia Perini

    2014-01-01

    Aim of study: The diversity of ectomycorrhizal fungal communities (EM) above (EMFb) and below (EMMt) ground associated with Quercus cerris L., Q. pubescens Willd., and Pinus nigra J.F.Arnold was analyzed.Area of study: A 20 year-old orchard that produces Tuber aestivum truffles, located a few kilometers from Chiusi della Verna (latitude 43° 41’ 53’’; longitude 11° 56’ 9’’) in Tuscany (central Italy) was observed.Material and Methods: This investigation combined analyses of EMFb, EMMt, T. aest...

  20. 转座子PiggyBac在哺乳动物中的应用%PiggyBac Transposon for Mammalian Applications

    Institute of Scientific and Technical Information of China (English)

    马元武; 张连峰

    2012-01-01

    As a tool of genetic engineering, DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Till now, transposons active in mammalian include; 1) hAT-like Tol2; 2) , two Te1-like transposons, Sleeping Beauty (SB) and Frog Prince ( FP) ; 3) PiggyBac family. Among those transposons, the moth-derived transposon piggybac appears to be the most promising genetic tool due to its higher efficient transposition and higher cargo capacity. Therefore, it can be used for transgenesis, discovery of cancer gene and tumor suppressor gene, and gene therapy. The traceless excision transposition property of the PB transposon can be used to generate transgene-free iPS cells while maintaining an unaltered genome. In this review we will discuss the application and the future of PB transposon in mammalian.%DNA转座子作为一种遗传工程工具已广泛应用于多物种的转基因及产生插入突变等研究.目前,在哺乳动物中有转座活性的转座子可分为三类:1)hAT样转座子;2)Tcl样转座子包括Sleeping Beauty和Frog Prince;3) PiggyBac转座子家族.其中甘蓝蠖度尺蛾(Cabbage looper moth Trichoplusia ni)来源的PiggyBac转座子是目前在哺乳动物中活性最高的转座子,并且可以携带十几kb的外源基因转座而不影响其效率,使其在哺乳动物的转基因、癌基因的发现、基因治疗研究方面具有巨大的应用潜力.此外,PB的无痕迹转座对于无转基因、无遗传物质改变的诱导多潜能干细胞(iPS)研究也具有非常重要的意义.本文主要对针对PB在哺乳动物中的应用现状及前景作一介绍.

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

    Directory of Open Access Journals (Sweden)

    Halder Georg

    2008-07-01

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

  2. The impact of Ty3-gypsy group LTR retrotransposons Fatima on B-genome specificity of polyploid wheats

    Directory of Open Access Journals (Sweden)

    Huneau Cecile

    2011-06-01

    Full Text Available Abstract Background Transposable elements (TEs are a rapidly evolving fraction of the eukaryotic genomes and the main contributors to genome plasticity and divergence. Recently, occupation of the A- and D-genomes of allopolyploid wheat by specific TE families was demonstrated. Here, we investigated the impact of the well-represented family of gypsy LTR-retrotransposons, Fatima, on B-genome divergence of allopolyploid wheat using the fluorescent in situ hybridisation (FISH method and phylogenetic analysis. Results FISH analysis of a BAC clone (BAC_2383A24 initially screened with Spelt1 repeats demonstrated its predominant localisation to chromosomes of the B-genome and its putative diploid progenitor Aegilops speltoides in hexaploid (genomic formula, BBAADD and tetraploid (genomic formula, BBAA wheats as well as their diploid progenitors. Analysis of the complete BAC_2383A24 nucleotide sequence (113 605 bp demonstrated that it contains 55.6% TEs, 0.9% subtelomeric tandem repeats (Spelt1, and five genes. LTR retrotransposons are predominant, representing 50.7% of the total nucleotide sequence. Three elements of the gypsy LTR retrotransposon family Fatima make up 47.2% of all the LTR retrotransposons in this BAC. In situ hybridisation of the Fatima_2383A24-3 subclone suggests that individual representatives of the Fatima family contribute to the majority of the B-genome specific FISH pattern for BAC_2383A24. Phylogenetic analysis of various Fatima elements available from databases in combination with the data on their insertion dates demonstrated that the Fatima elements fall into several groups. One of these groups, containing Fatima_2383A24-3, is more specific to the B-genome and proliferated around 0.5-2.5 MYA, prior to allopolyploid wheat formation. Conclusion The B-genome specificity of the gypsy-like Fatima, as determined by FISH, is explained to a great degree by the appearance of a genome-specific element within this family for Ae

  3. Variability of blood alcohol content (BAC) determinations: the role of measurement uncertainty, significant figures, and decision rules for compliance assessment in the frame of a multiple BAC threshold law.

    Science.gov (United States)

    Zamengo, Luca; Frison, Giampietro; Tedeschi, Gianpaola; Frasson, Samuela; Zancanaro, Flavio; Sciarrone, Rocco

    2014-10-01

    The measurement of blood-alcohol content (BAC) is a crucial analytical determination required to assess if an offence (e.g. driving under the influence of alcohol) has been committed. For various reasons, results of forensic alcohol analysis are often challenged by the defence. As a consequence, measurement uncertainty becomes a critical topic when assessing compliance with specification limits for forensic purposes. The aims of this study were: (1) to investigate major sources of variability for BAC determinations; (2) to estimate measurement uncertainty for routine BAC determinations; (3) to discuss the role of measurement uncertainty in compliance assessment; (4) to set decision rules for a multiple BAC threshold law, as provided in the Italian Highway Code; (5) to address the topic of the zero-alcohol limit from the forensic toxicology point of view; and (6) to discuss the role of significant figures and rounding errors on measurement uncertainty and compliance assessment. Measurement variability was investigated by the analysis of data collected from real cases and internal quality control. The contribution of both pre-analytical and analytical processes to measurement variability was considered. The resulting expanded measurement uncertainty was 8.0%. Decision rules for the multiple BAC threshold Italian law were set by adopting a guard-banding approach. 0.1 g/L was chosen as cut-off level to assess compliance with the zero-alcohol limit. The role of significant figures and rounding errors in compliance assessment was discussed by providing examples which stressed the importance of these topics for forensic purposes.

  4. Development of Health Education Learning Module in Bac.TSE-LDPE Programme in TTI: Needs Analysis Study

    Science.gov (United States)

    Ujang, Alijah; Alias, Norlidah; Siraj, Saedah

    2015-01-01

    This study is to explore the need to develop learning modules of health education for trainee teachers in the Bachelor Of Teaching (Hons)(Special Education-Learning Disabilities For Primary Education) Programme (Bac.TSE-LDPE) in the Teacher Training Institute (TTI). The questionnaire uses the Likert scale with the close ended questions analysed by…

  5. BacT/ALERT 3D检测系统在结核病诊治中的运用

    Institute of Scientific and Technical Information of China (English)

    刘子龙; 曹剑昆

    2010-01-01

    目的 评估BacT/ALERT 3D仪在结核病诊治中的运用价值.方法 采用BacT/ALERT 3D仪对432份临床标本进行分支杆菌快速检测,阳性者进行分支杆菌菌型鉴定和药敏试验,并与罗氏培养法进行比较.结果 BacT/ALERT 3D快速培养分支杆菌的阳性率为38.2%,明显高于罗氏法的28.0%(P<0.01);培养报告时间仅14.24 d,明显短于罗氏法的29.61 d;涂片阳性及阴性组快速培养的阳性率均高于罗氏法(P<0.01).结论 BacT/ALERT 3D快速培养检测系统有助于结核病的诊断和治疗,是一种快速、有效的检测方法.

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

    Science.gov (United States)

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

  7. Enriched Oxygen BAC Method in Advanced Treatment of Textile Dyeing-printing & Alkali-peeling Wastewater

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A novel Pressurized Enriched Oxygen Biological Activated Carbon (PRBAC) method in treating secondary effluent of textile dying-printing & alkali peeling wastewater was configured. The PRBAC reactor simply increased reactor pressure to create an enriched dissolved oxygen (DO) environment to stimulate the bioactivities of microbes on GAC surface for removing refractory organic matter. Rapid SmallScaled Column Test (RSSCT) was carried out to evaluate the adsorption characteristics of target stream constituents, and over 80% COD components were poorly adsorbable while about 82.5% color inducing matter and 85% UV254 surrogated matter were readily adsorbable. Compared with performances of normal BAC reactor under conventional DO condition, PRBAC achieved 20%, 10% and 50% more removal in COD, color and NH3-N abatement.

  8. NIG_MoG: a mouse genome navigator for exploring intersubspecific genetic polymorphisms.

    Science.gov (United States)

    Takada, Toyoyuki; Yoshiki, Atsushi; Obata, Yuichi; Yamazaki, Yukiko; Shiroishi, Toshihiko

    2015-08-01

    The National Institute of Genetics Mouse Genome database (NIG_MoG; http://molossinus.lab.nig.ac.jp/msmdb/) primarily comprises the whole-genome sequence data of two inbred mouse strains, MSM/Ms and JF1/Ms. These strains were established at NIG and originated from the Japanese subspecies Mus musculus molossinus. NIG_MoG provides visualized genome polymorphism information, browsing single-nucleotide polymorphisms and short insertions and deletions in the genomes of MSM/Ms and JF1/Ms with respect to C57BL/6J (whose genome is predominantly derived from the West European subspecies M. m. domesticus). This allows users, especially wet-lab biologists, to intuitively recognize intersubspecific genome divergence in these mouse strains using visual data. The database also supports the in silico screening of bacterial artificial chromosome (BAC) clones that contain genomic DNA from MSM/Ms and the standard classical laboratory strain C57BL/6N. NIG_MoG is thus a valuable navigator for exploring mouse genome polymorphisms and BAC clones that are useful for studies of gene function and regulation based on intersubspecific genome divergence.

  9. Comparative genomics of chondrichthyan Hoxa clusters

    Directory of Open Access Journals (Sweden)

    Zhong Ying-Fu

    2009-09-01

    Full Text Available Abstract Background The chondrichthyan or cartilaginous fish (chimeras, sharks, skates and rays occupy an important phylogenetic position as the sister group to all other jawed vertebrates and as an early lineage to diverge from the vertebrate lineage following two whole genome duplication events in vertebrate evolution. There have been few comparative genomic analyses incorporating data from chondrichthyan fish and none comparing genomic information from within the group. We have sequenced the complete Hoxa cluster of the Little Skate (Leucoraja erinacea and compared to the published Hoxa cluster of the Horn Shark (Heterodontus francisci and to available data from the Elephant Shark (Callorhinchus milii genome project. Results A BAC clone containing the full Little Skate Hoxa cluster was fully sequenced and assembled. Analyses of coding sequences and conserved non-coding elements reveal a strikingly high level of conservation across the cartilaginous fish, with twenty ultraconserved elements (100%,100 bp found between Skate and Horn Shark, compared to three between human and marsupials. We have also identified novel potential non-coding RNAs in the Skate BAC clone, some of which are conserved to other species. Conclusion We find that the Little Skate Hoxa cluster is remarkably similar to the previously published Horn Shark Hoxa cluster with respect to sequence identity, gene size and intergenic distance despite over 180 million years of separation between the two lineages. We suggest that the genomes of cartilaginous fish are more highly conserved than those of tetrapods or teleost fish and so are more likely to have retained ancestral non-coding elements. While useful for isolating homologous DNA, this complicates bioinformatic approaches to identify chondrichthyan-specific non-coding DNA elements

  10. Dicty_cDB: VHA155 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available e 1061005864011 3', genomic survey sequence. 46 1.6 1 DQ767612 |DQ767612.1 Triticum aestivum clone BAC TA105...go maydis genomic clone 1204168, genomic survey sequence. 44 6.2 1 DX756422 |DX756422.1 2365937 VV03 Ustilag...o maydis genomic clone 1025158, genomic survey sequence. 44 6.2 1 DX733440 |DX733...440.1 2315736 VV03 Ustilago maydis genomic clone 1006812, genomic survey sequence. 44 6.2 1 DX722040 |DX7220...40.1 2310775 VV03 Ustilago maydis genomic clone 1002065, genomic survey sequence. 44 6.2 1 DX660090 |DX66009

  11. What’s in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

    Science.gov (United States)

    Mannhaupt, Gertrud; Montrone, Corinna; Haase, Dirk; Mewes, H. Werner; Aign, Verena; Hoheisel, Jörg D.; Fartmann, Berthold; Nyakatura, Gerald; Kempken, Frank; Maier, Josef; Schulte, Ulrich

    2003-01-01

    The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes. PMID:12655011

  12. Novel SSR markers from BAC-end sequences, DArT arrays and a comprehensive genetic map with 1,291 marker loci for chickpea (Cicer arietinum L..

    Directory of Open Access Journals (Sweden)

    Mahendar Thudi

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

  13. Tethering of the conserved piggyBac transposase fusion protein CSB-PGBD3 to chromosomal AP-1 proteins regulates expression of nearby genes in humans.

    Directory of Open Access Journals (Sweden)

    Lucas T Gray

    2012-09-01

    Full Text Available The CSB-PGBD3 fusion protein arose more than 43 million years ago when a 2.5-kb piggyBac 3 (PGBD3 transposon inserted into intron 5 of the Cockayne syndrome Group B (CSB gene in the common ancestor of all higher primates. As a result, full-length CSB is now coexpressed with an abundant CSB-PGBD3 fusion protein by alternative splicing of CSB exons 1-5 to the PGBD3 transposase. An internal deletion of the piggyBac transposase ORF also gave rise to 889 dispersed, 140-bp MER85 elements that were mobilized in trans by PGBD3 transposase. The CSB-PGBD3 fusion protein binds MER85s in vitro and induces a strong interferon-like innate antiviral immune response when expressed in CSB-null UVSS1KO cells. To explore the connection between DNA binding and gene expression changes induced by CSB-PGBD3, we investigated the genome-wide DNA binding profile of the fusion protein. CSB-PGBD3 binds to 363 MER85 elements in vivo, but these sites do not correlate with gene expression changes induced by the fusion protein. Instead, CSB-PGBD3 is enriched at AP-1, TEAD1, and CTCF motifs, presumably through protein-protein interactions with the cognate transcription factors; moreover, recruitment of CSB-PGBD3 to AP-1 and TEAD1 motifs correlates with nearby genes regulated by CSB-PGBD3 expression in UVSS1KO cells and downregulated by CSB rescue of mutant CS1AN cells. Consistent with these data, the N-terminal CSB domain of the CSB-PGBD3 fusion protein interacts with the AP-1 transcription factor c-Jun and with RNA polymerase II, and a chimeric CSB-LacI construct containing only the N-terminus of CSB upregulates many of the genes induced by CSB-PGBD3. We conclude that the CSB-PGBD3 fusion protein substantially reshapes the transcriptome in CS patient CS1AN and that continued expression of the CSB-PGBD3 fusion protein in the absence of functional CSB may affect the clinical presentation of CS patients by directly altering the transcriptional program.

  14. 普通小麦-簇毛麦易位系T6BS·6BL-2VS的选育%Identification of Triticum aestivum-Haynaldia villosa Translocation Line T6BS·6BL-2VS

    Institute of Scientific and Technical Information of China (English)

    陈全战; 张边江; 周峰; 吴梅; 李华春

    2008-01-01

    [Objective] The aim of experiment was to provide a new germplasm for wheat breeding by further using desirable genes in 2V chromosome of Haynaldia villosa.[Method] Through hybridization between common wheat(Triticum aestivum)-Haynaldia villosa disomic substitution line and common wheat Nonglin26-3C chromosome of Aegilops triuncialis disomic addition line, the analysis methods such as chromosome C-banding, genomic in situ hybridization and molecular marker technique were comprehensively applied and combined characters investigation.[Result] The wheat-Haynaldia villosa translocation line(T6BS·6BL-2VS) was selected from hybrid progenies to conduct characters investigation,which found some bristles on glume ridge of T6BS·6BL-2VS.[Conclusion] The translocation line induced by gametocidal chromosome was a small segment translocation line and the gene of bristle on glume ridge of Haynaldia villosa was located between the middle and the terminal of 2VS.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1999-03-24

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

  16. Structure, variation and expression analysis of glutenin gene promoters from Triticum aestivum cultivar Chinese Spring shows the distal region of promoter 1Bx7 is key regulatory sequence.

    Science.gov (United States)

    Wang, Kai; Zhang, Xue; Zhao, Ying; Chen, Fanguo; Xia, Guangmin

    2013-09-25

    In this study, ten glutenin gene promoters were isolated from model wheat (Triticum aestivum L. cv. Chinese Spring) using a genomic PCR strategy with gene-specific primers. Six belonged to high-molecular-weight glutenin subunit (HMW-GS) gene promoters, and four to low-molecular-weight glutenin subunit (LMW-GS). Sequence lengths varied from 1361 to 2,554 bp. We show that the glutenin gene promoter motifs are conserved in diverse sequences in this study, with HMW-GS and LMW-GS gene promoters characterized by distinct conserved motif combinations. Our findings show that HMW-GS promoters contain more functional motifs in the distal region of the glutenin gene promoter (> -700 bp) compared with LMW-GS. The y-type HMW-GS gene promoters possess unique motifs including RY repeat and as-2 box compared to the x-type. We also identified important motifs in the distal region of HMW-GS gene promoters including the 5'-UTR Py-rich stretch motif and the as-2 box motif. We found that cis-acting elements in the distal region of promoter 1Bx7 enhanced the expression of HMW-GS gene 1Bx7. Taken together, these data support efforts in designing molecular breeding strategies aiming to improve wheat quality. Our results offer insight into the regulatory mechanisms of glutenin gene expression.

  17. Molecular characterization of LMW-GS genes from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to exploit the evolution and find novel low-molecular-weight glutenin subunit(LMW-GS)for improvement of common wheat quality,thirteen variants from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum cv.Jinan 177(JN177)and Agropyron elongatum were characterized via genomic PCR.Four clones were pseudogenes because they contained an internal stop codon.The remaining nine variants contained intact open reading frames(ORFs).Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously.However,they have some unique modifications in the structures.For example,EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain.Both EU159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain,but not in the N-conserved domain traditionally.These structural alterations may have positive contributions to wheat flour quality.The results of phylogeny showed that most LMW-GS variances from Ⅱ-12 were homologous to those from parent JN 177 and other wheat lines.The reason for quick evolution of LMW-GS in Ⅱ-12 was discussed.

