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Sample records for artificial chromosome libraries

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was compos...

  2. Recombination walking: genetic selection of clones from pooled libraries of yeast artificial chromosomes by homologous recombination.

    PubMed Central

    Miller, A M; Savinelli, E A; Couture, S M; Hannigan, G M; Han, Z; Selden, R F; Treco, D A

    1993-01-01

    Recombination walking is based on the genetic selection of specific human clones from a yeast artificial chromosome (YAC) library by homologous recombination. The desired clone is selected from a pooled (unordered) YAC library, eliminating labor-intensive steps typically used in organizing and maintaining ordered YAC libraries. Recombination walking represents an efficient approach to library screening and is well suited for chromosome-walking approaches to the isolation of genes associated with common diseases. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8367472

  3. Construction, characterization and FISH mapping of a bacterial artificial chromosome library of Chinese pangolin (Manis pentadactyla).

    PubMed

    Che, J; Wang, J; Su, W; Ye, J; Wang, Y; Nie, W; Yang, F

    2008-01-01

    Chinese pangolins as a representative species in the order Pholidota have highly specified morphological characters and occupy an important place in the mammalian phylogenetic tree. To obtain genomic data for this species, we have constructed a bacterial artificial chromosome (BAC) library of Chinese pangolin. The library contains 208,272 clones with an average insert size of 122.1 kb and represents approximately eight times the Chinese pangolin haploid genome (if we assume that the Chinese pangolins have a genome size similar to human). One hundred and twenty randomly-selected BAC clones were mapped onto Chinese pangolin chromosomes by fluorescence in situ hybridization (FISH), showing a largely unbiased chromosomal distribution. Several clones containing repetitive DNA and ribosomal DNA genes were also found. The BAC library and FISH mapped BAC clones are useful resources for comparative genomics and cytogenetics of mammals and in particular, the ongoing genome sequencing project of Chinese pangolins. PMID:18931486

  4. Construction, characterization and FISH mapping of a bacterial artificial chromosome library of Chinese pangolin (Manis pentadactyla).

    PubMed

    Che, J; Wang, J; Su, W; Ye, J; Wang, Y; Nie, W; Yang, F

    2008-01-01

    Chinese pangolins as a representative species in the order Pholidota have highly specified morphological characters and occupy an important place in the mammalian phylogenetic tree. To obtain genomic data for this species, we have constructed a bacterial artificial chromosome (BAC) library of Chinese pangolin. The library contains 208,272 clones with an average insert size of 122.1 kb and represents approximately eight times the Chinese pangolin haploid genome (if we assume that the Chinese pangolins have a genome size similar to human). One hundred and twenty randomly-selected BAC clones were mapped onto Chinese pangolin chromosomes by fluorescence in situ hybridization (FISH), showing a largely unbiased chromosomal distribution. Several clones containing repetitive DNA and ribosomal DNA genes were also found. The BAC library and FISH mapped BAC clones are useful resources for comparative genomics and cytogenetics of mammals and in particular, the ongoing genome sequencing project of Chinese pangolins.

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

    NASA Astrophysics Data System (ADS)

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

    2008-05-01

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

  6. Construction and characterization of a soybean bacterial artificial chromosome library and use of multiple complementary libraries for genome physical mapping.

    PubMed

    Wu, C-C; Nimmakayala, P; Santos, F A; Springman, R; Scheuring, C; Meksem, K; Lightfoot, D A; Zhang, H-B

    2004-09-01

    Two plant-transformation-competent large-insert binary clone bacterial artificial chromosome (hereafter BIBAC) libraries were previously constructed for soybean cv. Forrest, using BamHI or HindIII. However, they are not well suited for clone-based genomic sequencing due to their larger ratio of vector to insert size (27.6 kbp:125 kbp). Therefore, we developed a larger-insert bacterial artificial chromosome (BAC) library for the genotype in a smaller vector (pECBAC1), using EcoRI. The BAC library contains 38,400 clones; about 99.1% of the clones have inserts; the average insert size is 157 kbp; and the ratio of vector to insert size is much smaller (7.5 kbp:157 kbp). Colony hybridization with probes derived from several chloroplast and mitochondrial genes showed that 0.89% and 0.45% of the clones were derived from the chloroplast and mitochondrial genomes, respectively. Considering these data, the library represents 5.4 haploid genomes of soybean. The library was hybridized with six RFLP marker probes, 5S rDNA and 18S-5.8S-25S rDNA, respectively. Each RFLP marker hybridized to about six clones, and the 5S and 18S-5.8S-25S rDNA probes collectively hybridized to 402 BACs--about 1.05% of the clones in the library. The BAC library complements the existing soybean Forrest BIBAC libraries by using different restriction enzymes and vector systems. Together, the BAC and BIBAC libraries encompass 13.2 haploid genomes, providing the most comprehensive clone resource for a single soybean genotype for public genome research. We show that the BAC library has enhanced the development of the soybean whole-genome physical map and use of three complementary BAC libraries improves genome physical mapping by fingerprint analysis of most of the clones of the library. The rDNA-containing clones were also fingerprinted to evaluate the feasibility of constructing contig maps of the rDNA regions. It was found that physical maps for the rDNA regions could not be readily constructed by

  7. Mapping of low-frequency chimeric yeast artificial chromosome libraries from human chromosomes 16 and 21 by fluorescence in situ hybridization and quantitative image analysis

    SciTech Connect

    Marrone, B.L.; Campbell, E.W.; Anzick, S.L.; Shera, K.; Campbell, M.; Yoshida, T.M.; McCormick, M.K.; Deaven, L. )

    1994-05-01

    Yeast artificial chromosome (YAC) clones from low-frequency chimeric libraries of human chromosomes 16 and 21 were mapped onto human diploid fibroblast metaphase chromosomes using fluorescence in situ hybridization (FISH) and digital imaging microscopy. YACs mapped onto chromosome 21 were selected to provide subregional location and ordering of known and unknown markers on the long arm of chromosome 21, particularly in the Down syndrome region (q22). YACs mapped onto chromosome 16 were selected to overlap regions spanning chromosome 16 cosmid maps. YAC clones were indirectly labeled with fluorescein, and the total DNA of the chromosome was counterstained with propidium iodide. A single image containing both the FISH signal and the whole chromosome was acquired for each chromosome of interest containing the fluorescent probe signal in a metaphase spread. From the digitized image, the fluorescence intensity profile through the long axis of the chromosome gave the total chromosome length and the probe position. The map position of the probe was expressed as the fractional length (FL) of the total chromosome relative to the end of the short arm (Flpter). From each clone hybridized, 20-40 chromosome images were analyzed. Thirty-eight YACs were mapped onto chromosome 16, and their FLs were distributed along the short and long arms. On chromosome 21, 47 YACs were mapped, including 12 containing known markers. To confirm the order of a dense population of YACs within the Down syndrome region, a two-color mapping strategy was used in which an anonymous YAC was located relative to one or two known markers on the metaphase chromosome. The chromosome FL maps have a 1- to 2-Mb resolution, and the FL measurement of each probe has a typical standard error of 0.5-1 Mb. 14 refs., 3 figs., 3 tabs.

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

    PubMed

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

    2014-03-07

    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.

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

    PubMed Central

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

    2014-01-01

    Bacterial artificial chromosome (BAC) libraries are extremely valuable for the genome-wide genetic dissection of complex organisms. The Siberian tiger, one of the most well-known wild primitive carnivores in China, is an endangered animal. In order to promote research on its genome, a high-redundancy BAC library of the Siberian tiger was constructed and characterized. The library is divided into two sub-libraries prepared from blood cells and two sub-libraries prepared from fibroblasts. This BAC library contains 153,600 individually archived clones; for PCR-based screening of the library, BACs were placed into 40 superpools of 10 × 384-deep well microplates. The average insert size of BAC clones was estimated to be 116.5 kb, representing approximately 6.46 genome equivalents of the haploid genome and affording a 98.86% statistical probability of obtaining at least one clone containing a unique DNA sequence. Screening the library with 19 microsatellite markers and a SRY sequence revealed that each of these markers were present in the library; the average number of positive clones per marker was 6.74 (range 2 to 12), consistent with 6.46 coverage of the tiger genome. Additionally, we identified 72 microsatellite markers that could potentially be used as genetic markers. This BAC library will serve as a valuable resource for physical mapping, comparative genomic study and large-scale genome sequencing in the tiger. PMID:24608928

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

    SciTech Connect

    Jean-Michael H. Vos

    1999-12-09

    Collections of human DNA fragments are maintained for research purposes as clones in bacterial host cells. However for unknown reasons, some regions of the human genome appear to be unclonable or unstable in bacteria. Their team has developed a system using episomes (extrachromosomal, autonomously replication DNA) that maintains large DNA fragments in human cells. This human artificial episomal chromosomal (HAEC) system may prove useful for coverage of these especially difficult regions. In the broader biomedical community, the HAEC system also shows promise for use in functional genomics and gene therapy. Recent improvements to the HAEC system and its application to mapping, sequencing, and functionally studying human and mouse DNA are summarized. Mapping and sequencing the human genome and model organisms are only the first steps in determining the function of various genetic units critical for gene regulation, DNA replication, chromatin packaging, chromosomal stability, and chromatid segregation. Such studies will require the ability to transfer and manipulate entire functional units into mammalian cells.

  11. New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas).

    PubMed

    Cunningham, Charles; Hikima, Jun-ichi; Jenny, Matthew J; Chapman, Robert W; Fang, Guang-Chen; Saski, Chris; Lundqvist, Mats L; Wing, Rod A; Cupit, Pauline M; Gross, Paul S; Warr, Greg W; Tomkins, Jeff P

    2006-01-01

    Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at (www.genome.clemson.edu). PMID:16896533

  12. Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (Manihot esculenta Crantz).

    PubMed

    Tomkins, J; Fregene, M; Main, D; Kim, H; Wing, R; Tohme, J

    2004-11-01

    Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop's biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55, 296 clones with an insert size range of 40-150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25-250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2-3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2,301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and

  13. Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

    PubMed Central

    Anand, R; Villasante, A; Tyler-Smith, C

    1989-01-01

    A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a 'waltzer' apparatus to remove small DNA fragments increased the mean YAC size to congruent to 220kb or congruent to 370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4 degrees C and routinely yielded transformation efficiencies of greater than 700 colonies/micrograms. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source. Images PMID:2542900

  14. Construction and application of a bacterial artificial chromosome (BAC) library of Prunus armeniaca L. for the identification of clones linked to the self-incompatibility locus.

    PubMed

    Vilanova, S; Romero, C; Abernathy, D; Abbott, A G; Burgos, L; Llacer, G; Badenes, M L

    2003-08-01

    To facilitate gene discovery in the Rosaceae, a bacterial artificial chromosome (BAC) library was constructed using high-molecular-weight (HMW) DNA from apricot leaves (Prunus armeniaca L.). The library contains 101,376 clones (264, 384-well plates) with an average insert size of 64 kb, equivalent to 22-fold genome coverage. In the first application of this library, high-density filters were screened for self-incompatibility genes using apricot DNA probes. Eight positive BAC clones were detected and fingerprinted to determine clone relationships and assemble contigs. These results demonstrate the suitability of this library for gene identification and physical mapping of the apricot genome.

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

    PubMed

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

    2009-01-01

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

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

    PubMed

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

    2009-01-01

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

  17. Construction and characterization of a bacterial artificial chromosome library of the causal agent of Black Sigatoka fungal leaf spot disease of banana and plantain, Mycosphaerella fijiensis.

    PubMed

    Canto-Canché, Blondy; Guillén-Maldonado, Diana Karina; Peraza-Echeverría, Leticia; Conde-Ferráez, Laura; James-Kay, Andrew

    2007-05-01

    A bacterial artificial chromosome library of the causal agent of the Black Sigatoka leaf spot disease of banana and plantain, Mycosphaerella fijiensis, has been constructed using a non-sphaeroplasting technique and characterized using both homologous and heterologous probes. After first and a second size selection of PFGE-fractionated DNA, a ligation was obtained using a 1:4 molar ratio (insert:vector). One hundred random clones were analyzed, and the mean insert size was estimated to be 90 kb. The range of the insert sizes was between 40 and 160 kb. The highest percentage of inserts belonged to the range between 80 and 100 kb; 32% of the inserts had 2 or 3 internal NotI sites. This library consists of 1920 clones, if the genomic size is at least 35 Mb, then this represents 4.9 x genome equivalents, which was supported by hybridization results with homologous and heterologous probes. PMID:17827540

  18. Construction and characterization of a bacterial artificial chromosome library of the causal agent of Black Sigatoka fungal leaf spot disease of banana and plantain, Mycosphaerella fijiensis.

    PubMed

    Canto-Canché, Blondy; Guillén-Maldonado, Diana Karina; Peraza-Echeverría, Leticia; Conde-Ferráez, Laura; James-Kay, Andrew

    2007-05-01

    A bacterial artificial chromosome library of the causal agent of the Black Sigatoka leaf spot disease of banana and plantain, Mycosphaerella fijiensis, has been constructed using a non-sphaeroplasting technique and characterized using both homologous and heterologous probes. After first and a second size selection of PFGE-fractionated DNA, a ligation was obtained using a 1:4 molar ratio (insert:vector). One hundred random clones were analyzed, and the mean insert size was estimated to be 90 kb. The range of the insert sizes was between 40 and 160 kb. The highest percentage of inserts belonged to the range between 80 and 100 kb; 32% of the inserts had 2 or 3 internal NotI sites. This library consists of 1920 clones, if the genomic size is at least 35 Mb, then this represents 4.9 x genome equivalents, which was supported by hybridization results with homologous and heterologous probes.

  19. Analysis of chromosome 21 yeast artificial chromosome (YAC) clones

    SciTech Connect

    Tassone, F. A. Gemelli School of Medicine, Rome ); Cheng, S.; Gardiner, K. )

    1992-12-01

    Chromosome 21 contains genes relevant to several important diseases. Yeast artificial chromosome (YAC) clones, because they span >100 kbp, will provide attractive material for initiating searches for such genes. Twenty-two YAC clones, each of which maps to a region of potential relevance either to aspects of the Down syndrome phenotype or to one of the other chromosome 21-associated genetic diseases, have been analyzed in detail. Clones total [approximately]6,000 kb and derive from all parts of the long arm. Rare restriction-site maps have been constructed for each clone and have been used to determine regional variations in clonability, methylation frequency, CpG island density, and CpG island frequency versus gene density. This information will be useful for the isolation and mapping of new genes to chromosome 21 and for walking in YAC libraries. 48 refs., 3 figs., 4 tabs.

  20. Construction, Analysis, and β-Glucanase Screening of a Bacterial Artificial Chromosome Library from the Large-Bowel Microbiota of Mice†

    PubMed Central

    Walter, Jens; Mangold, Marco; Tannock, Gerald W.

    2005-01-01

    A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for β-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with β-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species. PMID:15870321

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

    PubMed

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

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

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

    PubMed

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

    2016-01-01

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

  3. Pure chromosome-specific PCR libraries from single sorted chromosomes.

    PubMed Central

    VanDevanter, D R; Choongkittaworn, N M; Dyer, K A; Aten, J; Otto, P; Behler, C; Bryant, E M; Rabinovitch, P S

    1994-01-01

    Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases. Images PMID:8016078

  4. Streptomyces autolyticus JX-47 large-insert bacterial artificial chromosome library construction and identification of clones covering geldanamycin biosynthesis gene cluster.

    PubMed

    Dai, Shikun; Ouyang, Yongchang; Wang, Guanghua; Li, Xiang

    2011-07-01

    Geldanamycin belongs to benzoquinone ansamycin antibiotic and has potent antitumor activities. In this study, a bacterial artificial chromosome (BAC) library with an average insert size of up to 150 kb was constructed from genomic DNA of Streptomyces autolyticus JX-47. A genetic-screening strategy was established using BAC end-sequencing and three pairs of primers designed to target the remote regions, gdmA1, gdmA3 and gdmRI, of the geldanamycin gene cluster. Three clones covering geldanamycin biosynthesis gene cluster were obtained, which together spanned a 250-kb genomic region, and a 150227-bp insert in the clone p4E9 was sequenced. Comparison with the reported geldanamycin gene cluster sequences from S. hygroscopicus revealed that it had the same gene arrangement and high gene homology in the polyketide synthase (PKS) region and its downstream with 84-100% DNA identity and 81-100% amino acid (AA) identity. Its DNA homology with the whole gene cluster sequence from S. hygroscopicus strain 17997 reached 99% identity. However, upstream of the PKS region exhibited great diversity, where only ORF16 was conserved, and the other genes including gdmL and gdmX were displaced.

  5. Construction of human chromosome 21-specific yeast artificial chromosomes.

    PubMed

    McCormick, M K; Shero, J H; Cheung, M C; Kan, Y W; Hieter, P A; Antonarakis, S E

    1989-12-01

    Chromosome 21-specific yeast artificial chromosomes (YACs) have been constructed by a method that performs all steps in agarose, allowing size selection by pulsed-field gel electrophoresis and the use of nanogram to microgram quantities of DNA. The DNA sources used were hybrid cell line WAV-17, containing chromosome 21 as the only human chromosome and flow-sorted chromosome 21. The transformation efficiency of ligation products was similar to that obtained in aqueous transformations and yielded YACs with sizes ranging from 100 kilobases (kb) to greater than 1 megabase when polyamines were included in the transformation procedure. Twenty-five YACs containing human DNA have been obtained from a mouse-human hybrid, ranging in size from 200 to greater than 1000 kb, with an average size of 410 kb. Ten of these YACs were localized to subregions of chromosome 21 by hybridization of RNA probes (corresponding to the YAC ends recovered in Escherichia coli) to a panel of somatic cell hybrid DNA. Twenty-one human YACs, ranging in size from 100 to 500 kb, with an average size of 150 kb, were obtained from approximately equal to 50 ng of flow-sorted chromosome 21 DNA. Three were localized to subregions of chromosome 21. YACs will aid the construction of a physical map of human chromosome 21 and the study of disorders associated with chromosome 21 such as Alzheimer disease and Down syndrome.

  6. Chromosome region-specific libraries for human genome analysis

    SciTech Connect

    Kao, Fa-Ten.

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  7. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

  8. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination. PMID:27349114

  9. Construction of DNA libraries from flow sorted human chromosomes

    SciTech Connect

    Deaven, L.L.; McCormick, M.K.; Grady, D.L.

    1994-09-01

    We have constructed a series of DNA libraries from flow-sorted chromosomes. Small insert, complete digest libraries cloned into the EcoRI insertion site of Charon 21A are available from the American Type Culture Collection, Rockville, MD. Partial digest libraries cloned into cosmid (sCos1) or phage (Charon 40) vectors have been constructed for chromosomes 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, X and Y. Purity estimates by in situ analysis of sorted chromosomes, flow karyotype analysis, and plaque or colony hybridization indicate that most of these libraries are 90-95% pure. Additional cosmid library constructions, 5-10X arrays of libraries into microtiter plates, and high density membrane arrays of libraries are in progress. Recently, we have completed YAC libraries for chromosomes 5, 9, 16, and 21. These libraries are made from complete DNA digests using the rare cutters Clal, SacII, EagI, or NotI/NheI. The average insert size is {similar_to}200 kb, and chimera frequencies are low (1-10%). Libraries have also been constructed using M13 or bluescript vectors (chromosomes 5, 7, 17) to generate STS markers for the selection of chromosome-specific inserts from total genomic AC libraries. Because of the advantages of insert size and stability associated with BAC and PAC cloning systems, we are currently attempting to adapt pBAC108L and pCYPAC1 vectors for use with flow-sorted chromosomal DNA.

  10. The Oryza bacterial artificial chromosome library resource: Construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza

    PubMed Central

    Ammiraju, Jetty S.S.; Luo, Meizhong; Goicoechea, José L.; Wang, Wenming; Kudrna, Dave; Mueller, Christopher; Talag, Jayson; Kim, HyeRan; Sisneros, Nicholas B.; Blackmon, Barbara; Fang, Eric; Tomkins, Jeffery B.; Brar, Darshan; MacKill, David; McCouch, Susan; Kurata, Nori; Lambert, Georgina; Galbraith, David W.; Arumuganathan, K.; Rao, Kiran; Walling, Jason G.; Gill, Navdeep; Yu, Yeisoo; SanMiguel, Phillip; Soderlund, Carol; Jackson, Scott; Wing, Rod A.

    2006-01-01

    Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%–4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed. PMID:16344555

  11. The transfer of human artificial chromosomes via cryopreserved microcells.

    PubMed

    Uno, Narumi; Uno, Katsuhiro; Zatti, Susi; Ueda, Kana; Hiratsuka, Masaharu; Katoh, Motonobu; Oshimura, Mitsuo

    2013-10-01

    Microcell-mediated chromosome transfer (MMCT) technology enables a single and intact mammalian chromosome or megabase-sized chromosome fragments to be transferred from donor to recipient cells. The conventional MMCT method is performed immediately after the purification of microcells. The timing of the isolation of microcells and the preparation of recipient cells is very important. Thus, ready-made microcells can improve and simplify the process of MMCT. Here, we established a cryopreservation method to store microcells at -80 °C, and compared these cells with conventionally- (immediately-) prepared cells with respect to the efficiency of MMCT and the stability of a human artificial chromosome (HAC) transferred to human HT1080 cells. The HAC transfer in microcell hybrids was confirmed by FISH analysis. There was no significant difference between the two methods regarding chromosome transfer efficiency and the retention rate of HAC. Thus, cryopreservation of ready-to-use microcells provides an improved and simplified protocol for MMCT.

  12. Human chromosome-specific DNA libraries: construction and availability

    SciTech Connect

    Van Dilla, M.A.; Deaven, L.L.; Albright, K.L.; Allen, N.A.; Aubuchon, M.R.; Bartholdi, M.F.; Brown, N.C.; Campbell, E.W.; Carrano, A.V.; Clark, L.M.; Cram, L.S.

    1986-06-01

    The goal of the National Laboratory Gene Library Project at the Los Alamos and Lawrence Livermore National Laboratories is the production of chromosome-specific human gene libraries and their distribution to the scientific community for studies of the molecular biology of genes and chromosomes, and for the study and diagnosis of genetic disease. The specific aim of Phase I of the project is the production of complete digest (4 kb average insert size) libraries from each of the 24 human chromosomal types purified by flow sorting. The bacteriophage vector is Charon 21A, which has both Eco R1 and Hind III insertion sites accommodating human DNA fragments up to 9.1 kb in size. Each laboratory has undertaken production of a complete set of chromosome-specific libraries, Los Alamos with Eco R1 and Livermore with Hind III; most of this task has now been accomplished. Close to 1200 library aliquots have been sent to about 300 laboratories world-wide through February 1986, at which time repository and distribution functions were transferred to the American Type Culture Collection, Rockville, MD. Following Phase I, libraries will be constructed with large inserts in a more advanced, recently developed bacteriophage vector (about 20 kb inserts) or in a cosmid vector (about 40 kb inserts), and with characteristics better suited to basic studies of gene structure and function.

  13. Isolation, characterization, and regional mapping of microclones from a human chromosome 21 microdissection library

    SciTech Connect

    Yu, J.; Hartz, J.; Yisheng Xu; Gemmill, R.M.; Patterson, D.; Kao, Faten ); Gemmill, R.M.; Patterson, D.; Kao, Fa-Ten ); Korenberg, J.R. )

    1992-08-01

    Thirty-four unique-sequence microclones were isolated from a previously described microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions. The mapping results showed that the microclones were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 21q21.3 and 21qter. The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm. This finding is consistent with those reported in other chromosome 21 libraries. Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences of genes. The microclones were also characterized for insert size and were used to identify the corresponding genomic fragments generated by HindIII. In addition, the authors demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromosome) clones with large inserts, for increased genomic coverage for high-resolution physical mapping. They also used 200 unique-sequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region. Thus, generation of unique-sequence microclones from chromosome 21 appears to be useful to isolate and regionally map many cDNA clones, among which will be candidate genes for important diseases on chromosome 21, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, and one form of epilepsy.

  14. Preparative in situ hybridization: Selection of chromosome region-specific libraries on mitotic chromosomes

    SciTech Connect

    Hozier, J.; Graham, R.; Westfall, T.; Davis, L. ); Siebert, P. )

    1994-02-01

    The authors have developed preparative in situ hybridization (Prep-ISH) of complex DNA populations to mitotic chromosomes as a means of generating chromosome region-specific DNA subpopulations. Prep-ISH is a combination of two cytogenetic techniques: in situ hybridization of DNA molecules to mitotic chromosomes and chromosome microdissection. Here, they present test cases demonstrating the feasibility of this approach on mouse and human genomes, using single nuclei, single chromosomes, or single chromosomal subregions to assess sensitivity, specificity, and representation of the Prep-ISH technique. Prep-ISH has a number of applications in studies of gene expression and genome organization, including efficient cytogenetic sorting of tissue-specific cDNAs and genomic DNA libraries. In addition, Prep-ISH is likely to dramatically reduce the number of candidate genes to aid in gene discovery efforts and to improve efficiency of developing transcription maps and YAC and cosmid contigs through defined cytogenetic regions. 34 refs., 4 figs.

  15. Isolation of mini- and microsatellite loci from chromosome 19 library

    SciTech Connect

    Prosnyak, M.I.; Belajeva, O.V.; Polukarova, L.G.

    1994-09-01

    Mini- and microsatellite sequences are abundant in the human genome and are very useful as genetic markers. We report the isolation of a panel of clones containing marker sequences from chromosome 19. We screened 10,000 clones from the chromosome 19 cosmid library for the presence of di-(CA)n, tri-(TCC)n, (CAC)n microsatellites and M13-like minisatellite sequences. For this we have used synthetic oligonucleotides and polynucleotides, including micro- (CA, TCC, CAC) and minisatellite (M13 core) sequences. Preliminary results indicated that the chromosome 19 cosmid library contained both human and hamster clones. In order to identify human sequences from this library we have developed the technique of colony and blot hybridization with Alu-PCR, L1-PCR and B1-PCR probes. Dozens of clones have been selected, some of which were analyzed by conventional Southern blot analysis and non-radioactive in situ hybridization of chromosomes. Highly informative markers derived from these clones will be used for physical and genetic mapping of chromosome 19.

  16. Integrative bacterial artificial chromosomes for DNA integration into the Bacillus subtilis chromosome.

    PubMed

    Juhas, Mario; Ajioka, James W

    2016-06-01

    Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.

  17. Drought-tolerant rice germplasm developed from an Oryza officinalis transformation-competent artificial chromosome clone.

    PubMed

    Liu, R; Zhang, H H; Chen, Z X; Shahid, M Q; Fu, X L; Liu, X D

    2015-10-29

    Oryza officinalis has proven to be a natural gene reservoir for the improvement of domesticated rice as it carries many desirable traits; however, the transfer of elite genes to cultivated rice by conventional hybridization has been a challenge for rice breeders. In this study, the conserved sequence of plant stress-related NAC transcription factors was selected as a probe to screen the O. officinalis genomic transformation-competent artificial chromosome library by Southern blot; 11 positive transformation-competent artificial chromosome clones were subsequently detected. By Agrobacterium-mediated transformation, an indica rice variety, Huajingxian 74 (HJX74), was transformed with a TAC clone harboring a NAC gene-positive genomic fragment from O. officinalis. Molecular analysis revealed that the O. officinalis genomic fragment was integrated into the genome of HJX74. The transgenic lines exhibited high tolerance to drought stress. Our results demonstrate that the introduction of stress-related transformation-competent artificial chromosome clones, coupled with a transgenic validation approach, is an effective method of transferring agronomically important genes from O. officinalis to cultivated rice.

  18. Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

    PubMed Central

    Kuspa, A; Loomis, W F

    1996-01-01

    High resolution gene maps of the six chromosomes of Dictyostelium discoideum have been generated by a combination of physical mapping techniques. A set of yeast artificial chromosome clones has been ordered into overlapping arrays that cover >98% of the 34-magabase pair genome. Clones were grouped and ordered according to the genes they carried, as determined by hybridization analyses with DNA fragments from several hundred genes. Congruence of the gene order within each arrangement of clones with the gene order determined from whole genome restriction site mapping indicates that a high degree of confidence can be placed on the clone map. This clone-based description of the Dictyostelium chromosomes should be useful for the physical mapping and subcloning of new genes and should facilitate more detailed analyses of this genome. cost of silicon-based construction and in the efficient sample handling afforded by component integration. PMID:8643615

  19. A Yeast Artificial Chromosome Clone Map of the Drosophila Genome

    PubMed Central

    Cai, H.; Kiefel, P.; Yee, J.; Duncan, I.

    1994-01-01

    We describe the mapping of 979 randomly selected large yeast artificial chromosome (YAC) clones of Drosophila DNA by in situ hybridization to polytene chromosomes. Eight hundred and fifty-five of the clones are euchromatic and have primary hybridization sites in the banded portions of the polytene chromosomes, whereas 124 are heterochromatic and label the chromocenter. The average euchromatic clone contains about 211 kb and, at its primary site, labels eight or nine contiguous polytene bands. Thus, the extent as well as chromosomal position of each clone has been determined. By direct band counts, we estimate our clones provide about 76% coverage of the euchromatin of the major autosomes, and 63% coverage of the X. When previously reported YAC mapping data are combined with ours, euchromatic coverage is extended to about 90% for the autosomes and 82% for the X. The distribution of gap sizes in our map and the coverage achieved are in good agreement with expectations based on the assumption of random coverage, indicating that euchromatic clones are essentially randomly distributed. However, certain gaps in coverage, including the entire fourth chromosome euchromatin, may be significant. Heterochromatic sequences are underrepresented among the YAC clones by two to three fold. This may result, at least in part, from underrepresentation of heterochromatic sequences in adult DNA (the source of most of the clones analyzed), or from clone instability. PMID:8013915

  20. The Neurospora crassa genome: cosmid libraries sorted by chromosome.

    PubMed Central

    Kelkar, H S; Griffith, J; Case, M E; Covert, S F; Hall, R D; Keith, C H; Oliver, J S; Orbach, M J; Sachs, M S; Wagner, J R; Weise, M J; Wunderlich, J K; Arnold, J

    2001-01-01

    A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification. PMID:11238388

  1. HACking the centromere chromatin code: insights from human artificial chromosomes.

    PubMed

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations. PMID:22825423

  2. HACking the centromere chromatin code: insights from human artificial chromosomes.

    PubMed

    Bergmann, Jan H; Martins, Nuno M C; Larionov, Vladimir; Masumoto, Hiroshi; Earnshaw, William C

    2012-07-01

    The centromere is a specialized chromosomal region that serves as the assembly site of the kinetochore. At the centromere, CENP-A nucleosomes form part of a chromatin landscape termed centrochromatin. This chromatin environment conveys epigenetic marks regulating kinetochore formation. Recent work sheds light on the intricate relationship between centrochromatin state, the CENP-A assembly pathway and the maintenance of centromere function. Here, we review the emerging picture of how chromatin affects mammalian kinetochore formation. We place particular emphasis on data obtained from Human Artificial Chromosome (HAC) biology and the targeted engineering of centrochromatin using synthetic HACs. We discuss implications of these findings, which indicate that a delicate balance of histone modifications and chromatin state dictates both de novo centromere formation and the maintenance of centromere identity in dividing cell populations.

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

    SciTech Connect

    Kao, Fa-Ten

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  4. Selection of chromosomal DNA libraries using a multiplex CRISPR system.

    PubMed

    Ryan, Owen W; Skerker, Jeffrey M; Maurer, Matthew J; Li, Xin; Tsai, Jordan C; Poddar, Snigdha; Lee, Michael E; DeLoache, Will; Dueber, John E; Arkin, Adam P; Cate, Jamie H D

    2014-08-19

    The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function.

  5. Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors.

    PubMed Central

    Neil, D L; Villasante, A; Fisher, R B; Vetrie, D; Cox, B; Tyler-Smith, C

    1990-01-01

    The suitability of yeast artificial chromosome vectors (YACs) for cloning human Y chromosome tandemly repeated DNA sequences has been investigated. Clones containing DYZ3 or DYZ5 sequences were found in libraries at about the frequency anticipated on the basis of their abundance in the genome, but clones containing DYZ1 sequences were under-represented and the three clones examined contained junctions between DYZ1 and DYZ2. One DYZ3 clone was quite stable and had a long-range structure corresponding to genomic DNA. All other clones had long-range structures which either did not correspond to genomic DNA, or were too unstable to allow a simple comparison. The effects of the transformation process and host genotype on YAC structural stability were investigated. Gross structural rearrangements were often associated with re-transformation of yeast by a YAC. rad1-deficient yeast strains showed levels of instability similar to wild-type for all YAC clones tested. In rad52-deficient strains, DYZ5 containing YACs were as unstable as in the wild-type host, but DYZ1/DYZ2 or DYZ3 containing YACs were more stable. Thus the use of rad52 hosts for future library construction is recommended, but some sequences will still be unstable. Images PMID:2183192

  6. Human cytomegalovirus: bacterial artificial chromosome (BAC) cloning and genetic manipulation.

    PubMed

    Paredes, Anne M; Yu, Dong

    2012-02-01

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

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

    PubMed Central

    Paredes, Anne M.; Yu, Dong

    2011-01-01

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

  8. Physical mapping of the human t-cell receptor beta gene complex, using yeast artificial chromosomes

    SciTech Connect

    Hashim, Y.; So, A.K.; Kearney, L.

    1995-04-01

    Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell {beta} chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280, 110, 90, and 125 kbp and was in accordance with published data. In addition, comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600-620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Fluorescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences. 32 refs., 5 figs., 1 tab.

  9. Chromosome region-specific libraries for human genome analysis

    SciTech Connect

    Kao, Fa-Ten.

    1991-01-01

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

  10. Genetic manipulation of poxviruses using bacterial artificial chromosome recombineering.

    PubMed

    Cottingham, Matthew G

    2012-01-01

    Traditional methods for genetic manipulation of poxviruses rely on low-frequency natural recombination in virus-infected cells. Although these powerful systems represent the technical foundation of current knowledge and applications of poxviruses, they require long (≥ 500 bp) flanking sequences for homologous recombination, an efficient viral selection method, and burdensome, time-consuming plaque purification. The beginning of the twenty-first century has seen the application of bacterial artificial chromosome (BAC) technology to poxviruses as an alternative method for their genetic manipulation, following the invention of a long-sought-after method for deriving a BAC clone of vaccinia virus (VAC-BAC) by Arban Domi and Bernard Moss. The key advantages of the BAC system are the ease and versatility of performing genetic manipulation using bacteriophage λ Red recombination (recombineering), which requires only ∼50 bp homology arms that can be easily created by PCR, and which allows seamless mutations lacking any marker gene without having to perform transient-dominant selection. On the other hand, there are disadvantages, including the significant setup time, the risk of contamination of the cloned genome with bacterial insertion sequences, and the nontrivial issue of removal of the BAC cassette from derived viruses. These must be carefully weighed to decide whether the use of BACs will be advantageous for a particular application, making pox-BAC systems likely to complement, rather than supplant, traditional methods in most laboratories. PMID:22688760

  11. Construction of a yeast artificial chromosome contig encompassing the chromosome 14 Alzheimer`s disease locus

    SciTech Connect

    Sharma, V.; Bonnycastle, L.; Poorkai, P.

    1994-09-01

    We have constructed a yeast artificial chromosome (YAC) contig of chromosome 14q24.3 which encompasses the chromosome 14 Alzheimer`s disease locus (AD3). Determined by linkage analysis of early-onset Alzheimer`s disease kindreds, this interval is bounded by the genetic markers D14S61-D14S63 and spans approximately 15 centimorgans. The contig consists of 29 markers and 74 YACs of which 57 are defined by one or more sequence tagged sites (STSs). The STS markers comprise 5 genes, 16 short tandem repeat polymorphisms and 8 cDNA clones. An additional number of genes, expressed sequence tags and cDNA fragments have been identified and localized to the contig by hybridization and sequence analysis of anonymous clones isolated by cDNA direct selection techniques. A minimal contig of about 15 YACs averaging 0.5-1.5 megabase in length will span this interval and is, at first approximation, in rough agreement with the genetic map. For two regions of the contig, our coverage has relied on L1/THE fingerprint and Alu-PCR hybridization data of YACs provided by CEPH/Genethon. We are currently developing sequence tagged sites from these to confirm the overlaps revealed by the fingerprint data. Among the genes which map to the contig are transforming growth factor beta 3, c-fos, and heat shock protein 2A (HSPA2). C-fos is not a candidate gene for AD3 based on the sequence analysis of affected and unaffected individuals. HSPA2 maps to the proximal edge of the contig and Calmodulin 1, a candidate gene from 4q24.3, maps outside of the region. The YAC contig is a framework physical map from which cosmid or P1 clone contigs can be constructed. As more genes and cDNAs are mapped, a highly resolved transcription map will emerge, a necessary step towards positionally cloning the AD3 gene.

  12. Selection of chromosomal DNA libraries using a multiplex CRISPR system

    PubMed Central

    Ryan, Owen W; Skerker, Jeffrey M; Maurer, Matthew J; Li, Xin; Tsai, Jordan C; Poddar, Snigdha; Lee, Michael E; DeLoache, Will; Dueber, John E; Arkin, Adam P; Cate, Jamie HD

    2014-01-01

    The directed evolution of biomolecules to improve or change their activity is central to many engineering and synthetic biology efforts. However, selecting improved variants from gene libraries in living cells requires plasmid expression systems that suffer from variable copy number effects, or the use of complex marker-dependent chromosomal integration strategies. We developed quantitative gene assembly and DNA library insertion into the Saccharomyces cerevisiae genome by optimizing an efficient single-step and marker-free genome editing system using CRISPR-Cas9. With this Multiplex CRISPR (CRISPRm) system, we selected an improved cellobiose utilization pathway in diploid yeast in a single round of mutagenesis and selection, which increased cellobiose fermentation rates by over 10-fold. Mutations recovered in the best cellodextrin transporters reveal synergy between substrate binding and transporter dynamics, and demonstrate the power of CRISPRm to accelerate selection experiments and discoveries of the molecular determinants that enhance biomolecule function. DOI: http://dx.doi.org/10.7554/eLife.03703.001 PMID:25139909

  13. Human Artificial Chromosomes for Gene Delivery and the Development of Animal Models

    PubMed Central

    Kazuki, Yasuhiro; Oshimura, Mitsuo

    2011-01-01

    Random integration of conventional gene delivery vectors such as viruses, plasmids, P1 phage-derived artificial chromosomes, bacterial artificial chromosomes and yeast artificial chromosomes can be associated with transgene silencing. Furthermore, integrated viral sequences can activate oncogenes adjacent to the insertion site resulting in cancer. Various human artificial chromosomes (HACs) exhibit several potential characteristics desired for an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci with their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. HACs have been generated mainly using either a “top-down approach” (engineered chromosomes), or a “bottom-up approach” (de novo artificial chromosomes). The recent emergence of stem cell–based tissue engineering has opened up new avenues for gene and cell therapies. This review describes the lessons learned and prospects identified mainly from studies in the construction of HACs and HAC-mediated gene expression systems in cultured cells, as well as in animals. PMID:21750534

  14. Construction of a chromosome specific library of human MARs and mapping of matrix attachment regions on human chromosome 19.

    PubMed

    Nikolaev, L G; Tsevegiyn, T; Akopov, S B; Ashworth, L K; Sverdlov, E D

    1996-04-01

    Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well.

  15. Construction of a chromosome specific library of human MARs and mapping of matrix attachment regions on human chromosome 19.

    PubMed Central

    Nikolaev, L G; Tsevegiyn, T; Akopov, S B; Ashworth, L K; Sverdlov, E D

    1996-01-01

    Using a novel procedure a representative human chromosome 19-specific library was constructed of short sequences, which bind preferentially to the nuclear matrix (matrix attachment regions, or MARs). Judging by 20 clones sequenced so far, the library contains > 50% of human inserts, about 90% of which are matrix-binding by the in vitro test. Computer analysis of sequences of eight human MARs did not reveal any significant homologies with the EMBL Nucleotide Data Base entries as well as between MARs themselves. Eight MARs were assigned to individual positions on the chromosome 19 physical map. The library constructed can serve as a good source of MAR sequences for comparative analysis and classification and for further chromosome mapping of MARs as well. PMID:8614638

  16. Phage p1-derived artificial chromosomes facilitate heterologous expression of the FK506 gene cluster.

    PubMed

    Jones, Adam C; Gust, Bertolt; Kulik, Andreas; Heide, Lutz; Buttner, Mark J; Bibb, Mervyn J

    2013-01-01

    We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L(-1) to 5.5 mg L(-1)) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.

  17. Survey of Artificial Intelligence and Expert Systems in Library and Information Science Literature.

    ERIC Educational Resources Information Center

    Hsieh, Cynthia C.; Hall, Wendy

    1989-01-01

    Examines the definition and history of artificial intelligence (AI) and investigates the body of literature on AI found in "Library Literature" and "Library and Information Science Abstracts." The results reported include the number of articles by year and per journal, and the percentage of articles dealing with library applications. (nine…

  18. The Arabidopsis TAC Position Viewer: a high-resolution map of transformation-competent artificial chromosome (TAC) clones aligned with the Arabidopsis thaliana Columbia-0 genome.

    PubMed

    Hirose, Yoshitsugu; Suda, Kunihiro; Liu, Yao-Guang; Sato, Shusei; Nakamura, Yukino; Yokoyama, Koji; Yamamoto, Naoki; Hanano, Shigeru; Takita, Eiji; Sakurai, Nozomu; Suzuki, Hideyuki; Nakamura, Yasukazu; Kaneko, Takakazu; Yano, Kentaro; Tabata, Satoshi; Shibata, Daisuke

    2015-09-01

    We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression. PMID:26227242

  19. Construction and availability of human chromosome-specific gene libraries

    SciTech Connect

    Fuscoe, J.C.; Van Dilla, M.A.; Deaven, L.L.

    1985-06-14

    This report briefly describes Phase I of the project, the production of complete digest fibraries. Each laboratory is currently in the process of sorting individual human chromosomes from normal human fibroblasts or human X hamster hybrids. The goal of 4 x 10/sup 6/ chromosomes for cloning purposes has been achieved. Each laboratory is also in the process of cloning the chromosomal DNA, after complete digestion with a 6-cutter, into the bacteriophage vector Charon 21A. 3 refs.

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

    PubMed Central

    Ostermann, Eleonore; Spohn, Michael; Indenbirken, Daniela

    2016-01-01

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

  1. Highly stable maintenance of a mouse artificial chromosome in human cells and mice.

    PubMed

    Kazuki, Kanako; Takehara, Shoko; Uno, Narumi; Imaoka, Natsuko; Abe, Satoshi; Takiguchi, Masato; Hiramatsu, Kei; Oshimura, Mitsuo; Kazuki, Yasuhiro

    2013-12-01

    Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) display several advantages as gene delivery vectors, such as stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts including the regulatory elements. Previously, we showed that a MAC vector developed from a natural mouse chromosome by chromosome engineering was more stably maintained in adult tissues and hematopoietic cells in mice than HAC vectors. In this study, to expand the utility for a gene delivery vector in human cells and mice, we investigated the long-term stability of the MACs in cultured human cells and transchromosomic mice. We also investigated the chromosomal copy number-dependent expression of genes on the MACs in mice. The MAC was stably maintained in human HT1080 cells in vitro during long-term culture. The MAC was stably maintained at least to the F8 and F4 generations in ICR and C57BL/6 backgrounds, respectively. The MAC was also stably maintained in hematopoietic cells and tissues derived from old mice. Transchromosomic mice containing two or four copies of the MAC were generated by breeding. The DNA contents were comparable to the copy number of the MACs in each tissue examined, and the expression of the EGFP gene on the MAC was dependent on the chromosomal copy number. Therefore, the MAC vector may be useful not only for gene delivery in mammalian cells but also for animal transgenesis.

  2. Comparative study of artificial chromosome centromeres in human and murine cells

    PubMed Central

    Moralli, Daniela; Jefferson, Andrew; Valeria Volpi, Emanuela; Larin Monaco, Zoia

    2013-01-01

    Human artificial chromosomes (HAC) are a valuable tool in the analysis of complex chromatin structures such as the human centromere because of their small size and relative simplicity compared with normal human chromosomes. This report includes a comprehensive study of the centromere and chromatin composition of HAC, expressing human genes, generated in human cells and transferred to murine cells. The analysis involved chromatin immuno-precipitation and immuno-FISH on metaphase chromosomes and chromatin fibres. In both the cell types, the HAC consisted of alphoid and non-alphoid DNA and were mainly euchromatic in composition, although a pericentromeric heterochromatic region was present on all the HAC. Fibre-FISH and chromatin immuno-precipitation data indicated that the position of the centromere differed between HAC in human cells and in murine cells. Our work highlights the importance and utilisation of HAC for understanding the epigenetic aspects of chromosome biology. PMID:23403904

  3. Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: their potential use as reference libraries.

    PubMed Central

    Nizetić, D; Zehetner, G; Monaco, A P; Gellen, L; Young, B D; Lehrach, H

    1991-01-01

    We have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains greater than 30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at -70 degrees C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. In addition, multiple sets of two membranes containing 4 chromosome equivalents of the human X chromosome, and one membrane containing 3 chromosome equivalents of chromosome 21, have been distributed to other interested laboratories as part of a system of reference libraries. This system allows other groups easy access to the clones and offers an efficient protocol to combine results generated in different laboratories using these libraries. Here we describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome. Images PMID:2014245

  4. Six rice chromosome segment substitution lines libraries with O. rufipogon introgressions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosome segment substitution lines (CSSLs) are a powerful tool for identifying naturally occurring, favorable alleles in unadapted germplasm. Six CSSL libraries in rice (Oryza sativa) were developed from crosses between three different accessions ('Khao Pa', W1944, IRGC105567) of the rice progeni...

  5. Exploring the power of rice (O. sativa x O. rufipogon) chromosome segment substitution line libraries

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgressive variation was reported as an increase in grain yield for several rice (Oryza sativa x O. rufipogon) advanced backcross mapping populations. The objective of this study was to develop chromosome segment substitution line (CSSL) libraries to further dissect the reported transgressive var...

  6. Gene recovery microdissection (GRM) a process for producing chromosome region-specific libraries of expressed genes

    SciTech Connect

    Christian, A T; Coleman, M A; Tucker, J D

    2001-02-08

    Gene Recovery Microdissection (GRM) is a unique and cost-effective process for producing chromosome region-specific libraries of expressed genes. It accelerates the pace, reduces the cost, and extends the capabilities of functional genomic research, the means by which scientists will put to life-saving, life-enhancing use their knowledge of any plant or animal genome.

  7. Development of inter-specific chromosomes segment substitution libraries (CSSL) in rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six libraries of inter-specific Chromosome Segment Substitution Lines (CSSLs) of rice are being developed as pre-breeding materials and genetic stocks. Three accessions of O. rufipogon were selected as donors, based on phylogenetic, geographical and morphological divergence, and crossed with two rec...

  8. Novel method to load multiple genes onto a mammalian artificial chromosome.

    PubMed

    Tóth, Anna; Fodor, Katalin; Praznovszky, Tünde; Tubak, Vilmos; Udvardy, Andor; Hadlaczky, Gyula; Katona, Robert L

    2014-01-01

    Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe's disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest.

  9. Novel Method to Load Multiple Genes onto a Mammalian Artificial Chromosome

    PubMed Central

    Tóth, Anna; Fodor, Katalin; Praznovszky, Tünde; Tubak, Vilmos; Udvardy, Andor; Hadlaczky, Gyula; Katona, Robert L.

    2014-01-01

    Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest. PMID:24454889

  10. Construction, arraying, and high-density screening of large insert libraries of human chromosomes X and 21: Their potential use as reference libraries

    SciTech Connect

    Nizetic, D.; Zehetner, G.; Monaco, A.P.; Gellen, L.; Lehrach, H. ); Young, B.D. )

    1991-04-15

    The authors have constructed cosmid libraries from flow-sorted human chromosomes X and 21, each of which contains {gt}30 genome equivalents, and have developed systems allowing permanent storage of primary clones, easy screening of libraries in high-density filter formats, and the simultaneous generation of fingerprinting and mapping data on the same set of cosmid clones. Clones are picked into microtiter plate wells and stored at {minus}70C. A semiautomatic robot system allows the generation of filter replicas containing up to 10,000 clones per membrane. Sets of membranes containing 15-20 chromosome equivalents of both chromosomes will be used for the construction of ordered clone libraries by hybridization fingerprinting protocols. The authors describe the construction of the libraries and demonstrate the use of high-density screening filters in oligonucleotide probe hybridizations and the isolation of cosmids by hybridization with probes from the X chromosome.

  11. Construction of a general human chromosome jumping library, with application to cystic fibrosis

    SciTech Connect

    Collins, F.S.; Drumm, M.L.; Cole, J.L.; Lockwood, W.K.; Woude, G.F.V.; Iannuzzi, M.C.

    1987-02-27

    In many genetic disorders, the responsible gene and its protein product are unknown. The technique known as reverse genetics, in which chromosomal map positions and genetically linked DNA markers are used to identify and clone such genes, is complicated by the fact that the molecular distances from the closest DNA markers to the gene itself are often too large to traverse by standard cloning techniques. To address this situation, a general human chromosome jumping library was constructed that allows the cloning of DNA sequences approximately 100 kilobases away from any starting point in genomic DNA. As an illustration of its usefulness, this library was searched for a jumping clone, starting at the met oncogene, which is a marker tightly linked to the cystic fibrosis gene that is located on human chromosome 7. Mapping of the new genomic fragment by pulsed field gel electrophoresis confirmed that it resides on chromosome 7 within 240 kilobases downstream of the met gene. The use of chromosome jumping should be applicable to any genetic locus for which a closely linked DNA marker is available.

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

    SciTech Connect

    Hubert, R.S.; Chen, X.N.; Mitchell, S.

    1994-09-01

    As the Human Genome Project progresses, large insert cloning vectors such as BACs, P1, and P1 Artificial Chromosomes (PACs) will be required to complement the YAC mapping efforts. The value of the BAC vector for physical mapping lies in the stability of the inserts, the lack of chimerism, the length of inserts (up to 300 kb), the ability to obtain large amounts of pure clone DNA and the ease of BAC manipulation. These features helped us design two approaches for generating physical mapping reagents for human genetic studies. The first approach is a whole genome strategy in which randomly selected BACs are mapped, using FISH, to specific chromosomal bands. To date, 700 BACs have been mapped to single chromosome bands at a resolution of 2-5 Mb in addition to BACs mapped to 14 different centromeres. These BACs represent more than 90 Mb of the genome and include >70% of all human chromosome bands at the 350-band level. These data revealed that >97% of the BACs were non-chimeric and have a genomic distribution covering most gaps in the existing YAC map with excellent coverage of gene-rich regions. In the second approach, we used YACs to identify BACs on chromosome 21. A 1.5 Mb contig between D21S339 and D21S220 nears completion within the Down syndrome congenital heart disease (DS-CHD) region. Seventeen BACs ranging in size from 80 kb to 240 kb were ordered using 14 STSs with FISH confirmation. We have also used 40 YACs spanning 21q to identify, on average, >1 BAC/Mb to provide molecular cytogenetic reagents and anchor points for further mapping. The contig generated on chromosome 21 will be helpful in isolating the genes for DS-CHD. The physical mapping reagents generated using the whole genome approach will provide cytogenetic markers and mapped genomic fragments that will facilitate positional cloning efforts and the identification of genes within most chromosomal bands.