  18. Next-generation sequencing strategies for characterizing the turkey genome.

    Science.gov (United States)

    Dalloul, Rami A; Zimin, Aleksey V; Settlage, Robert E; Kim, Sungwon; Reed, Kent M

    2014-02-01

    The turkey genome sequencing project was initiated in 2008 and has relied primarily on next-generation sequencing (NGS) technologies. Our first efforts used a synergistic combination of 2 NGS platforms (Roche/454 and Illumina GAII), detailed bacterial artificial chromosome (BAC) maps, and unique assembly tools to sequence and assemble the genome of the domesticated turkey, Meleagris gallopavo. Since the first release in 2010, efforts to improve the genome assembly, gene annotation, and genomic analyses continue. The initial assembly build (2.01) represented about 89% of the genome sequence with 17X coverage depth (931 Mb). Sequence contigs were assigned to 30 of the 40 chromosomes with approximately 10% of the assembled sequence corresponding to unassigned chromosomes (ChrUn). The sequence has been refined through both genome-wide and area-focused sequencing, including shotgun and paired-end sequencing, and targeted sequencing of chromosomal regions with low or incomplete coverage. These additional efforts have improved the sequence assembly resulting in 2 subsequent genome builds of higher genome coverage (25X/Build3.0 and 30X/Build4.0) with a current sequence totaling 1,010 Mb. Further, BAC with end sequences assigned to the Z/W and MG18 (MHC) chromosomes, ChrUn, or not placed in the previous build were isolated, deeply sequenced (Hi-Seq), and incorporated into the latest build (5.0). To aid in the annotation and to generate a gene expression atlas of major tissues, a comprehensive set of RNA samples was collected at various developmental stages of female and male turkeys. Transcriptome sequencing data (using Illumina Hi-Seq) will provide information to enhance the final assembly and ultimately improve sequence annotation. The most current sequence covers more than 95% of the turkey genome and should yield a much improved gene level of annotation, making it a valuable resource for studying genetic variations underlying economically important traits in poultry.

  19. Gene expression, cellular localisation and function of glutamine synthetase isozymes in wheat ( Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Bernard, Stéphanie M; Møller, Anders Laurell Blom; Dionisio, Giuseppe;

    2008-01-01

    We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2...

  20. SALT ACCLIMATION OF TRITICUM-AESTIVUM BY CHOLINE CHLORIDE - PLANT-GROWTH, MINERAL-CONTENT, AND CELL-PERMEABILITY

    NARCIS (Netherlands)

    MANSOUR, MM; STADELMANN, EJ; LEESTADELMANN, OY

    1993-01-01

    Seedlings of a salt sensitive line of Triticum aestivum were grown in Hoagland solution supplemented with 100 mM NaCl following a pretreatment with choline chloride (ChCl). Changes in growth, mineral content of roots and shoots, and passive permeability of the cell membrane were measured. Relative g

  1. TOXICITY OF METHYL-TERT BYTYL ETHER (MTBE) TO PLANTS (AVENA SATIVA, ZEA MAYS, TRITICUM AESTIVUM, AND LACTUCA SATIVA)

    Science.gov (United States)

    Effects of Methyl tert-butyl ether (MTBE) on the germination of seeds and growth of the plant were studied in some laboratory experiments. Test plants were wild oat (Avena sative), sweet corn (Zea mays), wheat (Triticum aestivum), and lettuce (Lactuca sativa). Seed germination,...

  2. Development of T. aestivum L.-H. californicum alien chromosome lines and assignment of homoeologous groups of Hordeum californicum chromosomes.

    Science.gov (United States)

    Fang, Yuhui; Yuan, Jingya; Wang, Zhangjun; Wang, Haiyan; Xiao, Jin; Yang, Zhixi; Zhang, Ruiqi; Qi, Zengjun; Xu, Weigang; Hu, Lin; Wang, Xiu-E

    2014-08-20

    Hordeum californicum (2n = 2x = 14, HH) is resistant to several wheat diseases and tolerant to lower nitrogen. In this study, a molecular karyotype of H. californicum chromosomes in the Triticum aestivum L. cv. Chinese Spring (CS)-H. californicum amphidiploid (2n = 6x = 56, AABBDDHH) was established. By genomic in situ hybridization (GISH) and multicolor fluorescent in situ hybridization (FISH) using repetitive DNA clones (pTa71, pTa794 and pSc119.2) as probes, the H. californicum chromosomes could be differentiated from each other and from the wheat chromosomes unequivocally. Based on molecular karyotype and marker analyses, 12 wheat-alien chromosome lines, including four disomic addition lines (DAH1, DAH3, DAH5 and DAH6), five telosomic addition lines (MtH7L, MtH1S, MtH1L, DtH6S and DtH6L), one multiple addition line involving H. californicum chromosome H2, one disomic substitution line (DSH4) and one translocation line (TH7S/1BL), were identified from the progenies derived from the crosses of CS-H. californicum amphidiploid with common wheat varieties. A total of 482 EST (expressed sequence tag) or SSR (simple sequence repeat) markers specific for individual H. californicum chromosomes were identified, and 47, 50, 45, 49, 21, 51 and 40 markers were assigned to chromosomes H1, H2, H3, H4, H5, H6 and H7, respectively. According to the chromosome allocation of these markers, chromosomes H2, H3, H4, H5, and H7 of H. californicum have relationship with wheat homoeologous groups 5, 2, 6, 3, and 1, and hence could be designated as 5H(c), 2H(c), 6H(c), 3H(c) and 1H(c), respectively. The chromosomes H1 and H6 were designated as 7H(c) and 4H(c), respectively, by referring to SSR markers located on rye chromosomes.

  3. BACs-on-Beads:5所产前诊断实验室的前瞻性研究”点评

    Institute of Scientific and Technical Information of China (English)

    刘鹏飞; 王谢桐

    2012-01-01

    <正>1 原文摘要 Objective We previously reported on the validation of Prenatal BACs-on-Beads TM on retrospectively selected and prospective prenatal samples.This bead-based multiplex assay detects chromosome 13 , 18 , 21and X / Y aneuploidies and the nine most frequent microdeletion syndromes. We demonstrated that Prenatal BACs-on-Beads TM is a newgeneration , prenatal screening tool.Here , we describe the experience of five European prenatal diagnosis laboratories concerning the ongoing use of Prenatal BACs- on-Beads TM . Methods Some 1653samples were analyzed.

  4. Nicotine addiction phenotypes in a BAC transgenic mouse model overexpressing the CHRNA5/A3/B4 genomic cluster

    OpenAIRE

    Molas Casacuberta, Susanna, 1985-

    2012-01-01

    El cluster gen??mic CHRNA5/A3/B4 codifica per les subunitats alfa5, alfa3 i beta4dels receptors d???acetilcolina (nAChRs). Estudis de gen??tica humana han revelat quevariants en aquesta regi?? gen??mica estan significativament associats a la dependencia anicotina. Malauradament, els mecanismes pels quals la sobreexpressi?? d???aquestes tressubunitats influencia comportaments relacionats amb el consum de tabac no s??n del totconeguts. Per tal d???entendre els possibles mecanismes, hem utilitza...

  5. Natural production of Tuber aestivum in central Spain: Pinus spp. versus Quercus spp. brûlés

    Directory of Open Access Journals (Sweden)

    Luis G. Garcia-Montero

    2014-08-01

    Full Text Available Aim of study: Tuber aestivum is the most widespread edible truffle, with increasing commercial interest. This species can produce carpophores with conifer hosts, in contrast with the inability of Pinus spp. to induce fruiting in other truffle species such as Tuber melanosporum. Therefore the objective is to compare the characteristics and carpophore production of T. aestivum brûlés associated with Pinus spp. versus Quercus spp.Area of study: We studied the natural habitats of T. aestivum in the Alto Tajo Nature Reserve in central Spain.Material and methods: During 5 years, we monitored the production of carpophores and brûlé size of 145 T. aestivum brûlés associated with Pinus nigra subsp. salzmanni and P. sylvestris and Quercus ilex subsp. ballota and Q. faginea hosts. Statistical treatment was performed using the Statistica Program v. 6.Main Results: The size of brûlés associated with Pinus was significantly smaller than that of brûlés associated with Quercus. However, carpophore production per brûlé, and especially for brûlés of similar size, was greater when the host plant was a pine. After accounting for brûlé size, the production of brûlés associated with Pinus spp. was 2.23 (95% CI, between 1.35 and 3.69 and 1.61 (95% CI, between 1.02 and 2.54 times greater than the production of brûlés associated with Quercus faginea and Q. ilex subsp. ballota, respectively.Research highlights: The considerable ability of Pinus nigra subsp. salzmanni and P. sylvestris to form effective brûlés and to produce carpophores of Tuber aestivum in natural conditions was clearly demonstrated, and suggest that those species can be of use in the culture of T. aestivum.Key words: Summer truffle; Tuber aestivum; truffle culture; truffle ecology; Pinus spp.; Quercus spp.

  6. Insights into the Bamboo Genome: Syntenic Relationships to Rice and Sorghum

    Institute of Scientific and Technical Information of China (English)

    Yi-Jie Gui; Nai-Xun Ma; Tian-Zhen Zhang; Long-Jiang Fan; Yan Zhou; Yu Wang; Sheng Wang; Sheng-Yue Wang; Yan Hu; Shi-Ping Bo; Huan Chen; Chang-Ping Zhou

    2010-01-01

    Bamboo occupies an important phylogenetic node in the grass family and plays a significant role in the forest industry.We produced 1.2 Mb of tetraploid moso bamboo(Phyllostachys pubescens E.Mazel ex H.de Leh.)sequences from 13 bacterial artificial chromosome(BAC)clones,and these are the largest genomic sequences available so far from the subfamily Bambusoideae.The content of repetitive elements(36.2%)in bamboo is similar to that in rice.Both rice and sorghum exhibit high genomic synteny with bamboo,which suggests that rice and sorghum may be useful as models for decoding Bambusoideae genomes.

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

    Science.gov (United States)

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

    2004-03-01

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

  8. Construction of recombinant pseudorabies viruses by using PRV BACs deficient in IE180 or pac sequences: Application of vBAC90D recombinant virus to production of PRV amplicons.

    Science.gov (United States)

    Lerma, L; Muñoz, A L; Wagner, S; Dinu, M; Martín, B; Tabarés, E

    2016-02-01

    We describe a simple and efficient method to obtain recombinant pseudorabies virus (PRV) in mammalian cells by using the PRV BACs, PBAC80 deficient in pac sequences and PBAC90 deficient in the IE180 gene. These essential viral sequences were used as targets to obtain viable recombinant viruses. PBAC80 was constructed, confirmed to encode a copy of the IE180 gene regulated by the inducible Ptet promoter, and used to obtain recombinant attenuated PRV viruses that express the EGFP protein (PRV-BT80GF virus). PBAC90 was used to obtain the vBAC90D virus, deficient in IE180 and free of replication-competent revertants, and which can be used as a helper in the production of PRV amplicons. PMID:26756577

  9. Integration of Lupinus angustifolius L. (narrow-leafed lupin) genome maps and comparative mapping within legumes.

    Science.gov (United States)

    Wyrwa, Katarzyna; Książkiewicz, Michał; Szczepaniak, Anna; Susek, Karolina; Podkowiński, Jan; Naganowska, Barbara

    2016-09-01

    Narrow-leafed lupin (Lupinus angustifolius L.) has recently been considered a reference genome for the Lupinus genus. In the present work, genetic and cytogenetic maps of L. angustifolius were supplemented with 30 new molecular markers representing lupin genome regions, harboring genes involved in nitrogen fixation during the symbiotic interaction of legumes and soil bacteria (Rhizobiaceae). Our studies resulted in the precise localization of bacterial artificial chromosomes (BACs) carrying sequence variants for early nodulin 40, nodulin 26, nodulin 45, aspartate aminotransferase P2, asparagine synthetase, cytosolic glutamine synthetase, and phosphoenolpyruvate carboxylase. Together with previously mapped chromosomes, the integrated L. angustifolius map encompasses 73 chromosome markers, including 5S ribosomal DNA (rDNA) and 45S rDNA, and anchors 20 L. angustifolius linkage groups to corresponding chromosomes. Chromosomal identification using BAC fluorescence in situ hybridization identified two BAC clones as narrow-leafed lupin centromere-specific markers, which served as templates for preliminary studies of centromere composition within the genus. Bioinformatic analysis of these two BACs revealed that centromeric/pericentromeric regions of narrow-leafed lupin chromosomes consisted of simple sequence repeats ordered into tandem repeats containing the trinucleotide and pentanucleotide simple sequence repeats AGG and GATAC, structured into long arrays. Moreover, cross-genus microsynteny analysis revealed syntenic patterns of 31 single-locus BAC clones among several legume species. The gene and chromosome level findings provide evidence of ancient duplication events that must have occurred very early in the divergence of papilionoid lineages. This work provides a strong foundation for future comparative mapping among legumes and may facilitate understanding of mechanisms involved in shaping legume chromosomes. PMID:27168155

  10. Modelling human regulatory variation in mouse: finding the function in genome-wide association studies and whole-genome sequencing.

    Directory of Open Access Journals (Sweden)

    Jean-François Schmouth

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

  11. Advancing Eucalyptus genomics: cytogenomics reveals conservation of Eucalyptus genomes

    Directory of Open Access Journals (Sweden)

    Teresa Mousinho Resina Ribeiro

    2016-04-01

    Full Text Available The genus Eucalyptus encloses several species with high ecological and economic value, being the subgenus Symphyomyrtus one of the most important. Species such as E. grandis and E. globulus are well characterized at the molecular level but knowledge regarding genome and chromosome organization is very scarce. Here we characterized and compared the karyotypes of three economically important species, E. grandis, E. globulus and E. calmadulensis, and three with ecological relevance, E. pulverulenta, E. cornuta and E. occidentalis, through an integrative approach including genome size estimation, fluorochrome banding, rDNA FISH and BAC landing comprising genes involved in lignin biosynthesis. All karyotypes show a high degree of conservation with pericentromeric 35S and 5S rDNA loci in the first and third pairs, respectively. GC-rich heterochromatin was restricted to the 35S locus while the AT-rich het pattern was species-specific. The slight differences in karyotype formulas and distribution of AT-rich het, along with genome sizes estimations, supports the idea of Eucalyptus genome evolution by local expansions of heterochromatin clusters. The unusual co-localization of both rDNA with AT-rich het was attributed mainly to the presence of silent transposable elements in those loci. The cinnamoyl CoA reductase gene (CCR1 previously assessed to linkage group 10 (LG10 was clearly localized distally at the long arm of chromosome 9 establishing an unexpected correlation between the cytogenetic chromosome 9 and the LG10. Our work is novel and contributes to the understanding of Eucalyptus genome organization which is essential to develop successful advanced breeding strategies for this genus.

  12. Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8′-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition

    Science.gov (United States)

    Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

    2013-01-01

    Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8′-hydroxyase gene which was highly expressed during seed development (TaABA8′OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8′OH1 on the D genome (TaABA8′OH1-D) was identified in Japanese cultivars including ‘Tamaizumi’. However, a single mutation in TaABA8′OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8′OH1 on the A genome (TaABA8′OH1-A), TM1833, was identified from gamma-ray irradiation lines of ‘Tamaizumi’. TM1833 (a double mutant in TaABA8′OH1-A and TaABA8′OH1-D) showed lower TaABA8′OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in ‘Tamaizumi’ (a single mutant in TaABA8′OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8′OH1 may be effective in germination inhibition in field-grown wheat. PMID:23641187

  13. Isolation of a wheat (Triticum aestivum L.) mutant in ABA 8'-hydroxylase gene: effect of reduced ABA catabolism on germination inhibition under field condition.

    Science.gov (United States)

    Chono, Makiko; Matsunaka, Hitoshi; Seki, Masako; Fujita, Masaya; Kiribuchi-Otobe, Chikako; Oda, Shunsuke; Kojima, Hisayo; Kobayashi, Daisuke; Kawakami, Naoto

    2013-03-01

    Pre-harvest sprouting, the germination of mature seeds on the mother plant under moist condition, is a serious problem in cereals. To investigate the effect of reduced abscisic acid (ABA) catabolism on germination in hexaploid wheat (Triticum aestivum L.), we cloned the wheat ABA 8'-hydroxyase gene which was highly expressed during seed development (TaABA8'OH1) and screened for mutations that lead to reduced ABA catabolism. In a screen for natural variation, one insertion mutation in exon 5 of TaABA8'OH1 on the D genome (TaABA8'OH1-D) was identified in Japanese cultivars including 'Tamaizumi'. However, a single mutation in TaABA8'OH1-D had no clear effect on germination inhibition in double haploid lines. In a screen for a mutation, one deletion mutant lacking the entire TaABA8'OH1 on the A genome (TaABA8'OH1-A), TM1833, was identified from gamma-ray irradiation lines of 'Tamaizumi'. TM1833 (a double mutant in TaABA8'OH1-A and TaABA8'OH1-D) showed lower TaABA8'OH1 expression, higher ABA content in embryos during seed development under field condition and lower germination than those in 'Tamaizumi' (a single mutant in TaABA8'OH1-D). These results indicate that reduced ABA catabolism through mutations in TaABA8'OH1 may be effective in germination inhibition in field-grown wheat.

  14. A genetic linkage map with 178 SSR and 1 901 SNP markers constructed using a RIL population in wheat (Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    ZHAI Hui-jie; FENG Zhi-yu; LIU Xin-ye; CHENG Xue-jiao; PENG Hui-ru; YAO Ying-yin; SUN Qi-xin; NI Zhong-fu

    2015-01-01

    The construction of high density genetic linkage map provides a powerful tool to detect and map quantitative trait loci (QTLs) controlling agronomically important traits. In this study, simple sequence repeat (SSR) markers and Illumina 9K iSelect single nucleotide polymorphism (SNP) genechip were employed to construct one genetic linkage map of common wheat (Triticum aestivum L.) using 191 recombinant inbred lines (RILs) derived from cross Yu 8679xJing 411. This map included 1 901 SNP loci and 178 SSR loci, covering 1 659.9 cM and 1 000 marker bins, with an average interval distance of 1.66 cM. A, B and D genomes covered 719.1,703.5 and 237.3 cM, with an average interval distance of 1.66, 1.45 and 2.9 cM, respectively. Notably, the genetic linkage map covered 20 chromosomes, with the exception of chromosome 5D. Bioinformatics analysis revealed that 1 754 (92.27%) of 1 901 mapped SNP loci could be aligned to 1 215 distinct wheat unigenes, among which 1 184 (97.4%) were located on one single chromosome, and the rest 31 (2.6%) were located on 2 to 3 chromosomes. By performing in silico comparison, 214 chromosome deletion bin-mapped expressed sequence tags (ESTs), 1 043 Brachypodium genes and 1 033 rice genes were further added onto the genetic linkage map. This map not only integrated genetic and physical maps, SSR and SNP loci, respectively, but also provided the information of Brachypodium and rice genes corresponding to 1 754 SNP loci. Therefore, it will be a useful tool for comparative genomics analysis, fine mapping of QTL/gene controlling agronomically important traits and marker-assisted selection breeding in wheat.