  13. A pathway from chromosome transfer to engineering resulting in human and mouse artificial chromosomes for a variety of applications to bio-medical challenges.

    PubMed

    Oshimura, Mitsuo; Uno, Narumi; Kazuki, Yasuhiro; Katoh, Motonobu; Inoue, Toshiaki

    2015-02-01

    Microcell-mediated chromosome transfer (MMCT) is a technique to transfer a chromosome from defined donor cells into recipient cells and to manipulate chromosomes as gene delivery vectors and open a new avenue in somatic cell genetics. However, it is difficult to uncover the function of a single specific gene via the transfer of an entire chromosome or fragment, because each chromosome or fragment contains a set of numerous genes. Thus, alternative tools are human artificial chromosome (HAC) and mouse artificial chromosome (MAC) vectors, which can carry a gene or genes of interest. HACs/MACs have been generated mainly by either a "top-down approach" (engineered creation) or a "bottom-up approach" (de novo creation). HACs/MACs with one or more acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. The MMCT technique is also applied for manipulating HACs and MACs in donor cells and delivering them to recipient cells. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs, and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. New avenues for a variety of applications to bio-medical challenges are also proposed.

  14. Characterization of four human YAC libraries for clone size, chimerism and X chromosome sequence representation.

    PubMed Central

    Nagaraja, R; Kere, J; MacMillan, S; Masisi, M J; Johnson, D; Molini, B J; Halley, G R; Wein, K; Trusgnich, M; Eble, B

    1994-01-01

    Four collections of human X-specific YACs, derived from human cells containing supernumerary X chromosomes or from somatic cell hybrids containing only X human DNA were characterized. In each collection, 80-85% of YAC strains contained a single X YAC. Five thousand YACs from the various libraries were sized, and cocloning was assessed in subsets by the fraction of YAC insert-ends with non-X sequences. Cocloning was substantial, ranging up to 50% for different collections; and in agreement with previous indications, in all libraries the larger the YACs, the higher the level of cocloning. In libraries made from human-hamster hybrid cells, expected numbers of clones were recovered by STS-based screening; but unexpectedly, the two collections from cells with 4 or 5 X chromosomes yielded numbers of YACs corresponding to an apparent content of only about two X equivalents. Thus it is possible that the DNA of inactive X chromosomes is poorly cloned into YACs, speculatively perhaps because of its specialized chromatin structure. Images PMID:8078777

  15. Bacterial artificial chromosome transgenesis through pronuclear injection of fertilized mouse oocytes.

    PubMed

    Vintersten, Kristina; Testa, Giuseppe; Naumann, Ronald; Anastassiadis, Konstantinos; Stewart, A Francis

    2008-01-01

    In the mouse, conventional transgenes often produced unpredictable results mainly because they were too small to recapitulate a natural gene context. Bacterial artificial chromosomes (BACs) are large enough to encompass the natural context of most mammalian genes and consequently deliver more reliable recapitulations of their endogenous counterparts. Furthermore, recombineering methods now make it easy to engineer precise changes in a BAC transgene. Consequently, BACs have become the preferred vehicle for mouse transgenesis. Here, we detail methods for BAC transgenesis through pronuclear injection of fertilized oocytes. PMID:18370149

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

    SciTech Connect

    Kao, F.T.

    1994-04-01

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

  17. PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries.

    PubMed

    Potvin, Marianne; Lovejoy, Connie

    2009-01-01

    Environmental clone libraries constructed using small subunit ribosomal RNA (rRNA) or other gene-specific primers have become the standard molecular approach for identifying microorganisms directly from their environment. This technique includes an initial polymerase chain reaction (PCR) amplification step of a phylogenetically useful marker gene using universal primers. Although it is acknowledged that such primers introduce biases, there have been few studies if any to date systematically examining such bias in eukaryotic microbes. We investigated some implications of such bias by constructing clone libraries using several universal primer pairs targeting rRNA genes. Firstly, we constructed artificial libraries using a known mix of small cultured pelagic arctic algae with representatives from five major lineages and secondly we investigated environmental samples using several primer pairs. No primer pair retrieved all of the original algae in the artificial clone libraries and all showed a favorable bias toward the dinoflagellate Polarella glacialis and a bias against the prasinophyte Micromonas and a pennate diatom. Several other species were retrieved by only one primer pair tested. Despite this, sequences from nine environmental libraries were diverse and contained representatives from all major eukaryotic clades expected in marine samples. Further, libraries from the same sample grouped together using Bray-Curtis clustering, irrespective of primer pairs. We conclude that environmental PCR-based techniques are sufficient to compare samples, but the total diversity will probably always be underestimated and relative abundance estimates should be treated with caution.

  18. Construction of a DNA library representing 15q11-13 by subtraction of two flow sorted marker chromosome-specific libraries

    SciTech Connect

    Blennow, E.; Werelius, B.; Nordenskjoeld, M.

    1994-09-01

    Constitutional extra {open_quotes}marker chromosomes{close_quotes} are found in {approx}0.5/1000 of newborns. Of these, 50% are inverted duplications of the pericentromeric region of chromosome 15, including two variants; (1) inv dup(15)(pter{yields}q11:q11{yields}pter) and (2) inv dup(15) (pter{yields}q12-13::q12-13{yields}pter). Variant (1) is found in phenotypically normal individuals, whereas variant (2) will produce a typical clinical picture including mental retardation, autism, hyperactivity and discrete dysmorphic features. Fluorescence in situ hybridization (FISH) using single copy probes from the Prader-Willi region confirms these observations as well as chromosome painting using a flow-sorted marker chromosome-specific library from a variant (1) marker, hybridized to the chromosomes of a patient with a variant (2) marker chromosome. Followingly, a flow-sorted biotinylated variant (1) library was subtracted from a non-labeled variant (2) library using magnetic beads and subsequent amplification by degenerate oligonucleotide-primed PCR (DOP-PCR). The successful result was demonstrated by using the amplified material for chromosome painting on chromosome slides from variant (1) and variant (2) patients. We have constructed a library from 15q11-13. This region contains genes producing a specific abnormal phenotype when found in a tri- or tetrasomic state. The region also contains the genes responsible for the Prader-Willi and Angelman syndromes when the paternal/maternal copy is missing, respectively. It is therefore a region where parental imprinting plays an important role. The isolated library may be used to isolate single copy clones which will allow further investigations of this region.

  19. TOPAAS, a Tomato and Potato Assembly Assistance System for Selection and Finishing of Bacterial Artificial Chromosomes1[W

    PubMed Central

    Peters, Sander A.; van Haarst, Jan C.; Jesse, Taco P.; Woltinge, Dennis; Jansen, Kim; Hesselink, Thamara; van Staveren, Marjo J.; Abma-Henkens, Marleen H.C.; Klein-Lankhorst, René M.

    2006-01-01

    We have developed the software package Tomato and Potato Assembly Assistance System (TOPAAS), which automates the assembly and scaffolding of contig sequences for low-coverage sequencing projects. The order of contigs predicted by TOPAAS is based on read pair information; alignments between genomic, expressed sequence tags, and bacterial artificial chromosome (BAC) end sequences; and annotated genes. The contig scaffold is used by TOPAAS for automated design of nonredundant sequence gap-flanking PCR primers. We show that TOPAAS builds reliable scaffolds for tomato (Solanum lycopersicum) and potato (Solanum tuberosum) BAC contigs that were assembled from shotgun sequences covering the target at 6- to 8-fold coverage. More than 90% of the gaps are closed by sequence PCR, based on the predicted ordering information. TOPAAS also assists the selection of large genomic insert clones from BAC libraries for walking. For this, tomato BACs are screened by automated BLAST analysis and in parallel, high-density nonselective amplified fragment length polymorphism fingerprinting is used for constructing a high-resolution BAC physical map. BLAST and amplified fragment length polymorphism analysis are then used together to determine the precise overlap. Assembly onto the seed BAC consensus confirms the BACs are properly selected for having an extremely short overlap and largest extending insert. This method will be particularly applicable where related or syntenic genomes are sequenced, as shown here for the Solanaceae, and potentially useful for the monocots Brassicaceae and Leguminosea. PMID:16524981

  20. Preparation of PAC libraries. Final technical report

    SciTech Connect

    Pieter J. de Jong

    1997-12-31

    The goals of this project were to create P1 Artificial Chromosome (PAC) cloning vectors and use these vectors to generate, characterize, and distribute both human and mouse genomic PAC libraries to the scientific community.

  1. Human Xq24-Xq28: approaches to mapping with yeast artificial chromosomes.

    PubMed Central

    Wada, M; Little, R D; Abidi, F; Porta, G; Labella, T; Cooper, T; Della Valle, G; D'Urso, M; Schlessinger, D

    1990-01-01

    One hundred twenty-seven yeast strains with artificial chromosomes containing Xq24-Xqter human DNA were obtained starting from a human/hamster somatic cell hybrid. The clones were characterized with respect to their insert size, stability, and representation of a set of Xq24-Xqter DNA probes. The inserts of the clones add up to 19.3 megabase (Mb) content, or about 0.4 genomic equivalents of that portion of the X chromosome, with a range of 40-650 kb in individual YACs. Eleven clones contained more than one YAC, the additional ones usually having hamster DNA inserts; the individual YACs could be separated by extracting the total DNA from such strains and using it to retransform yeast cells. One of the YACs, containing the probe for the DXS49 locus, was grossly unstable, throwing off smaller versions of an initial 300-kb YAC during subculture; the other YACs appeared to breed true on subculture. Of 52 probes tested, 12 found cognate YACs; the YACs included one with the glucose-6-phosphate dehydrogense gene and another containing four anonymous probe sequences (DX13, St14, cpx67, and cpx6). Xq location of YACs is being verified by in situ hybridization to metaphase chromosomes, and fingerprinting and hybridization methods are being used to detect YACs that overlap. Images Figure 3 Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:2294758

  2. Preparation and characterization of ordered libraries of transcribable sequences from human chromosome 19 from hybrid human-hamster cells

    SciTech Connect

    Obradovick, D.; Borodin, A.M.; Kopantsev, E.P.

    1994-08-20

    Improvements in the preparation of chromosome-specific libraries of transcribable sequences from the human genome using somatic hybrid cells are described. One of the main advantages of the new method is the enrichment of the starting material with human-specific heterogeneous nuclear RNA (hnRNA) sequences at each of the three steps in library construction. The method was used to prepare a chromosome-specific library from a hybrid cell line. The resulting ranged library was characterized. The primary structures of 80 randomly selected clones were determined, and these were analyzed. Some 95% of the clones were found to be human-specific and to have arisen from hnRNA, and the chromosomal localizations of a number of clones were identified. The advantages and disadvantages of the method are discussed. 34 refs., 3 figs., 2 tabs.

  3. [New method of construction of artificial translational-coupled operons in bacterial chromosome].

    PubMed

    Gulevich, A Iu; Skorokhodova, A Iu; Ermishev, V Iu; Krylov, A A; Minaeva, N I; Polonskaia, Z M; Zimenkov, D V; Biriukova, I V; Mashko, S V

    2009-01-01

    The new method of translational-coupled operons construction in bacterial chromosome has been developed on the basis of recombineering approach. It includes construction in vitro of the artificial operon with efficiently translated proximal cistron followed by its insertion E. coli chromosome, modification of the operon due to Red-driven insertion of the special "Junction" with excisable selective marker in the intercistronic region of the initial operon and excising the marker. The structure of this Junction has been designed and tested in the present investigation. It consists of: 1) E. coli rplC-rplD intercistronic region for placing the TAA-codon of the proximal operon's gene in the SD-sequence (TAAGGAG) of rplD; 2) Cm(R)-gene flanked by lambdaattL/R-sites in such a fashion that after lambdaInt/Xis-driven excision of the marker the residual lambdaattB-site would not contain the termination codons in frame with ATG of rplD; 3) E. coli trpE-trpD intercistronic region for location of ATG of trpD at the position of initiation codon of the distal gene of original operon. The general design of desired construction provides the conversion of the original two-cistronic operon into three-cistronic operon with translational-coupled genes, where the coupling of the artificial ORF (rplD'-lambdaattB-'trpE) with the proximal gene is occurred due to rplC-rplD intercistronic region and the coupling of this ORF with the distal gene--due to trpE-trpD. The experimental implementation of the described strategy was showed by construction of artificial operon P(tac-aroG4-serA5, where expression optimization of the distal serA5 gene was achieved via construction of three-cistronic operon with translational-coupled genes. PMID:19548541

  4. Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes

    SciTech Connect

    Perou, C.M.; Pryor, R.J.; Kaplan, J.; Justice, M.J.

    1996-06-11

    Chediak-Higashi syndrome in man and the beige mutation of mice are phenotypically similar disorders that have profound effects upon lysosome and melansosome morphology and function. We isolated two murine yeast artificial chromosomes (YACs) that, when introduced into beige mouse fibroblasts, complement the beige mutation. The complementing YACs exist as extrachromosomal elements that are amplified in high concentrations of G418. When YAC-complemented beige cells were fused to human Chediak-Higashi syndrome or Aleutian mink fibroblasts, complementation of the mutant phenotype also occurred. These results localize the beige gene to a 500-kb interval and demonstrate that the same or homologous genes are defective in mice, minks, and humans. 16 refs., 5 figs.

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

    PubMed

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

    2016-01-01

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

  6. Cloning of a very virulent plus, 686 strain of Marek’s disease virus as a bacterial artificial chromosome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek’s disease virus (MDV) is a highly oncogenic herpe...

  7. Plant artificial chromosome technology and its potential application in genetic engineering.

    PubMed

    Yu, Weichang; Yau, Yuan-Yeu; Birchler, James A

    2016-05-01

    Genetic engineering with just a few genes has changed agriculture in the last 20 years. The most frequently used transgenes are the herbicide resistance genes for efficient weed control and the Bt toxin genes for insect resistance. The adoption of the first-generation genetically engineered crops has been very successful in improving farming practices, reducing the application of pesticides that are harmful to both human health and the environment, and producing more profit for farmers. However, there is more potential for genetic engineering to be realized by technical advances. The recent development of plant artificial chromosome technology provides a super vector platform, which allows the management of a large number of genes for the next generation of genetic engineering. With the development of other tools such as gene assembly, genome editing, gene targeting and chromosome delivery systems, it should become possible to engineer crops with multiple genes to produce more agricultural products with less input of natural resources to meet future demands.

  8. Plant artificial chromosome technology and its potential application in genetic engineering.

    PubMed

    Yu, Weichang; Yau, Yuan-Yeu; Birchler, James A

    2016-05-01

    Genetic engineering with just a few genes has changed agriculture in the last 20 years. The most frequently used transgenes are the herbicide resistance genes for efficient weed control and the Bt toxin genes for insect resistance. The adoption of the first-generation genetically engineered crops has been very successful in improving farming practices, reducing the application of pesticides that are harmful to both human health and the environment, and producing more profit for farmers. However, there is more potential for genetic engineering to be realized by technical advances. The recent development of plant artificial chromosome technology provides a super vector platform, which allows the management of a large number of genes for the next generation of genetic engineering. With the development of other tools such as gene assembly, genome editing, gene targeting and chromosome delivery systems, it should become possible to engineer crops with multiple genes to produce more agricultural products with less input of natural resources to meet future demands. PMID:26369910

  9. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

    PubMed Central

    Mandegar, Mohammad A.; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y.L.; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P.; Volpi, Emanuela V.; Thrasher, Adrian J.; James, William; Monaco, Zoia L.

    2011-01-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  10. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells.

    PubMed

    Mandegar, Mohammad A; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y L; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P; Volpi, Emanuela V; Thrasher, Adrian J; James, William; Monaco, Zoia L

    2011-08-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  11. Artificial decoy spectral libraries for false discovery rate estimation in spectral library searching in proteomics.

    PubMed

    Lam, Henry; Deutsch, Eric W; Aebersold, Ruedi

    2010-01-01

    The challenge of estimating false discovery rates (FDR) in peptide identification from MS/MS spectra has received increased attention in proteomics. The simple approach of target-decoy searching has become popular with traditional sequence (database) searching methods, but has yet to be practiced in spectral (library) searching, an emerging alternative to sequence searching. We extended this target-decoy searching approach to spectral searching by developing and validating a robust method to generate realistic, but unnatural, decoy spectra. Our method involves randomly shuffling the peptide identification of each reference spectrum in the library, and repositioning each fragment ion peak along the m/z axis to match the fragment ions expected from the shuffled sequence. We show that this method produces decoy spectra that are sufficiently realistic, such that incorrect identifications are equally likely to match real and decoy spectra, a key assumption necessary for decoy counting. This approach has been implemented in the open-source library building software, SpectraST.

  12. A cosmid and yeast artificial chromosome contig containing the complete ryanodine receptor (RYR1) gene

    SciTech Connect

    Rouquier, S.; Giorgi, D.; Trask, B.; Bergmann, A.; De Jong, P. ); Phillips, M.S.; MacLennan, D.H. )

    1993-08-01

    The ryanodine receptor (RYR1) gene is responsible for some forms of malignant hyperthermia and has been localized to 19q13.1. Central core disease is a genetic myopathy that is genetically linked to RYR1. The authors have identified an overlapping set of cosmid and YAC clones that spans more than 800 kb and includes the RYR1 gene ([approximately]205 kb). Cosmids from this region were identified by screening three chromosome 19 cosmid libraries (11-fold coverage) with six subclones representing the entire RYR1 cDNA. Genomic sequences from positive cosmids were then used as probes to identify additional cosmids. A minimally overlapping set of 23 cosmids was assembled into two contigs on the basis of restriction fragment analysis and hybridization data. Three YAC clones were isolated by screening a human YAC library with selected cosmid inserts. Overlaps among these YACs and the cosmid contigs were determined by hybridizing YAC Alu-PCR products to cosmid DNAs. The YACs bridged the gap between the cosmid contigs and extended the contig on both sides. Fluorescence in situ hybridization experiments positioned the RYR1 contig between GPI, MAG, and D19S191 on the proximal side and D19S190, CYP2A, CYP2F, SNRPA, BCKDHA, and other markers on the distal side. The 800-kb contig of cloned reagents will facilitate the detailed characterization of the RYR1 gene and other loci that may be closely related to central core disease. 62 refs., 3 figs., 3 tabs.

  13. Chromosome

    MedlinePlus

    Chromosomes are structures found in the center (nucleus) of cells that carry long pieces of DNA. DNA ... is the building block of the human body. Chromosomes also contain proteins that help DNA exist in ...

  14. Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

    PubMed Central

    Zabarovsky, E R; Boldog, F; Thompson, T; Scanlon, D; Winberg, G; Marcsek, Z; Erlandsson, R; Stanbridge, E J; Klein, G; Sümegi, J

    1990-01-01

    Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed. Images PMID:2243778

  15. Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.

    PubMed

    Aliyeva-Schnorr, Lala; Beier, Sebastian; Karafiátová, Miroslava; Schmutzer, Thomas; Scholz, Uwe; Doležel, Jaroslav; Stein, Nils; Houben, Andreas

    2015-10-01

    Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single-copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination-poor region is preferentially marked by a heterochromatin-typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin-typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.

  16. Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.

    PubMed

    Yoshimura, Yuki; Nakamura, Kazuomi; Endo, Takeshi; Kajitani, Naoyo; Kazuki, Kanako; Kazuki, Yasuhiro; Kugoh, Hiroyuki; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2015-08-01

    The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10(-6)). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50% in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.

  17. Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.

    PubMed

    Yoshimura, Yuki; Nakamura, Kazuomi; Endo, Takeshi; Kajitani, Naoyo; Kazuki, Kanako; Kazuki, Yasuhiro; Kugoh, Hiroyuki; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2015-08-01

    The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10(-6)). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50% in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis. PMID:26055730

  18. Development of a Safeguard System Using an Episomal Mammalian Artificial Chromosome for Gene and Cell Therapy

    PubMed Central

    Uno, Narumi; Uno, Katsuhiro; Komoto, Shinya; Suzuki, Teruhiko; Hiratsuka, Masaharu; Osaki, Mitsuhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo

    2015-01-01

    The development of a safeguard system to remove tumorigenic cells would allow safer clinical applications of stem cells for the treatment of patients with an intractable disease including genetic disorders. Such safeguard systems should not disrupt the host genome and should have long-term stability. Here, we attempted to develop a tumor-suppressing mammalian artificial chromosome containing a safeguard system that uses the immune rejection system against allogeneic tissue from the host. For proof-of-concept of the safeguard system, B16F10 mouse melanoma cells expressing the introduced H2-K(d) major histocompatibility complex (MHC class I)-allogenic haplotype were transplanted into recipient C57BL/6J mice expressing MHC H2-K(b). Subcutaneous implantation of B16F10 cells into C57BL/6J mice resulted in high tumorigenicity. The volume of tumors derived from B16F10 cells expressing allogenic MHC H2-K(d) was decreased significantly (P < 0.01). Suppression of MHC H2-K(d)-expressing tumors in C57BL/6J mice was enhanced by immunization with MHC H2-K(d)-expressing splenocytes (P < 0.01). These results suggest that the safeguard system is capable of suppressing tumor formation by the transplanted cells. PMID:26670279

  19. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    SciTech Connect

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  20. Integration-Free iPS Cells Engineered Using Human Artificial Chromosome Vectors

    PubMed Central

    Hiratsuka, Masaharu; Uno, Narumi; Ueda, Kana; Kurosaki, Hajime; Imaoka, Natsuko; Kazuki, Kanako; Ueno, Etsuya; Akakura, Yutaro; Katoh, Motonobu; Osaki, Mitsuhiko; Kazuki, Yasuhiro; Nakagawa, Masato; Yamanaka, Shinya; Oshimura, Mitsuo

    2011-01-01

    Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system. PMID:21998730

  1. A bacterial artificial chromosome transgenic mouse model for visualization of neurite growth.

    PubMed

    Tao, Tao; Chen, Chen; Sun, Jie; Peng, YaJing; Zhu, MinSheng

    2015-04-01

    Class III β-tubulin (Tubb3) is a component of the microtubules in neurons and contributes to microtubule dynamics that are required for axon outgrowth and guidance during neuronal development. We here report a novel bacterial artificial chromosome (BAC) transgenic mouse line that expresses Class III β-tubulin fused to mCherry, an improved monomeric red fluorescent protein, for the visualization of microtubules during neuronal development. A BAC containing Tubb3 gene was modified by insertion of mCherry complementary DNA downstream of Tubb3 coding sequence via homologous recombination. mCherry fusion protein was expressed in the nervous system and testis of the transgenic animal, and the fluorescent signal was observed in the neurons that located in the olfactory bulb, cerebral cortex, hippocampal formation, cerebellum, as well as the retina. Besides, Tubb3-mCherry fusion protein mainly distributed in neurites and colocalized with endogenous Class III β-tubulin. The fusion protein labels Purkinje cell dendrites during cerebellar circuit formation. Therefore, this transgenic line might be a novel tool for scientific community to study neuronal development both in vitro and in vivo.

  2. Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome

    PubMed Central

    Gillen, Austin E.; Lucas, Catherine A.; Haussecker, Pei Ling; Kosak, Steven T.; Harris, Ann

    2013-01-01

    Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization (FISH), we observed that the integrated BAC forms extended, megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use. PMID:23749207

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

    PubMed

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

    2016-07-01

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

  4. Delivery of bacterial artificial chromosomes into mammalian cells with psoralen-inactivated adenovirus carrier.

    PubMed Central

    Baker, A; Cotten, M

    1997-01-01

    Molecular biology has many applications where the introduction of large (>100 kb) DNA molecules is required. The current methods of large DNA transfection are very inefficient. We reasoned that two limits to improving transfection methods with these large DNA molecules were the difficulty of preparing workable quantities of clean DNA and the lack of rapid assays to determine transfection success. We have used bacterial artificial chromosomes (BACs) based on the Escherichia coli F factor plasmid system, which are simple to manipulate and purify in microgram quantities. Because BAC plasmids are kept at one to two copies per cell, the problems of rearrangement observed with YACs are eliminated. We have generated two series of BAC vectors bearing marker genes for luciferase and green fluorescent protein (GFP). Using these reagents, we have developed methods of delivering BACs of up to 170 kb into mammalian cells with transfection efficiency comparable to 5-10 kb DNA. Psoralen-inactivated adenovirus is used as the carrier, thus eliminating the problems associated with viral gene expression. The delivered DNA is linked to the carrier virus with a condensing polycation. Further improvements in gene delivery were obtained by replacing polylysine with low molecular weight polyethylenimine (PEI) as the DNA condensing agent. PMID:9115362

  5. Yeast artificial chromosome cloning in the glycerol kinase and adrenal hypoplasia congenita region of Xp21

    SciTech Connect

    Worley, K.C.; Ellison, K.A.; Zhang, Y.H.; Wang, D.F.; Mason, J.; Roth, E.J.; Adams, V.; Fogt, D.D.; Zhu, X.M.; Towbin, J.A.