  15. A genetic linkage map with 178 SSR and 1 901 SNP markers constructed using a RIL population in wheat (Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    ZHAI Hui-jie; FENG Zhi-yu; LIU Xin-ye; CHENG Xue-jiao; PENG Hui-ru; YAO Ying-yin; SUN Qi-xin; NI Zhong-fu

    2015-01-01

    The construction of high density genetic linkage map provides a powerful tool to detect and map quantitative trait loci (QTLs) control ing agronomical y important traits. In this study, simple sequence repeat (SSR) markers and Il umina 9K iSelect single nucleotide polymorphism (SNP) genechip were employed to construct one genetic linkage map of common wheat (Triticum aestivum L.) using 191 recombinant inbred lines (RILs) derived from cross Yu 8679×Jing 411. This map included 1 901 SNP loci and 178 SSR loci, covering 1 659.9 cM and 1 000 marker bins, with an average interval distance of 1.66 cM. A, B and D genomes covered 719.1, 703.5 and 237.3 cM, with an average interval distance of 1.66, 1.45 and 2.9 cM, respectively. Notably, the genetic linkage map covered 20 chromosomes, with the exception of chromosome 5D. Bioinformatics analysis revealed that 1 754 (92.27%) of 1 901 mapped SNP loci could be aligned to 1 215 distinct wheat unigenes, among which 1 184 (97.4%) were located on one single chromosome, and the rest 31 (2.6%) were located on 2 to 3 chromosomes. By performing in silico comparison, 214 chromosome deletion bin-mapped expressed sequence tags (ESTs), 1 043 Brachypodium genes and 1 033 rice genes were further added onto the genetic linkage map. This map not only integrated genetic and physical maps, SSR and SNP loci, respectively, but also provided the information of Brachypodium and rice genes corresponding to 1 754 SNP loci. Therefore, it wil be a useful tool for comparative genomics analysis, ifne mapping of QTL/gene control ing agronomical y important traits and marker-assisted selection breeding in wheat.

  16. BORON-NITROGEN RELATIONSHIP IN WHEAT (Triticum aestivum L. GROWN WITH THE NUTRIENT SOLUTION

    Directory of Open Access Journals (Sweden)

    Mehmet ALPASLAN

    1996-03-01

    Full Text Available The effects of boron applied 0.01, 0.1, 1.0, and 10.0 µg B/ml levels as boric acid (H3 BO3, and nitrogen applied 25, 100, 200, and 400 µg N/ml as ammonium nitrate (NH4 NO3, respectively, on the amount of dry matter yield, and boron, nitrogen and nitrate contents of wheat (Triticum aestivum L. grown in perlite medium with Ruakura nutrient solution under greenhouse conditions were investigated. Dry matter yield and nitrate contents of wheat were decreased and boron content was increased with increasing boron application. Controversially, application of nitrogen increased dry matter yield, nitrogen and nitrate contents of wheat, while decreases in boron contents. Those effects of boron and nitrogen were found to be statistically significant (P

  17. [CdCl2-induced morphogenetic variation of Triticum aestivum cultivars].

    Science.gov (United States)

    Chunetova, Zh Zh; Omirbekova, N Zh; Shulembaeva, K K

    2008-11-01

    The effect cadmium chloride on released local cultivars of soft spring wheat (Triticum aestivum) has been studied under laboratory and field conditions in order to widen the variation spectrum of this plant. It has been found that treatment of grains with a 0.01% aqueous solution of CdCl2 induces the appearance of tall, strong plants with productive bushiness in the M1 generation that are characterized by various morphological changes: elongated ears, scales, and grains; increased number of grains per ear and mass of 1000 grains; anthocyan pigmentation of the stem and leaf axil; etc. Study of meiosis showed chromosome aggregation, displacement of the mitotic spindle of the metaphase plate, and empty (sterile) cells in anaphases (AI and AII). The altered characters of M1 plants are preserved in the M2-M4 generations. PMID:19137733

  18. Improved fluorimetric measurement of uranium uptake and distribution in spring wheat (Triticum aestivum L.)

    Energy Technology Data Exchange (ETDEWEB)

    Borcia, Catalin [' ' Alexandru Ioan Cuza' ' Univ., Iasi (Romania). Dept. of Physics; Popa, Karin; Cecal, Alexandru [' ' Alexandru Ioan Cuza' ' Univ., Iasi (Romania). Dept. of Chemistry; Murariu, Manuela [' ' Petru Poni' ' Institute of Macromolecular Chemistry, Iasi (Romania)

    2016-08-01

    Uranium uptake and (radio)toxicity was tested on spring wheat (Triticum aestivum L.) in a laboratory study using differently concentrated uranium nitrate solutions. Within these experiments, two analytical assays of uranium were comparatively tested: a fast and improved fluorimetric assay and the classical colorimetric (U(IV)-arsenazo(III) complexation) one. During the germination, the wheat seeds and plantlets supported well the uranium solutions of treatment within the entire concentration range (1 x 10{sup -4} -5 x 10{sup -3} M). Uranium proved to be non (radio)toxic to wheat as compared with other natural and anthropogenic radiocations, probably because its uptake by spring wheat during the germination is low. Indeed, only a small fraction of uranium administered was located within the roots, whereas the uranium content of the stems was negligible. A high correlation between the results obtained by two analytical methods was found. However, the fluorimetric assay proved to be more reliable and fast, and accurate.

  19. Contributions of Bacău to the economic literature and culture – The life and work of Professor Vasile Pătruţ

    Directory of Open Access Journals (Sweden)

    Mihai Deju

    2012-12-01

    Full Text Available The economic culture and accounting theory in Bacău area has its beginnings in the setting up of the first practical school of agriculture, by Ion Ionescu de la Brad, who included in the curriculum an accounting course, as well. Over the years, the economic education of Bacău area education has evolved from “The School of Accounting and Co-operative Education” (1919 to the modern economic higher education, in our days. During an important period of the evolution of education and culture in Bacău area, Professor Vasile Pătruţ is present, having a significant contribution to the accounting theory, teaching and practice in Bacău.

  20. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum.

    Directory of Open Access Journals (Sweden)

    Wei Gao

    Full Text Available The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC imaging method for cadmium ions (Cd2+ was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

  1. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice.

    Science.gov (United States)

    Cubells, Joseph F; Schroeder, Jason P; Barrie, Elizabeth S; Manvich, Daniel F; Sadee, Wolfgang; Berg, Tiina; Mercer, Kristina; Stowe, Taylor A; Liles, L Cameron; Squires, Katherine E; Mezher, Andrew; Curtin, Patrick; Perdomo, Dannie L; Szot, Patricia; Weinshenker, David

    2016-01-01

    Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a "C" or a "T" at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency

  2. Driving performance on the descending limb of blood alcohol concentration (BAC in undergraduate students: a pilot study.

    Directory of Open Access Journals (Sweden)

    Mathieu Tremblay

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

  3. Molecular verification of the integration of Tripsacum dactyloides DNA into wheat genome through wide hybridization

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RAPD and RFLP analyses of double haploid lines which derived from hybridization between hexaploid wheat (Triticum aestivum L.2n=42) and eastern gamagrass (Tripsacum dactyloides L.2n=4x=72) are reported.Two of the 340 Operon primers have been screened,which stably amplified Tripsacum dactyloides (male parent) specific bands in the double haploid lines.These results confirm the fact that Tripsacum dactyloides DNA has been integrated into wheat genome by sexual hybridization at molecular level.This idea has been further testified by RFLP analysis.Application and potentials of transferring Tripsacum dactyloides DNA into wheat genome by sexual hybridization in wheat breeding are discussed.

  4. Sur la filabilité d'une laine teinte en bac-ouvert et d'une laine teinte à la continue

    NARCIS (Netherlands)

    Werker, W.; Mulder, D.; Stomph, J.; Schartman, A.F.

    1966-01-01

    Afin de constater, si la teinture selon le procédé à la continue ou selon le procédé en bac-ouvert influence les résultats dans Ia filature, on a teint deux lots homogènes à 18OO kg par lot en quatre parties: 450 kg, procédé à la continue, teints en rouge, 450 kg, procédé bac-ouvert, teints en rouge

  5. Evaluation von Screeningverfahren zum Schnellnachweis von Methicillin-resistenten Staphylococcus aureus (MRSA)-Isolaten:Vergleich der Testsysteme BacLite Rapid MRSA+ und BD GeneOhm MRSA

    OpenAIRE

    Maas, D. (Dominik)

    2015-01-01

    Zwei Schnellverfahren mit unterschiedlichem methodischem Ansatz wurden in dieser Studie evaluiert. Das auf Kurzkultur basierende Testsystem BacLite Rapid MRSA+, und das auf molekulargenetischem Nachweis basierende BD GeneOhm MRSA-Testsystem. Als Referenzmethode diente das klassische Kulturverfahren. Von 497 getesteten Patienten wurden klassisch kulturell bei insgesamt fuenf Patienten eine MRSA Besiedlung entdeckt. Das BacLite Rapid MRSA+ konnte davon zwei MRSA-Traeger erkennen und zeigte eine...

  6. Array comparative genomic hybridisation (aCGH) analysis of premenopausal breast cancers from a nuclear fallout area and matched cases from Western New York

    OpenAIRE

    Varma, G.; Varma, R.; Huang, H.; Pryshchepava, A; Groth, J.; Fleming, D; Nowak, N. J.; McQuaid, D; Conroy, J.; Mahoney, M; Moysich, K.; Falkner, K L; Geradts, J

    2005-01-01

    High-resolution array comparative genomic hybridisation (aCGH) analysis of DNA copy number aberrations (CNAs) was performed on breast carcinomas in premenopausal women from Western New York (WNY) and from Gomel, Belarus, an area exposed to fallout from the 1986 Chernobyl nuclear accident. Genomic DNA was isolated from 47 frozen tumour specimens from 42 patients and hybridised to arrays spotted with more than 3000 BAC clones. In all, 20 samples were from WNY and 27 were from Belarus. In total,...

  7. Insights into the Musa genome: Syntenic relationships to rice and between Musa species

    Directory of Open Access Journals (Sweden)

    Althoff Ryan

    2008-01-01

    Full Text Available Abstract Background Musa species (Zingiberaceae, Zingiberales including bananas and plantains are collectively the fourth most important crop in developing countries. Knowledge concerning Musa genome structure and the origin of distinct cultivars has greatly increased over the last few years. Until now, however, no large-scale analyses of Musa genomic sequence have been conducted. This study compares genomic sequence in two Musa species with orthologous regions in the rice genome. Results We produced 1.4 Mb of Musa sequence from 13 BAC clones, annotated and analyzed them along with 4 previously sequenced BACs. The 443 predicted genes revealed that Zingiberales genes share GC content and distribution characteristics with eudicot and Poaceae genomes. Comparison with rice revealed microsynteny regions that have persisted since the divergence of the Commelinid orders Poales and Zingiberales at least 117 Mya. The previously hypothesized large-scale duplication event in the common ancestor of major cereal lineages within the Poaceae was verified. The divergence time distributions for Musa-Zingiber (Zingiberaceae, Zingiberales orthologs and paralogs provide strong evidence for a large-scale duplication event in the Musa lineage after its divergence from the Zingiberaceae approximately 61 Mya. Comparisons of genomic regions from M. acuminata and M. balbisiana revealed highly conserved genome structure, and indicated that these genomes diverged circa 4.6 Mya. Conclusion These results point to the utility of comparative analyses between distantly-related monocot species such as rice and Musa for improving our understanding of monocot genome evolution. Sequencing the genome of M. acuminata would provide a strong foundation for comparative genomics in the monocots. In addition a genome sequence would aid genomic and genetic analyses of cultivated Musa polyploid genotypes in research aimed at localizing and cloning genes controlling important agronomic

  8. Nanog reporter system in mouse embryonic stem cells based on highly efficient BAC homologous recombination

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nanog is a novel transcription factor specifically expressed in mouse embryonic stem cells (mES cells). It has been reported that Nanog plays an essential role in maintaining multi-potency of ES cells. The expression of Nanog is very sensitive to ES cells differentiation, making Nanog one of the best markers to indicate the status of ES cells. In this study, we developed an efficient method to construct Nanog promoter driven EGFP reporter system based on the BAC homologous recombination. We further generated a Nanog-EGFP reporter mES cell line. This reporter mES cell line exhibited features similar to those of normal mES cells, and the EGFP reporter efficiently reflected the expression of Nanog, indicating the differentiation status of mES cells. We achieved a reliable experimental reporter system to research self-renewal and differentiation of mES cells. The system could facilitate research on culture system of mES cells and researches on the expression and regulation of Nanog and other related factors in mES cells.

  9. C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD.

    Science.gov (United States)

    O'Rourke, Jacqueline G; Bogdanik, Laurent; Muhammad, A K M G; Gendron, Tania F; Kim, Kevin J; Austin, Andrew; Cady, Janet; Liu, Elaine Y; Zarrow, Jonah; Grant, Sharday; Ho, Ritchie; Bell, Shaughn; Carmona, Sharon; Simpkinson, Megan; Lall, Deepti; Wu, Kathryn; Daughrity, Lillian; Dickson, Dennis W; Harms, Matthew B; Petrucelli, Leonard; Lee, Edward B; Lutz, Cathleen M; Baloh, Robert H

    2015-12-01

    Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (∼100-1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients. PMID:26637796

  10. Geotechnical aspects for the optimization of dump design at Chinh Bac Mine waste dump in Vietnam

    Energy Technology Data Exchange (ETDEWEB)

    Fuchsschwanz, M.; Ziegler, M. [Aachen Univ., Aachen (Germany). Dept. of Geotechnical Engineering; Ahmad, S.; Fernandez, J.B.P.; Martens, P.N. [Aachen Univ., Aachen (Germany). Inst. of Mining Engineering; Deissmann, G. [Brenk Systemplanung GmbH, Aachen (Germany)

    2009-07-01

    Vietnam's Quang Ninh province is one of the country's most important coal producing regions. Several open pit mines are being operated in the area by Nui Beo Coal Company (NBCC). The construction of large waste dumps for overburden removed by blasting have led to environmental problems at the mining sites, including dust emissions from mining and dumping operations; ground and surface water contamination by acid mine drainage; and slope stability problems caused by heavy rainfall and dump movements. This paper discussed investigations regarding the influence of the dump layout on slope stability and erosion. The paper described the project site and ongoing activities for the development of optimized stabilization and rehabilitation concepts with a particular focus on geotechnical aspects. The site was described in terms of coal and waste rock production; Chinh Bac waste rock dump; crack mapping; material properties of dumped material; density; and settlements. Ongoing activities focus on the effect of benches on slope stability; influence of benches on erosion; and layered dumping. 7 refs., 4 figs.

  11. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    Science.gov (United States)

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  12. Geotechnical aspects for the optimization of dump design at Chinh Bac Mine waste dump in Vietnam

    International Nuclear Information System (INIS)

    Vietnam's Quang Ninh province is one of the country's most important coal producing regions. Several open pit mines are being operated in the area by Nui Beo Coal Company (NBCC). The construction of large waste dumps for overburden removed by blasting have led to environmental problems at the mining sites, including dust emissions from mining and dumping operations; ground and surface water contamination by acid mine drainage; and slope stability problems caused by heavy rainfall and dump movements. This paper discussed investigations regarding the influence of the dump layout on slope stability and erosion. The paper described the project site and ongoing activities for the development of optimized stabilization and rehabilitation concepts with a particular focus on geotechnical aspects. The site was described in terms of coal and waste rock production; Chinh Bac waste rock dump; crack mapping; material properties of dumped material; density; and settlements. Ongoing activities focus on the effect of benches on slope stability; influence of benches on erosion; and layered dumping. 7 refs., 4 figs.

  13. A Sox2 BAC transgenic approach for targeting adult neural stem cells.

    Directory of Open Access Journals (Sweden)

    Wenfei Kang

    Full Text Available The transcription factor gene Sox2 is expressed in embryonic neural stem/progenitor cells and previous evidence suggests that it is also expressed in adult neural stem cells. To target Sox2-expressing neural stem/progenitor cells in a temporal manner, we generated a bacterial artificial chromosome (BAC transgenic mouse line, in which an inducible form of Cre, CreER™, is expressed under Sox2 regulatory elements. Inducible Cre activity in these mice was characterized using floxed reporters. During development, the Sox2-CreER transgenic mice show inducible Cre activity specifically in CNS stem/progenitor cells, making them a useful tool to regulate the expression of floxed genes temporally in embryonic neural stem/progenitor cells. In the adult, we examined the cell-specific expression of Sox2 and performed long-term lineage tracing. Four months after the transient induction of Cre activity, recombined GFAP+ stem-like cells and DCX+ neuroblasts were still abundant in the neurogenic regions including the subventricular zone (SVZ, rostral migratory stream (RMS, and subgranular zone (SGZ of the dentate gyrus. These results provide definitive in vivo evidence that Sox2 is expressed in neural stem cells (NSC in both the SVZ and SGZ that are capable of self-renewal and long-term neurogenesis. Therefore, Sox2-CreER mice should be useful in targeting floxed genes in adult neural stem cells.

  14. Minimus: a fast, lightweight genome assembler

    Directory of Open Access Journals (Sweden)

    Salzberg Steven L

    2007-02-01

    Full Text Available Abstract Background Genome assemblers have grown very large and complex in response to the need for algorithms to handle the challenges of large whole-genome sequencing projects. Many of the most common uses of assemblers, however, are best served by a simpler type of assembler that requires fewer software components, uses less memory, and is far easier to install and run. Results We have developed the Minimus assembler to address these issues, and tested it on a range of assembly problems. We show that Minimus performs well on several small assembly tasks, including the assembly of viral genomes, individual genes, and BAC clones. In addition, we evaluate Minimus' performance in assembling bacterial genomes in order to assess its suitability as a component of a larger assembly pipeline. We show that, unlike other software currently used for these tasks, Minimus produces significantly fewer assembly errors, at the cost of generating a more fragmented assembly. Conclusion We find that for small genomes and other small assembly tasks, Minimus is faster and far more flexible than existing tools. Due to its small size and modular design Minimus is perfectly suited to be a component of complex assembly pipelines. Minimus is released as an open-source software project and the code is available as part of the AMOS project at Sourceforge.

  15. Isolation and characterization of bovine herpesvirus 4 (BoHV-4 from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome

    Directory of Open Access Journals (Sweden)

    Cavirani Sandro

    2009-08-01

    Full Text Available Abstract Background Bovine herpesvirus 4 (BoHV-4 is a gammaherpesvirus with a Worldwide distribution in cattle and is often isolated from the uterus of animals with postpartum metritis or pelvic inflammatory disease. Virus strain adaptation to an organ, tissue or cell type is an important issue for the pathogenesis of disease. To explore the mechanistic role of viral strain variation for uterine disease, the present study aimed to develop a tool enabling precise genetic discrimination between strains of BoHV-4 and to easily manipulate the viral genome. Methods A strain of BoHV-4 was isolated from the uterus of a persistently infected cow and designated BoHV-4-U. The authenticity of the isolate was confirmed by RFLP-PCR and sequencing using the TK and IE2 loci as genetic marker regions for the BoHV-4 genome. The isolated genome was cloned as a Bacterial Artificial Chromosome (BAC and manipulated through recombineering technology Results The BoHV-4-U genome was successfully cloned as a BAC, and the stability of the pBAC-BoHV-4-U clone was confirmed over twenty passages, with viral growth similar to the wild type virus. The feasibility of using BoHV-4-U for mutagenesis was demonstrated using the BAC recombineering system. Conclusion The analysis of genome strain variation is a key method for investigating genes associated with disease. A resource for dissection of the interactions between BoHV-4 and host endometrial cells was generated by cloning the genome of BoHV-4 as a BAC.