    1993-05-01

    The adrenal hypoplasia congenita (AHC) and glycerol kinase (GK) loci are telomeric to the Duchenne muscular dystrophy locus in Xp21. The authors developed a pair of yeast artificial chromosome (YAC) contigs spanning at least 1.2 Mb and encompassing the region from the telomeric end of the Duchenne muscular dystrophy (DMD) locus to beyond YHX39 (DXS727), including the genes for AHC and GK. The centromeric contig consists of 13 YACs reaching more than 600 kb from DMD through GK. The telomeric contig group consists of 8 YACs containing more than 600 kb including the markers YHX39 (DXS727) and QST-59 (DXS319). Patient deletion breakpoints in the region of the two YAC contigs define at least eight intervals, and seven deletion breakpoints are contained within these contigs. In addition to the probes developed from YAC ends, they have mapped eight Alu-PCR probes amplified from a radiation-reduced somatic cell hybrid, two anonymous DNA probes, and one Alu-PCR product amplified from a cosmid end, for a total of 26 new markers within this region of 2 Mb or less. One YAC in the centromeric contig contains an insert encompassing the minimum interval for GK deficiency defined by patient deletion breakpoints, and this clone includes all or part of the GK gene. 33 refs., 3 figs., 5 tabs.

  6. The selection and use of sorghum (Sorghum propinquum) bacterial artificial chromosomes as cytogenetic FISH probes for maize (Zea mays L.).

    PubMed

    Figueroa, Debbie M; Davis, James D; Strobel, Cornelia; Conejo, Maria S; Beckham, Katherine D; Ring, Brian C; Bass, Hank W

    2011-01-01

    The integration of genetic and physical maps of maize is progressing rapidly, but the cytogenetic maps lag behind, with the exception of the pachytene fluorescence in situ hybridization (FISH) maps of maize chromosome 9. We sought to produce integrated FISH maps of other maize chromosomes using Core Bin Marker loci. Because these 1 Kb restriction fragment length polymorphism (RFLP) probes are below the FISH detection limit, we used BACs from sorghum, a small-genome relative of maize, as surrogate clones for FISH mapping. We sequenced 151 maize RFLP probes and compared in silico BAC selection methods to that of library filter hybridization and found the latter to be the best. BAC library screening, clone verification, and single-clone selection criteria are presented along with an example of transgenomic BAC FISH mapping. This strategy has been used to facilitate the integration of RFLP and FISH maps in other large-genome species. PMID:21234422

  7. Genetic Stability of Bacterial Artificial Chromosome-Derived Human Cytomegalovirus during Culture In Vitro

    PubMed Central

    Murrell, Isa; Wilkie, Gavin S.; Davison, Andrew J.; Statkute, Evelina; Fielding, Ceri A.; Tomasec, Peter; Wilkinson, Gavin W. G.

    2016-01-01

    ABSTRACT Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagated in vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique long b′ (UL/b′) region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generated in vitro by deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L. IMPORTANCE Researchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagated in vitro, becoming less cell associated, altered in

  8. Bacterial artificial chromosome-derived molecular markers for early bolting in sugar beet.

    PubMed

    Gaafar, R M; Hohmann, U; Jung, C

    2005-04-01

    Early bolting in sugar beet (Beta vulgaris L.) is controlled by the dominant gene B. From an incomplete physical map around the B gene, 18 bacterial artificial chromosomes (BACs) were selected for marker development. Three BACs were shotgun-sequenced, and 61 open reading frames (ORFs) were identified. Together with 104 BAC ends from 54 BACs, a total number of 55,464 nucleotides were sequenced. Of these, 37 BAC ends and 12 ORFs were selected for marker development. Thirty-one percent of the sequences were found to be single copy and 24%, low copy. From these sequences, 15 markers from ten different BACs were developed. Ten polymorphisms were determined by simple agarose gel electrophoresis of either restricted or non-restricted PCR products. Another five markers were determined by tetra-primer amplification refractory mutation system-PCR. In order to select candidate BACs for cloning the gene, genetic linkage between seven markers and the bolting gene was calculated using 1,617 plants from an F2 population segregating for early bolting. The recombination values ranged between 0.0033 and 0.0201. In addition, a set of 41 wild and cultivated Beta accessions differing in their early bolting character was genotyped with seven markers. A common haplotype encompassing two marker loci and the b allele was found in all sugar beet varieties, indicating complete linkage disequilibrium between these loci. This suggests that the bolting gene is located in close vicinity to these markers, and the corresponding BACs can be used for cloning the gene.

  9. Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

    PubMed Central

    Yun, Sang-Im; Song, Byung-Hak; Kim, Jin-Kyoung; Lee, Young-Min

    2015-01-01

    Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase. PMID:26780115

  10. Identification of the LHRH locus in a flow-sorted human chromosome 8 cosmid library

    SciTech Connect

    Bruskiewich, R.; Schertzer, M.; Wood, S.

    1994-09-01

    Genomic sequence for luteinizing hormone releasing hormone (LHRH) gene has been published recently. The LHRH locus has been mapped to 8p21-8p11.2 using tritium labelling for in situ hybridization. Our goal is to determine a more precise localization for for LHRH. We have identified a genomic clone, 37F12, within the LA08NC01 flow-sorted human chromosome 8 cosmid library using PCR amplification of primary and secondary pools of mixed cosmids. The 37F12 cosmid was screened for novel sequence polymorphisms suitable for linkage analysis and preliminary data indicates that 37F12 contains a CA repeat sequence. The possible role of LHRH in human disease phenotypes mapping to 8p is unknown. Currently the only marker reported for LHRH is an RFLP that exhibits 11% heterozygosity. Development of a highly informative PCR marker will facilitate family studies where this locus is being excluded as a candidate locus for a disease phenotype.

  11. 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

    SciTech Connect

    Chen, X.N.; Gonsky, R.; Korenberg, J.R.; Knauf, J.A.; Fagin, J.A.; Chissoe, S.

    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.

  12. Narrowing the genetic interval and yeast artificial chromosome map in the branchio-oto-renal region on chromosome 8q

    SciTech Connect

    Kumar, Shrawan; Kimberling, W.J.; Pinnt, J.

    1996-01-01

    Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by branchial abnormality, hearing loss, and renal anomalies. Recently, the disease gene has been localized to chromosome 8q. Here, we report genetic studies that further refine the disease gene region to a smaller interval and identify several YACs from the critical region. We studied two large, clinically well-characterized BOR families with a set of 13 polymorphic markers spanning the D8S165-D8S275 interval from the chromosome 8q region. Based on multipoint analysis, the highest likelihood for the location of the BOR gene is between markers D8S543 and D8S530, a distance of about 2 cM. YACs that map in the BOR critical region have been identified and characterized by fluorescence in situ hybridization and pulsed-field gel electrophoresis. A YAC contig, based on the STS content map, that covers a minimum of 4 Mb of human DNA in the critical region of BOR is assembled. This lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in BOR syndrome. 40 refs., 4 figs., 1 tab.

  13. Telomere length homeostasis and telomere position effect on a linear human artificial chromosome are dictated by the genetic background.

    PubMed

    Weuts, An; Voet, Thierry; Verbeeck, Jelle; Lambrechts, Nathalie; Wirix, Evelyne; Schoonjans, Luc; Danloy, Sophie; Marynen, Peter; Froyen, Guy

    2012-12-01

    Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression. Previous studies suggested that TPE depends on genetic background. As these analyses were performed on different chromosomes, cell types and species, it remains unclear whether TPE represents a chromosome-rather than genetic background-specific regulation. We describe the development of a Linear Human Artificial Chromosome (L-HAC) as a new tool for telomere studies. The L-HAC was generated through the Cre-loxP-mediated addition of telomere ends to an existing circular HAC (C-HAC). As it can be transferred to genetically distinct cell lines and animal models the L-HAC enables the study of TPE in an unprecedented manner. The HAC was relocated to four telomerase-positive cell lines via microcell-mediated chromosome transfer and subsequently to mice via blastocyst injection of L-HAC(+)-ES-cells. We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice. Expression of the subtelomeric neomycin gene was inversely correlated with telomere length and subtelomeric methylation. We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent.

  14. Use of laser microdissection for the construction of Humulusjaponicus Siebold et Zuccarini, 1846 (Cannabaceae) sex chromosome-specific DNA library and cytogenetics analysis.

    PubMed

    Yakovin, Nickolay A; Divashuk, Mikhail G; Razumova, Olga V; Soloviev, Alexander A; Karlov, Gennady I

    2014-01-01

    Dioecy is relatively rare among plant species, and distinguishable sex chromosomes have been reported in few dioecious species. The multiple sex chromosome system (XX/XY1Y2) of Humulusjaponicus Siebold et Zuccarini, 1846 differs from that of other members of the family Cannabaceae, in which the XX/XY chromosome system is present. Sex chromosomes of Humulusjaponicus were isolated from meiotic chromosome spreads of males by laser microdissection with the P.A.L.M. MicroLaser system. The chromosomal DNA was directly amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). Fast fluorescence in situ hybridization (FAST-FISH) using a labeled, chromosome-specific DOP-PCR product as a probe showed preferential hybridization to sex chromosomes. In addition, the DOP-PCR product was used to construct a short-insert, Humulusjaponicus sex chromosomes-specific DNA library. The randomly sequenced clones showed that about 12% of them have significant homology to Humuluslupulus and 88% to Cannabissativa Linnaeus, 1753 sequences from GenBank database. Forty-four percent of the sequences show homology to plant retroelements. It was concluded that laser microdissection is a useful tool for isolating the DNA of sex chromosomes of Humulusjaponicus and for the construction of chromosome-specific DNA libraries for the study of the structure and evolution of sex chromosomes. The results provide the potential for identifying unique or sex chromosome-specific sequence elements in Humulusjaponicus and could aid in the identification of sex chromosome-specific repeat and coding regions through chromosome isolation and genome complexity reduction. PMID:25610546

  15. Use of laser microdissection for the construction of Humulus japonicus Siebold et Zuccarini, 1846 (Cannabaceae) sex chromosome-specific DNA library and cytogenetics analysis

    PubMed Central

    Yakovin, Nickolay A.; Divashuk, Mikhail G.; Razumova, Olga V.; Soloviev, Alexander A.; Karlov, Gennady I.

    2014-01-01

    Abstract Dioecy is relatively rare among plant species, and distinguishable sex chromosomes have been reported in few dioecious species. The multiple sex chromosome system (XX/XY1Y2) of Humulus japonicus Siebold et Zuccarini, 1846 differs from that of other members of the family Cannabaceae, in which the XX/XY chromosome system is present. Sex chromosomes of Humulus japonicus were isolated from meiotic chromosome spreads of males by laser microdissection with the P.A.L.M. MicroLaser system. The chromosomal DNA was directly amplified by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). Fast fluorescence in situ hybridization (FAST-FISH) using a labeled, chromosome-specific DOP-PCR product as a probe showed preferential hybridization to sex chromosomes. In addition, the DOP-PCR product was used to construct a short-insert, Humulus japonicus sex chromosomes-specific DNA library. The randomly sequenced clones showed that about 12% of them have significant homology to Humulus lupulus and 88% to Cannabis sativa Linnaeus, 1753 sequences from GenBank database. Forty-four percent of the sequences show homology to plant retroelements. It was concluded that laser microdissection is a useful tool for isolating the DNA of sex chromosomes of Humulus japonicus and for the construction of chromosome-specific DNA libraries for the study of the structure and evolution of sex chromosomes. The results provide the potential for identifying unique or sex chromosome-specific sequence elements in Humulus japonicus and could aid in the identification of sex chromosome-specific repeat and coding regions through chromosome isolation and genome complexity reduction. PMID:25610546

  16. Mapping and cloning of the critical region for the spineocerebellar ataxia Type 1 gene (SCA1) in a yeast artificial chromosome contig spanning 1. 2 Mb

    SciTech Connect

    Banfi, S.; McCall, A.E.; Zoghbi, H.Y.; Kwiatkowski, T.J. Jr.; Chinault, A.C. ); Ranum, L.P.W.; Orr, H.T. )

    1993-12-01

    The gene responsible for spinocerebellar ataxia type 1 (SCA1) has been localized to a 6.7-cM region between the centromeric marker D6S109 and the telomeric marker D6S89. The authors screened two yeast artificial chromosome (YAC) libraries using sequence-tagged sites at D6S89 and at newly identified markers in 6p22-p23. Fifty YAC clones were identified and 34 insert termini were isolated from some of these YACs for detailed overlap mapping and long-range restriction analysis. A large YAC contig estimated to span 2.5 Mb was developed and genetic analysis in five large SCA1 kindreds using highly informative dinucleotide repeat polymorphisms mapped to this contig allowed the identification of D6S274 as the closest centromeric flanking marker for SCA1. Long-range restriction analysis determined the size for the critical SCA1 region, as defined by the two flanking markers D6S274 and D6S89, to be 1.2 Mb. This region is spanned by a minimum set of four nonchimeric YAC clones. The development of a 2.5-Mb YAC contig in 6p22-p23 provides valuable reagents for characterization of this genomic region and for the cloning of the SCA1 gene. 34 refs., 4 figs., 2 tabs.

  17. Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity

    PubMed Central

    Endo, Takeshi; Noda, Natsumi; Kuromi, Yasushi; Kokura, Kenji; Kazuki, Yasuhiro; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2016-01-01

    Background Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. Methods We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse Hprt promoter fused with green-emitting luciferase (SLG) coding sequence. The Hprt-SLG vector was loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase in mouse A9 cells. We established three independent clones. Results The established cell lines had similar levels of expression of the Hprt-SLG reporter gene. Hprt-SLG activity increased proportionately under growth conditions and decreased under cytotoxic conditions after blasticidin or cisplatin administration. Similar increases and decreases in the SLG luminescent were observed under growth and cytotoxic conditions, respectively, to those in the fluorescent obtained using the commercially available reagent, alamarBlue. Conclusion By employing a reliable and stable expression system in a mammalian artificial chromosome, the activity of an Hprt-SLG reporter can reflect cell numbers under cell growth condition and cell viability in the evaluation of cytotoxic conditions. PMID:27493490

  18. Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology.

    PubMed

    Mallin, Hendrik; Hestericová, Martina; Reuter, Raphael; Ward, Thomas R

    2016-05-01

    Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d. PMID:27031496

  19. Telomere length homeostasis and telomere position effect on a linear human artificial chromosome are dictated by the genetic background

    PubMed Central

    Weuts, An; Voet, Thierry; Verbeeck, Jelle; Lambrechts, Nathalie; Wirix, Evelyne; Schoonjans, Luc; Danloy, Sophie; Marynen, Peter; Froyen, Guy

    2012-01-01

    Telomere position effect (TPE) is the influence of telomeres on subtelomeric epigenetic marks and gene expression. Previous studies suggested that TPE depends on genetic background. As these analyses were performed on different chromosomes, cell types and species, it remains unclear whether TPE represents a chromosome—rather than genetic background-specific regulation. We describe the development of a Linear Human Artificial Chromosome (L-HAC) as a new tool for telomere studies. The L-HAC was generated through the Cre-loxP-mediated addition of telomere ends to an existing circular HAC (C-HAC). As it can be transferred to genetically distinct cell lines and animal models the L-HAC enables the study of TPE in an unprecedented manner. The HAC was relocated to four telomerase-positive cell lines via microcell-mediated chromosome transfer and subsequently to mice via blastocyst injection of L-HAC+-ES-cells. We could show consistent genetic background-dependent adaptation of telomere length and telomere-associated de novo subtelomeric DNA methylation in mouse ES-R1 cells as well as in mice. Expression of the subtelomeric neomycin gene was inversely correlated with telomere length and subtelomeric methylation. We thus provide a new tool for functional telomere studies and provide strong evidence that telomere length, subtelomeric chromatin marks and expression of subtelomeric genes are genetic background dependent. PMID:23066103

  20. Extending long-range phasing and haplotype library imputation methods to impute genotypes on sex chromosomes

    PubMed Central

    2013-01-01

    AlphaImpute is a flexible and accurate genotype imputation tool that was originally designed for the imputation of genotypes on autosomal chromosomes. In some species, sex chromosomes comprise a large portion of the genome. For example, chromosome Z represents approximately 8% of the chicken genome and therefore is likely to be important in determining genetic variation in a population. When breeding programs make selection decisions based on genomic information, chromosomes that are not represented on the genotyping platform will not be subject to selection. Therefore imputation algorithms should be able to impute genotypes for all chromosomes. The objective of this research was to extend AlphaImpute so that it could impute genotypes on sex chromosomes. The accuracy of imputation was assessed using different genotyping strategies in a real commercial chicken population. The correlation between true and imputed genotypes was high in all the scenarios and was 0.96 for the most favourable scenario. Overall, the accuracy of imputation of the sex chromosome was slightly lower than that of autosomes for all scenarios considered. PMID:23617460

  1. A yeast artificial chromosome contig of the critical region for cri-du-chat syndrome

    SciTech Connect

    Goodart, S.A.; Rojas, K.; Overhauser, J.

    1994-11-01

    Cri-du-chat is a chromosomal deletion syndrome characterized by partial deletion of the short arm of chromosome 5. The clinical symptoms include growth and mental retardation, microcephaly, hypertelorism, epicanthal folds, hyptonia, and a high-pitched monochromatic cry that is usually considered diagnostic for the syndrome. Recently, a correlation between clinical features and the extent of the chromosome 5 deletions has identified two regions of the short arm that appear to be critical for the abnormal development manifested in this syndrome. Loss of a small region in 5p15.2 correlates with all of the clinical features of cri-du-chat with the exception of the cat-like cry, which maps to 5p15.3. Here the authors report the construction of a YAC contig that spans the chromosomal region in 5p15.2 that plays a major role in the etiology of the cri-du-chat syndrome. YACs that span the 2-Mb cri-du-chat critical region have been identified and characterized. This YAC contig lays the groundwork for the construction of a transcriptional map of this region and the eventual identification of genes involved in the clinical features associated with the cri-du-chat syndrome. It also provides a new diagnostic tool for cri-du-chat in the shape of a YAC clone that may span the entire critical region. 24 refs., 4 figs., 2 tabs.

  2. Development of a novel DNA-launched dengue virus type 2 infectious clone assembled in a bacterial artificial chromosome.

    PubMed

    Usme-Ciro, Jose A; Lopera, Jaime A; Enjuanes, Luis; Almazán, Fernando; Gallego-Gomez, Juan C

    2014-02-13

    Major progress in Dengue virus (DENV) biology has resulted from the use of infectious clones obtained through reverse genetics. The construction of these clones is commonly based on high- or low-copy number plasmids, yeast artificial chromosomes, yeast-Escherichia coli shuttle vectors, and bacterial artificial chromosomes (BACs). Prokaryotic promoters have consistently been used for the transcription of these clones. The goal of this study was to develop a novel DENV infectious clone in a BAC under the control of the cytomegalovirus immediate-early promoter and to generate a virus with the fusion envelope-green fluorescent protein in an attempt to track virus infection. The transfection of Vero cells with a plasmid encoding the DENV infectious clone facilitated the recovery of infectious particles that increased in titer after serial passages in C6/36 cells. The plaque size and syncytia phenotypes of the recombinant virus were similar to those of the parental virus. Despite the observation of autonomous replication and the detection of low levels of viral genome after two passages, the insertion of green fluorescent protein and Renilla luciferase reporter genes negatively impacted virus rescue. To the best of our knowledge, this is the first study using a DENV infectious clone under the control of the cytomegalovirus promoter to facilitate the recovery of recombinant viruses without the need for in vitro transcription. This novel molecular clone will be useful for establishing the molecular basis of replication, assembly, and pathogenesis, evaluating potential antiviral drugs, and the development of vaccine candidates for attenuated recombinant viruses.

  3. Three-region specific microdissection libraries for the long arm of human chromosome 2, regions q33-q35, q31-q32, and q23-q24

    SciTech Connect

    Yu, J.; Tong, S.; Whittier, A.

    1995-09-01

    Three region-specific libraries have been constructed from the long arm of human chromosome 2, including regions 2q33-35 (2Q2 library), 2q31-32 (2Q3) and 2q23-24 (2Q4). Chromosome microdissection and the MboI linker-adaptor microcloning techniques were used in constructing these libraries. The libraries comprised hundreds of thousands of microclones in each library. Approximately half of the microclones in the library contained unique or low-copy number sequence inserts. The insert sizes ranged between 50 and 800 bp, with a mean of 130-190 bp. Southern blot analysis of individual unique sequence microclones showed that 70-94% of the microclones were derived from the dissected region. 31 unique sequence microclones from the 2Q2 library, 31 from 2Q3, and 30 from 2Q4, were analyzed for insert sizes, the hybridizing genomic HindIII fragment sizes, and cross-hybridization to rodent species. These libraries and the short insert microclones derived from the libraries should be useful for high resolution physical mapping, sequence-ready reagents for large scale genomic sequencing, and positional cloning of disease-related genes assigned to these regions, e.g. the recessive familial amyotrophic lateral sclerosis assigned to 2q33-q35, and a type I diabetes susceptibility gene to 2q31-q33. 17 refs., 5 figs., 2 tabs.

  4. Artifically inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The long terminal repeat (LTR) sequence of reticuloendotheliosis virus (REV) was inserted into the very virulent Marek’s disease virus (MDV) Md5 bacterial artificial chromosome clone. The insertion site was nearly identical to the REV LTR that was naturally inserted into the JM/102W strain of MDV fo...

  5. Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle-alpha-actin gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Smooth muscle a actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells(vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a ...

  6. Use of a Human Artificial Chromosome for Delivering Trophic Factors in a Rodent Model of Amyotrophic Lateral Sclerosis

    PubMed Central

    Watanabe, Yasuhiro; Kazuki, Yasuhiro; Kazuki, Kanako; Ebiki, Mitsutaka; Nakanishi, Mami; Nakamura, Kazuomi; Yoshida Yamakawa, Miho; Hosokawa, Hiroyuki; Ohbayashi, Tetsuya; Oshimura, Mitsuo; Nakashima, Kenji

    2015-01-01

    A human artificial chromosome (HAC) is maintained as an episome within a cell and avoids random integration into the host genome. It can transfer multiple and/or large transgenes along with their regulatory elements thereby resembling native chromosomes. Using this HAC system, we established mesenchymal stem cells (MSCs) that simultaneously expressed hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1, termed HAC-MSCs. This cell line provides an opportunity for stable transplantation and thorough analyses. We then introduced the cells for the treatment of a neurodegenerative disorder, amyotrophic lateral sclerosis. The HAC-MSCs were transplanted via the fourth cerebral ventricle (CV) or intravenous (i.v.) infusion at various ages of recipient mice. Littermate- and sex-matched mice underwent a sham procedure. Compared to the controls, there was an encouraging trend of increased life span via CV transplantation and delayed onset in i.v. infusion 60 days after transplantation. Further, we confirmed a statistically significant increase in life span via CV transplantation at 100 days. This effect was not seen in mice transplanted with MSCs lacking the HAC. We successfully enhanced the trophic potential of the MSCs using the HAC. This strategy could be a promising direction for the treatment of neurodegenerative disorders. PMID:26440597

  7. A yeast artificial chromosome (YAC) contig encompassing the critical region of the X-linked lymphoproliferative disease (XLP) locus.

    PubMed

    Lanyi, A; Li, B; Li, S; Talmadge, C B; Brichacek, B; Davis, J R; Kozel, B A; Trask, B; van den Engh, G; Uzvolgyi, E; Stanbridge, E J; Nelson, D L; Chinault, C; Heslop, H; Gross, T G; Seemayer, T A; Klein, G; Purtilo, D T; Sumegi, J

    1997-01-01

    X-linked lymphoproliferative disease (XLP) is characterized by a marked vulnerability to Epstein-Barr virus (EBV) infection. Infection of XLP patients with EBV invariably results in fatal mononucleosis, agammaglobulinemia, or malignant lymphoma. Initially the XLP gene was assigned to a 10-cM region in Xq25 between DXS42 and DXS37. Subsequently, an interstitial, cytogenetically visible deletion in Xq25 was identified in one XLP family, 43. In this study we estimated the deletion in XLP patient 43-004 by dual-laser flow karyotyping to involve 2% of the X chromosome, or approximately 3 Mb of DNA sequence. From a human chromosome Xq25-specific yeast artificial chromosome (YAC) sublibrary, five YACs containing DNA sequences deleted in patient 43-004 have been isolated. Sequence-tagged sites (STSs) from these YACs have been used to identify interstitial deletions in unrelated XLP patients. Three more families with interstitial deletions were found. Two of the patients (63-003 and 73-032) carried an interstitial deletion of 3.0 Mb overlapping the 43-004 deletion. In one XLP patient (30-011) who exhibited the characteristic postinfectious mononucleosis phenotype of XLP with hypogammaglobulinemia and malignant lymphoma, a deletion of approximately 250 kb was detected overlapping the deletion detected in patients 43-004, 63-003, and 73-032. A YAC contig of 2.2 Mb spanning the XLP critical region, whose orientation on chromosome X was determined by double-color fluorescence in situ hybridization and which consists of 15 overlapping YAC clones, has been constructed. A detailed restriction enzyme map of the region has been constructed. YAC insert sizes were determined by counter-clamped homogenous electric field gel electrophoresis. Chimerism of YACs was determined by FISH and restriction mapping. On the basis of lambda subclones, YAC end-derived plasmids, and STSs with an average spacing of 100 kb, a long-range physical map was constructed using 5 rare-cutter restriction

  8. Library+

    ERIC Educational Resources Information Center

    Merrill, Alex

    2011-01-01

    This article discusses possible future directions for academic libraries in the post Web/Library 2.0 world. These possible directions include areas such as data literacy, linked data sets, and opportunities for libraries in support of digital humanities. The author provides a brief sketch of the background information regarding the topics and…

  9. A novel bacterial artificial chromosome-transgenic podoplanin-cre mouse targets lymphoid organ stromal cells in vivo.

    PubMed

    Onder, Lucas; Scandella, Elke; Chai, Qian; Firner, Sonja; Mayer, Christian T; Sparwasser, Tim; Thiel, Volker; Rülicke, Thomas; Ludewig, Burkhard

    2011-01-01

    Stromal cells provide the structural foundation of secondary lymphoid organs (SLOs), and regulate leukocyte access and cell migration within the different compartments of spleen and lymph nodes (LNs). Furthermore, several stromal cell subsets have been implied in shaping of T cell responses through direct presentation of antigen. Despite significant gain of knowledge on the biology of different SLO-resident stromal cell subsets, their molecular and functional characterization has remained incomplete. To address this need, we have generated a bacterial artificial chromosome-transgenic mouse model that utilizes the podoplanin (pdpn) promoter to express the Cre-recombinase exclusively in stromal cells of SLOs. The characterization of the Pdpn-Cre mouse revealed transgene expression in subsets of fibroblastic reticular cells and lymphatic endothelial cells in LNs. Furthermore, the transgene facilitated the identification of a novel splenic perivascular stromal cell subpopulation that forms web-like structures around central arterioles. Assessment of the in vivo antigen expression in the genetically tagged stromal cells in Pdpn-Cre mice revealed activation of both MHC I and II-restricted TCR transgenic T cells. Taken together, stromal pdpn-Cre expression is well-suited to characterize the phenotype and to dissect the function of lymphoid organ stromal cells.

  10. Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

    PubMed

    Reddy, Sanjay M; Sun, Aijun; Khan, Owais A; Lee, Lucy F; Lupiani, Blanca

    2013-06-01

    Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

  11. Gata3 Hypomorphic Mutant Mice Rescued with a Yeast Artificial Chromosome Transgene Suffer a Glomerular Mesangial Cell Defect.

    PubMed

    Moriguchi, Takashi; Yu, Lei; Otsuki, Akihito; Ainoya, Keiko; Lim, Kim-Chew; Yamamoto, Masayuki; Engel, James Douglas

    2016-09-01

    GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generated Gata3 hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kb Gata3 yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kb Gata3 YAC transgene recapitulated Gata3 expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease. PMID:27296697

  12. Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

    SciTech Connect

    Sathish, Narayanan; Yuan Yan

    2010-11-25

    A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-{Delta}65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

  13. Sequence analysis of bacterial artificial chromosome clones from the apospory-specific genomic region of Pennisetum and Cenchrus.

    PubMed

    Conner, Joann A; Goel, Shailendra; Gunawan, Gunawati; Cordonnier-Pratt, Marie-Michele; Johnson, Virgil Ed; Liang, Chun; Wang, Haiming; Pratt, Lee H; Mullet, John E; DeBarry, Jeremy; Yang, Lixing; Bennetzen, Jeffrey L; Klein, Patricia E; Ozias-Akins, Peggy

    2008-07-01

    Apomixis, asexual reproduction through seed, is widespread among angiosperm families. Gametophytic apomixis in Pennisetum squamulatum and Cenchrus ciliaris is controlled by the apospory-specific genomic region (ASGR), which is highly conserved and macrosyntenic between these species. Thirty-two ASGR bacterial artificial chromosomes (BACs) isolated from both species and one ASGR-recombining BAC from P. squamulatum, which together cover approximately 2.7 Mb of DNA, were used to investigate the genomic structure of this region. Phrap assembly of 4,521 high-quality reads generated 1,341 contiguous sequences (contigs; 730 from the ASGR and 30 from the ASGR-recombining BAC in P. squamulatum, plus 580 from the C. ciliaris ASGR). Contigs containing putative protein-coding regions unrelated to transposable elements were identified based on protein similarity after Basic Local Alignment Search Tool X analysis. These putative coding regions were further analyzed in silico with reference to the rice (Oryza sativa) and sorghum (Sorghum bicolor) genomes using the resources at Gramene (www.gramene.org) and Phytozome (www.phytozome.net) and by hybridization against sorghum BAC filters. The ASGR sequences reveal that the ASGR (1) contains both gene-rich and gene-poor segments, (2) contains several genes that may play a role in apomictic development, (3) has many classes of transposable elements, and (4) does not exhibit large-scale synteny with either rice or sorghum genomes but does contain multiple regions of microsynteny with these species. PMID:18508959

  14. Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

    SciTech Connect

    Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko; Hiratsuka, Masaharu; Sano, Akiko; Osawa, Kanako; Okazaki, Akiyo; Katoh, Motonobu; Kazuki, Yasuhiro; Oshimura, Mitsuo; Tomizuka, Kazuma

    2008-05-09

    Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-{beta}-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.

  15. Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

  16. Comparative fluorescence in situ hybridization mapping of a 431-kb Arabidopsis thaliana bacterial artificial chromosome contig reveals the role of chromosomal duplications in the expansion of the Brassica rapa genome.

    PubMed

    Jackson, S A; Cheng, Z; Wang, M L; Goodman, H M; Jiang, J

    2000-10-01

    Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative physical mapping between Arabidopsis thaliana and Brassica rapa. A set of six bacterial artificial chromosomes (BACs) representing a 431-kb contiguous region of chromosome 2 of A. thaliana was mapped on both chromosomes and DNA fibers of B. rapa. This DNA fragment has a single location in the A. thaliana genome, but hybridized to four to six B. rapa chromosomes, indicating multiple duplications in the B. rapa genome. The sizes of the fiber-FISH signals from the same BACs were not longer in B. rapa than those in A. thaliana, suggesting that this genomic region is duplicated but not expanded in the B. rapa genome. The comparative fiber-FISH mapping results support that chromosomal duplications, rather than regional expansion due to accumulation of repetitive sequences in the intergenic regions, played the major role in the evolution of the B. rapa genome.

  17. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice

    PubMed Central

    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

  18. Functional characterization of Kaposi's sarcoma-associated herpesvirus open reading frame K8 by bacterial artificial chromosome-based mutagenesis.

    PubMed

    Wang, Yan; Sathish, Narayanan; Hollow, Charles; Yuan, Yan

    2011-03-01

    The open reading frame K8 of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a basic leucine zipper (bZip) protein that binds to the origin of viral DNA replication and is an integral component of viral lytic DNA replication complex. Moreover, K8 physically interacts with replication and transcription activator (RTA) and represses its transactivation activity on several viral promoters. To investigate the role of this protein in viral life cycle, we constructed two K8-null recombinant mutant viruses (BAC-ΔK8 and BAC-stopK8) by using a bacterial artificial chromosome (BAC) system. Latent viral infection can be reconstituted in 293T and BJAB cells with wild-type and the K8-null recombinant viruses by introducing the cloned viral genomes into the cells. When the cells carrying these viruses were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, no significant difference was seen in overall viral gene expression between wild-type and K8-null viruses, with lytic DNA replication still active in the latter. However, 293T cells harboring K8-null mutant viruses, either BAC-ΔK8 or BAC-stopK8, displayed lower copy numbers of latent KSHV genome in comparison with wild-type viruses. Furthermore, although K8 deficiency appeared to not affect infectivity when K8-null viruses were used to infect 293T, primary human microvascular dermal endothelial and human foreskin fibroblast cells, they exhibited much lower viral genome copy numbers in all types of cell compared to wild-type viruses. Taken together, these data suggest a possible role of K8 in abortive lytic DNA replication occurring in early stages of de novo infection or in the maintenance of latent viral genomes. PMID:21159864

  19. Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice.

    PubMed

    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

  20. Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

    PubMed Central

    2013-01-01

    Background The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome’) strategy to expand our understanding of human gene regulation in vivo. Results In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. Conclusions We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression. PMID:24124870

  1. A method for producing transgenic cells using a multi-integrase system on a human artificial chromosome vector.

    PubMed

    Yamaguchi, Shigeyuki; Kazuki, Yasuhiro; Nakayama, Yuji; Nanba, Eiji; Oshimura, Mitsuo; Ohbayashi, Tetsuya

    2011-01-01

    The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3-96.8%. Additionally, we observed homogenous gene expression in 77.3-87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression. PMID:21390305

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

    PubMed

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

    2011-01-01

    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. PMID:21314965

  3. Construction and Characterization of a Repetitive DNA Library in Parodontidae (Actinopterygii: Characiformes): A Genomic and Evolutionary Approach to the Degeneration of the W Sex Chromosome

    PubMed Central

    Oliveira, Jordana Inácio Nascimento; Nogaroto, Viviane; Almeida, Mara Cristina; Artoni, Roberto Ferreira; Cestari, Marta Margarete; Moreira-Filho, Orlando; Vicari, Marcelo Ricardo

    2014-01-01

    Abstract Repetitive DNA sequences, including tandem and dispersed repeats, comprise a large portion of eukaryotic genomes and are important for gene regulation, sex chromosome differentiation, and karyotype evolution. In Parodontidae, only the repetitive DNAs WAp and pPh2004 and rDNAs were previously studied using fluorescence in situ hybridization. This study aimed to build a library of repetitive DNA in Parodontidae. We isolated 40 clones using Cot-1; 17 of these clones exhibited similarity to repetitive DNA sequences, including satellites, minisatellites, microsatellites, and class I and class II transposable elements (TEs), from Danio rerio and other organisms. The physical mapping of the clones to chromosomes revealed the presence of a satellite DNA, a Helitron element, and degenerate short interspersed element (SINE), long interspersed element (LINE), and tc1-mariner elements on the sex chromosomes. Some clones exhibited dispersed signals; other sequences were not detected. The 5S rDNA was detected on an autosomal pair. These elements likely function in the molecular degeneration of the W chromosome in Parodontidae. Thus, the location of these elements on the chromosomes is important for understanding the function of these repetitive DNAs and for integrative studies with genome sequencing. The presented data demonstrate that an intensive invasion of TEs occurred during W sex chromosome differentiation in the Parodontidae. PMID:25122415

  4. Artificial Intelligence and Expert Systems: Will They Change the Library? Papers Presented at the Annual Clinic on Library Applications of Data Processing (27th, Urbana, Illinois, March 25-27, 1990). Illinois, March 25-27, 1990).

    ERIC Educational Resources Information Center

    Lancaster, F. W., Ed.; Smith, Linda C., Ed.

    Some of the 12 conference papers presented in this proceedings focus on the present and potential capabilities of artificial intelligence and expert systems as they relate to a wide range of library applications, including descriptive cataloging, technical services, collection development, subject indexing, reference services, database searching,…

  5. Construction of a yeast artificial chromosome contig encompassing the human acidic fibroblast growth factor (FGF1) gene: Toward the cloning of the ANLL/MDS tumor-suppressor gene

    SciTech Connect

    Chiu, Ing-Ming; Gilmore, E.C.; Liu, Yang; Payson, R.A. )

    1994-02-01

    The region surrounding the human acidic fibroblast growth factor (FGF1) locus on chromosome 5q31 is of particular interest since it represents a critical region consistently lost in acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) patients who have a demonstrable deletion of the distal portion of the long arm of chromosome 5. It is proposed that an ANLL/MDS leukemia suppressor gene resides on 5q31. The authors have previously shown that the gene is most likely localized between FGF1 and PDGFRB/CSF1R loci. The region has also been linked to at least four other genetic diseases, Treacher Collins syndrome, diastrophic dysplasia, limb-girdle muscular dystrophy, and an autosomal dominant deafness, by linkage analysis. Here, they describe yeast artificial chromosomes (YAC) spanning 450 kb around the FGF1 gene. Six YAC clones were isolated from a human YAC library and their restriction enzyme maps were determined. The overlap of the clones with each other and with FGF1 cosmid and phage clones was characterized. Three of the YAC clones were found to contain the entire FGF1 gene, which spans more than 100 kb. Proximal and distal ends of several of these YAC clones were isolated for further overlap cloning. The proximal ends of both Y2 and Y4 were localized to previously isolated FGF1 DNA by sequence analysis. The distal ends of these two clones also hybridized to a human-hamster hybrid containing chromosome 5 as the only human genetic material. These results suggest that these YAC clones represent colinear DNA around the FGF1 locus. None of the YAC clones were found to contain the CD 14 and GRL genes, the closest known proximal and distal markers (relative to the centromere) to the FGF1 gene, respectively. This contig is useful for the overlap cloning of the 5q31 region and for reverse genetic strategies for the isolation of disease genes in the region. 46 refs., 7 figs., 5 tabs.

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

    SciTech Connect

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

    1995-12-31

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

    PubMed Central

    Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

    2013-01-01

    We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties. PMID:24014183

  9. Progress towards complete genetic and physical maps of chromosome 3

    SciTech Connect

    Naylor, S.L.; Munoz, C.; Badura, M.

    1994-09-01

    Human chromosome 3 contains 210 million base pairs and approximately 7,000 genes. Our center is directed towards making genetic and physical maps of chromosome 3 with the goal of a YAC contig of the chromosome and 2500 STSs. Markers are first binned into 23 regions of chromosome 3 using framework somatic cell hybrids. To date, STSs for 259 microsatellite and 63 genes have been placed into bins. 125 polymorphic simple sequences repeat (SSR) markers were genetically mapped to chromosome 3 using PCR-based genotyping of the CEPH reference families. This genetic map spans 271 cM (sex averaged). A chromosome 3-specific radiation-reduced somatic cell hybrid panels has been constructed to obtain higher resolution map order information. With the map locations of these markers as a guide, 226 loci were screened using PCR-based hierarchial screening of the CEPH Mark I and Mark IV-VII yeast artificial chromosome (YAC) libraries. YAC contigs which are ordered on the chromosome consist of 33 multi-locus contigs covering 80 cM and single locus contigs accounting for 62 cM. We have isolated approximately 1000 YACs by this method covering 65% of chromosome 3. IRS-PCR screening of the CEPH YAC library has yielded several thousand more chromosome 3 YACs which are being screened with the STSs to integrate them into contigs. FISH analysis on a random sampling of 60 YACs from the long arm was performed to assess the degree of chimerism. 40% of the YACs hybridized to the map locations predicted by the STSs used in their isolation. 30% were obviously chimeric while 40% hybridized uniquely to the predicted region as well as nonspecifically to centromeres and telomeres. This integrated approach to constructing a map of chromosome 3 will result in a reliably ordered YAC contig for this chromosome.

  10. Artificially inserting a reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of Marek's disease virus (MDV) alters expression of nearby MDV genes.

    PubMed

    Kim, Taejoong; Mays, Jody; Fadly, Aly; Silva, Robert F

    2011-06-01

    Researchers reported that co-cultivating the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in an REV long terminal repeat (LTR) being inserted into the internal repeat short (IRS) region of JM/102W. When the resulting recombinant virus was serially passed in cell culture, the initial LTR was duplicated and a second LTR spontaneously appeared in the terminal repeat short (TRS) region of the MDV genome. The virus, designated RM1, was significantly attenuated but still induced severe bursal and thymic atrophy (Isfort et al. PNAS 89:991-995). To determine whether the altered phenotype was due solely to the LTR, we cloned the LTR from the RM1 IRS region and inserted it into the IRS region of a very virulent bacterial artificial clone (BAC) of the Md5 strain of MDV, which we designated rMd5-RM1-LTR. During blind passage in duck embryo fibroblast cultures, the initial LTR in the rMd5-RM1-LTR was also duplicated, with LTRs appearing in both IRS and TRS regions of the MDV genome. The inserted LTR sequences and transcripts associated with the MDV open reading frames MDV085, MDV086, SORF2, US1, and US10 were molecularly characterized. The parental Md5 BAC contains a family of transcripts of 3, 2, and 1 kb that all terminate at the end of the US10 gene. The rMd5-RM1-LTR and RM1 viruses both express an additional 4 kb transcript that originates in the LTR and also terminates after US10. Collectively, the data suggest that our engineered rMd5-RM1-LTR virus very closely resembles the RM1 virus in its structure and transcription patterns.

  11. Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering in Saccharomyces cerevisiae.

    PubMed

    Sasano, Yu; Yamagishi, Kazuo; Tanikawa, Marie; Nakazawa, Toshimasa; Sugiyama, Minetaka; Kaneko, Yoshinobu; Harashima, Satoshi

    2015-05-01

    Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected-a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.

  12. HIV gene expression from intact proviruses positioned in bacterial artificial chromosomes at integration sites previously identified in latently infected T cells

    SciTech Connect

    Eipers, Peter G.; Salazar-Gonzalez, Jesus F.; Morrow, Casey D.

    2011-02-05

    HIV integration predominantly occurs in introns of transcriptionally active genes. To study the impact of the integration site on HIV gene expression, a complete HIV-1 provirus (with GFP as a fusion with Nef) was inserted into bacterial artificial chromosomes (BACs) at three sites previously identified in latent T cells of patients: topoisomerase II (Top2A), DNA methyltransferase 1 (DNMT1), or basic leucine transcription factor 2 (BACH2). Transfection of BAC-HIV into 293 T cells resulted in a fourfold difference in production of infectious HIV-1. Cell lines were established that contained BAC-Top2A, BAC-DNMT1, or BAC-BACH2, but only BAC-DNMT1 spontaneously produced virus, albeit at a low level. Stimulation with TNF-{alpha} resulted in virus production from four of five BAC-Top2A and all BAC-DNMT1 cell lines, but not from the BAC-BACH2 lines. The results of these studies highlight differences between integration sites identified in latent T cells to support virus production and reactivation from latency.

  13. Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

    SciTech Connect

    Somboonthum, Pranee; Koshizuka, Tetsuo; Okamoto, Shigefumi; Matsuura, Masaaki; Gomi, Yasuyuki; Takahashi, Michiaki; Yamanishi, Koichi; Mori, Yasuko

    2010-06-20

    Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZ{alpha}-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cells as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.

  14. Transgenic mice containing a 248-kb yeast artificial chromosome carrying the human beta-globin locus display proper developmental control of human globin genes.

    PubMed Central

    Peterson, K R; Clegg, C H; Huxley, C; Josephson, B M; Haugen, H S; Furukawa, T; Stamatoyannopoulos, G

    1993-01-01

    Transgenic mice were generated using a purified 248-kb yeast artificial chromosome (YAC) bearing an intact 82-kb human beta-globin locus and 148 kb of flanking sequence. Seventeen of 148 F0 pups were transgenic. RNase protection analysis of RNA isolated from the blood of 13 gamma- and beta-globin-positive founders showed that only the human beta-globin gene was expressed in the adult founders. Studies of F1 and F2 fetuses demonstrated that the genes of the beta-locus YAC displayed the proper developmental switches in beta-like globin gene expression. Expression of epsilon- and gamma-globin, but not beta-globin, was observed in the yolk sac, there was only minor gamma and mostly beta expression in the 14-day liver, and only beta mRNA in the blood of the adult animals. Structural data showed that the locus was intact. These results indicate that it is now possible to dissect regulatory mechanisms within the context of an entire locus in vivo by using the ability to perform mutagenesis efficiently in yeast via homologous recombination, followed by purification of the altered YAC and its introduction into mice. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8356061

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

    PubMed

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

    2005-12-01

    We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome 4 and 5, respectively, and BAC clones from the KBrH library, we studied conservation and microsynteny corresponding to the Arabidopsis regions in B. rapa ssp. pekinensis. The BAC contigs assembled according to the Arabidopsis homoeologues revealed triplication and rearrangements in the Chinese cabbage. In general, collinearity of genes in the paralogous segments was maintained, but gene contents were highly variable with interstitial losses. We also used representative BAC clones, from the assembled contigs, as probes and hybridized them on mitotic (metaphase) and/or meiotic (leptotene/pachytene/metaphase I) chromosomes of Chinese cabbage using bicolor fluorescence in situ hybridization. The hybridization pattern physically identified the paralogous segments of the Arabidopsis homoeologues on B. rapa ssp. pekinensis chromosomes. The homoeologous segments corresponding to chromosome 4 of Arabidopsis were located on chromosomes 2, 8 and 7, whereas those of chromosome 5 were present on chromosomes 6, 1 and 4 of B. rapa ssp. pekinensis.

  16. Cloning of replication-incompetent herpes simplex viruses as bacterial artificial chromosomes to facilitate development of vectors for gene delivery into differentiated neurons.

    PubMed

    Schmeisser, Falko; Weir, Jerry P

    2006-01-01

    We have previously described the adaptation of a tetracycline-regulated system of gene expression for herpes simplex virus (HSV) vectors and demonstrated that such a system was capable of inducible foreign gene expression in irreversibly differentiated neurons. These studies suggested that such gene delivery vectors would be especially useful for studying the neuron in vitro. Here, we describe the cloning of a replication-incompetent HSV vector as a bacterial artificial chromosome (BAC) to facilitate vector construction. Using prokaryotic genetic techniques for allele replacement, we demonstrate the ease of manipulation of the BAC-containing vector, including the construction of vector mutations for which there is no simple phenotypic selection. Such constructions include the insertion of a tetracycline-regulated gene cassette into the UL41 gene for regulated gene expression and the mutation of the UL48 gene to reduce vector toxicity. In addition, HSV vectors cloned as BACs can be sequentially modified to make multiple changes to the vector platform. Finally, using the BAC system, we constructed an HSV vector that expressed an inducible human superoxide dismutase-1 (SOD1) gene for delivery into differentiated human NT-neurons (cells of the human embryonal carcinoma cell line NT2, which differentiate irreversibly into postmitotic neuron-like cells after treatment with retinoic acid). The results indicated that there is appreciable expression of SOD1 from this HSV vector in the presence of doxycycline and that vector-expressed SOD1 interacts with endogenous SOD1. Thus, the BAC system provides a practicable platform for construction and manipulation of HSV vectors that are suitable for gene delivery into postmitotic neurons in vitro.

  17. Insertion of reticuloendotheliosis virus long terminal repeat into a bacterial artificial chromosome clone of a very virulent Marek's disease virus alters its pathogenicity.

    PubMed

    Mays, Jody K; Silva, Robert F; Kim, Taejoong; Fadly, Aly

    2012-01-01

    Co-cultivation of the JM/102W strain of Marek's disease virus (MDV) with reticuloendotheliosis virus (REV) resulted in the generation of a recombinant MDV containing the REV long terminal repeat (LTR) named the RM1 strain of MDV, a strain that was highly attenuated for oncogenicity but induced severe bursal and thymic atrophy. We hypothesize that the phenotypic changes were solely due to the LTR insertion. Furthermore, we hypothesize that insertion of REV LTR into an analogous location in a different MDV would result in a similar phenotypic change. To test these hypotheses, we inserted the REV LTR into a bacterial artificial chromosome (BAC) clone of a very virulent strain of MDV, Md5, and designated the virus rMd5-RM1-LTR. The rMd5-RM1-LTR virus and the rMd5 virus were passaged in duck embryo fibroblast cells for up to 40 passages before pathogenicity studies. Susceptible chickens were inoculated intra-abdominally at hatch with the viruses rMd5-RM1-LTR, rMd5 BAC parental virus, wild-type strain Md5, or strain RM1 of MDV. The rMd5-RM1-LTR virus was attenuated at cell culture passage 40, whereas the rMd5 BAC without RM1 LTR retained its pathogenicity at cell culture passage 40. Using polymerase chain analysis, the RM1 LTR insert was detected in MDV isolated from buffy coat cells collected from chickens inoculated with rMd5-RM1-LTR, but only at 1 week post inoculation. The data suggest that the presence of the RM1 LTR insert within MDV genome for 1 week post inoculation with virus at hatch is sufficient to cause a reduction in pathogenicity of strain Md5 of MDV.

  18. The isolation of a human Ig V lambda gene from a recombinant library of chromosome 22 and estimation of its copy number.

    PubMed Central

    Anderson, M L; Szajnert, M F; Kaplan, J C; McColl, L; Young, B D

    1984-01-01

    We report the first isolation and characterisation of a human Ig V lambda gene. The gene was isolated from a recombinant phage library of human chromosome 22 using a mouse Ig lambda cDNA as probe. DNA sequence analysis predicts a short leader peptide interrupted by an intron of 88 nucleotides, and a mature polypeptide of 96-97 amino acids which shares 61% homology with mouse V lambda I chains. Comparison with the amino acid sequence of known human lambda chains of all six subgroups shows agreement at 22/25 low variance positions. However the maximum homology with human chains is 49%, so we conclude that this sequence represents a new IgV lambda subgroup. The coding region is followed by the conserved heptamer, CACAGTG, and nonamer, ACATAAACC, sequences which have been implicated in V-J segment recombination. This gene has the hallmarks of an active V lambda gene including recently identified transcriptional controlling sequences. Probing genomic DNA with the subcloned V lambda gene detects a family of about 10 cross hybridizing members at low stringency and 2 at high stringency. There is limited polymorphism of the V lambda locus. Images PMID:6091030

  19. De Novo Designed Proteins from a Library of Artificial Sequences Function in Escherichia Coli and Enable Cell Growth

    PubMed Central

    Fisher, Michael A.; McKinley, Kara L.; Bradley, Luke H.; Viola, Sara R.; Hecht, Michael H.

    2011-01-01

    A central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >106 de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains – ΔserB, ΔgltA, ΔilvA, and Δfes – were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo. PMID:21245923

  20. Mapping and ordered cloning of the human X chromosome

    SciTech Connect

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  1. [Developing a physical map of human chromosome 22]. Progress report

    SciTech Connect

    Simon, M.I.

    1991-12-31

    We have developed bacterial F-factor based systems for cloning large fragments of human DNA in E. coli. In addition to large size, these systems are capable of maintaining human DNA with a high degree of stability. The cosmid size clones are called Fosmids and the clones containing larger inserts (100--200 kb) are called bacterial artificial chromosomes (BACs). The ultimate test of the effectiveness of cloning and mapping technology is the degree to which it can be efficiently applied to solve complex mapping problems. We, therefore, plan to use the large fragment cloning procedure as well as a variety of other approaches to generate a complete map of overlapping clones corresponding to human chromosome 22. We have thus far prepared two human chromosome 22 specific Fosmid libraries and we are in the process of constructing a chromosome 22 specific BAC library composed of fragments larger than 100 kb. We will further optimize the technology so that libraries of fragments larger than 200 kb can be readily prepared.