  16. Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes

    Directory of Open Access Journals (Sweden)

    McGuire Patrick E

    2010-12-01

    Full Text Available Abstract Background A genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD and wild tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat. Results Nucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed. Conclusions In a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large

  17. Observing copepods through a genomic lens

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    Johnson Stewart C

    2011-09-01

    Full Text Available Abstract 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

  18. From amplification to gene in thyroid cancer: A high-resolution mapped bacterial-artificial-chromosome resource for cancer chromosome aberrations guides gene discovery after comparative genome hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X.N.; Gonsky, R.; Korenberg, J.R. [UCLA School of Medicine, Los Angeles, CA (United States). Cedars-Sinai Research Inst.; Knauf, J.A.; Fagin, J.A. [Univ. of Cincinnati, OH (United States). Div. of Endocrinology/Metabolism; Wang, M.; Lai, E.H. [Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Pharmacology; Chissoe, S. [Washington Univ. School of Medicine, St. Louis, MO (United States). Genome Sequencing

    1998-08-01

    Chromosome rearrangements associated with neoplasms provide a rich resource for definition of the pathways of tumorigenesis. The power of comparative genome hybridization (CGH) to identify novel genes depends on the existence of suitable markers, which are lacking throughout most of the genome. The authors now report a general approach that translates CGH data into higher-resolution genomic-clone data that are then used to define the genes located in aneuploid regions. They used CGH to study 33 thyroid-tumor DNAs and two tumor-cell-line DNAs. The results revealed amplifications of chromosome band 2p21, with less-intense amplification on 2p13, 19q13.1, and 1p36 and with least-intense amplification on 1p34, 1q42, 5q31, 5q33-34, 9q32-34, and 14q32. To define the 2p21 region amplified, a dense array of 373 FISH-mapped chromosome 2 bacterial artificial chromosomes (BACs) was constructed, and 87 of these were hybridized to a tumor-cell line. Four BACs carried genomic DNA that was amplified in these cells. The maximum amplified region was narrowed to 3--6 Mb by multicolor FISH with the flanking BACs, and the minimum amplicon size was defined by a contig of 420 kb. Sequence analysis of the amplified BAC 1D9 revealed a fragment of the gene, encoding protein kinase C epsilon (PKC{epsilon}), that was then shown to be amplified and rearranged in tumor cells. In summary, CGH combined with a dense mapped resource of BACs and large-scale sequencing has led directly to the definition of PKC{epsilon} as a previously unmapped candidate gene involved in thyroid tumorigenesis.

  19. Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine

    International Nuclear Information System (INIS)

    Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors. A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk) and the monomeric red fluorescent protein (mRFP1) under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV) system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used as an efficient tool for gene therapy in ovarian cancer

  20. Europe, a continent with high potential for the cultivation of the Burgundy truffle (Tuber aestivum/uncinatum)

    OpenAIRE

    Gérard Chevalier

    2013-01-01

    The Burgundy truffle (Tuber aestivum/uncinatum) grows, in Europe, in the most varied soils from a physical and chemical viewpoint. The only common point is the presence of a minimum level of exchangeable calcium in the soil. The truffle soils in Europe can be classified in two categories: the soils coming directly from the parent rock, and those formed from deposits covering the parent rock. The first group corresponds to most traditional truffle areas , France, Italy and Spain. It is also tr...

  1. Ultrastructural studies on the sporogenous tissue and anther wall of Leucojum aestivum (amaryllidaceae) in different developmental stages.

    Science.gov (United States)

    Ekici, Nuran; Dane, Feruzan

    2012-12-01

    In this study, ultrastructures of anther wall and sporogenous tissue of Leucojum aestivum were investigated during different developmental stages. Cytomictic channels were seen between pollen mother cells during prophase I. Polar distribution was described in the organelle content of pollen mother cells and microspores in early phases of microsporogenesis and also in pollen mitosis. Active secretion was observed in tapetal cells. Previous reports about developmental stages of male gametophyte were compared with the results of this study.

  2. An Investigation of the Effects of Cationic, Anionic and Nonionic Surfactants on Germination Rates of Triticum Aestivum L

    OpenAIRE

    Yılmaz, Gülden

    2015-01-01

    In this study, the effects of different concentrations of a cationic surfactant (CTAB) cetrymonium bromid, cetyltrimethylammonium bromide, hexadecyltrimethylammonium bromide (CTAB), an anionic surfactant sodium dodecyl sulphate (SDS) and a nonionic surfactant Triton X-100 on germination rates of Triticum aestivum L. are observed. Surfactants are compounds that lower the surface tension of a liquid, the interfacial tension between two liquids, or between a liquid and a solid. Detergents, wetti...

  3. Auxin secretion by Bacillus amyloliquefaciens FZB42 both stimulates root exudation and limits phosphorus uptake in Triticum aestivum

    OpenAIRE

    Talboys, Peter J.; Owen, Darren W; Healey, John R; Withers, Paul JA; Jones, David L.

    2014-01-01

    Background The use of auxin-producing rhizosphere bacteria as agricultural products promises increased root production and therefore greater phosphate (Pi) uptake. Whilst such bacteria promote root production in vitro, the nature of the bacteria-plant interaction in live soil, particularly concerning any effects on nutrient uptake, are not known. This study uses Bacillus amyloliquefaciens FZB42, an auxin-producing rhizobacterium, as a dressing on Triticum aestivum seeds. It then examines the ...

  4. Primers for the Amplification of the Circular Chloroplast DNA from the A-genome Group of Cultivated Cotton

    Institute of Scientific and Technical Information of China (English)

    IBRAHIM Rashid Ismael Hag; AZUMA Jun-Ichi; SAKAMOTO Masahiro

    2008-01-01

    @@ The availability of the plastid genome sequences is one of the bases for comparative,functional,and structural genomic studies of plastid-containing living organisms,in addition to the application of plastid genetic engineering technology.The past efforts to sequence plastid genomes involve complicated preparation protocols.One procedure starts with the isolation of plastids,which was tiresome and time wasting that followed by a second step to extract plastid DNA from the isolated plastids,then finally the build up of plasmid or bacterial artificial chromosome (BAC) library.

  5. Sequencing of bovine herpesvirus 4 v.test strain reveals important genome features

    Directory of Open Access Journals (Sweden)

    Gillet Laurent

    2011-08-01

    Full Text Available Abstract Background Bovine herpesvirus 4 (BoHV-4 is a useful model for the human pathogenic gammaherpesviruses Epstein-Barr virus and Kaposi's Sarcoma-associated Herpesvirus. Although genome manipulations of this virus have been greatly facilitated by the cloning of the BoHV-4 V.test strain as a Bacterial Artificial Chromosome (BAC, the lack of a complete genome sequence for this strain limits its experimental use. Methods In this study, we have determined the complete sequence of BoHV-4 V.test strain by a pyrosequencing approach. Results The long unique coding region (LUR consists of 108,241 bp encoding at least 79 open reading frames and is flanked by several polyrepetitive DNA units (prDNA. As previously suggested, we showed that the prDNA unit located at the left prDNA-LUR junction (prDNA-G differs from the other prDNA units (prDNA-inner. Namely, the prDNA-G unit lacks the conserved pac-2 cleavage and packaging signal in its right terminal region. Based on the mechanisms of cleavage and packaging of herpesvirus genomes, this feature implies that only genomes bearing left and right end prDNA units are encapsulated into virions. Conclusions In this study, we have determined the complete genome sequence of the BAC-cloned BoHV-4 V.test strain and identified genome organization features that could be important in other herpesviruses.

  6. 应用随机多肽文库分析BAC5单抗相关的抗原表位%Analysis of BAC5 mcAb-Related Epitope Using Random Peptide library

    Institute of Scientific and Technical Information of China (English)

    肖锡宾; 张昌卿; 张颖; 张如华; 李经略; 冯凯涛; 孙韵; 叶永照

    2003-01-01

    Objective To identify epitope relating to BAC5 mcAb, a kind of monoclonal antibody (mcAb) located on the surface of nasopha-ryngeal carcinoma (NPC) cells.Methods Using BAC5 mcAb as a selected target, the 3 rounds of biopanning to a 12 mer random peptide library (RPL) presen-ted by M13 phages were carried out. The positive M13 phage clones were chosen and confirmed with sandwich ELISA for antibodycapture and competitive assay. The exogenous DNA fragments in the positive/negative M13 phages were sequenced to deduce andcompare the order of the amino acids of exogenous peptides among the phage clones.Results 77% (35/45) of the phages eluted from the 3rd round of biopnning could be captured by BAC5 mcAb. The 3 kinds ofthe peptides were displayed by M13 phages from the 8 positive clones identified with competitive assay. The same character of "-P-V-"structure existed near N-terminus of the 3 different peptides, i.e. -H-Q-S-H-Y-P-Y-P-V-V-S-L- (4/8) -Q-N-Q-A-W-F-S-Q-P-V-R-M- (3/8) and T-Q-A-Y-K-G-F-P-V-L-P-S- (1/8) in comparison with the peptide" -N-H-Q-S-T-F-W-Q-K-W-T-A-" dis-played by M13 phages from the negative clones (6/6).Conclusion BAC5 mcAb can recognize the 3 kinds of the peptides with-P-V-structure near N-terminus. These peptides mimic thestructure of the epitope on the surface of NPC cells recognized by BAC5 mcAb.%目的 鉴定能被BAC5单抗识别的定位于鼻咽癌细胞表面的抗原表位.方法 应用BAC5单抗作为靶抗体对噬菌体呈现的随机12肽文库进行3轮生物淘洗,用抗体捕获和竞争试验的夹心ELISA方法选择和鉴定阳性噬菌体克隆,对阳性和阴性噬菌体的外源性DNA片段进行序列分析,推导和比较由这些噬菌体所呈现的多肽氨基酸序列.结果 通过3轮生物淘洗能被抗体捕获的噬菌体克隆为77%(35/45).用竞争试验从所捕获的克隆中测得8个阳性克隆.来自这8个克隆的噬菌体呈现三种外源多肽,即-H-Q-S-H-Y-P-Y-P-V-V-S-L-(4/8)-Q-N-Q-A

  7. Complexity of genome evolution by segmental rearrangement in Brassica rapa revealed by sequence-level analysis

    Directory of Open Access Journals (Sweden)

    Paterson Andrew H

    2009-11-01

    Full Text Available Abstract Background The Brassica species, related to Arabidopsis thaliana, include an important group of crops and represent an excellent system for studying the evolutionary consequences of polyploidy. Previous studies have led to a proposed structure for an ancestral karyotype and models for the evolution of the B. rapa genome by triplication and segmental rearrangement, but these have not been validated at the sequence level. Results We developed computational tools to analyse the public collection of B. rapa BAC end sequence, in order to identify candidates for representing collinearity discontinuities between the genomes of B. rapa and A. thaliana. For each putative discontinuity, one of the BACs was sequenced and analysed for collinearity with the genome of A. thaliana. Additional BAC clones were identified and sequenced as part of ongoing efforts to sequence four chromosomes of B. rapa. Strikingly few of the 19 inter-chromosomal rearrangements corresponded to the set of collinearity discontinuities anticipated on the basis of previous studies. Our analyses revealed numerous instances of newly detected collinearity blocks. For B. rapa linkage group A8, we were able to develop a model for the derivation of the chromosome from the ancestral karyotype. We were also able to identify a rearrangement event in the ancestor of B. rapa that was not shared with the ancestor of A. thaliana, and is represented in triplicate in the B. rapa genome. In addition to inter-chromosomal rearrangements, we identified and analysed 32 BACs containing the end points of segmental inversion events. Conclusion Our results show that previous studies of segmental collinearity between the A. thaliana, Brassica and ancestral karyotype genomes, although very useful, represent over-simplifications of their true relationships. The presence of numerous cryptic collinear genome segments and the frequent occurrence of segmental inversions mean that inference of the positions

  8. Removing organic and nitrogen content from a highly saline municipal wastewater reverse osmosis concentrate by UV/H2O2-BAC treatment.

    Science.gov (United States)

    Pradhan, Shovana; Fan, Linhua; Roddick, Felicity A

    2015-10-01

    Reverse osmosis (RO) concentrate (ROC) streams generated from RO-based municipal wastewater reclamation processes pose potential health and environmental risks on their disposal to confined water bodies such as bays. A UV/H2O2 advanced oxidation process followed by a biological activated carbon (BAC) treatment was evaluated at lab-scale for the removal of organic and nutrient content from a highly saline ROC (TDS 16 g L(-1), EC 23.5 mS cm(-1)) for its safe disposal to the receiving environment. Over the 230-day operation of the UV/H2O2-BAC process, the colour and UV absorbance (254 nm) of the ROC were reduced to well below those of the influent to the reclamation process. The concentrations of DOC and total nitrogen (TN) were reduced by approximately 60% at an empty bed contact time (EBCT) of 60 min. The reduction in ammonia nitrogen by the BAC remained high under all conditions tested (>90%). Further investigation confirmed that the presence of residual peroxide in the UV/H2O2 treated ROC was beneficial for DOC removal, but markedly inhibited the activities of the nitrifying bacteria (i.e., nitrite oxidising bacteria) in the BAC system and hence compromised total nitrogen removal. This work demonstrated that the BAC treatment could be acclimated to the very high salinity environment, and could be used as a robust method for the removal of organic matter and nitrogen from the pre-oxidised ROC under optimised conditions.

  9. Proteomic analysis reveals key proteins and phosphoproteins upon seed germination of wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Kun eDong

    2015-11-01

    Full Text Available Wheat (Triticum aestivum L. is one of the oldest cultivated crops and the second most important food crop in the world. Seed germination is the key developmental process in plant growth and development, and poor germination directly affects plant growth and subsequent grain yield. In this study, we performed the first dynamic proteome analysis of wheat seed germination using a two-dimensional differential gel electrophoresis (2D-DIGE-based proteomic approach. A total of 166 differentially expressed protein (DEP spots representing 73 unique proteins were identified, which are mainly involved in storage, stress/defense/detoxification, carbohydrate metabolism, photosynthesis, cell metabolism, and transcription/translation/ transposition. The identified DEPs and their dynamic expression profiles generally correspond to three distinct seed germination phases after imbibition: storage degradation, physiological processes/morphogenesis, and photosynthesis. Some key DEPs involved in storage substance degradation and plant defense mechanisms, such as globulin 3, sucrose synthase type I, serpin, beta-amylase, and plastid ADP-glucose pyrophosphorylase (AGPase small subunit, were found to be phosphorylated during seed germination. Particularly, the phosphorylation site Ser355 was found to be located in the enzyme active region of beta-amylase, which promotes substrate binding. Phosphorylated modification of several proteins could promote storage substance degradation and environmental stress defense during seed germination. The central metabolic pathways involved in wheat seed germination are proposed herein, providing new insights into the molecular mechanisms of cereal seed germination.

  10. Molecular markers validation to drought resistance in wheat meal (Triticum aestivum L. under greenhouse conditions

    Directory of Open Access Journals (Sweden)

    Gabriel Julio

    2013-08-01

    Full Text Available With the aim to study the genetic resistance to drought and validate molecular markers co-localized with genes/QTLs for this factor, 16 varieties were evaluated as well as advanced lines of wheat meal (Triticum aestivum in two stages of crop development. Physiological parameters were considered: amount of chlorophyll (clo, wilting or severity degree (SEV and recovery (reco, morphological parameters: foliage dry matter (FDM and root dry matter (RDM, the integrated resistance mechanisms: water use efficiency (WUE, other parameters: number of grains (Ngrain and grain weight (Wgrain, biochemical parameters: Catalaza (CAT, Ascorbate Peroxidase (APX and Guaiacol Peroxidase (POX and three microsatellite markers (Xwmc603, Xwmc596, Xwmc9. Results showed significant differences for MSR and Ngrain. It was observed that Anzaldo, ERR2V.L-20, EARII2V.L-5, EARIZV.L-11, ERR2V.L-11 and EE2V.L-19 were the most resistant to drough water stress. There was a highly significant negative correlation between the MSR and Ngrain. All other variables showed low and non-significant correlations. In biochemical analyzes, the Anzaldo variety showed an increased enzymatic activity compared to controls in all cases (CAT-APX and POX, being the most resistant to water stress by drought. Finally, it was found that SSR markers (Xwmc596 and Xwmc9 are co-located with the gene / QTL of drought resistance and can be used for marker-assisted selection.

  11. Nitrogen and Photosynthesis in the Flag Leaf of Wheat (Triticum aestivum L.).

    Science.gov (United States)

    Evans, J R

    1983-06-01

    Wheat (Triticum aestivum L. cv Yecora 70) plants were grown with various concentrations of nitrate nitrogen available to the roots. Sampling of flag leaves began after they had reached full expansion and continued throughout senescence. Rates of gas exchange, ribulose-1,5-bisphosphate (RuP(2)) carboxylase activity, and the amounts of chlorophyll, soluble protein, nitrogen, and phosphorus were determined for each flag leaf. Rate of CO(2) assimilation was uniquely related to total leaf nitrogen irrespective of nutrient treatment, season, and leaf age. Assimilation rate increased with leaf nitrogen, but the slope of the relationship declined markedly when leaf nitrogen exceeded 125 millimoles nitrogen per square meter. Chlorophyll content and RuP(2) carboxylase activity were approximately proportional to leaf nitrogen content. As leaves aged, RuP(2) carboxylase activity and calculated Hill activity declined in parallel. With normal ambient partial pressure of CO(2), the intercellular partial pressure of CO(2) was always such that rate of assimilation appeared colimited by RuP(2) carboxylation and RuP(2) regeneration capacity.The initial slope of rate of CO(2) assimilation against intercellular partial pressure of CO(2) varied nonlinearly with carboxylase activity. It is suggested that this was due to a finite conductance to CO(2) diffusion in the wall and liquid phase which causes a drop in CO(2) partial pressure between the intercellular spaces and the site of carboxylation. A double reciprocal plot was used to obtain an estimate of the transfer conductance.

  12. Performance of Wheat Varieties (Triticum aestivum L. under Conservation Tillage Practices in Organic Agriculture

    Directory of Open Access Journals (Sweden)

    Dimitrios BILALIS

    2011-11-01

    Full Text Available Field experiments were conducted to determine the effects of tillage systems and varieties on growth, yield and quality of wheat crop (Triticum aestivum L.. The experiments conducted at two sites were laid out in a split-plot design with four replicates, three main plots [conventional tillage (CT, no-tillage (NT and minimum tillage (MT] and four sub-plots (�Siette�, �Panifor�, �Myrto�, �Estero�. The soil porosity and total nitrogen were higher in soils subjected to conservation tillage systems (NT and MT than under conventional tillage. There were no differences in root growth neither between the tillage systems nor among the varieties. Yield was influenced by the tillage system and variety. The highest grain yield (421-459 kg ha-1 was found under the CT system with �Siette�, �Myrto� and �Estero� varieties. In contrast, the highest grain yield was observed under conservation tillage (NT and MT with �Panifor� variety. There were no significant differences between the tillage systems concerning the protein content and Zeleny value. In contrast, the highest Hagberg falling number was found with MT. Zeleny value was positively and significantly correlated with protein content. High flour quality, as demonstrated by high protein content and Zeleny value, and low Hagberg falling number, was produced in the �Estero� variety.