  2. (Developing a physical map of human chromosome 22)

    SciTech Connect

    Simon, M.I.

    1991-01-01

    We have developed bacterial F-factor based systems for cloning large fragments of human DNA in E. coli. In addition to large size, these systems are capable of maintaining human DNA with a high degree of stability. The cosmid size clones are called Fosmids and the clones containing larger inserts (100--200 kb) are called bacterial artificial chromosomes (BACs). The ultimate test of the effectiveness of cloning and mapping technology is the degree to which it can be efficiently applied to solve complex mapping problems. We, therefore, plan to use the large fragment cloning procedure as well as a variety of other approaches to generate a complete map of overlapping clones corresponding to human chromosome 22. We have thus far prepared two human chromosome 22 specific Fosmid libraries and we are in the process of constructing a chromosome 22 specific BAC library composed of fragments larger than 100 kb. We will further optimize the technology so that libraries of fragments larger than 200 kb can be readily prepared.

  3. Isolation of chromosome-specific DNA sequences from an Alu polymerase chain reaction library to define the breakpoint in a patient with a constitutional translocation t(1;13) (q22;q12) and ganglioneuroblastoma.

    PubMed

    Michalski, A J; Cotter, F E; Cowell, J K

    1992-08-01

    We describe the cytogenetic and molecular characterization of a t(1;13)(q22;q12) constitutional rearrangement occurring in a patient with a relatively benign form of neuroblastoma, called ganglioneuroblastoma. Somatic cell hybrids were generated between mouse 3T3 cells and a lymphoblastoid cell line from this patient, D.G. One isolated subclone, DGF27C11, contained the derivative chromosome, 1pter-q22::13q12-qter, but no other material from either chromosome 1 or 13. Using available DNA probes the 13 breakpoint was assigned proximal to all reported markers. In order to generate flanking markers to define this translocation further, an Alu polymerase chain reaction library was constructed from a somatic cell hybrid containing only the proximal, 13pter-13q14, region of chromosome 13. Seven unique sequences have been isolated from the library, three of which lie below and four of which lie above the 13q12 breakpoint. More precise mapping of the distal markers was achieved using a panel of somatic cell hybrids with overlapping deletions of chromosome 13. The paucity of probes in the 1q22 region has made a precise assignment of this breakpoint difficult, however it has been shown to lie distal to c-SKI and proximal to APOA2. This refined characterization of the breakpoint is a prerequisite for its cloning, which may yield genes important in the pathogenesis of ganglioneuroblastoma.

  4. Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

    SciTech Connect

    Antonarakis, S.E.

    1991-09-01

    The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

  5. Possible mechanisms responsible for absence of a retrotransposon family on a plant Y chromosome.

    PubMed

    Kubat, Zdenek; Zluvova, Jitka; Vogel, Ivan; Kovacova, Viera; Cermak, Tomas; Cegan, Radim; Hobza, Roman; Vyskot, Boris; Kejnovsky, Eduard

    2014-04-01

    Some transposable elements (TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat (LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S. latifolia genome but surprisingly absent on the Y chromosome. Bacterial artificial chromosome (BAC) library analysis and fluorescence in situ hybridization (FISH) were used to determine Ogre structure and chromosomal localization. Next generation sequencing (NGS) data were analysed to assess the transcription level and abundance of small RNAs. Methylation of Ogres was determined by bisulphite sequencing. Phylogenetic analysis was used to determine mobilization time and selection forces acting on Ogre elements. We characterized three Ogre families ubiquitous in the S. latifolia genome. One family is nearly absent on the Y chromosome despite all the families having similar structures and spreading mechanisms. We showed that Ogre retrotransposons evolved before sex chromosomes appeared but were mobilized after formation of the Y chromosome. Our data suggest that the absence of one Ogre family on the Y chromosome may be caused by 24-nucleotide (24-nt) small RNA-mediated silencing leading to female-specific spreading. Our findings highlight epigenetic silencing mechanisms as potentially crucial factors in sex-specific spreading of some TEs, but other possible mechanisms are also discussed.

  6. Possible mechanisms responsible for absence of a retrotransposon family on a plant Y chromosome.

    PubMed

    Kubat, Zdenek; Zluvova, Jitka; Vogel, Ivan; Kovacova, Viera; Cermak, Tomas; Cegan, Radim; Hobza, Roman; Vyskot, Boris; Kejnovsky, Eduard

    2014-04-01

    Some transposable elements (TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat (LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S. latifolia genome but surprisingly absent on the Y chromosome. Bacterial artificial chromosome (BAC) library analysis and fluorescence in situ hybridization (FISH) were used to determine Ogre structure and chromosomal localization. Next generation sequencing (NGS) data were analysed to assess the transcription level and abundance of small RNAs. Methylation of Ogres was determined by bisulphite sequencing. Phylogenetic analysis was used to determine mobilization time and selection forces acting on Ogre elements. We characterized three Ogre families ubiquitous in the S. latifolia genome. One family is nearly absent on the Y chromosome despite all the families having similar structures and spreading mechanisms. We showed that Ogre retrotransposons evolved before sex chromosomes appeared but were mobilized after formation of the Y chromosome. Our data suggest that the absence of one Ogre family on the Y chromosome may be caused by 24-nucleotide (24-nt) small RNA-mediated silencing leading to female-specific spreading. Our findings highlight epigenetic silencing mechanisms as potentially crucial factors in sex-specific spreading of some TEs, but other possible mechanisms are also discussed. PMID:24456522

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

    PubMed

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

    2013-08-01

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

  8. A yeast artificial chromosome contig that spans the RB1-D13S31 interval on human chromosome 13 and encompasses the frequently deleted region in B-cell chronic lymphocytic leukemia

    SciTech Connect

    Hawthorn, L.; Roberts, T.; Cowell, J.K.

    1995-12-10

    Abnormalities involving chromosome 13 have been a reported as the only cytogenetic change in B-cell chronic lymphocytic leukemia (BCLL). Deletions are the most common cytogenetic abnormality and always involve 13q14, but when translocations are seen, the consistent breakpoint is always in 13q14. It is now established that deletions, distal to the RB1 gene in 13q14, are invariably associated with these translocations. We have recently described the smallest such deletion from a series of rearrangements from these tumors isolated in somatic cell hybrids, which spans approximately 1 Mb. In this report, we present the results of a series of a chromosome walking experiments using YACs and have been able to span this small deletion, which must contain the gene that is frequently deleted in BCLL. Four probes from 13q14 (RB1-mgg15-D13S25-D13S31) were used to isolate corresponding YACs for each of the markers. The chromosomal location of these YACs was verified using FISH, which also demonstrated their nonchimeric nature. Vectorette end rescue was then used to demonstrate the overlap of the YACs and to isolate new clones to complete the contig. The extremes of the contig were shown to cross the chromosome 13 translocation breakpoints isolated in somatic cell hybrids that carry the derivatives of chromosome 13 involved in the smallest BCLL deletion. This YAC contig covers the entire deletion and will prove a valuable resource to begin isolating genes from this region. In addition, we have isolated YACs corresponding to the RB1 locus, which extends the contig over a 3.8-cM distance on the chromosome. 25 refs., 1 fig., 1 tab.

  9. The Chromosome Microdissection and Microcloning Technique.

    PubMed

    Zhang, Ying-Xin; Deng, Chuan-Liang; Hu, Zan-Min

    2016-01-01

    Chromosome microdissection followed by microcloning is an efficient tool combining cytogenetics and molecular genetics that can be used for the construction of the high density molecular marker linkage map and fine physical map, the generation of probes for chromosome painting, and the localization and cloning of important genes. Here, we describe a modified technique to microdissect a single chromosome, paint individual chromosomes, and construct single-chromosome DNA libraries. PMID:27511173

  10. Amplification of chromosomal DNA in situ

    DOEpatents

    Christian, Allen T.; Coleman, Matthew A.; Tucker, James D.

    2002-01-01

    Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

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

    2016-01-01

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

  13. Chromosomal Conditions

    MedlinePlus

    ... 150 babies is born with a chromosomal condition. Down syndrome is an example of a chromosomal condition. Because ... all pregnant women be offered prenatal tests for Down syndrome and other chromosomal conditions. A screening test is ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  15. Mapping and ordered cloning of the human X chromosome. Progress report, September 1991--November 1992

    SciTech Connect

    Caskey, C.T.; Nelson, D.L.

    1992-12-01

    Progress is reported on gathering X chromosome specific libraries and integrating those with the library produced in this project. Further studies on understanding Fragile X Syndrome and other hereditary diseases related to the X chromosome are described. (DT)

  16. Human chromosome 22.

    PubMed Central

    Kaplan, J C; Aurias, A; Julier, C; Prieur, M; Szajnert, M F

    1987-01-01

    The acrocentric chromosome 22, one of the shortest human chromosomes, carries about 52 000 kb of DNA. The short arm is made up essentially of heterochromatin and, as in other acrocentric chromosomes, it contains ribosomal RNA genes. Ten identified genes have been assigned to the long arm, of which four have already been cloned and documented (the cluster of lambda immunoglobulin genes, myoglobin, the proto-oncogene c-sis, bcr). In addition, about 10 anonymous DNA segments have been cloned from chromosome 22 specific DNA libraries. About a dozen diseases, including at least four different malignancies, are related to an inherited or acquired pathology of chromosome 22. They have been characterised at the phenotypic or chromosome level or both. In chronic myelogenous leukaemia, with the Ph1 chromosome, and Burkitt's lymphoma, with the t(8;22) variant translocation, the molecular pathology is being studied at the DNA level, bridging for the first time the gap between cytogenetics and molecular genetics. PMID:3550088

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

    PubMed

    Gindullis, F; Dechyeva, D; Schmidt, T

    2001-10-01

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

  18. Genetic and physical mapping of the bovine X chromosome.

    PubMed

    Yeh, C C; Taylor, J F; Gallagher, D S; Sanders, J O; Turner, J W; Davis, S K

    1996-03-01

    Three hundred eighty reciprocal backcross and F(2) full sib progeny from 33 families produced by embryo transfer from 77 Angus (Bos taurus), Brahman (Bos indicus), and F1 parents and grandparents were used to construct genetic maps of the bovine X and Y chromosomes. Ml individuals were scored for 15 microsatellite loci, with an average of 608 informative meioses per locus. The length of the bovine X chromosome genetic map was 118.7 cM (female only) and of the pseudoautosomal region was 13.0 cM (male only). The 15-marker framework map in Kosambi centimorgans is [BM6017-6.1 -TGLA89-35.8-TEXAN13-3.4-TGLA128-1.3 -BM2713 -21.1 -BM4604-2.4-BR215 - 12.9-TGLA68-10.0-BM4321 - 1.0-HEL14-4.9-TGLA15-2.3-INRA12O- 12.5-TGLA325- 1.6-MAF45-3.2-INRA3O], with an average interval of 7.91 cM. Clones containing pseudoautosomal or sex-linked microsatellites were isolated from a bovine bacterial artificial chromosome library and were physically mapped to bovine metaphase chromosomes by fluorescence in situ hybridization to orient the X and Y chromosome maps. BAC57, containing the pseudoautosomal microsatellite INRA3O, mapped to the distal end of the long arm of the X chromosome at q42-ter and to the short arm of the Y chromosome at p13-ter. This confirms the published assignment of this region to Ypl2-ter, but challenges the published assignment of Xpl4-ter and thus reorients the X chromosome physical map. BAC2O4, containing the X-linked microsatellite BM4604, mapped to the middle of the long arm of the X chromosome at q26-q31. The position of the physically mapped markers indicates either a lack of microsatellite markers for a large (30 to 50 cM) region of the short arm of the X chromosome or heterogeneity of recombination along the X chromosome. PMID:8833151

  19. Combination of Multiple Spectral Libraries Improves the Current Search Methods Used to Identify Missing Proteins in the Chromosome-Centric Human Proteome Project.

    PubMed

    Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Kim, Kwang-Youl; Kwon, Kyung-Hoon; Yoo, Jong Shin; Omenn, Gilbert S; Baker, Mark S; Hancock, William S; Paik, Young-Ki

    2015-12-01

    Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ~15% of all proteins, have no or very weak proteomic evidence and are still missing. Missing proteins may be present in rare samples in very low abundance or be only temporarily expressed, causing problems in their detection and protein profiling. In particular, some technical limitations cause missing proteins to remain unassigned. For example, current mass spectrometry techniques have high limits and error rates for the detection of complex biological samples. An insufficient proteome coverage in a reference sequence database and spectral library also raises major issues. Thus, the development of a better strategy that results in greater sensitivity and accuracy in the search for missing proteins is necessary. To this end, we used a new strategy, which combines a reference spectral library search and a simulated spectral library search, to identify missing proteins. We built the human iRefSPL, which contains the original human reference spectral library and additional peptide sequence-spectrum match entries from other species. We also constructed the human simSPL, which contains the simulated spectra of 173 907 human tryptic peptides determined by MassAnalyzer (version 2.3.1). To prove the enhanced analytical performance of the combination of the human iRefSPL and simSPL methods for the identification of missing proteins, we attempted to reanalyze the placental tissue data set (PXD000754). The data from each experiment were analyzed using PeptideProphet, and the results were combined using iProphet. For the quality control, we applied the class-specific false-discovery rate filtering method. All of the results were filtered at a false-discovery rate of <1% at the peptide and protein levels. The quality-controlled results were then cross-checked with the neXtProt DB (2014-09-19 release). The two

  20. A Dense Single-Nucleotide Polymorphism-Based Genetic Linkage Map of Grapevine (Vitis vinifera L.) Anchoring Pinot Noir Bacterial Artificial Chromosome Contigs

    PubMed Central

    Troggio, Michela; Malacarne, Giulia; Coppola, Giuseppina; Segala, Cinzia; Cartwright, Dustin A.; Pindo, Massimo; Stefanini, Marco; Mank, Rolf; Moroldo, Marco; Morgante, Michele; Grando, M. Stella; Velasco, Riccardo

    2007-01-01

    The construction of a dense genetic map for Vitis vinifera and its anchoring to a BAC-based physical map is described: it includes 994 loci mapped onto 19 linkage groups, corresponding to the basic chromosome number of Vitis. Spanning 1245 cM with an average distance of 1.3 cM between adjacent markers, the map was generated from the segregation of 483 single-nucleotide polymorphism (SNP)-based genetic markers, 132 simple sequence repeats (SSRs), and 379 AFLP markers in a mapping population of 94 F1 individuals derived from a V. vinifera cross of the cultivars Syrah and Pinot Noir. Of these markers, 623 were anchored to 367 contigs that are included in a physical map produced from the same clone of Pinot Noir and covering 352 Mbp. On the basis of contigs containing two or more genetically mapped markers, region-dependent estimations of physical and recombinational distances are presented. The markers used in this study include 118 SSRs common to an integrated map derived from five segregating populations of V. vinifera. The positions of these SSR markers in the two maps are conserved across all Vitis linkage groups. The addition of SNP-based markers introduces polymorphisms that are easy to database, are useful for evolutionary studies, and significantly increase the density of the map. The map provides the most comprehensive view of the Vitis genome reported to date and will be relevant for future studies on structural and functional genomics and genetic improvement. PMID:17603124

  1. Ribosomal protein gene mapping and human chromosomal disorders

    SciTech Connect

    Kenmochi, N.; Goodman, N.; Page, D.C.

    1994-09-01

    In Drosophila, the Minute phenotype (reduced body size, diminished viability and fertility, and short, thin bristles) results from heterozygous deficiencies (deletions) at any one of 50 loci scattered about the genome. A handful of these Minute loci have been molecularly characterized, and all have been found to encode ribosomal proteins. Thus, the Minute phenotype appears to result from reduced protein synthetic capacity in flies with one rather than two copies of a given ribosomal protein (rp) gene. We are pursuing the possibility that similar reductions in protein synthetic capacity--again resulting from rp gene deficiencies--might underlie phenotypes associated with certain chromosomal disorders in humans. We and our colleagues have reported findings consistent with a role for RPS4 deficiency in the etiology of certain features of Turner syndrome, a complex human disorder classically associated with an XO karyotype. We are intrigued by the possibility that deficiencies of other human rp genes might cause phenotypic abnormalities similar to those seen in Turner syndrome--just as deficiencies of any of a number of Drosophila rp genes cause the Minute phenotype. We must first learn the chromosomal map position of each of the estimated 83 human rp genes. The task of mapping the functional (intron-containing) rp genes is complicated by the existence of processed pseudogenes elsewhere in the genome. To date, we have assigned (or confirmed the previous assignment of) 38 rp genes to individual human chromosomes by PCR analysis of human-rodent somatic cell hybrids containing subsets of human chromosomes, with all but four chromosomes carrying at least one rp gene. We have also identified more than 100 large-insert human YAC (yeast artificial chromosome) clones that contain individual rp genes. Such screening of YAC libraries will result in precise positioning of the rp genes on the emerging physical map of the human genome.

  2. Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

    PubMed

    Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

    2015-12-01

    A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.

  3. In Pursuit of Artificial Intelligence.

    ERIC Educational Resources Information Center

    Watstein, Sarah; Kesselman, Martin

    1986-01-01

    Defines artificial intelligence and reviews current research in natural language processing, expert systems, and robotics and sensory systems. Discussion covers current commercial applications of artificial intelligence and projections of uses and limitations in library technical and public services, e.g., in cataloging and online information and…

  4. Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle

    SciTech Connect

    Masumoto, Hiroshi; Sugimoto, Kenji; Okazaki, Tuneko )

    1989-03-01

    In this study, the authors have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenicity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library they found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, they have found perfect overlapping of the alphoid DNA sites with the centromere antigen in both metaphase chromosomes and nuclei at various stages in the cell cycle. They have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.

  5. Chromosome specific repetitive DNA sequences

    DOEpatents

    Moyzis, Robert K.; Meyne, Julianne

    1991-01-01

    A method is provided for determining specific nucleotide sequences useful in forming a probe which can identify specific chromosomes, preferably through in situ hybridization within the cell itself. In one embodiment, chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family me This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).

  6. Losing Libraries, Saving Libraries

    ERIC Educational Resources Information Center

    Miller, Rebecca

    2010-01-01

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

  7. Constructing chromosome- and region-specific cosmid maps of the human genome.

    PubMed

    Carrano, A V; de Jong, P J; Branscomb, E; Slezak, T; Watkins, B W

    1989-01-01

    A chromosome-specific ordered set of cosmids would be a significant contribution toward understanding human chromosome structure and function. We are developing two parallel approaches for creating an ordered cosmid library of human chromosome 19 and other selected subregions of the human genome. The "bottom up" approach is used to establish sets of overlapping cosmids as islands or "contigs" along the chromosome, while the "top down" approach, using pulsed-field gel electrophoresis and yeast cloning, will establish a large-fragment map and close the inevitable gaps remaining from the "bottom up" approach. Source DNA consists of a single homolog of chromosome 19 from a hamster--human hybrid cell and human fragments cloned in yeast artificial chromosomes. We have constructed cosmid libraries in a vector that facilitates cloning small amounts of DNA, allows transcription of the insert termini, and contains unique sites for partial-digest mapping. Computer simulations of cosmid contig building suggest that near-optimal efficiency can be achieved with high-density restriction fragment digest schemes that can detect 20-30% overlap between cosmids. We developed the chemistry and data analysis tools to compare the ordering efficiencies of several cosmid restriction digest fingerprinting strategies. Restriction fragments from a four-cutter digest are labeled with a fluorochrome, separated by polyacrylamide gel electrophoresis, and detected after laser excitation as they traverse a fixed point in the gel. We have also developed the software to rapidly process the output signal to define and analyze the fragment peaks. Up to three cosmids (or three different digests of the same cosmid) plus a size standard are analyzed simultaneously in a single gel lane.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2698823

  8. Chromosomal Flexibility

    ERIC Educational Resources Information Center

    Journal of College Science Teaching, 2005

    2005-01-01

    Scientists have shown that a genetic element on one chromosome may direct gene activity on another. Howard Hughes Medical Institute (HHMI) researchers report that a multitasking master-control region appears to over-see both a set of its own genes and a related gene on a nearby chromosome. The findings reinforce the growing importance of location…

  9. Cloning of the Epstein-Barr virus-related rhesus lymphocryptovirus as a bacterial artificial chromosome: a loss-of-function mutation of the rhBARF1 immune evasion gene.

    PubMed

    Ohashi, Makoto; Orlova, Nina; Quink, Carol; Wang, Fred

    2011-02-01

    Rhesus macaques are naturally infected with a gammaherpesvirus which is in the same lymphocryptovirus (LCV) genus as and closely related to Epstein-Barr virus (EBV). The rhesus macaque LCV (rhLCV) contains a repertoire of genes identical to that of EBV, and experimental rhLCV infection of naive rhesus macaques accurately models acute and persistent EBV infection of humans. We cloned the LCL8664 rhLCV strain as a bacterial artificial chromosome to create recombinant rhLCV for investigation in this animal model system. A recombinant rhLCV (clone 16 rhLCV) carrying a mutation in the putative immune evasion gene rhBARF1 was created along with a rescued wild-type (rWT) rhLCV in which the rhBARF1 open reading frame (ORF) was repaired. The rWT rhLCV molecular clone demonstrated viral replication and B-cell immortalization properties comparable to those of the naturally derived LCL8664 rhLCV. Qualitatively, clone 16 rhLCV carrying a mutated rhBARF1 was competent for viral replication and B-cell immortalization, but quantitative assays showed that clone 16 rhLCV immortalized B cells less efficiently than LCL8664 and rWT rhLCV. Functional studies showed that rhBARF1 could block CSF-1 cytokine signaling as well as EBV BARF1, whereas the truncated rhBARF1 from clone 16 rhLCV was a loss-of-function mutant. These recombinant rhLCV can be used in the rhesus macaque animal model system to better understand how a putative viral immune evasion gene contributes to the pathogenesis of acute and persistent EBV infection. The development of a genetic system for making recombinant rhLCV constitutes a major advance in the study of EBV pathogenesis in the rhesus macaque animal model.

  10. Meiotic Crossing over between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast

    PubMed Central

    Jinks-Robertson, S.; Sayeed, S.; Murphy, T.

    1997-01-01

    Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover events have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura(+) random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation. PMID:9136001

  11. Chromosome Abnormalities

    MedlinePlus

    ... decade, newer techniques have been developed that allow scientists and doctors to screen for chromosomal abnormalities without using a microscope. These newer methods compare the patient's DNA to a normal DNA ...

  12. Creating Library Spaces: Libraries 2040.

    ERIC Educational Resources Information Center

    Bruijnzeels, Rob

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

  13. Original Research: Generation of non-deletional hereditary persistence of fetal hemoglobin β-globin locus yeast artificial chromosome transgenic mouse models: -175 Black HPFH and -195 Brazilian HPFH

    PubMed Central

    Braghini, Carolina A; Costa, Flavia C; Fedosyuk, Halyna; Neades, Renee Y; Novikova, Lesya V; Parker, Matthew P; Winefield, Robert D

    2016-01-01

    Fetal hemoglobin is a major genetic modifier of the phenotypic heterogeneity in patients with sickle cell disease and certain β-thalassemias. Normal levels of fetal hemoglobin postnatally are approximately 1% of total hemoglobin. Patients who have hereditary persistence of fetal hemoglobin, characterized by elevated synthesis of γ-globin in adulthood, show reduced disease pathophysiology. Hereditary persistence of fetal hemoglobin is caused by β-globin locus deletions (deletional hereditary persistence of fetal hemoglobin) or γ-globin gene promoter point mutations (non-deletional hereditary persistence of fetal hemoglobin). Current research has focused on elucidating the pathways involved in the maintenance/reactivation of γ-globin in adult life. To better understand these pathways, we generated new β-globin locus yeast artificial chromosome transgenic mice bearing the Aγ-globin -175 T > C or -195 C > G hereditary persistence of fetal hemoglobin mutations to model naturally occurring hereditary persistence of fetal hemoglobin. Adult -175 and -195 mutant β-YAC mice displayed a hereditary persistence of fetal hemoglobin phenotype, as measured at the mRNA and protein levels. The molecular basis for these phenotypes was examined by chromatin immunoprecipitation of transcription factor/co-factor binding, including YY1, PAX1, TAL1, LMO2, and LDB1. In -175 HPFH versus wild-type samples, the occupancy of LMO2, TAL1 and LDB1 proteins was enriched in HPFH mice (5.8-fold, 5.2-fold and 2.7-fold, respectively), a result that concurs with a recent study in cell lines showing that these proteins form a complex with GATA-1 to mediate long-range interactions between the locus control region and the Aγ-globin gene. Both hereditary persistence of fetal hemoglobin mutations result in a gain of Aγ-globin activation, in contrast to other hereditary persistence of fetal hemoglobin mutations that result in a loss of repression. The mice provide additional tools to study

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

    PubMed

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

    2011-01-01

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

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

    PubMed

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

    2011-01-01

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

  16. Cytogenetical anchoring of sheep linkage map and syntenic groups using a sheep BAC library.

    PubMed

    Tabet-Aoul, K; Oustry-Vaiman, A; Vaiman, D; Saïdi-Mehtar, N; Cribiu, E P; Lantier, F

    2000-01-01

    In order to simultaneously integrate linkage and syntenic groups to the ovine chromosomal map, a sheep bacterial artificial chromosome (BAC) library was screened with previously assigned microsatellites using a sheep-hamster hybrid panel and genetic linkage. Thirty-three BACs were obtained, fluorescently labelled and hybridised on sheep-goat hybrid metaphases (2n = 57). This study allowed us, (i), to anchor all linkage groups on sheep chromosomes, (ii), to give information on the probable position of the centromere on the linkage map for the centromeric chromosomes, (iii), to contradict the previous orientation of the ovine X linkage group by the mapping of BMS1008 on OARXq38. Concerning our somatic cell hybrid panel, this study resulted in the assignment of all the previously unassigned groups to ovine chromosomes and a complete characterisation of the hybrid panel. In addition, since hybridisations were performed on a sheep-goat hybrid, new marker/anchoring points were added to the caprine cytogenetic map. PMID:14736389

  17. Artificial Intelligence.

    ERIC Educational Resources Information Center

    Waltz, David L.