  13. EVALUATION OF TECHNOLOGICAL AND ANTIOXIDANT PROPERTIES OF TRITICUM AESTIVUM L. AND TRITICUM DURUM L. VARIETIES

    Directory of Open Access Journals (Sweden)

    Ján Mareček

    2014-02-01

    Full Text Available The study deals with the evaluation of technological and antioxidant characteristics of selected varieties of Triticum aestivum and Triticum durum grown in Slovakia and Serbia. Research was conducted during the two years 2009 and 2010. Measured values of water activity were in the range 0.4 - 0.5. Optimal activity of alpha-amylase was measured in Serbian varieties Etida (210 seconds, Pobeda (218 seconds and Renesansa (272 seconds. The highest sedimentation capacity expressed as sedimentation index by Zeleny had variety Karpatia (60 cm3. The high content of insoluble protein (gluten was measured in a variety Rusija (36.6%. Nitrogen content was in the range 12.7 - 13.9% of dry matter, starch content in the range 56.6 - 61.6% of dry matter. Antioxidant activity measured by DPPH method ranged in wheat varieties from 44 to 49%. The highest content of polyphenols was measured in a variety Etida (0.464 mg of catechin/g of sample. Durum wheat varieties have a higher content of polyphenols in general. The production of semolina flour from durum wheat may have the positive antioxidant effect according to gained measurements.

  14. Toxicological effects of rare earth yttrium on wheat seedlings (Triticum aestivum)

    Institute of Scientific and Technical Information of China (English)

    冯秀娟; 朱国才; 李亚宁

    2013-01-01

    This study examined the biochemical responses of wheat (Triticum aestivum) to the stress of rare earth yttrium (Y) and showed that 25-100 mg/kg Y treatments evidently increased the biomass (root mass, shoot mass and leaf mass), accompanied by a significant (p<0.05) increase in the chlorophyll (CHL) content in wheat leaves. Increased malondialdehyde (MDA) levels were de-tected in wheat shoots (stem and leaf) and roots too, indicating the presence of poisoning active oxygen species (AOS). The MDA content in wheat roots increased with the augmentation of Y concentration. These results indicated that there was a dose-dependent effect of Y on the changes of MDA content in wheat roots. Although the activities of superoxide dismutases (SOD), peroxidases (POD) and catalases (CAT) in wheat shoots and roots irregularly fluctuated with the increase in Y concentration, 25-100 mg/kg Y significantly (p<0.01) increased the activities of SOD and POD. In general, the dose-dependent effects of Y on the activity of anti-oxidant enzymes were insignificant. Our data also indicated that the increase in SOD and POD activities could be used as a good bio-marker for the stress induced by low concentrations of Y.

  15. Joint stress of chlorimuron-ethyl and cadmium on wheat Triticum aestivum at biochemical levels

    International Nuclear Information System (INIS)

    Biochemical responses to joint stress of chlorimuron-ethyl and cadmium (Cd) in wheat Triticum aestivum were examined. The joint action of chlorimuron-ethyl and Cd weakened the inhibition of Cd or chlorimuron-ethyl on the formation of chlorophyll. It was deduced that wheat plants had the capability to protect themselves by increasing the activity of the antioxidant enzyme peroxidase (POD) with the exposure time. The joint effect of chlorimuron-ethyl and Cd on the superoxide dismutase (SOD) activity in leaves was additive, while the joint effect on the SOD activity in roots was determined by the interaction of chlorimuron-ethyl and Cd in wheat. It was also concluded that the change of malondialdehyde (MDA) content in wheat might not be a good biomarker in the oxidative damage by chlorimuron-ethyl, while a decrease in the soluble protein content and POD activity in roots could be considered as a biomarker in the damage of wheat by chlorimuron-ethyl and Cd. - Soluble protein content and peroxidase activity in seedlings were the biomarkers indicating joint stress of chemicals

  16. Toxic effect of perfluorooctanoic acid (PFOA) on germination and seedling growth of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Zhou, Lina; Xia, Mengjie; Wang, Li; Mao, Hui

    2016-09-01

    As a persistent organic pollutant in the environment, perfluorooctanoic acid (PFOA) has been extensively investigated. It can accumulate in food chains and in the human body. This work investigated the effect of PFOA on wheat (Triticum aestivum L.) germination and seedling growth by conducting a germination trial and a pot trial. A stimulatory effect of PFOA on seedling growth and root length of wheat was found at 800 mg kg(-1) PFOA inhibited germination rate, index, and root and shoot growth. In the pot trial, PFOA concentration in root was double that in the shoot. Soil and plant analyzer development (SPAD) and plant height of wheat seedling were inhibited by adding 200 mg kg(-1) PFOA. Proline content and POD activity in wheat seedlings increased as PFOA increased, while CAT activity decreased. Using logarithmic equations, proline content was selected as the most sensitive index by concentration for 50% of maximal effect (EC50). Hence, the tolerance of wheat seedlings to PFOA levels could be evaluated on the basis of the physiological index. PMID:27337433

  17. Insights Into Triticum aestivum Seedling Root Rot Caused by Fusarium graminearum.

    Science.gov (United States)

    Wang, Qing; Vera Buxa, Stefanie; Furch, Alexandra; Friedt, Wolfgang; Gottwald, Sven

    2015-12-01

    Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum), known for causing devastating spike infections and grain yield damage. F. graminearum is a typical soil-borne pathogen that builds up during consecutive cereal cropping. Speculation on systemic colonization of cereals by F. graminearum root infection have long existed but have not been proven. We have assessed the Fusarium root rot disease macroscopically in a diverse set of 12 wheat genotypes and microscopically in a comparative study of two genotypes with diverging responses. Here, we show a 'new' aspect of the F. graminearum life cycle, i.e., the head blight fungus uses a unique root-infection strategy with an initial stage typical for root pathogens and a later stage typical for spike infection. Root colonization negatively affects seedling development and leads to systemic plant invasion by tissue-adapted fungal strategies. Another major outcome is the identification of partial resistance to root rot. Disease severity assessments and histological examinations both demonstrated three distinct disease phases that, however, proceeded differently in resistant and susceptible genotypes. Soil-borne inoculum and root infection are considered significant components of the F. graminearum life cycle with important implications for the development of new strategies of resistance breeding and disease control.

  18. Identification, Characterization, and Expression Analysis of the TaNF-YB3 Gene from Triticum aestivum

    Directory of Open Access Journals (Sweden)

    Xiuchun DONG

    2011-11-01

    Full Text Available A full-length cDNA encoding a nuclear factor-YB (NF-YB/HAP3/CCAAT binding factor-A (CBF-A subunit of a CCAAT-box binding complex, designated as TaNF-YB3 was isolated from Triticum aestivum. Sequence analysis indicated that the full-length cDNA was 809�bp long, including an open reading frame (ORF of 597�bp, which encoded a deduced polypeptide of 199 amino acids and is located in chromosome 3D. The deduced protein contained conserved structural domains and showed high identity to other plant NF-YBs. TaNF-YB3 was expressed in various organs, especially in the leaves and stamens; it was also regulated by salt, mannitol, abscisic acid, wounding, and cold. Moreover, TaNF-YB3 was down-regulated by short days and vernalization, and sensitive to the transfer of day length. It was mainly induced by light and exhibited a similar diurnal rhythmic expression pattern with the CCT-domain family gene VRN2 (TaZCCT1 and TaZCCT2, but not with CO (WCO1 and TaHd1. Overall, the results suggested that TaNF-YB3, aside from having a role in regulating day length and vernalization responses, might integrate signals from other environmental stresses to perform its functions in winter wheat adaptability and development.

  19. Anticancer Effect of Rutin Isolated from the Methanolic Extract of Triticum aestivum Straw in Mice

    Directory of Open Access Journals (Sweden)

    Savita Dixit

    2014-10-01

    Full Text Available Rutin is the bioactive flavanoid isolated from the straw part of Triticum aestivum and possess various pharmacological applications. The aim of this study is to evaluate the chemopreventive potential of rutin in an experimental skin carcinogenesis mice model system. Skin tumor was induced by topical application of 7,12-dimethyl benz(a anthracene (DMBA and promoted by croton oil in Swiss albino mice. To assess the chemopreventive potential of rutin, it was orally administered at a concentration of (200 mg/kg and 400 mg/kg body weight continued three times weekly for 16th weeks. The development of skin carcinogenesis was assessed by histopathological analysis. Reductions in tumor size and cumulative number of papillomas were seen due to rutin treatment. Average latent period was significantly increased as compared to carcinogen treated control. Rutin produced significant decrease in the activity of serum enzyme serum glutamate oxalate transaminase (SGOT, serum glutamate pyruvate transaminase (SGPT, alkaline phosphatase (ALP and bilirubin when compared with the control. They significantly increased the levels of enzyme involved in oxidative stress glutathione (GSH, superoxide dismutase (SOD and catalase. The elevated level of lipid peroxidase in the control group was significantly inhibited by rutin administration. The results from the present study suggest the chemopreventive effect of rutin in DMBA and croton oil induced skin carcinogenesis in swiss albino mice and one of the probable reasons would be its antioxidant potential.

  20. Composition of cuticular waxes coating flag leaf blades and peduncles of Triticum aestivum cv. Bethlehem.

    Science.gov (United States)

    Racovita, Radu C; Hen-Avivi, Shelly; Fernandez-Moreno, Josefina-Patricia; Granell, Antonio; Aharoni, Asaph; Jetter, Reinhard

    2016-10-01

    The work herein presents comprehensive analyses of the cuticular wax mixtures covering the flag leaf blade and peduncle of bread wheat (Triticum aestivum) cv. Bethlehem. Overall, Gas Chromatography-Mass Spectrometry and Flame Ionization Detection revealed a wax coverage of flag leaf blades (16 μg/cm(2)) a third that of peduncles (49 μg/cm(2)). Flag leaf blade wax was dominated by 1-alkanols, while peduncle wax contained primarily β-diketone and hydroxy-β-diketones, thus suggesting differential regulation of the acyl reduction and β-diketone biosynthetic pathways in the two analyzed organs. The characteristic chain length distributions of the various wax compound classes are discussed in light of their individual biosynthetic pathways and biosynthetic relationships between classes. Along with previously reported wheat wax compound classes (fatty acids, 1-alkanols, 1-alkanol esters, aldehydes, alkanes, β-diketone, hydroxy-β-diketones, alkylresorcinols and methyl alkylresorcinols), esters of 2-alkanols and three types of aromatic esters (benzyl, phenethyl and p-hydroxyphenethyl) are also reported. In particular, 2-heptanol esters were identified. Detailed analyses of the isomer distributions within 1-alkanol and 2-alkanol ester homologs revealed distinct patterns of esterified acids and alcohols, suggesting several wax ester synthases with very different substrate preferences in both wheat organs. Terpenoids, including two terpenoid esters, were present only in peduncle wax. PMID:27264640

  1. Arsenate (As) uptake by and distribution in two cultivars of winter wheat (Triticum aestivum L.).

    Science.gov (United States)

    Geng, Chun-Nu; Zhu, Yong-Guan; Tong, Yi-Ping; Smith, Sally E; Smith, F A

    2006-01-01

    Two cultivars of winter wheat (Triticum aestivum L.) (Jing 411 and Lovrin 10) were used to investigate arsenate (As) uptake and distribution in plants grown in hydroponic culture and in the soil. Results showed that without As addition, Lovrin 10 had higher biomass than Jing 411 in the soil pot experiment; in the hydroponic experiment Lovrin 10 had similar root biomass to and lower shoot biomass than Jing 411. Increasing P supply from 32 to 161 microM resulted in lower tissue As concentrations, and increasing As supply from 0 to 2,000 microM resulted in lower tissue P concentrations. Increasing P supply tended to increase shoot-to-root ratios of As concentrations, and increasing As supply tended to decrease shoot-to-root ratios of As concentrations. Both cultivars invested more in root production under P deficient conditions than under P sufficient conditions. Lovrin 10 invested more biomass production to roots than Jing 411, which might be partly responsible for higher shoot P and As concentrations and higher shoot-to-root ratios of As concentrations. Moreover, Lovrin 10 allocated less As to roots than Jing 411 and the difference disappeared with decreasing P supply. PMID:16081139

  2. Search for diagnostic proteins to prove authenticity of organic wheat grains (Triticum aestivum L.).

    Science.gov (United States)

    Zörb, Christian; Betsche, Thomas; Langenkämper, Georg

    2009-04-01

    Research comparing the biochemical composition of wheat grains from organic or conventional agriculture has used the targeted analytical approach. To obtain a more comprehensive record of the food's composition, we employed protein profiling techniques. Levels of 1049 proteins were recorded in wheat grains (Triticum aestivum L., cv. Titlis) of two growing seasons from a rigorously controlled field trial in Switzerland, containing organic and conventional plots. Levels of 25 proteins were different between organic and conventional wheat in both years. Storage proteins, enzymes of carbohydrate metabolism, a peroxidase, and proteins of unknown function were affected by the agricultural regime. Total protein content was lower in organic wheat. We consider these differences negligible with regard to nutrition in an average diet and propose that food quality of conventional and organic wheat grown in the field trial was equal. Applying various filters and calculations, one of which takes seasonal influences into account, 16 of the 25 proteins with different levels in organic and conventional wheat were retained. These 16 "diagnostic" proteins have the potential to afford a signature to prove authenticity of organic wheat.

  3. Quantitative Trait Loci Mapping of Dark-Induced Senescence in Winter Wheat (Triticum aestivum)

    Institute of Scientific and Technical Information of China (English)

    Hongwei Li; Fanyun Lin; Gui Wang; Ruilian Jing; Qi Zheng; Bin Li; Zhensheng Li

    2012-01-01

    In order to explore the genetics of dark-induced senescence in winter wheat (Triticum aestivum L.),aquantitative trait loci (QTL) analysis was carried out in a doubled haploid population developed from across between the varieties Hanxuan 10 (HX) and Lumai 14 (LM).The senescence parameters chlorophyll content (Chl a+b,Chl a,and Chl b),original fluorescence (Fo),maximum fluorescence level (Fm),maximum photochemical efficiency (Fv/Fm),and ratio of variable fluorescence to original fluorescence (Fv/Fo) were evaluated in the second leaf of whole three-leaf seedlings subjected to 7 d of darkness.A total of 43 QTLs were identified that were associated with dark-induced senescence using composite interval mapping.These QTLs were mapped to 20 loci distributed on 11 chromosomes:1B,1D,2A,2B,3B,3D,5D,6A,6B,7A,and 7B.The phenotypic variation explained by each QTL ranged from 7.5% to 19.4%.Eleven loci coincided with two or more of the analyzed parameters.In addition,14 loci co-located or were linked with previously reported QTLs regulating flag leaf senescence,tolerance to high light stress,and grain protein content (Gpc),separately.

  4. Metabolic pathways of the wheat (Triticum aestivum endosperm amyloplast revealed by proteomics

    Directory of Open Access Journals (Sweden)

    Dupont Frances M

    2008-04-01

    Full Text Available Abstract Background By definition, amyloplasts are plastids specialized for starch production. However, a proteomic study of amyloplasts isolated from wheat (Triticum aestivum Butte 86 endosperm at 10 days after anthesis (DPA detected enzymes from many other metabolic and biosynthetic pathways. To better understand the role of amyloplasts in food production, the data from that study were evaluated in detail and an amyloplast metabolic map was outlined. Results Analysis of 288 proteins detected in an amyloplast preparation predicted that 178 were amyloplast proteins. Criteria included homology with known plastid proteins, prediction of a plastid transit peptide for the wheat gene product or a close homolog, known plastid location of the pathway, and predicted plastid location for other members of the same pathway. Of these, 135 enzymes were arranged into 18 pathways for carbohydrate, lipid, amino acid, nucleic acid and other biosynthetic processes that are critical for grain-fill. Functions of the other proteins are also discussed. Conclusion The pathways outlined in this paper suggest that amyloplasts play a central role in endosperm metabolism. The interacting effects of genetics and environment on starch and protein production may be mediated in part by regulatory mechanisms within this organelle.

  5. Functional study of a salt-inducible TaSR gene in Triticum aestivum.

    Science.gov (United States)

    Ma, Xiao-Li; Cui, Wei-Na; Zhao, Qian; Zhao, Jing; Hou, Xiao-Na; Li, Dong-Yan; Chen, Zhao-Liang; Shen, Yin-Zhu; Huang, Zhan-Jing

    2016-01-01

    The gene expression chip of a salt-tolerant wheat mutant under salt stress was used to clone a salt-induced gene with unknown functions. This gene was designated as TaSR (Triticum aestivum salt-response gene) and submitted to GenBank under accession number EF580107. Quantitative polymerase chain reaction (PCR) analysis showed that gene expression was induced by salt stress. Arabidopsis and rice (Oryza sativa) plants expressing TaSR presented higher salt tolerance than the controls, whereas AtSR mutant and RNA interference rice plants were more sensitive to salt. Under salt stress, TaSR reduced Na(+) concentration and improved cellular K(+) and Ca(2+) concentrations; this gene was also localized on the cell membrane. β-Glucuronidase (GUS) staining and GUS fluorescence quantitative determination were conducted through fragmentation cloning of the TaSR promoter. Salt stress-responsive elements were detected at 588-1074 bp upstream of the start codon. GUS quantitative tests of the full-length promoter in different tissues indicated that promoter activity was highest in the leaf under salt stress. Bimolecular fluorescence complementation and yeast two-hybrid screening further showed the correlation of TaSR with TaPRK and TaKPP. In vitro phosphorylation of TaSR and TaPRK2697 showed that TaPRK2697 did not phosphorylate TaSR. This study revealed that the novel TaSR may be used to improve plant tolerance to salt stress. PMID:25855206

  6. EVALUATING THE DROUGHT STRESS TOLERANCE EFFICIENCY OF WHEAT (TRITICUM AESTIVUM L. CULTIVARS

    Directory of Open Access Journals (Sweden)

    M.A. Sammar Raza

    2012-12-01

    Full Text Available The study was conducted at the Nuclear Institute for Agriculture and Biology (NIAB, Faisalabad, Pakistan to evaluate the drought stress tolerance efficiency of wheat cultivars. Eight wheat (Triticum aestivum L. genotypes namely (Pasban-90, Inqalab-91, Auqab-2000, AS-2002, Sahar-2006, Shafaq-2006, Lasani-2008, and FSD-2008 were used for screening in laboratory. Three techniques were used for the screening. These techniques are (I seed germination at -0.6 MPa external water potential (PEG-6000, (2 seedling growth after 14 days at -0.6 MPa (PEG-6000, and (3 plant water relations of seven-week-old pot-grown plants. Significantly highest germination stress tolerance index, root length stress tolerance index and the water content was recorded in Lasani-2008 and lowest was recorded in Auqab-2000. Dry matter percentage was recorded significantly highest in pasban-90 while the lowest dry matter percentage was observed in auqab-2000. FSD-2008 gained maximum plant height and was recorded minimum in Inqilab-91. Relative saturation deficit was significantly highest in Auqab-2000 while the significantly lowest relative saturation deficit was recorded in case of Lasani-2008. It was concluded from the results that Lasani-2008 performed better under drought and Auqab-2000 was recorded most drought sensitive variety.

  7. Nitric oxide mediates alginate oligosaccharides-induced root development in wheat (Triticum aestivum L.).

    Science.gov (United States)

    Zhang, Yunhong; Liu, Hang; Yin, Heng; Wang, Wenxia; Zhao, Xiaoming; Du, Yuguang

    2013-10-01

    Alginate oligosaccharides (AOS), which are marine oligosaccharides, are involved in regulating plant root growth, but the promotion mechanism for AOS remains unclear. Here, AOS (10-80 mg L(-1)) were found to induce the generation of nitric oxide (NO) in the root system of wheat (Triticum aestivum L.), which promoted the formation and elongation of wheat roots in a dose-dependent manner. NO inhibitors suggested that nitrate reductase (NR), rather than nitric oxide synthase (NOS), was essential for AOS-induced root development. Further studies confirmed that AOS-induced NO generation in wheat roots by up-regulating the gene expression and enzyme activity of NR at the post-transcriptional level. The anatomy and RT-PCR results showed that AOS accelerated the division and growth of stele cells, leading to an increase in the ratio of stele area to root transverse area. This could be inhibited by the NR inhibitor, sodium tungstate, which indicated that NO catalyzed by the NR was involved in AOS regulation of root development. Taken together, in the early stage of AOS-induced root development, NO generation was a novel mechanism by which AOS regulated plant growth. The results also showed that this marine resource could be widely used for crop development.

  8. Effects of Auxins and Media on Callus Induction of Chinese Spring Wheat( Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    WU Li-min; WEI Yu-ming; ZHENG You-liang

    2005-01-01

    The effects of auxins and media on callus induction from the mature and immature embryos of Chinese spring wheat(Triticum aestivum L.) varieties were investigated. It was found that genotype, medium, auxin source and concentration had the significant effects on the induction of embryogenic callus, explants germination and the increment of callus fresh weight. For immature embryos cultured on MS medium, 2 mg L-1of 2, 4-D was optimal, and the highest frequency of embryogenic callus (33.50%) was observed. For the mature embryos on N6 medium, 4 mg L-1 of 2, 4-D was optimal. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. These results indicated that 2, 4, 5-T was superior to 2, 4-D and NAA in the culture of immature embryos. This is the first report about the effect of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail.

  9. Acid Carboxypeptidases in Grains and Leaves of Wheat, Triticum aestivum L.

    Science.gov (United States)

    Mikola, L

    1986-07-01

    Extracts of resting and germinating (3 days at 20 degrees C) wheat (Triticum aestivum L. cv Ruso) grains rapidly hydrolyzed various benzyloxycarbonyldipeptides (Z-dipeptides) at pH 4 to 6. Similar activities were present in extracts of mature flag leaves. Fractionation by chromatography on CM-cellulose and on Sephadex G-200 showed that the activities in germinating grains were due to five acid carboxypeptidases with different and complementary substrate specificities. The wheat enzymes appeared to correspond to the five acid carboxypeptidases present in germinating barley (L Mikola 1983 Biochim Biophys Acta 747: 241-252). The enzymes were designated wheat carboxypeptidases I to V and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 120,000; II, Z-Ala-Arg, 120,000; III, Z-Ala-Phe, 40,000; IV, Z-Pro-Ala, 165,000; and V, Z-Pro-Ala, 150,000. Resting grains contained carboxypeptidase II as a series of three isoenzymes and low activities of carboxypeptidases IV and V. During germination the activity of carboxypeptidase II decreased, those of carboxypeptidases IV and V increased, and high activities of carboxypeptidases I and III appeared. The flag leaves contained high activity of carboxypeptidase I and lower activities of carboxypeptidases II, IV, and V, whereas carboxypeptidase III was absent.

  10. Acid Carboxypeptidases in Grains and Leaves of Wheat, Triticum aestivum L

    Science.gov (United States)

    Mikola, Leena

    1986-01-01

    Extracts of resting and germinating (3 days at 20°C) wheat (Triticum aestivum L. cv Ruso) grains rapidly hydrolyzed various benzyloxycarbonyldipeptides (Z-dipeptides) at pH 4 to 6. Similar activities were present in extracts of mature flag leaves. Fractionation by chromatography on CM-cellulose and on Sephadex G-200 showed that the activities in germinating grains were due to five acid carboxypeptidases with different and complementary substrate specificities. The wheat enzymes appeared to correspond to the five acid carboxypeptidases present in germinating barley (L Mikola 1983 Biochim Biophys Acta 747: 241-252). The enzymes were designated wheat carboxypeptidases I to V and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 120,000; II, Z-Ala-Arg, 120,000; III, Z-Ala-Phe, 40,000; IV, Z-Pro-Ala, 165,000; and V, Z-Pro-Ala, 150,000. Resting grains contained carboxypeptidase II as a series of three isoenzymes and low activities of carboxypeptidases IV and V. During germination the activity of carboxypeptidase II decreased, those of carboxypeptidases IV and V increased, and high activities of carboxypeptidases I and III appeared. The flag leaves contained high activity of carboxypeptidase I and lower activities of carboxypeptidases II, IV, and V, whereas carboxypeptidase III was absent. PMID:16664910

  11. Hydrogeological investigations for mineral water and CO2 gas in the village of Bac, Bitola, The Republic of Macedonia

    OpenAIRE

    Mircovski, Vojo; Spasovski, Orce; Petrov, Dimitar

    2005-01-01

    Tree exploration drill holes were dug in the Neogene Middle Pliocene sediments in the vicinity of the village of Bac for the investigation of the mineral water and CO2 gas. The first drill hole (B 1) was dug 30 m in depth indicated a water bearing horizon in o to 26.4 m thick grey marly sandstone with a free level aquifer and ground water 2.2 m at depth and yield of 10l/s. The second and third drill holes (ED-1 and ED-2) were dug 301 m to depth and determined a free level aquifer, a sub ar...

  12. Development of a database system for mapping insertional mutations onto the mouse genome with large-scale experimental data

    OpenAIRE

    Yang, Wenwei; Jin, Ke; Xie, Xing; Li, Dongsheng; Yang, Jigang; Wang, Li; Gu, Ning; Zhong, Yang; Sun, Ling V.

    2009-01-01

    Background Insertional mutagenesis is an effective method for functional genomic studies in various organisms. It can rapidly generate easily tractable mutations. A large-scale insertional mutagenesis with the piggyBac (PB) transposon is currently performed in mice at the Institute of Developmental Biology and Molecular Medicine (IDM), Fudan University in Shanghai, China. This project is carried out via collaborations among multiple groups overseeing interconnected experimental steps and gene...

  13. Whole genome comparative studies between chicken and turkey and their implications for avian genome evolution

    Directory of Open Access Journals (Sweden)

    Carré Wilfrid

    2008-04-01

    Full Text Available Abstract Background Comparative genomics is a powerful means of establishing inter-specific relationships between gene function/location and allows insight into genomic rearrangements, conservation and evolutionary phylogeny. The availability of the complete sequence of the chicken genome has initiated the development of detailed genomic information in other birds including turkey, an agriculturally important species where mapping has hitherto focused on linkage with limited physical information. No molecular study has yet examined conservation of avian microchromosomes, nor differences in copy number variants (CNVs between birds. Results We present a detailed comparative cytogenetic map between chicken and turkey based on reciprocal chromosome painting and mapping of 338 chicken BACs to turkey metaphases. Two inter-chromosomal changes (both involving centromeres and three pericentric inversions have been identified between chicken and turkey; and array CGH identified 16 inter-specific CNVs. Conclusion This is the first study to combine the modalities of zoo-FISH and array CGH between different avian species. The first insight into the conservation of microchromosomes, the first comparative cytogenetic map of any bird and the first appraisal of CNVs between birds is provided. Results suggest that avian genomes have remained relatively stable during evolution compared to mammalian equivalents.

  14. Construction of BAC Physical Map on Chro moso me A9 in Brassica juncea and Co mparative Geno me Analysis%芥菜型油菜A9染色体BAC物理图谱构建及比较基因组分析

    Institute of Scientific and Technical Information of China (English)

    刘芳瑛; 陆赢; 王卓; 刘显军; 胡学芳; 刘旭东; 徐海鹏; 刘忠松

    2016-01-01

    To abtain a precise physical map of Brassica juncea chromosome A9,the markers located on the A9 chromosome were used in this study to screen Brassica juncea BAC library by three-dimensional mixed PCR screening. A total of 3200 positive BAC were screened out;constructing a physical map composed of 16 contigs and 4 singletons. According to the principle of the minimum path,435 BACs were chosen as seed BACs to represent the whole physical map and were subject to end sequencing,at last,862 qualified sequences were received. As the average insert size of BAC is 126 kb,the contigs accounted up to 52. 08 M,covering 1. 37 times of chromosome A9. By BLAST,the seed BAC end sequences were mapped to chromosome A9 pseudomolecule of Brassica rapa and Brassica napus respectively,results showed that the physical map covered 36. 4 Mb and 31. 0 Mb,equivalent to 93. 9%、91. 8% of the whole genome assemble length. Among the 862 BAC end sequences,702 sequences (81. 4%)were located on Brassica rapa A9 chromosome and 646 sequences(74. 9%)to Brassica napus A9 chromosome,which means Brassica juncea A9 chromosome have higher homology with Brassica rapa A9 chromosome than Brassica napus A9 chromosome. Compared with Brassica napus A9 pseudomolecule and BAC end se-quence of Brassica juncea A9 chromosome,there are two chromosome inversions about 1. 4 Mb、3. 6 Mb in length at the po-sition of 6413031-7827177 and 14856710-18458808 in Brassica rapa A9. While compared with BAC end sequence of Bras-sica juncea A9,there is a inserted mutation about 0. 98 Mb in length at the position of 15676847-16064302 of Brassica na-pus A9 pseudomolecule,which lie in one of the two chromosome inversions and was consist with the predicted position of centromere of Brassica napus A9 chromosome remained to assembled.%为获得高精确度的芥菜型油菜A9染色体物理图,本研究利用定位在A9染色体上的标记对芥菜型油菜BAC文库进行三维混合PCR筛选,共得到阳性BAC 3200

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

    Directory of Open Access Journals (Sweden)

    Kudrna Dave

    2010-06-01

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

  16. Cancer genomics

    DEFF Research Database (Denmark)

    Norrild, Bodil; Guldberg, Per; Ralfkiær, Elisabeth Methner

    2007-01-01

    Almost all cells in the human body contain a complete copy of the genome with an estimated number of 25,000 genes. The sequences of these genes make up about three percent of the genome and comprise the inherited set of genetic information. The genome also contains information that determines whe...

  17. A SINE in the genome of the cephalochordate amphioxus is an Alu element

    Directory of Open Access Journals (Sweden)

    2006-04-01

    Full Text Available Transposable elements of about 300 bp, termed “short interspersed nucleotide elements or SINEs are common in eukaryotes. However, Alu elements, SINEs containing restriction sites for the AluI enzyme, have been known only from primates. Here I report the first SINE found in the genome of the cephalochordate, amphioxus. It is an Alu element of 375 bp that does not share substantial identity with any genomic sequences in vertebrates. It was identified because it was located in the FoxD regulatory region in a cosmid derived from one individual, but absent from the two FoxD alleles of BACs from a second individual. However, searches of sequences of BACs and genomic traces from this second individual gave an estimate of 50-100 copies in the amphioxus genome. The finding of an Alu element in amphioxus raises the question of whether Alu elements in amphioxus and primates arose by convergent evolution or by inheritance from a common ancestor. Genome-wide analyses of transposable elements in amphioxus and other chordates such as tunicates, agnathans and cartilaginous fishes could well provide the answer.

  18. The Potential Of High-Resolution BAC-FISH In Banana Breeding

    NARCIS (Netherlands)

    Capdeville, De G.; Souza, M.T.; Szinay, D.; Eugenio Cardamone Diniz, L.; Wijnker, T.G.; Swennen, R.; Kema, G.H.J.; Jong, de J.H.S.G.M.

    2009-01-01

    Abstract The genetic complexity in the genus Musa has been subject of study in many breeding programs worldwide. Parthenocarpy, female sterility, polyploidy in different cultivars and limited amount of genetic and genomic information make the production of new banana cultivars difficult and time con

  19. Comparison of Mycobacterium tuberculosis susceptibility testing performed with BACTEC 460TB (Becton Dickinson) and MB/BacT (Organon Teknika) systems.

    Science.gov (United States)

    Tortoli, E; Mattei, R; Savarino, A; Bartolini, L; Beer, J

    2000-10-01

    The recently introduced automated culture systems MB/BacT (Organon Teknika, Belgium) was compared with radiometric BACTEC 460TB (Becton Dickinson, USA) to test antimicrobial susceptibility of Mycobacterium tuberculosis to first line drugs. On 113 strains 97.5% agreement was obtained, with the difference being not significant. Concordance was practically complete for the most important drugs, isoniazid and rifampin. The two methods however significantly differed for the time needed to complete the test; in fact MB/BacT required on the average five days more than BACTEC 460TB. Despite the delay in the completion of the test, the excellent reliability along with the elimination of radioactivity and full automation make MB/BacT an attractive alternative for susceptibility testing of M. tuberculosis.

  20. C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD.

    Science.gov (United States)

    Liu, Yuanjing; Pattamatta, Amrutha; Zu, Tao; Reid, Tammy; Bardhi, Olgert; Borchelt, David R; Yachnis, Anthony T; Ranum, Laura P W

    2016-05-01

    To define how the C9orf72 GGGGCC expansion mutation causes ALS/FTD and to facilitate therapy development, a mouse model that recapitulates the molecular and phenotypic features of the disease is urgently needed. Two groups recently reported BAC mouse models that produce RNA foci and RAN proteins but, surprisingly, do not develop the neurodegenerative or behavioral features of ALS/FTD. We now report a BAC mouse model of C9orf72 ALS/FTD that shows decreased survival, paralysis, muscle denervation, motor neuron loss, anxiety-like behavior, and cortical and hippocampal neurodegeneration. These mice express C9orf72 sense transcripts and upregulated antisense transcripts. In contrast to sense RNA foci, antisense foci preferentially accumulate in ALS/FTD-vulnerable cell populations. RAN protein accumulation increases with age and disease, and TDP-43 inclusions are found in degenerating brain regions in end-stage animals. The ALS/FTD phenotypes in our mice provide a unique tool that will facilitate developing therapies targeting pathways that prevent neurodegeneration and increase survival. PMID:27112499

  1. EVALUACIÓN DE UNA COLECCIÓN DEL GÉNERO Triticum: TRIGO HARINERO (Triticum aestivum ssp aestivum, TRIGO DURO (Triticum turgidum ssp. durum Y TRITICALE (X Triticum secale Wittmack EN LAS CONDICIONES DEL OCCIDENTE DE CUBA

    Directory of Open Access Journals (Sweden)

    R. Plana

    2006-01-01

    Full Text Available En el Instituto Nacional de Ciencias Agrícolas (INCA se evaluó una colección del género Triticum, con el objetivo de conocer el comportamiento de diferentes especies en las condiciones del occidente de Cuba. Las especies estudiadas fueron: Triticum aestivum ssp. aestivum, Triticum turgidum ssp. durum y X Triticum secale Wittmack. Se utilizó un diseño experimental de bloques al azar con cuatro repeticiones. Los caracteres analizados fueron altura de la planta (cm, longitud de las espigas (cm, masa de mil granos (g y rendimiento agrícola (t.ha-1. Se utilizó la estadística descriptiva para cada una de las variables estudiadas, estimándose la media y desviación típica por especie. La variedad cubana de trigo harinero CC-204 fue utilizada como control. La matriz de datos obtenida (genotipo x variable fue procesada mediante la técnica multivariada de Componentes Principales. Los datos para todos los análisis estadísticos se ejecutaron por el paquete estadístico SPSS 11. 0 para Windows. La mayoría de los genotipos tuvieron un comportamiento medio general, aunque se separaron del resto algunos genotipos de trigo harinero y triticale. Los valores de la media y desviación estándar para las variables analizadas muestran que no fue posible distinguir diferencias por especie, aunque las plantas de trigo duro fueron las de menor altura, siendo los mejores genotipos: 12 y 54 de la especie T. aestivum (trigo harinero; 17, 18 y 25 de T. durum (trigo duro; 29 y 37 del X Triticum secale (tritic

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

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

    2004-12-01

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

  4. Contributions of Bacău to the economic literature and culture – The life and work of Professor Vasile Pătruţ

    OpenAIRE

    Mihai Deju

    2012-01-01

    The economic culture and accounting theory in Bacău area has its beginnings in the setting up of the first practical school of agriculture, by Ion Ionescu de la Brad, who included in the curriculum an accounting course, as well. Over the years, the economic education of Bacău area education has evolved from “The School of Accounting and Co-operative Education” (1919) to the modern economic higher education, in our days. During an important period of the evolution of education and culture in B...

  5. Effect of Temperature on Triticum aestivum L. Seedlings Growth and Phytohormone Balance

    Directory of Open Access Journals (Sweden)

    I.V. Kosakivska

    2015-12-01

    Full Text Available The study aimed to determine the effect of short-term heat and cold stresses on growth, accumulation and distribution of abscisic acid (ABA and indole-3-acetic acid (IAA in 7 and 14-day-old Triticum aestivum L. seedlings of the frost resistant cultivar Volodarka. A high performance liquid chromatography method has been used to provide a qualitative and quantitative analysis of hormones in leaves and roots. Biometric analysis showed that at the early stage of vegetation (7 days seedlings were more resistant to temperature stresses, especially to cold, than at the later ones (14 and 21 days, that correlates with the frost resistance of cultivar. The results indicated that at the early stages of growth, after a short-term cold stress (2 h., +20C, the amount of free ABA substantially increased in roots, while at the later ones, it occurred after a short-term heat stress (2 h., +400C in leaves. After a heat stress the leaves of 7-day-old seedlings accumulated a conjugated form of IAA. Heat stress caused the accumulation of free IAA in roots of 14-day-old seedlings. Cross stress (cold+heat caused some increase in the pool of endogenous ABA both in roots and leaves while the amount of free IAA increased only in leaves. Сhanges in the accumulation of the free and conjugated forms of ABA and IAA depended both on the organs and the age of seedlings, and the type of stress and correlated with frost resistance of the cultivar Volodarka.

  6. New isoforms and assembly of glutamine synthetase in the leaf of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Wang, Xiaochun; Wei, Yihao; Shi, Lanxin; Ma, Xinming; Theg, Steven M

    2015-11-01

    Glutamine synthetase (GS; EC 6.3.1.2) plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Here, three developmentally regulated isoforms of GS holoenzyme in the leaf of wheat (Triticum aestivum L.) seedlings are described using native-PAGE with a transferase activity assay. The isoforms showed different mobilities in gels, with GSII>GSIII>GSI. The cytosolic GSI was composed of three subunits, GS1, GSr1, and GSr2, with the same molecular weight (39.2kDa), but different pI values. GSI appeared at leaf emergence and was active throughout the leaf lifespan. GSII and GSIII, both located in the chloroplast, were each composed of a single 42.1kDa subunit with different pI values. GSII was active mainly in green leaves, while GSIII showed brief but higher activity in green leaves grown under field conditions. LC-MS/MS experiments revealed that GSII and GSIII have the same amino acid sequence, but GSII has more modification sites. With a modified blue native electrophoresis (BNE) technique and in-gel catalytic activity analysis, only two GS isoforms were observed: one cytosolic and one chloroplastic. Mass calibrations on BNE gels showed that the cytosolic GS1 holoenzyme was ~490kDa and likely a dodecamer, and the chloroplastic GS2 holoenzyme was ~240kDa and likely a hexamer. Our experimental data suggest that the activity of GS isoforms in wheat is regulated by subcellular localization, assembly, and modification to achieve their roles during plant development.