    1982-01-01

    Describes kinds of results achieved by computer programs in artificial intelligence. Topics discussed include heuristic searches, artificial intelligence/psychology, planning program, backward chaining, learning (focusing on Winograd's blocks to explore learning strategies), concept learning, constraint propagation, language understanding…

  18. Artificial Limbs

    MedlinePlus

    ... you are missing an arm or leg, an artificial limb can sometimes replace it. The device, which is ... activities such as walking, eating, or dressing. Some artificial limbs let you function nearly as well as before.

  19. Generation of a human chromosome 18-specific YAC clone collection and mapping of 55 unique YACs by FISH and fingerprinting

    SciTech Connect

    Chang, E.; Giacalone, J.; Welch, S.; Luna, J.; Francke, U. )

    1993-08-01

    A yeast artificial chromosome (YAC) library was constructed from a somatic cell hybrid line in which the human chromosome content had been reduced by repeated subcloning to one or two copies of chromosome 18. Screening of 4700 primary yeast transformants generated 74 clones containing a YAC with a human DNA insert averaging 190 kb in size. The human YACs were localized to subregions of chromosome 18 by in situ hybridization of biotin-labeled Alu-PCR products obtained using total yeast DNA as a template. Comparisons of interspersed repetitive sequence-PCR and restriction fragment fingerprint patterns identified five sets of identical and three sets of overlapping YACs. Dual-label fluorescence in situ hybridization interphase mapping was used to determine the order of some nonoverlapping YACs. STS (sequence-tagged site) content mapping was carried out with PCR primers for 56 chromosome 18-specific markers. The identification of YACs containing four known genes - encoding the pituitary adenylate cyclase activating polypeptide (PA-CAP), the myelin basic protein (MBP), ferrochelatase (FECH), and SSAV1, an endogenous retroviral element related to the SSAV virus - provides a precise cytological map position for the respective loci. The final collection of 55 randomly isolated, unique, and regionally localized YACs (D18S107-D18S161) is distributed over the entire chromosome and collectively covers approximately 12 Mb, i.e., 16% of the estimated 77 Mb of DNA in euchromatin of chromosome 18. These YACs provide reagents for isolation of genes in these regions and represent nucleation points for the generation of STS to increase coverage of the chromosome by YAC contigs.

  20. Chromosome and cell genetics

    SciTech Connect

    Sharma, A.K.; Sharma, A.

    1985-01-01

    This book contains 11 chapters. Some of the titles are: Chromosomes in differentiation; Chromosome axis; Nuclear and organelle split genes; Chemical mutagenesis; and Chromosome architecture and additional elements.

  1. Inexpensive multiplexed library preparation for megabase-sized genomes.

    PubMed

    Baym, Michael; Kryazhimskiy, Sergey; Lieberman, Tami D; Chung, Hattie; Desai, Michael M; Kishony, Roy

    2015-01-01

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

  2. Inexpensive Multiplexed Library Preparation for Megabase-Sized Genomes

    PubMed Central

    Desai, Michael M.; Kishony, Roy

    2015-01-01

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

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

    SciTech Connect

    Jang Songhun; Zhou Fang; Xia Laixin; Zhao Wei; Cheng Hanhua . E-mail: hhcheng@whu.edu.cn; Zhou Rongjia . E-mail: rjzhou@whu.edu.cn

    2006-09-22

    A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes.

  4. Artificial Intelligence.

    ERIC Educational Resources Information Center

    Information Technology Quarterly, 1985

    1985-01-01

    This issue of "Information Technology Quarterly" is devoted to the theme of "Artificial Intelligence." It contains two major articles: (1) Artificial Intelligence and Law" (D. Peter O'Neill and George D. Wood); (2) "Artificial Intelligence: A Long and Winding Road" (John J. Simon, Jr.). In addition, it contains two sidebars: (1) "Calculating and…

  5. Human genome libraries. Final progress report, February 1, 1994--August 31, 1997

    SciTech Connect

    Kao, Fa-Ten

    1998-01-01

    The goal of this program is to use a novel technology of chromosome microdissection and microcloning to construct chromosome region-specific libraries as resources for various human genome program studies. Region specific libraries have been constructed for the entire human chromosomes 2 and 18.

  6. Chromosome Microarray.

    PubMed

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed. PMID:27276104

  7. Chromosome Analysis

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Perceptive Scientific Instruments, Inc., provides the foundation for the Powergene line of chromosome analysis and molecular genetic instrumentation. This product employs image processing technology from NASA's Jet Propulsion Laboratory and image enhancement techniques from Johnson Space Center. Originally developed to send pictures back to earth from space probes, digital imaging techniques have been developed and refined for use in a variety of medical applications, including diagnosis of disease.

  8. Isolation of high molecular weight DNA from marine sponge bacteria for BAC library construction.

    PubMed

    Ouyang, Yongchang; Dai, Shikun; Xie, Lianwu; Ravi Kumar, M S; Sun, Wei; Sun, Huimin; Tang, Danling; Li, Xiang

    2010-06-01

    Metagenomics is a powerful tool for mining the genetic repositories from environmental microorganisms. Bacteria associated with marine sponges (phylum Porifera) are rich sources of biologically active natural products. However, to date, few compounds are discovered from the sponge metagenomic libraries, and the main reason might be the difficulties in recovery of high molecular weight (HMW) DNA from sponge symbionts to construct large insert libraries. Here, we describe a method to recover HMW bacterial DNA from diverse sponges with high quality for bacterial artificial chromosome (BAC) library construction. Microorganisms concentrated from sponges by differential centrifugation were embedded in agarose plugs to lyse out the HMW DNA for recovery. DNA fragments over 436 kb size were recovered from three different types of sponges, Halichondria sp., Haliclona sp., and Xestospongia sp. To evaluate the recovered DNA quality, the diversity of bacterial DNA comprised in the HMW DNA derived from sponge Halichondria sp. was analyzed, and this HMW DNA sample was also cloned into a shuttle BAC vector between Escherichia coli and Streptomyces sp. The results showed that more than five types of bacterial DNA, i.e., Proteobacteria, Nitrospirae, Cyanobacteria, Planctomycetes, and unidentified bacteria, had been recovered by this method, and an average 100 kb size insert DNA in a constructed BAC library demonstrated that the recovered HMW DNA is suitable for metagenomic library construction.

  9. Identification and mapping of expressed genes, simple sequence repeats and transposable elements in centromeric regions of rice chromosomes.

    PubMed

    Mizuno, Hiroshi; Ito, Kazue; Wu, Jianzhong; Tanaka, Tsuyoshi; Kanamori, Hiroyuki; Katayose, Yuichi; Sasaki, Takuji; Matsumoto, Takashi

    2006-12-31

    The genomic sequences derived from rice centromeric regions were analyzed to facilitate the comprehensive understanding of the rice genome. A rice centromere-specific satellite sequence, RCS2/TrsD/CentO, was used to screen P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) genomic libraries derived from Oryza sativa L. ssp. japonica cultivar Nipponbare. Physical maps of the centromeric regions were constructed by DNA fingerprinting methods and the aligned clones were analyzed by end sequencing. BLAST analysis revealed the composition of genes, centromeric satellites and other repetitive elements, such as RIRE7/CRR, RIRE8, Squiq, Anaconda, CACTA and miniature inverted-repeat transposable elements. Fiber-fluorescent in situ hybridization analysis also indicated the presence of distinct clusters of RCS2/TrsD/CentO satellite interspersed with other elements, instead of a long homogeneous region. Several expressed genes, sequences representative of ancestral organellar insertions, relatively long simple sequence repeats (SSRs), and sequences corresponding to 5S and 45S ribosomal RNA genes were also identified. Thirty-one gene sequences showed high-similarity to rice full-length cDNA sequences that had not been matched to the published rice genome sequence in silico. These results suggest the presence of expressed genes within and around the clusters of RCS2/TrsD/CentO satellites in unsequenced centromeric regions of the rice chromosomes.

  10. FISH probes for mouse chromosome identification

    SciTech Connect

    Shi, Yu-Ping; Mohapatra, G.; Hanahan, D.; Miller, J.

    1997-10-01

    P1 clones near the telomeres and centromeres of each mouse chromosome except Y have been selected from a mouse genomic library and mapped using fluorescence in situ hybridization (FISH). Each clone was selected to contain a genetically mapped polymorphic DNA sequence as close as possible to the centromere or telomere of a chromosome. The genetic distance from the various P1 clones to the most distal genetically mapped polymorphic sequence ranged from 0 for about half of the clones to 6.7 cM for the probe at the telomere of chromosome 14. The average distance to the most distal or proximal chromosome marker was 1.5 cM. The use of FISH with these probes for mouse chromosome identification during comparative genomic hybridization is illustrated. 17 refs., 2 figs., 2 tabs.

  11. Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements.

    SciTech Connect

    Leem, Sun-Hee; Kouprina, Natalay; Grimwood, Jane; Kim, Jung-Hyun; Mullokandov, Michael; Yoon, Young-Ho; Chae, Ji-Youn; Morgan, Jenna; Lucas, Susan; Richardson, Paul; Detter, Chris; Glavina, Tijana; Rubin, Eddy; Barrett, J. Carl; Larionov, Vladimir

    2003-09-01

    The reported human genome sequence includes about 400 gaps of unknown sequence that were not found in the bacterial artificial chromosome (BAC) and cosmid libraries used for sequencing of the genome. These missing sequences correspond to {approx} 1 percent of euchromatic regions of the human genome. Gap filling is a laborious process because it relies on analysis of random clones of numerous genomic BAC or cosmid libraries. In this work we demonstrate that closing the gaps can be accelerated by a selective recombinational capture of missing chromosomal segments in yeast. The use of both methodologies allowed us to close the four remaining gaps on the human chromosome 19. Analysis of the gap sequences revealed that they contain several abnormalities that could result in instability of the sequences in microbe hosts, including large blocks of micro- and minisatellites and a high density of Alu repeats. Sequencing of the gap regions, in both BAC and YAC forms, allowed us to generate a complete sequence of four genes, including the neuronal cell signaling gene SCK1/SLI. The SCK1/SLI gene contains a record number of minisatellites, most of which are polymorphic and transmitted through meiosis following a Mendelian inheritance. In conclusion, the use of the alternative recombinational cloning system in yeast may greatly accelerate work on closing the remaining gaps in the human genome (as well as in other complex genomes) to achieve the goal of annotation of all human genes.

  12. Artificial intelligence

    SciTech Connect

    Firschein, O.

    1984-01-01

    This book presents papers on artificial intelligence. Topics considered include knowledge engineering, expert systems, applications of artificial intelligence to scientific reasoning, planning and problem solving, error recovery in robots through failure reason analysis, programming languages, natural language, speech recognition, map-guided interpretation of remotely-sensed imagery, and image understanding architectures.

  13. Artificial Intelligence.

    ERIC Educational Resources Information Center

    Thornburg, David D.

    1986-01-01

    Overview of the artificial intelligence (AI) field provides a definition; discusses past research and areas of future research; describes the design, functions, and capabilities of expert systems and the "Turing Test" for machine intelligence; and lists additional sources for information on artificial intelligence. Languages of AI are also briefly…

  14. Mosaic supernumerary ring chromosome 19 identified by comparative genomic hybridisation.

    PubMed Central

    Ghaffari, S R; Boyd, E; Connor, J M; Jones, A M; Tolmie, J L

    1998-01-01

    We report the use of comparative genomic hybridisation (CGH) to define the origin of a supernumerary ring chromosome which conventional cytogenetic banding and fluorescence in situ hybridisation (FISH) methods had failed to identify. Targeted FISH using whole chromosome 19 library arm and site specific probes then confirmed the CGH results. This study shows the feasibility of using CGH for the identification of supernumerary marker chromosomes, even in fewer than 50% of cells, where no clinical or cytogenetic clues are present. Images PMID:9783708

  15. Analysis of chromosome conservation in Lemur catta studied by chromosome paints and BAC/PAC probes.

    PubMed

    Cardone, Maria Francesca; Ventura, Mario; Tempesta, Sergio; Rocchi, Mariano; Archidiacono, Nicoletta

    2002-12-01

    A panel of human chromosome painting probes and bacterial and P1 artificial chromosome (BAC/PAC) clones were used in fluorescence in situ hybridization (FISH) experiments to investigate the chromosome conservation of the ring-tailed lemur (Lemur catta, LCA) with respect to human. Whole chromosome paints specific for human chromosomes 7, 9, 11, 13, 14, 17, 18, 20, 21, and X were found to identify a single chromosome or an uninterrupted chromosomal region in LCA. A large set of partial chromosome paints and BAC/PAC probes were then used to refine the characterization of the rearrangements differentiating the two karyotypes. The results were also used to reconstruct the ancestral Lemuridae karyotype. Lemur catta, indeed, can be used as an outgroup, allowing symplesiomorphic (ancestral) rearrangements to be distinguished from apomorphic (derived) rearrangements in lemurs. Some LCA chromosomes are difficult to distinguish morphologically. The 'anchorage' of most LCA chromosomes to specific probes will contribute to the standardization of the karyotype of this species. PMID:12474064

  16. National Libraries: An Analysis

    ERIC Educational Resources Information Center

    Burston, Godfrey

    1973-01-01

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

  17. Results of genetic screening of donors for artificial insemination.

    PubMed

    Czeizel, A; Szentesi, I; Horváth, L

    1983-08-01

    Only 47 of 61 (77%) medical students were selected as sperm donors for artificial insemination. Fourteen were excluded because of family history 1, eye disorders 7, oligospermia 5, permanent structural chromosome aberration 1. PMID:6616950

  18. Library Skills.

    ERIC Educational Resources Information Center

    Paul, Karin; Kuhlthau, Carol C.; Branch, Jennifer L.; Solowan, Diane Galloway; Case, Roland; Abilock, Debbie; Eisenberg, Michael B.; Koechlin, Carol; Zwaan, Sandi; Hughes, Sandra; Low, Ann; Litch, Margaret; Lowry, Cindy; Irvine, Linda; Stimson, Margaret; Schlarb, Irene; Wilson, Janet; Warriner, Emily; Parsons, Les; Luongo-Orlando, Katherine; Hamilton, Donald

    2003-01-01

    Includes 19 articles that address issues related to library skills and Canadian school libraries. Topics include information literacy; inquiry learning; critical thinking and electronic research; collaborative inquiry; information skills and the Big 6 approach to problem solving; student use of online databases; library skills; Internet accuracy;…

  19. Molecular mapping of chromosomes 17 and X

    SciTech Connect

    Barker, D.F.

    1991-01-15

    Progress toward the construction of high density genetic maps of chromosomes 17 and X has been made by isolating and characterizing a relatively large set of polymorphic probes for each chromosome and using these probes to construct genetic maps. We have mapped the same polymorphic probes against a series of chromosome breakpoints on X and 17. The probes could be assigned to over 30 physical intervals on the X chromosome and 7 intervals on 17. In many cases, this process resulted in improved characterization of the relative locations of the breakpoints with respect to each other and the definition of new physical intervals. The strategy for isolation of the polymorphic clones utilized chromosome specific libraries of 1--15 kb segments from each of the two chromosomes. From these libraries, clones were screened for those detecting restriction fragment length polymorphisms. The markers were further characterized, the chromosomal assignments confirmed and in most cases segments of the original probes were subcloned into plasmids to produce probes with improved signal to noise ratios for use in the genetic marker studies. The linkage studies utilize the CEPH reference families and other well-characterized families in our collection which have been used for genetic disease linkage work. Preliminary maps and maps of portions of specific regions of 17 and X are provided. We have nearly completed a map of the 1 megabase Mycoplasma arthritidis genome by applying these techniques to a lambda phage library of its genome. We have found bit mapping to be an efficient means to organize a contiguous set of overlapping clones from a larger genome.

  20. Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast.

    PubMed

    Belton, Jon-Matthew; Dekker, Job

    2015-06-01

    Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing.

  1. Relationships between chromosome structure and chromosomal aberrations

    NASA Astrophysics Data System (ADS)

    Eidelman, Yuri; Andreev, Sergey

    An interphase nucleus of human lymphocyte was simulated by the novel Monte Carlo tech-nique. The main features of interphase chromosome structure and packaging were taken into account: different levels of chromatin organisation; nonrandom localisation of chromosomes within a nucleus; chromosome loci dynamics. All chromosomes in a nucleus were modelled as polymer globules. A dynamic pattern of intra/interchromosomal contacts was simulated. The detailed information about chromosomal contacts, such as distribution of intrachromoso-mal contacts over the length of each chromosome and dependence of contact probability on genomic separation between chromosome loci, were calculated and compared to the new exper-imental data obtained by the Hi-C technique. Types and frequencies of simple and complex radiation-induced chromosomal exchange aberrations (CA) induced by X-rays were predicted with taking formation and decay of chromosomal contacts into account. Distance dependence of exchange formation probability was calculated directly. mFISH data for human lymphocytes were analysed. The calculated frequencies of simple CA agreed with the experimental data. Complex CA were underestimated despite the dense packaging of chromosome territories within a nucleus. Possible influence of chromosome-nucleus structural organisation on the frequency and spectrum of radiation-induced chromosome aberrations is discussed.

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

    PubMed Central

    Lufino, Michele M. P.; Edser, Pauline A. H.; Quail, Michael A.; Rice, Stephen; Adams, David J.; Wade-Martins, Richard

    2016-01-01

    Gene dosage plays a critical role in a range of cellular phenotypes, yet most cellular expression systems use heterologous cDNA-based vectors which express proteins well above physiological levels. In contrast, genomic DNA expression vectors generate physiologically-relevant levels of gene expression by carrying the whole genomic DNA locus of a gene including its regulatory elements. Here we describe the first genomic DNA expression library generated using the high-capacity herpes simplex virus-1 amplicon technology to deliver bacterial artificial chromosomes (BACs) into cells by viral transduction. The infectious BAC (iBAC) library contains 184,320 clones with an average insert size of 134.5 kb. We show in a Chinese hamster ovary (CHO) disease model cell line and mouse embryonic stem (ES) cells that this library can be used for genetic rescue studies in a range of contexts including the physiological restoration of Ldlr deficiency, and viral receptor expression. The iBAC library represents an important new genetic analysis tool openly available to the research community. PMID:27353647

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2013-06-01

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

  5. Human chromosome 8.

    PubMed Central

    Wood, S

    1988-01-01

    The role of human chromosome 8 in genetic disease together with the current status of the genetic linkage map for this chromosome is reviewed. Both hereditary genetic disease attributed to mutant alleles at gene loci on chromosome 8 and neoplastic disease owing to somatic mutation, particularly chromosomal translocations, are discussed. PMID:3070042

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

    PubMed

    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 <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 2.5.1.32)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced 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.

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

    PubMed

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

    2009-03-01

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

  8. Libraries program

    USGS Publications Warehouse

    2011-01-01

    The U.S. Congress authorized a library for the U.S. Geological Survey (USGS) in 1879. The library was formally established in 1882 with the naming of the first librarian and began with a staff of three and a collection of 1,400 books. Today, the USGS Libraries Program is one of the world's largest Earth and natural science repositories and a resource of national significance used by researchers and the public worldwide.

  9. Generating a physical map of chromosome 11

    SciTech Connect

    Nowak, N.; Oin, S.; Sait, S.

    1994-09-01

    A four-hit chromosome-specific YAC library is being used to construct an overlapping YAC clone physical map of human chromosome 11. Each of the 1824 YAC clones have been sized by pulsed-field gel analysis with a resulting average insert size of 350 kb. Three hundred of the clones have been localized to a chromosome band by fluorescence in situ hybridization (FISH). We are using two complementary approaches to assemble YAC contigs: hybridization-based screening with Alu-PCR products and STS-based screening. Both screening approaches use pooling strategies to uniquely address each of the well locations with a minimum number of DNA pools. Since our main source of YAC clones is a chromosome specific library, the pool complexity is an order of magnitude smaller in comparison to total genomic libraries. Alu products with three different Alu primers were generated from both the monochromosomal J1 hybrid and individual YAC clones. YAC clones were chosen to generate probes using a sample without replacement strategy. Greater than 1700 YAC clones have been assembled into contigs. These initial contigs are estimated to be on average 1 Mb in size. Each of the contigs have been binned into a chromosomal band by virture of it containing YACs localized by FISH or YACs positive for one or more mapped STSs. Confirmation of the contigs is being achieved with Line I fingerprinting. The initial contigs are currently being joined using probes derived from the YAC clone ends defining the outermost points in the contigs for hybridization of colony filters. Remaining gaps in the contigs will be joined using alternative large insert libraries.

  10. Chromosome-specific cDNAs/STSs

    SciTech Connect

    Soares, M.B.; Efstratiadis, A.

    1992-10-01

    This project seeks to construct high-quality normalized cDNA libraries from human tissues, to develop methods for isolation of chromosome-specific cDNAs, and to contribute sequence information to the expanding cDNA/EST database. A human infant brain cDNA library having a very high complexity, short poly (A) tails; long size inserts; undetectable co-cloning events; and low background of non-recombinants was previously described. Over 2,000 ESTs have already been successfully derived from this library, and so become one of the best so far characaterized. Having established the protocol to construct high quality cDNA libraries, a major effort was mounted to develope a method to normalize cDNA libraries constructed in phagemid vectors. Briefly, this method involves priming of the library in the form of single-stranded circles with a Not I-oligo (dT) primer and controlled extensions with Klenow in the presence of dNTPs and ddNTPS. After purification of the partial duplexes over HAP, melting and reannealing to a moderate Cot, unhybridized (normalized) single-stranded circles are purified by HAP and electroporated into bacteria, generating a normalized library. The extent of normalization of the infant brain cDNA library has been determined by a series of screenings with probes that represent mRNAs from the 3 frequency classes.

  11. Process of labeling specific chromosomes using recombinant repetitive DNA

    DOEpatents

    Moyzis, R.K.; Meyne, J.

    1988-02-12

    Chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family members and consensus sequences of the repetitive DNA families for the chromosome preferential sequences. The selected low homology regions are then hybridized with chromosomes to determine those low homology regions hybridized with a specific chromosome under normal stringency conditions.

  12. America's Star Libraries: Top-Rated Libraries

    ERIC Educational Resources Information Center

    Lance, Keith Curry; Lyons, Ray

    2009-01-01

    "Library Journal"'s national rating of public libraries, the "LJ" Index of Public Library Service 2009, Round 2, identifies 258 "star" libraries. Created by Keith Curry Lance and Ray Lyons and based on 2007 data from the IMLS, it rates 7,268 public libraries. The top libraries in each group get five, four, or three stars. All included libraries,…

  13. Molecular characterization of the pericentric inversion that causes differences between chimpanzee chromosome 19 and human chromosome 17.

    PubMed

    Kehrer-Sawatzki, Hildegard; Schreiner, Bettina; Tänzer, Simone; Platzer, Matthias; Müller, Stefan; Hameister, Horst

    2002-08-01

    A comparison of the human genome with that of the chimpanzee is an attractive approach to attempts to understand the specificity of a certain phenotype's development. The two karyotypes differ by one chromosome fusion, nine pericentric inversions, and various additions of heterochromatin to chromosomal telomeres. Only the fusion, which gave rise to human chromosome 2, has been characterized at the sequence level. During the present study, we investigated the pericentric inversion by which chimpanzee chromosome 19 differs from human chromosome 17. Fluorescence in situ hybridization was used to identify breakpoint-spanning bacterial artificial chromosomes (BACs) and plasmid artificial chromosomes (PACs). By sequencing the junction fragments, we localized breakpoints in intergenic regions rich in repetitive elements. Our findings suggest that repeat-mediated nonhomologous recombination has facilitated inversion formation. No addition or deletion of any sequence element was detected at the breakpoints or in the surrounding sequences. Next to the break, at a distance of 10.2-39.1 kb, the following genes were found: NGFR and NXPH3 (on human chromosome 17q21.3) and GUC2D and ALOX15B (on human chromosome 17p13). The inversion affects neither the genomic structure nor the gene-activity state with regard to replication timing of these genes.

  14. The Case for Artificial Intelligence in Medicine

    PubMed Central

    Reggia, James A.

    1983-01-01

    Current artificial intelligence (AI) technology can be viewed as producing “systematic artifacts” onto which we project an interpretation of intelligent behavior. One major benefit this technology could bring to medicine is help with handling the tremendous and growing volume of medical knowledge. The reader is led to a vision of the medical library of tomorrow, an interactive, artificially intelligent knowledge source that is fully and directly integrated with daily patient care.