  7. Characterization and Expression Analysis of Phytoene Synthase from Bread Wheat (Triticum aestivum L.)

    Science.gov (United States)

    Flowerika; Alok, Anshu; Kumar, Jitesh; Thakur, Neha; Pandey, Ashutosh; Pandey, Ajay Kumar; Upadhyay, Santosh Kumar; Tiwari, Siddharth

    2016-01-01

    Phytoene synthase (PSY) regulates the first committed step of the carotenoid biosynthetic pathway in plants. The present work reports identification and characterization of the three PSY genes (TaPSY1, TaPSY2 and TaPSY3) in wheat (Triticum aestivum L.). The TaPSY1, TaPSY2, and TaPSY3 genes consisted of three homoeologs on the long arm of group 7 chromosome (7L), short arm of group 5 chromosome (5S), and long arm of group 5 chromosome (5L), respectively in each subgenomes (A, B, and D) with a similarity range from 89% to 97%. The protein sequence analysis demonstrated that TaPSY1 and TaPSY3 retain most of conserved motifs for enzyme activity. Phylogenetic analysis of all TaPSY revealed an evolutionary relationship among PSY proteins of various monocot species. TaPSY derived from A and D subgenomes shared proximity to the PSY of Triticum urartu and Aegilops tauschii, respectively. The differential expression of TaPSY1, TaPSY2, and TaPSY3 in the various tissues, seed development stages, and stress treatments suggested their role in plant development, and stress condition. TaPSY3 showed higher expression in all tissues, followed by TaPSY1. The presence of multiple stress responsive cis-regulatory elements in promoter region of TaPSY3 correlated with the higher expression during drought and heat stresses has suggested their role in these conditions. The expression pattern of TaPSY3 was correlated with the accumulation of β-carotene in the seed developmental stages. Bacterial complementation assay has validated the functional activity of each TaPSY protein. Hence, TaPSY can be explored in developing genetically improved wheat crop. PMID:27695116

  8. Exogenous application of putrescine at pre-anthesis enhances the thermotolerance of wheat (Triticum aestivum L.).

    Science.gov (United States)

    Kumar, Ranjeet R; Sharma, Sushil K; Rai, Gyanendra K; Singh, Khushboo; Choudhury, Madhumanthi; Dhawan Gaurav; Singh, Gyaneshwar P; Goswami, Suneha; Pathak, Himanshu; Rai, Raj D

    2014-10-01

    Antioxidant enzymes, besides being involved in various developmental processes, are known to be important for environmental stress tolerance in plants. In this study, the effect of treatment of 2.5 mM putrescine (Put), heat stress (HS -42 degrees C for 2 h) and their combination on the expression and activity of antioxidant enzymes was studied at pre-anthesis in the leaves of two wheat (Triticum aestivum L.) cultivars--HDR77 (thermotolerant) and HD2329 (thermosusceptible). We observed that 2.5 mM Put before HS significantly enhanced the transcript levels of superoxide dismutase (SOD), catalase (CAT), cytoplasmic and peroxisomal ascorbate peroxidase (cAPX, pAPX) in both the cultivars. However, the activities of antioxidant enzymes (SOD, CAT, APX and GR), as well as accumulation of antioxidants (ascorbic acid and total thiol content) were higher in HDR77 than in HD2329 in response to the treatment 2.5 mM Put + HS. No significant change was observed in the proline accumulation in response to HS and combined treatment of 2.5 mM Put + HS. A decrease in the H2O2 accumulation, lipid peroxidation and increase in cell membrane stability (CMS) were observed in response to 2.5 mM Put + HS treatment, as compared to HS treatment alone in both the cultivars; HDR77 was, however, more responsive to 2.5 mM Put + HS treatment. Put (2.5 mM) treatment at pre-anthesis thus modulated the defense mechanism responsible for the thermotolerance capacity of wheat under the heat stress. Elicitors like Put, therefore, need to be further studied for temporarily manipulating the thermotolerance capacity of wheat grown under the field conditions in view of the impending global climate change. PMID:25630110

  9. Zinc and copper uptake by plants under two transpiration rates. Part I. Wheat (Triticum aestivum L.)

    International Nuclear Information System (INIS)

    To evaluate the environmental risk of irrigating crops with treated wastewater, an experiment was conducted using two growth chambers, each offering a different vapour pressure deficit (VPD) for high and low transpiration rates (TR), respectively. One of the two sets of 24 pots planted with 6 week old wheat (Triticum aestivum L.), was placed in each growth chamber, and irrigated in triplicates for 20 days with 8 Zn and Cu solutions (0 and 25 mg Zn/L combined with 0, 5, 15 and 30 mg Cu/L). Water losses from planted and non-planted pots served to measure evapo-transpiration and evaporation, respectively. Pots were monitored for Cu and Zn uptake by collecting three plants (shoot and grain)/pots after 0, 10 and 20 days, and roots in each pot after 20 days, and analyzing these plant parts for dry mass, and Cu and Zn levels. Transpiration rate was not affected by any Cu/Zn treatment, but Cu and Zn uptake increase with the time, irrigation solution level and higher TR, with the roots retaining most Cu and Zn, compared to the shoot followed by the grain. For the shoot and grain, Cu had a significant synergetic effect on Zn uptake, when Zn had slight but insignificant antagonistic effects on Cu uptake. For the roots, Cu and Zn had significant synergetic effect on each other. Regression equations obtained from the data indicate that Cu and Zn levels normally found in treated wastewater (0.08 mg/L) are 300 times lower than those used for the most concentrated experimental solutions (30 and 25 mg/L, respectively) and may, on a long term basis, be beneficial rather than toxic to wheat plants and do not acidify soil pH. - Cu and Zn levels normally found in treated wastewater may be beneficial rather than toxic to wheat plants

  10. A study on application of BacT/ALERT 3D liquid culture technology on tuberculosis drug resistance detection%BacT/ALERT 3D 技术在结核杆菌耐药检测中的应用研究

    Institute of Scientific and Technical Information of China (English)

    黄文忠; 王平平; 吴红照; 周燕珍; 胡真宝

    2014-01-01

    目的:比较 BacT/ALERT 3D 法与传统罗氏(L -J)比例法在耐药结核分枝杆菌检测中的差异,评价BacT/ALERT 3D 快速培养系统在结核分枝杆菌耐药检测中的应用价值。方法对219份固体培养结核杆菌阳性的培养物采用 BacT/ALERT 3D 液体培养技术和 L -J 比例法同时进行药敏试验,并进行比对分析。结果BacT/ALERT 3D 法平均检出时间(8.02±3.85)d,比 L -J 比例法检出时间平均缩短约20 d;BacT/ALERT 3D 法检出耐药菌60株,L -J 比例法检出耐药菌79株,差异无统计学意义(P >0.05);以 L -J 比例法结果为判定标准, BacT/ALERT 3D 法对 INH、RFP、EMB 和 SM 4种抗结核药耐药检测结果符合率分别为95.43%、92.69%、95.43%和92.24%。结论与 L -J 比例法比较,BacT/ALERT 3D 药敏检测方法对结核杆菌的耐药检出结果有较高的一致性,BacT/ALERT 3D 法检出时间明显短于 L -J 比例法。%Objective To evaluate the BacT/ALERT 3D liquid culture technology on the detection of drug resistance of Mycobacterium tuberculosis(MTB)and to compare the difference between this technology and Lowenstein -Jensen (L -J) proportion method.Methods BacT/ALERT 3D liquid culture technology and L -J proportion technology were applied to detect the drug resistance of tuberculosis from the positive cultures of 219 solid culture samples.Results The average detection time of BacT/ALERT 3D method was 8.02 ±3.85 d,which was about 20 days shorter than that of L -J proportion method.60 drug resistance strains were found using BacT/ALERT 3D technology,While 79 drug resistance strains were found using L -J proportion technology.There showed no significant difference (P >0.05).The compliance rate of BacT/ALERT 3D method and L -J proportion method on the anti -tuberculosis drugs INH,RFP,EMB and SMwas 95.43%,92.69%,95.43% and 92.24% respectively.Conclusion BacT/ALERT 3D liquid culture technology could detect drug resistant TB

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

    OpenAIRE

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

    2003-01-01

    P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniqu...

  12. The Potential Of High-Resolution BAC-FISH In Banana Breeding

    OpenAIRE

    Capdeville, De, G.; de Souza, M T; Szinay, D.; Eugenio Cardamone Diniz, L.; Wijnker, T.G.; Swennen, R.; Kema, G.H.J.; Jong, de, T.

    2009-01-01

    Abstract The genetic complexity in the genus Musa has been subject of study in many breeding programs worldwide. Parthenocarpy, female sterility, polyploidy in different cultivars and limited amount of genetic and genomic information make the production of new banana cultivars difficult and time consuming. In addition, it is known that part of the cultivars and related wild species in the genus contain numerous chromosomal rearrangements. In order to produce new cultivars more effectively bre...

  13. Functional and structural insights into novel DREB1A transcription factors in common wheat (Triticum aestivum L.)

    DEFF Research Database (Denmark)

    Kumar, Anuj; Kumar, Sanjay; Kumar, Upendra;

    2016-01-01

    -binding (DREB1A) factors, a class of transcription factors (TF) play an important role in combating drought stress. It is known that DREB1A specifically interacts with the dehydration responsive elements (DRE/CRT) inducing expression of genes involved in environmental stress tolerance in plants. Despite its......Triticum aestivum L. known as common wheat is one of the most important cereal crops feeding a large and growing population. Various environmental stress factors including drought, high salinity and heat etc. adversely affect wheat production in a significant manner. Dehydration-responsive element...

  14. Chromium uptake and toxicity effects on growth and metabolic activities in wheat, Triticum aestivum L. cv. UP 2003.

    Science.gov (United States)

    Sharma, D C; Sharma, C P

    1996-07-01

    Chromium (Cr) at graded levels when added in sand culture of wheat (T. aestivum L. cv. UP2003) under glasshouse conditions resulted in reduction in biomass, chlorophyll and activities of catalase and peroxidase while enhanced acid phosphatase and ribonuclease activities. Elevated levels of Cr supply significantly reduced the concentration of inorganic phosphorus. With an increase in Cr supply the uptake of chromium also increased significantly in different plant parts especially in roots. Above metabolic lesions due to Cr in wheat provided evidence that the element in nutrient medium if present in excess may be inhibitory to plant growth and development.

  15. The Effects of Different Carbon Dioxide Doses on Yield and Nutritional Values of Hydroponic Wheat (Triticum aestivum L.) Grass Juice

    OpenAIRE

    KARAŞAHİN, Muhammet

    2015-01-01

    This research has been conducted in the conditioning chamber of  Karabük University Eskipazar Vocational School Department Crop and Animal Production between the dates of 5.01.2015 and 01.08.2015 in order to identify the effects of different carbon dioxide doses on yield and nutritional values of hydroponic wheat (Triticum aestivum L.) grass juice. In the research, three different carbon dioxide doses (Control; 0, D1; 750, D2; 1500, D3; 2000 ppm) have been applied to growth medium and their e...

  16. A recessive gene controlling male sterility sensitive to short daylength/low temperature in wheat (Triticum aestivum L.)*

    OpenAIRE

    Chen, Xiao-Dong; Sun, Dong-Fa; Rong, De-fu; Peng, Jun-hua; Li, Cheng-dao

    2011-01-01

    Utilization of a two-line breeding system via photoperiod-thermo sensitive male sterility has a great potential for hybrid production in wheat (Triticum aestivum L.). 337S is a novel wheat male sterile line sensitive to both short daylength/low temperature and long daylength/high temperature. Five F2 populations derived from the crosses between 337S and five common wheat varieties were developed for genetic analysis. All F1’s were highly fertile while segregation occurred in the F2 population...

  17. Two quality-associated HMW glutenin subunits in a somatic hybrid line between Triticum aestivum and Agropyron elongatum

    OpenAIRE

    Feng, Deshun; Xia, Guangmin; Zhao, Shuangyi; Chen, Fanguo

    2004-01-01

    High-molecular-weight glutenin subunits (HMW-GSs) from hybrid line II-12 between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nivski were characterized with SDS-PAGE. Out of these HMW-GSs, two subunits, h1Bx and h1By, had mobilities similar to the subunits 1Bx13 and 1By16 from common wheat 4072, which was used as control. Polyclonal antibodies (pAbs) of h1Bx and h1By were prepared, and Western blotting showed that the pAbs had strong affinities for h1Bx and h1By, separately. Th...

  18. Genetic transformation of Indian bread (T. aestivum and pasta (T. durum wheat by particle bombardment of mature embryo-derived calli

    Directory of Open Access Journals (Sweden)

    Khurana Paramjit

    2003-09-01

    Full Text Available Abstract Background Particle bombardment has been successfully employed for obtaining transgenics in cereals in general and wheat in particular. Most of these procedures employ immature embryos which are not available throughout the year. The present investigation utilizes mature seeds as the starting material and the calli raised from the hexaploid Triticum aestivum and tetraploid Triticum durum display a high regeneration response and were therefore used as the target tissue for genetic transformation by the biolistic approach. Results Mature embryo-derived calli of bread wheat (Triticum aestivum, cv. CPAN1676 and durum wheat (T. durum, cv. PDW215 were double bombarded with 1.1 gold microprojectiles coated with pDM302 and pAct1-F at a target distance of 6 cm. Southern analysis using the bar gene as a probe revealed the integration of transgenes in the T0 transformants. The bar gene was active in both T0 and T1 generations as evidenced by phosphinothricin leaf paint assay. Approximately 30% and 33% primary transformants of T. aestivum and T. durum, respectively, were fertile. The transmission of bar gene to T1 progeny was demonstrated by PCR analysis of germinated seedlings with primers specific to the bar gene. Conclusions The transformation frequency obtained was 8.56% with T. aestivum and 10% with T. durum. The optimized protocol was subsequently used for the introduction of the barley gene encoding a late embryogenesis abundant protein (HVA1 in T. aestivum and T. durum. The presence of the HVA1 transgene was confirmed by Southern analysis in the T0 generation in case of Triticum aestivum, and T0 and T1 generation in Triticum durum.

  19. Application of BacT/ALERT 3D in Fast Culture and Drug Sensitive Test of Mycobacterium Tuberculosis%BacT/ALERT3D在结核分枝杆菌快速培养和药敏试验中的应用

    Institute of Scientific and Technical Information of China (English)

    徐东芳; 王庆

    2011-01-01

    目的 探讨BacT/ALERT 3D在结核分枝杆菌快速培养中的应用.方法 应用BacT/ALERT 3D检测结核分枝杆菌,并与传统L-J法比较.结果 523份痰标本BacT/ALERT 3D法检出阳性217份(41.52%),阳性报告时间平均14.05 d;L-J法检出阳性178份(34.03%).BacT/ALERT 法培养结核分枝杆菌的阳性率明显高于L-J法(P<0.05);阳性报告时间提前14.66 d,差异有统计学意义(P<0.05).BacT/ALERT 3D快速药敏法表明总耐药率为56.2%,药敏报告时间平均8.5 d,L-J法则为30 d,两种方法药敏符合率为96.38%(213/221); 使用BacT/ALERT 3D快速法进行结核分枝杆菌的培养和药敏试验,报告时间比L-J法总计提前35 d以上.结论 BacT/ALERT 3D法是目前较理想的一种快速检测结核分枝杆菌的方法.

  20. Evaluación de la calidad funcional y sensorial en cultivares de Triticum aestivum ssp. vulgare y ssp. spelta en cultivo ecológico

    OpenAIRE

    Vargas Kostiuk, María Eugenia

    2016-01-01

    En esta Tesis Doctoral se ha estudiado la influencia del cultivar sobre el comportamiento reológico y panadero de cinco cultivares de trigo sembrados en el mismo año y en el mismo ambiente, en condiciones de cultivo ecológico. Tres de ellos eran de trigo panadero (Triticum aestivum ssp. vulgare), ‘Bonpain’, ‘Craklin’ y ‘Sensas’ y los otros dos de trigo espelta (Triticum aestivum ssp. spelta), ‘Espelta Álava’ y ‘Espelta Navarra’. Actualmente, el alohexaploide trigo panadero (2n=6x=42 genomio A...

  1. Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants

    Science.gov (United States)

    Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversi...

  2. Dissecting miRNAs in Wheat D Genome Progenitor, Aegilops tauschii.

    Science.gov (United States)

    Akpinar, Bala A; Budak, Hikmet

    2016-01-01

    As the post-transcriptional regulators of gene expression, microRNAs or miRNAs comprise an integral part of understanding how genomes function. Although miRNAs have been a major focus of recent efforts, miRNA research is still in its infancy in most plant species. Aegilops tauschii, the D genome progenitor of bread wheat, is a wild diploid grass exhibiting remarkable population diversity. Due to the direct ancestry and the diverse gene pool, A. tauschii is a promising source for bread wheat improvement. In this study, a total of 87 Aegilops miRNA families, including 51 previously unknown, were computationally identified both at the subgenomic level, using flow-sorted A. tauschii 5D chromosome, and at the whole genome level. Predictions at the genomic and subgenomic levels suggested A. tauschii 5D chromosome as rich in pre-miRNAs that are highly associated with Class II DNA transposons. In order to gain insights into miRNA evolution, putative 5D chromosome miRNAs were compared to its modern ortholog, Triticum aestivum 5D chromosome, revealing that 48 of the 58 A. tauschii 5D miRNAs were conserved in orthologous T. aestivum 5D chromosome. The expression profiles of selected miRNAs (miR167, miR5205, miR5175, miR5523) provided the first experimental evidence for miR5175, miR5205 and miR5523, and revealed differential expressional changes in response to drought in different genetic backgrounds for miR167 and miR5175. Interestingly, while miR5523 coding regions were present and expressed as pre-miR5523 in both T. aestivum and A. tauschii, the expression of mature miR5523 was observed only in A. tauschii under normal conditions, pointing out to an interference at the downstream processing of pre-miR5523 in T. aestivum. Overall, this study expands our knowledge on the miRNA catalog of A. tauschii, locating a subset specifically to the 5D chromosome, with ample functional and comparative insight which should contribute to and complement efforts to develop drought tolerant

  3. Dissecting miRNAs in wheat D genome progenitor, Aegilops tauschii

    Directory of Open Access Journals (Sweden)

    Hikmet eBudak

    2016-05-01

    Full Text Available As the post-transcriptional regulators of gene expression, microRNAs or miRNAs comprise an integral part of understanding how genomes function. Although miRNAs have been a major focus of recent efforts, miRNA research is still in its infancy in most plant species. Aegilops tauschii, the D genome progenitor of bread wheat, is a wild diploid grass exhibiting remarkable population diversity. Due to the direct ancestry and the diverse gene pool, A. tauschii is a promising source for bread wheat improvement. In this study, a total of 87 Aegilops miRNA families, including 51 previously unknown, were computationally identified both at the subgenomic level, using flow-sorted A. tauschii 5D chromosome, and at the whole genome level. Predictions at the genomic and subgenomic levels suggested A. tauschii 5D chromosome as rich in pre-miRNAs that are highly associated with Class II DNA transposons. In order to gain insights into miRNA evolution, putative 5D chromosome miRNAs were compared to its modern ortholog, T. aestivum 5D chromosome, revealing that 48 of the 58 A. tauschii 5D miRNAs were conserved in orthologous T. aestivum 5D chromosome. The expression profiles of selected miRNAs (miR167, miR5205, miR5175, miR5523 provided the first experimental evidence for miR5175, miR5205 and miR5523, and revealed differential expressional changes in response to drought in different genetic backgrounds for miR167 and miR5175. Interestingly, while miR5523 coding regions were present and expressed as pre-miR5523 in both T. aestivum and A. tauschii, the expression of mature miR5523 was observed only in A. tauschii under normal conditions, pointing out to an interference at the downstream processing of pre-miR5523 in T. aestivum. Overall, this study expands our knowledge on the miRNA catalogue of Aegilops tauschii, locating a subset specifically to the 5D chromosome, with ample functional and comparative insight which should contribute to and complement efforts to

  4. Clinical evaluation of BacT/Alert and the factor for false positive%全自动血培养系统临床应用评价

    Institute of Scientific and Technical Information of China (English)

    张秀珍; 宣天芝; 胡云建

    1998-01-01

    目的评价全自动血培养系统BacT/Alert系统的功能及影响因素.方法 1 800份血标本应用BacT/Alert系统的分析结果对其分离阳性率、假阳性率、阳性最早出现时间及所分离出有意义的病原微生物进行讨论和评价.结果 1 800份血液标本用 BacT/Alert全自动血液培养仪检测,检出阳性标本 363份(20.2%);平均假阳性率为1.6%;系统总体阳性出现时间分别为≤10小时占20.8%;10~24小时占41.6%;24~48小时占35.6%;超过48小时报告阳性占2%.结论 BacT/Alert系统全自动血液分析仪具有自动化程度高、快速、正确等优点,严格控制各项操作条件是正确报告的关键.

  5. Visualization of the Epiblast and Visceral Endodermal Cells Using Fgf5-P2A-Venus BAC Transgenic Mice and Epiblast Stem Cells

    Science.gov (United States)

    Tsukiyama, Tomoyuki; Matsushita, Jun; Tsukiyama-Fujii, Setsuko; Takahashi, Satoru; Ema, Masatsugu

    2016-01-01

    Fibroblast growth factor 5 (Fgf5) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research. PMID:27409080

  6. Visualization of the Epiblast and Visceral Endodermal Cells Using Fgf5-P2A-Venus BAC Transgenic Mice and Epiblast Stem Cells.

    Directory of Open Access Journals (Sweden)

    Le Tran Phuc Khoa

    Full Text Available Fibroblast growth factor 5 (Fgf5 has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.

  7. Characteristics of DOM and Removal of DBPs Precursors across O3-BAC Integrated Treatment for the Micro-Polluted Raw Water of the Huangpu River

    Directory of Open Access Journals (Sweden)

    Xiwu Lu

    2013-09-01

    Full Text Available The aim of this study is to determine the impact upon dissolved organic matter (DOM and removal of disinfection by-product (DBP precursors of adding an ozone-biological activated carbon (O3-BAC process after the conventional treatment process (CTP using water from the Huangpu River in Shanghai, east China. Several metrics, including size fractionation, dissolved organic carbon (DOC and trihalomethane formation potential (THMFP, were employed to accomplish this goal. In the raw water collected from the Huangpu River, the low molecular weight (MW molecules (MW 10 kDa by 61% and the low MW molecules (MW <3 kDa by only 8%. The O3-BAC presented an accumulated DOC removal efficiency of approximately 50%, in particular, showing a high degree of the removal effectiveness of low MW molecules. Samplings from the CTP, ozone and BAC were subjected to excessive chlorination to determine the THMFP, and the measured concentrations were 211.89 ± 4.58 µg/L, 169.52 ± 4.55 µg/L, and 124.42 ± 4.27 µg/L, respectively. Therefore, coupled with the THMFP removal rate of 74%, the addition of the O3-BAC process after an existing CTP improved the water quality of the effluent, particularly, in terms of the improved reduction in the precursors of DBPs.

  8. BRAD, the genetics and genomics database for Brassica plants

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    Li Pingxia

    2011-10-01

    Full Text Available Abstract Background Brassica species include both vegetable and oilseed crops, which are very important to the daily life of common human beings. Meanwhile, the Brassica species represent an excellent system for studying numerous aspects of plant biology, specifically for the analysis of genome evolution following polyploidy, so it is also very important for scientific research. Now, the genome of Brassica rapa has already been assembled, it is the time to do deep mining of the genome data. Description BRAD, the Brassica database, is a web-based resource focusing on genome scale genetic and genomic data for important Brassica crops. BRAD was built based on the first whole genome sequence and on further data analysis of the Brassica A genome species, Brassica rapa (Chiifu-401-42. It provides datasets, such as the complete genome sequence of B. rapa, which was de novo assembled from Illumina GA II short reads and from BAC clone sequences, predicted genes and associated annotations, non coding RNAs, transposable elements (TE, B. rapa genes' orthologous to those in A. thaliana, as well as genetic markers and linkage maps. BRAD offers useful searching and data mining tools, including search across annotation datasets, search for syntenic or non-syntenic orthologs, and to search the flanking regions of a certain target, as well as the tools of BLAST and Gbrowse. BRAD allows users to enter almost any kind of information, such as a B. rapa or A. thaliana gene ID, physical position or genetic marker. Conclusion BRAD, a new database which focuses on the genetics and genomics of the Brassica plants has been developed, it aims at helping scientists and breeders to fully and efficiently use the information of genome data of Brassica plants. BRAD will be continuously updated and can be accessed through http://brassicadb.org.

  9. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  10. Arbuscular mycorrhizal fungi modify nutrient allocation and composition in wheat (Triticum aestivum L.) subjected to heat-stress

    DEFF Research Database (Denmark)

    Cabral, Carmina Falcato; Ravnskov, Sabine; Tringovska, Ivanka;

    2016-01-01

    Aims To evaluate the role of the AM symbiosis on nutrient allocation in Triticum aestivum L. cv. 1110 at different growth stages before and after heat-stress at anthesis. Methods Measurements of plant biomass and grain yield at anthesis, grain-filling and maturity; determination of macro- and mic......Aims To evaluate the role of the AM symbiosis on nutrient allocation in Triticum aestivum L. cv. 1110 at different growth stages before and after heat-stress at anthesis. Methods Measurements of plant biomass and grain yield at anthesis, grain-filling and maturity; determination of macro......- and micronutrient concentrations in aboveground biomass; evaluation of AM fungal structures in roots and assessment of light-use efficiency of plants. Results AM increased grain number in wheat under heat-stress, and altered nutrient allocation and tiller nutrient composition. Heat increased number of arbuscules...... in wheat root, whereas number of vesicles and total colonization were unaffected. Heat increased photosystem II yield and the electron transfer rate, whereas non-photochemical quenching decreased during the first 2 days of heat-stress. Conclusions Nutrient allocation and –composition in wheat grown under...

  11. A whole-genome, radiation hybrid mapping resource of hexaploid wheat.

    Science.gov (United States)

    Tiwari, Vijay K; Heesacker, Adam; Riera-Lizarazu, Oscar; Gunn, Hilary; Wang, Shichen; Wang, Yi; Gu, Young Q; Paux, Etienne; Koo, Dal-Hoe; Kumar, Ajay; Luo, Ming-Cheng; Lazo, Gerard; Zemetra, Robert; Akhunov, Eduard; Friebe, Bernd; Poland, Jesse; Gill, Bikram S; Kianian, Shahryar; Leonard, Jeffrey M

    2016-04-01

    Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole-genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics-based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole-genome using these approaches is nearly impossible. We developed a whole-genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high-density single nucleotide polymorphism (SNP) array. At the whole-genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR1500 with a total map length of 6866 cR1500 . The 7296 unique mapping bins provided a five- to eight-fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low-cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS-WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high-quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species. PMID:26945524

  12. A novel piggyBac transposon inducible expression system identifies a role for AKT signalling in primordial germ cell migration.

    Directory of Open Access Journals (Sweden)

    James D Glover

    Full Text Available In this work, we describe a single piggyBac transposon system containing both a tet-activator and a doxycycline-inducible expression cassette. We demonstrate that a gene product can be conditionally expressed from the integrated transposon and a second gene can be simultaneously targeted by a short hairpin RNA contained within the transposon, both in vivo and in mammalian and avian cell lines. We applied this system to stably modify chicken primordial germ cell (PGC lines in vitro and induce a reporter gene at specific developmental stages after injection of the transposon-modified germ cells into chicken embryos. We used this vector to express a constitutively-active AKT molecule during PGC migration to the forming gonad. We found that PGC migration was retarded and cells could not colonise the forming gonad. Correct levels of AKT activation are thus essential for germ cell migration during early embryonic development.

  13. Jícuri y bacánowa: Rituales de vida y muerte en la sierra tarahumara

    Directory of Open Access Journals (Sweden)

    Ángel Acuña Delgado

    2009-01-01

    Full Text Available En base a los datos obtenidos durante los once meses de experiencia de campo, entre 2001 y 2005, en varias comunidades rarámuris de la Alta y Baja Tarahumara, al suroeste del estado de Chihuahua (México, en el presente trabajo centramos la atención en dos rituales y al mismo tiempo formas de danza, ambos de carácter privado, que ocupan un papel esencial en la cosmovisión rarámuri. El hecho de haber participado como espectador invitado en tales prácticas nos permitirá describir el contexto etnográfico de primera mano; para finalizar destacando especialmente las funciones y significados que llevan asociados. Antes ofreceremos, no obstante, una serie de referencias bibliográficas que sitúan al jícuri y al bacánowa entre la historia y la leyenda.

  14. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available Bloodstream infection (BSI and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample, amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS. We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis, and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours.The IRIDICA BAC BSI Assay is not available in the United States.

  15. Characterization of constitutive promoters for piggyBac transposon-mediated stable transgene expression in mesenchymal stem cells (MSCs.

    Directory of Open Access Journals (Sweden)

    Sheng Wen

    Full Text Available Multipotent mesenchymal stem cells (MSCs can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40, one housekeeping gene promoter (UbC, and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH. CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.

  16. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood

    Science.gov (United States)

    Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Zhang, Sean X.; Avornu, Gideon D.; Rounds, Megan A.; Carolan, Heather E.; Toleno, Donna M.; Moore, David; Hall, Thomas A.; Massire, Christian; Richmond, Gregory S.; Gutierrez, Jose R.; Sampath, Rangarajan; Ecker, David J.; Blyn, Lawrence B.

    2016-01-01

    Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States. PMID:27384540

  17. ANTHOCYANIN PIGMENTATION IN TRITICUM AESTIVUM L.: GENETIC BASIS AND ROLE UNDER ABIOTIC STRESS CONDITIONS

    Directory of Open Access Journals (Sweden)

    Tereshchenko O.Yu.

    2012-08-01

    Full Text Available Anthocyanins are secondary metabolites of plants. They have a wide range of biological activity such as antioxidant, photoprotection, osmoregulation, heavy metal ions chelation, antimicrobial and antifungal activities, which help plants to survive under different stress conditions. Bread wheat (T. aestivum L. can have purple pigmentation provided by anthocyanin compounds in different organs, such as grain pericarp, coleoptile, culm, leaf blades, leaf sheaths, glumes and anthers. However, the genetic mechanisms underlying formation of these traits as well as contribution of the pigmentation to stress tolerance have not been widely studied in wheat. The aim of the current study was to investigate molecular-genetic mechanisms underlying anthocyanin pigmentation in different wheat organs and to estimate the role of the pigmentation under different abiotic stress conditions in wheat seedlings. In the current study, near-isogenic lines (NILs: cv. ‘Saratovskaya 29’ (‘S29’ and lines i:S29Pp1Pp2PF and i:S29Pp1Pp3P developed on the ‘S29’ background but having grain pericarp coloration (genes Pp and more intense coleoptile (Rc, culm (Pc, leaf blade (Plb, leaf sheath (Pls pigmentation in comparison with ‘S29’, were used. Comparative transcriptional analysis of the five structural genes Chs, Chi, F3h, Dfr, Ans, encoding enzymes participating in the anthocyanin biosynthesis, was performed in different organs of NILs. It was shown that the presence of the Rc, Pc, Plb, Pls and Pp alleles conferring strong anthocyanin pigmentation induced more intense transcription of the structural genes, suggesting the genes Rc, Pc, Plb, Pls and Pp to play a regulatory role in anthocyanin biosynthesis network. To evaluate the role of anthocyanins in stress response at the seedling stage, growth ability of the NILs and anthocyanin content in their coleoptiles were assessed after treatments with NaCl (100 and 200 mM, CdCl2 (25 and 50 μM and 15% PEG 6000

  18. GENETIC DETERMINATION OF THE NITROGEN SUPPLY OF SPRING WHEAT (TRITICUM AESTIVUM L.

    Directory of Open Access Journals (Sweden)

    Sitnikov M.N.

    2012-08-01

    Full Text Available The maximum grain productivity can be achieved only taking into account plant biological needs. The need in mineral nutrients depends on the plant hereditary nature and environmental conditions. The greatest demand for nitrogen is characteristic in cereals for spring and winter wheat, the lowest one for barley and rye. The use of mineral nutrients in amounts exceeding plant needs does not result in yield increasing and can worsen the production quality. We were studying reaction of spring bread wheat (Triticum aestivum L. genotypes to changes in nutrition soil conditions. In the experiment, ITMI mapping population consisting of 110 recombinant inbred lines was evaluated for a number of morphological, biological and economically important traits under different levels of the nitrogen supply. To create different soil nutrition level and to prevent leaching of fertilizers during the plant vegetation season we prepared trenches with depth 0.4 m, width 1 m and length 20 m; the bottoms of which were covered with plastic films. The trenches were filled with soil from the lower soil horizons. In first variant of the experiment, nutrient mixture on the basis of the physiological rate for cereals (N – 0.15 g, P - 0.1 g, K - 0.1 g of active substances per 1 kg of dry soil was applied. In the second variant, nitrogen dose was reduced half with the same phosphorus and potassium doses. Variant without fertilizers applying was used as a control. Thirty nine characters were analyzed during the all growing season. The combination of field and vegetation experiment conditions allowed approximating maximally to real conditions of the experiment and at the same time to control strictly plant vegetation. QTLs identified in our study can be differentiated as dependent and independent on environmental conditions. For example, some QTLs controlling such traits as a wax bloom, phenological phases, etc. are stable under different conditions of soil nutrition. QTLs of

  19. Identification of superior parents and hybrids from diallel crosses of bread wheat (triticum aestivum l.)

    International Nuclear Information System (INIS)

    Five parents of bread wheat (Triticum aestivum L.) viz. TD-1, SKD-1, Marvi, Moomal and Mehran were crossed in a half diallel design; hence 10 F1 hybrids were developed. Parents alongwith hybrids were evaluated for combining ability and heterosis for tillers/plant, spike length, spike density, grains/spike, grain yield/plant and seed index. The experiment was conducted in a randomized complete block design with four replications at Botanical Garden, Department of Plant Breeding and Genetics, Sindh Agriculture University,Tandojam, during 2010. The analysis of variance due to genotypes, parents, hybrids and parents vs. hybrids was significant for all the characters which revealed presence of significant amount of genetic variability in the material. The results also indicated significant differences among the parents for their general combining ability (GCA) and hybrids for specific combining ability (SCA) suggesting the importance of both additive and non-additive genes in the expression of traits studied. The greater magnitude of SCA variances over GCA were recorded for tillers/plant, grains/spike and grain yield/plant which indicated the importance of additive gene action while the involvement of non-additive genes was evident in the inheritance of spike length, spike density and seed index. Among the parents, generally TD-I, Mehran, Moomal and Marvi were the best general combiners for tillers/plant, spike length, spike density, grains/spike, grain yield/plant and seed index. Whereas, the hybrids like SKD-1 x Mehran, Marvi x Mehran, Marvix Moomal and TD-I x SKD-I were the best specific combiners for majority of yield traits. Positive heterosis was expressed by the hybrid SKD-1 x Moomal for tillers per plant; TD-I x Moomal for spike length; TD-1 x SKD-I for grains per spike; Marvi x Mehran for spike density and Marvi x Moomal for seed index. The best parents and hybrids could be effectively utilized in hybridization and selection programmes and also for hybrid crop

  20. Effect of dissolved organic matter on the toxicity of chlorotoluron to Triticum aestivum

    Institute of Scientific and Technical Information of China (English)

    SONG Ning-hui; YANG Zhi-min; ZHOU Li-xian; WU Xin; YANG Hong

    2006-01-01

    Response of two wheat cultivars (Triticum aestivum cv. YM 158 and NM 9) to the herbicide chlorotoluron and the effect of two forms of dissolved organic matter on the chlorotoluron toxicity to the plants were characterized. Treatment with chlorotoluron at 10-50 μg/ml inhibited the seed germination and a dose-response was observed. The inhibition of seed germination was correlated to the depression of α-amylase activities. To identify whether chlorotoluron induced oxidative damage to wheat plants, the malondlaldehyde (MDA) content and electrolyte leakage were measured. Results showed that both MDA content and electrolyte leakage in the chlorotoluron-treated roots significantly increased. Activities of several key enzymes were measured that operate in citric acid cycle and carbohydrate metabolic pathway. Inhibited activities of citrate synthase and NADP-isocitrate dehydrogenase were observed in the chlorotoluron-treated roots as compared to control plants. We also examined malate dehydrogenase and phosphoenolpyruvate carboxylase in wheat roots exposed to 30 μg/ml chlorotoluron. However, none of the enzymes showed significant changes in activities. Application of 160 μg/ml dissolved organic matter (DOM) extracted from non-treated sludge(NTS)and heat-expanded sludge (HES) in the medium with 30 μg/ml chlorotoluron induced an additive inhibition of seed germination and plant growth. The inhibition of growth due to the DOM treatment was associated with the depression of activities of α-amylase, citrate synthase and NADP-isocitrate dehydrogenase, as well as the increase in malondlaldehyde content and electrolyte leakage. These results suggested that the presence of DOM might enhance the uptake and accumulation of chlorotoluron, and thus resulted in greater toxicity in wheat plants. The two forms of DOM exhibited differences in regulation of chlorotoluron toxicity to the wheat plants.Treatments with DOM-NTS induced greater toxicity to plants as compared to those with