  15. Artificial rearing.

    PubMed

    Dominguez, Hector D; Thomas, Jennifer D

    2008-01-01

    Prenatal alcohol exposure disrupts development, leading to a range of effects referred to as fetal alcohol spectrum disorders (FASD). FASDs include physical, central nervous system, and behavioral alterations. Animal model systems are used to study the relationship between alcohol-related central nervous system damage and behavioral alterations, risk factors for FASD, mechanisms of alcohol-induced damage, as well as treatments and interventions. When using a rodent model, it is important to recognize that the timing of brain development relative to birth differs between humans and rodents. Thus, to model alcohol exposure during the third trimester equivalent, rats must be exposed during early postnatal development (postnatal days 4-9). Artificial rearing is one experimental paradigm that is used to expose neonatal rats to alcohol during this period of brain development. Neonatal rat pups are housed in an artificial rearing environment and automatically fed a milk diet substitute via an intragastric cannula to ensure adequate growth during the treatment period. Alcohol is delivered in the milk diet. This chapter provides a description of the methods needed for this administrative technique, including preparation of the artificial rearing environment, gastrostomy surgery, and care of artificially reared rat pups.

  16. Artificial Intelligence.

    ERIC Educational Resources Information Center

    Wash, Darrel Patrick

    1989-01-01

    Making a machine seem intelligent is not easy. As a consequence, demand has been rising for computer professionals skilled in artificial intelligence and is likely to continue to go up. These workers develop expert systems and solve the mysteries of machine vision, natural language processing, and neural networks. (Editor)

  17. Undetected sex chromosome aneuploidy by chromosomal microarray.

    PubMed

    Markus-Bustani, Keren; Yaron, Yuval; Goldstein, Myriam; Orr-Urtreger, Avi; Ben-Shachar, Shay

    2012-11-01

    We report on a case of a female fetus found to be mosaic for Turner syndrome (45,X) and trisomy X (47,XXX). Chromosomal microarray analysis (CMA) failed to detect the aneuploidy because of a normal average dosage of the X chromosome. This case represents an unusual instance in which CMA may not detect chromosomal aberrations. Such a possibility should be taken into consideration in similar cases where CMA is used in a clinical setting.

  18. Privatizing Libraries

    ERIC Educational Resources Information Center

    Jerrard, Jane; Bolt, Nancy; Strege, Karen

    2012-01-01

    This timely special report from ALA Editions provides a succinct but comprehensive overview of the "privatization" of public libraries. It provides a history of the trend of local and state governments privatizing public services and assets, and then examines the history of public library privatization right up to the California legislation…

  19. Library Advocacy

    ERIC Educational Resources Information Center

    Plunkett, Kate

    2010-01-01

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

  20. Library Legislation.

    ERIC Educational Resources Information Center

    Kavass, Igor

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

  1. Chromatin structure and ionizing-radiation-induced chromosome aberrations

    SciTech Connect

    Muehlmann-Diaz, M.C.

    1993-01-01

    The possible influence of chromatic structure or activity on chromosomal radiosensitivity was studied. A cell line was isolated which contained some 10[sup 5] copies of an amplified plasmid in a single large mosquito artificial chromosome (MAC). This chromosome was hypersensitive to DNase I. Its radiosensitivity was some three fold greater than normal mosquito chromosomes in the same cell. In cultured human cells irradiated during G[sub 0], the initial breakage frequency in chromosome 4, 19 and the euchromatic and heterochromatic portions of the Y chromosome were measured over a wide range of doses by inducing Premature Chromosome Condensation (PCC) immediately after irradiation with Cs-137 gamma rays. No evidence was seen that Y heterochromatin or large fragments of it remained unbroken. The only significant deviation from the expected initial breakage frequency per Gy per unit length of chromosome was that observed for the euchromatic portion of the Y chromosome, with breakage nearly twice that expected. The development of aberrations involving X and Y chromosomes at the first mitosis after irradation was also studied. Normal female cells sustained about twice the frequency of aberrations involving X chromosomes for a dose of 7.3 Gy than the corresponding male cells. Fibroblasts from individuals with supernumerary X chromosomes did not show any further increase in X aberrations for this dos. The frequency of aberrations involving the heterochromatic portion of the long arm of the Y chromosome was about what would be expected for a similar length of autosome, but the euchromatic portion of the Y was about 3 times more radiosensitive per unit length. 5-Azacytidine treatment of cultured human female fibroblasts or fibroblasts from a 49,XXXXY individual, reduced the methylation of cytosine residues in DNA, and resulted in an increased chromosomal radiosensitivity in general, but it did not increase the frequency of aberrations involving the X chromosomes.

  2. The precarious prokaryotic chromosome.

    PubMed

    Kuzminov, Andrei

    2014-05-01

    Evolutionary selection for optimal genome preservation, replication, and expression should yield similar chromosome organizations in any type of cells. And yet, the chromosome organization is surprisingly different between eukaryotes and prokaryotes. The nuclear versus cytoplasmic accommodation of genetic material accounts for the distinct eukaryotic and prokaryotic modes of genome evolution, but it falls short of explaining the differences in the chromosome organization. I propose that the two distinct ways to organize chromosomes are driven by the differences between the global-consecutive chromosome cycle of eukaryotes and the local-concurrent chromosome cycle of prokaryotes. Specifically, progressive chromosome segregation in prokaryotes demands a single duplicon per chromosome, while other "precarious" features of the prokaryotic chromosomes can be viewed as compensations for this severe restriction.

  3. B-chromosome evolution.

    PubMed Central

    Camacho, J P; Sharbel, T F; Beukeboom, L W

    2000-01-01

    B chromosomes are extra chromosomes to the standard complement that occur in many organisms. They can originate in a number of ways including derivation from autosomes and sex chromosomes in intra- and interspecies crosses. Their subsequent molecular evolution resembles that of univalent sex chromosomes, which involves gene silencing, heterochromatinization and the accumulation of repetitive DNA and transposons. B-chromosome frequencies in populations result from a balance between their transmission rates and their effects on host fitness. Their long-term evolution is considered to be the outcome of selection on the host genome to eliminate B chromosomes or suppress their effects and on the B chromosome's ability to escape through the generation of new variants. Because B chromosomes interact with the standard chromosomes, they can play an important role in genome evolution and may be useful for studying molecular evolutionary processes. PMID:10724453

  4. Rapid generation of region-specific probes by chromosome microdissection: Application to the identification of chromosomal rearrangements

    SciTech Connect

    Trent, J.M.; Guan, X.Y.; Zang, J.; Meltzer, P.S. )

    1993-01-01

    The authors present results using a novel strategy for chromosome microdissection and direct in vitro amplification of specific chromosomal regions, to identify cryptic chromosome alterations, and to rapidly generate region-specific genomic probes. First, banded chromosomes are microdissected and directly PCR amplified by a procedure which eliminates microchemistry (Meltzer, et al., Nature Genetics, 1:24, 1992). The resulting PCR product can be used for several applications including direct labeling for fluorescent in situ hybridization (FISH) to normal metaphase chromosomes. A second application of this procedure is the extremely rapid generation of chromosome region-specific probes. This approach has been successfully used to determine the derivation of chromosome segments unidentifiable by standard chromosome banding analysis. In selected instances these probes have also been used on interphase nuclei and provides the potential for assessing chromosome abnormalities in a variety of cell lineages. The microdissection probes (which can be generated in <24 hours) have also been utilized in direct library screening and provide the possibility of acquiring a significant number of region-specific probes for any chromosome band. This procedure extends the limits of conventional cytogenetic analysis by providing an extremely rapid source of numerous band-specific probes, and by enabling the direct analysis of essentially any unknown chromosome region.

  5. Artificial Intelligence.

    PubMed

    Lawrence, David R; Palacios-González, César; Harris, John

    2016-04-01

    It seems natural to think that the same prudential and ethical reasons for mutual respect and tolerance that one has vis-à-vis other human persons would hold toward newly encountered paradigmatic but nonhuman biological persons. One also tends to think that they would have similar reasons for treating we humans as creatures that count morally in our own right. This line of thought transcends biological boundaries-namely, with regard to artificially (super)intelligent persons-but is this a safe assumption? The issue concerns ultimate moral significance: the significance possessed by human persons, persons from other planets, and hypothetical nonorganic persons in the form of artificial intelligence (AI). This article investigates why our possible relations to AI persons could be more complicated than they first might appear, given that they might possess a radically different nature to us, to the point that civilized or peaceful coexistence in a determinate geographical space could be impossible to achieve.

  6. Artificial Intelligence.

    PubMed

    Lawrence, David R; Palacios-González, César; Harris, John

    2016-04-01

    It seems natural to think that the same prudential and ethical reasons for mutual respect and tolerance that one has vis-à-vis other human persons would hold toward newly encountered paradigmatic but nonhuman biological persons. One also tends to think that they would have similar reasons for treating we humans as creatures that count morally in our own right. This line of thought transcends biological boundaries-namely, with regard to artificially (super)intelligent persons-but is this a safe assumption? The issue concerns ultimate moral significance: the significance possessed by human persons, persons from other planets, and hypothetical nonorganic persons in the form of artificial intelligence (AI). This article investigates why our possible relations to AI persons could be more complicated than they first might appear, given that they might possess a radically different nature to us, to the point that civilized or peaceful coexistence in a determinate geographical space could be impossible to achieve. PMID:26957450

  7. Isolation and refined regional mapping of expressed sequences from human chromosome 21

    SciTech Connect

    Kao, F.T.; Yu, J.; Patterson, D.

    1994-10-01

    To increase candidate genes from human chromosome 21 for the analysis of Down syndrome and other genetic diseases localized on this chromosome, we have isolated and studied 9 cDNA clones encoded by chromosome 21. For isolating cDNAs, single-copy microclones from a chromosome 21 microdissection library were used in direct screening of various cDNA libraries. Seven of the cDNA clones have been regionally mapped on chromosome 21 using a comprehensive hybrid mapping panel comprising 24 cell hybrids that divide the chromosome into 33 subregions. These cDNA clones with refined mapping positions should be useful for identification and cloning of genes responsible for the specific component phenotypes of Down syndrome and other diseases on chromosome 21, including progressive myoclonus epilepsy in 21q22.3. 12 refs., 2 figs., 1 tab.

  8. Artificial vision.

    PubMed

    Zarbin, M; Montemagno, C; Leary, J; Ritch, R

    2011-09-01

    A number treatment options are emerging for patients with retinal degenerative disease, including gene therapy, trophic factor therapy, visual cycle inhibitors (e.g., for patients with Stargardt disease and allied conditions), and cell transplantation. A radically different approach, which will augment but not replace these options, is termed neural prosthetics ("artificial vision"). Although rewiring of inner retinal circuits and inner retinal neuronal degeneration occur in association with photoreceptor degeneration in retinitis pigmentosa (RP), it is possible to create visually useful percepts by stimulating retinal ganglion cells electrically. This fact has lead to the development of techniques to induce photosensitivity in cells that are not light sensitive normally as well as to the development of the bionic retina. Advances in artificial vision continue at a robust pace. These advances are based on the use of molecular engineering and nanotechnology to render cells light-sensitive, to target ion channels to the appropriate cell type (e.g., bipolar cell) and/or cell region (e.g., dendritic tree vs. soma), and on sophisticated image processing algorithms that take advantage of our knowledge of signal processing in the retina. Combined with advances in gene therapy, pathway-based therapy, and cell-based therapy, "artificial vision" technologies create a powerful armamentarium with which ophthalmologists will be able to treat blindness in patients who have a variety of degenerative retinal diseases.

  9. One-Step Generation of Chromosomal Rearrangements in Rice.

    PubMed

    Murata, Minoru; Kanatani, Asaka; Kashihara, Kazunari

    2016-01-01

    The combination of the DNA sequence-specific recombination system Cre/LoxP and the DNA transposon system Activator (Ac)/Dissociation (Ds) has been used for insertional and deletional mutagenesis, as well as for the generation of artificial ring chromosomes in model plants such as Arabidopsis and tobacco. However, it takes a long time to complete this process, even in Arabidopsis. To overcome this issue, a new binary vector, pDLHC, has been developed to induce chromosomal rearrangements for a short time in rice. pDLHC has been found to be effective in the induction of deletions between two LoxPs in the T2 generation of "Nihon bare" expressing Ac TPase. pDLHC has potential for the efficient generation of various types of chromosomal rearrangements including deletions, inversions, translocations and artificial ring chromosomes in plants, and the detailed protocol for rice is described here. PMID:27557686

  10. [The Presidential Libraries.

    ERIC Educational Resources Information Center

    Webb, John

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

  11. Chromosome Disorder Outreach

    MedlinePlus

    ... BLOG Join Us Donate You are not alone. Chromosome Disorder Outreach, Inc. is a non-profit organization, ... Support For all those diagnosed with any rare chromosome disorder. Since 1992, CDO has supported the parents ...

  12. Standards for British Libraries.

    ERIC Educational Resources Information Center

    Vaughan, Anthony

    1982-01-01

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

  13. Digital Libraries.

    ERIC Educational Resources Information Center

    Fox, Edward A.; Urs, Shalini R.

    2002-01-01

    Provides an overview of digital libraries research, practice, and literature. Highlights include new technologies; redefining roles; historical background; trends; creating digital content, including conversion; metadata; organizing digital resources; services; access; information retrieval; searching; natural language processing; visualization;…

  14. Callpath Library

    2013-11-09

    The "Callpath Library" is a software abstraction layer over a number of stack tracing utilities. It allows tool develoopers to conveniently represent and mNipulate call paths gathered fro U. Wisconsin's Stackwalker API and GNU Backtrace.

  15. Abnormal human sex chromosome constitutions

    SciTech Connect

    1993-12-31

    Chapter 22, discusses abnormal human sex chromosome constitution. Aneuploidy of X chromosomes with a female phenotype, sex chromosome aneuploidy with a male phenotype, and various abnormalities in X chromosome behavior are described. 31 refs., 2 figs.

  16. Mapping genes to human chromosome 19

    SciTech Connect

    Connolly, Sarah

    1996-05-01

    For this project, 22 Expressed Sequence Tags (ESTs) were fine mapped to regions of human chromosome 19. An EST is a short DNA sequence that occurs once in the genome and corresponds to a single expressed gene. {sup 32}P-radiolabeled probes were made by polymerase chain reaction for each EST and hybridized to filters containing a chromosome 19-specific cosmid library. The location of the ESTs on the chromosome was determined by the location of the ordered cosmid to which the EST hybridized. Of the 22 ESTs that were sublocalized, 6 correspond to known genes, and 16 correspond to anonymous genes. These localized ESTs may serve as potential candidates for disease genes, as well as markers for future physical mapping.

  17. Chromosomal Disorders and Autism.

    ERIC Educational Resources Information Center

    Gillberg, Christopher

    1998-01-01

    This paper reviews the literature on chromosomal aberrations in autism, especially possible gene markers. It notes that Chromosome 15 and numerical and structural abnormalities of the sex chromosomes have been most frequently reported as related to the genesis of autism. (Author/DB)

  18. Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers

    SciTech Connect

    Kandpal, R.P.; Kandpal, G.; Weissman, S.M. )

    1994-01-04

    The authors describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotde. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)[sub n] microsatellites with this approach contained clones with inserts containing CA repeats. They have also used this protocol for enrichment of (CAG)[sub n] and (AGAT)[sub n] sequence repeats and for Not I jumping clones. They have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.

  19. High-resolution physical mapping of a 250-kb region of human chromosome 11q24 by genomic sequence sampling (GSS)

    SciTech Connect

    Selleri, L.; Smith, M.W.; Holmsen, A.L.

    1995-04-10

    A physical map of the region of human chromosome 11q24 containing the FLI1 gene, disrupted by the t(11;22) translocation in Ewing sarcoma and primitive neuroectodermal tumors, was analyzed by genomic sequence sampling. Using a 4- to 5-fold coverage chromosome 11-specific library, 22 region-specific cosmid clones were identified by phenol emulsion reassociation hybridization, with a 245-kb yeast artificial chromosome clone containing the FLI1 gene, and by directed {open_quotes}walking{close_quotes} techniques. Cosmid contigs were constructed by individual clone fingerprinting using restriction enzyme digestion and assembly with the Genome Reconstruction and AsseMbly (GRAM) computer algorithm. The relative orientation and spacing of cosmid contigs with respect to the chromosome were determined by the structural analysis of cosmid clones and by direct visual in situ hybridization mapping. Each cosmid clone in the contig was subjected to {open_quotes}one-pass{close_quotes} end sequencing, and the resulting ordered sequence fragments represent {approximately}5% of the complete DNA sequence, making the entire region accessible by PCR amplification. The sequence samples were analyzed for putative exons, repetitive DNAs, and simple sequence repeats using a variety of computer algorithms. Based upon the computer predictions, Southern and Northern blot experiments led to the independent identification and localization of the FLI1 gene as well as a previously unknown gene located in this region of chromosome 11q24. This approach to high-resolution physical analysis of human chromosomes allows the assembly of detailed sequence-based maps. 62 refs., 7 figs.

  20. Sequence conservation on the Y chromosome

    SciTech Connect

    Gibson, L.H.; Yang-Feng, L.; Lau, C.

    1994-09-01

    The Y chromosome is present in all mammals and is considered to be essential to sex determination. Despite intense genomic research, only a few genes have been identified and mapped to this chromosome in humans. Several of them, such as SRY and ZFY, have been demonstrated to be conserved and Y-located in other mammals. In order to address the issue of sequence conservation on the Y chromosome, we performed fluorescence in situ hybridization (FISH) with DNA from a human Y cosmid library as a probe to study the Y chromosomes from other mammalian species. Total DNA from 3,000-4,500 cosmid pools were labeled with biotinylated-dUTP and hybridized to metaphase chromosomes. For human and primate preparations, human cot1 DNA was included in the hybridization mixture to suppress the hybridization from repeat sequences. FISH signals were detected on the Y chromosomes of human, gorilla, orangutan and baboon (Old World monkey) and were absent on those of squirrel monkey (New World monkey), Indian munjac, wood lemming, Chinese hamster, rat and mouse. Since sequence analysis suggested that specific genes, e.g. SRY and ZFY, are conserved between these two groups, the lack of detectable hybridization in the latter group implies either that conservation of the human Y sequences is limited to the Y chromosomes of the great apes and Old World monkeys, or that the size of the syntenic segment is too small to be detected under the resolution of FISH, or that homologeous sequences have undergone considerable divergence. Further studies with reduced hybridization stringency are currently being conducted. Our results provide some clues as to Y-sequence conservation across species and demonstrate the limitations of FISH across species with total DNA sequences from a particular chromosome.

  1. Artificial Intelligence. LC Science Tracer Bullet.

    ERIC Educational Resources Information Center

    Rodgers, Kay, Comp.

    Desgined to serve as a guide to resources on artificial intelligence (AI) and expert systems, this Library of Congress bulletin is divided into 18 sections that contain lists of books, journal articles, periodicals, associations, and other sources of information. A brief statement of the scope of the guide introduces the sections, which are listed…

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

    PubMed Central

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

    2003-01-01

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

  3. A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection

    PubMed Central

    Xu, Xu-Ping; Gan, Hai-Yan; Li, Fen-Xia; Tian, Qi; Zhang, Jun; Liang, Rong-Liang; Li, Ming

    2016-01-01

    Objective The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure. Methods Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction. Results A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B. Conclusion A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability. PMID:26765738

  4. Artificial Intelligence

    SciTech Connect

    Shirai, Y.; Tsujii, Jun-ichi

    1985-01-01

    Based on the Japanese 5th Generation Computer Program, this volume provides coverage of the fundamental concepts and various techniques in the different applications of Artificial Intelligence. Also presented are the methods which can be used to put these concepts and techniques into practice. Explanations are presented of all the basic topics in the field, including the representation of problems; searching techniques; the control of problem solving; programming languages for Al, such as LISP, PLANNER, CONNIVER, and PROLOG; the representation and utilization of knowledge; and the approach to human intelligence.

  5. Mapping strategies: Chromosome 16 workshop

    SciTech Connect

    Not Available

    1989-01-01

    The following topics from a workshop on chromosome 16 are briefly discussed: genetic map of chromosome 16; chromosome breakpoint map of chromosome 16; integrated physical/genetic map of chromosome 16; pulsed field map of the 16p13.2--p13.3 region (3 sheets); and a report of the HGM10 chromosome 16 committee.

  6. Structure, sequence, expression, and chromosomal localization of the human V{sub 1a} vasopressin receptor gene

    SciTech Connect

    Thibonnier, M.; Graves, M.K.; Wagner, M.S.

    1996-02-01

    We recently reported the structure and functional expression of a human V{sub 1a} vasopressin receptor (V{sub 1a}R) cDNA isolated from human liver cDNA libraries. To understand further the expression and regulation of the V{sub 1a}R, we now describe the genomic characteristics, tissue expression, chromosomal localization, and regional mapping of the human V{sub 1a}R gene, AVPR1A. Tissue distribution of the human V{sub 1a}R mRNA explored by Northern blot analysis of various human tissues or organs revealed the presence of a 5.5-kb mRNA transcript expressed in the liver and to a lesser degree in the heart, the kidney, and skeletal muscle. Screening of human genomic libraries revealed that the human AVPR1A gene is included entirely within a 6.4-kb rated by a 2.2-kb intron located before the corresponding seventh transmembrane domain of the receptor sequence. The first exon also contains 2 kb of 5{prime}-untranslated region, and the second exon includes 1 kb of 3{prime}-untranslated region. 5{prime}-RACE analysis of human liver mRNA by PCR localized the V{sub 1a}R mRNA transcription start site 1973 bp upstream of the translation the intron sequence were used as primers in polymerase chain reaction (PCR) analysis of human/rodent somatic cell hybrids. AVPR1A was localized by PCR analysis of a somatic cell hybrid panel to chromosome 12. Fluorescence in situ hybridization using a yeast artificial chromosome physically mapped AVPR1A to region 12q14-q15. 34 refs., 4 figs.

  7. America's Star Libraries

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2009-01-01

    "Library Journal"'s new national rating of public libraries, the "LJ" Index of Public Library Service, identifies 256 "star" libraries. It rates 7,115 public libraries. The top libraries in each group get five, four, or three Michelin guide-like stars. All included libraries, stars or not, can use their scores to learn from their peers and improve…

  8. University Libraries in Pakistan.

    ERIC Educational Resources Information Center

    Haider, Syed Jalaluddin

    1986-01-01

    This profile of university libraries in Pakistan covers history of higher education and role of the library; organization of library service (strong central library, decentralized library service, central library with department libraries); resources and collection building; technical processing; readers' services; administration and staffing;…

  9. High-Resolution BAC-Based Map of the Central Portion of Mouse Chromosome 5

    PubMed Central

    Crabtree, Jonathan; Wiltshire, Tim; Brunk, Brian; Zhao, Shaying; Schug, Jonathan; Stoeckert, Christian J.; Bucan, Maja

    2001-01-01

    The current strategy for sequencing the mouse genome involves the combination of a whole-genome shotgun approach with clone-based sequencing. High-resolution physical maps will provide a foundation for assembling contiguous segments of sequence. We have established a bacterial artificial chromosome (BAC)-based map of a 5-Mb region on mouse Chromosome 5, encompassing three gene families: receptor tyrosine kinases (PdgfraKit-Kdr), nonreceptor protein-tyrosine type kinases (Tec–Txk), and type-A receptors for the neurotransmitter GABA (Gabra2, Gabrb1, Gabrg1, and Gabra4). The construction of a BAC contig was initiated by hybridization screening the C57BL/6J (RPCI-23) BAC library, using known genes and sequence tagged sites (STSs). Additional overlapping clones were identified by searching the database of available restriction fingerprints for the RPCI-23 and RPCI-24 libraries. This effort resulted in the selection of >600 BAC clones, 251 kb of BAC-end sequences, and the placement of 40 known and/or predicted genes within this 5-Mb region. We use this high-resolution map to illustrate the integration of the BAC fingerprint map with a radiation-hybrid map via assembled expressed sequence tags (ESTs). From annotation of three representative BAC clones we demonstrate that up to 98% of the draft sequence for each contig could be ordered and oriented using known genes, BAC ends, consensus sequences for transcript assemblies, and comparisons with orthologous human sequence. For functional studies, annotation of sequence fragments as they are assembled into 50–200-kb stretches will be remarkably valuable. PMID:11591652

  10. A YAC contig of approximately 3 Mb from human chromosome 5q31 [yields] q33

    SciTech Connect

    Li, Xiang; Wang Jabs, E.; Hawkins, A.L.; Griffin, C.A. ); Wise, C.A.; Lovett, M. ); Le Paslier, D. ); Pittler, S.J. )

    1994-02-01

    The human chromosome 5q31-q33 region contains an interesting cluster of growth factor and receptor genes. In addition, several genetic disease loci have been localized within this region, but have not as yet been isolated as molecular clones. These include those loci involved in autosomal dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q-syndrome. A yeast artificial chromosome (YAC) contig of this region would assist in the further localization and isolation of these genes. The authors have used YACs isolated from the Washington University and Centre d'Etude du Polymorphisme Humain YAC libraries, including YACs from the large insert (mega) YAC library to build a contig greater than 3 Mb in size. An STS content strategy coupled with limited walking from YAC ends was used to isolate 22 overlapping YACs with as much as sixfold coverage. A total of 20 STSs, derived from genes, anonymous sequences, and vector Alu-PCR or inverse PCR products, were used to compile this contig. The order of loci, centromere-GRL-D5S207-D5S70-D5S545-D5S546-D5S547-D5S68-D5S548-D5S210-D5S549-D5S686- ADRB2-D5S559-CSF1R-D5S551-RPS14-D5S519-SPARC-telomere, was derived from the overlapping clones. This contig and clones derived from it will be useful substrates in selecting candidate cDNAs for the disease loci in this interval. 45 refs., 1 fig., 2 tabs.

  11. Chromosome-specific DNA Repeat Probes

    SciTech Connect

    Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

    2006-03-16

    In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

  12. Laser microdissection-based analysis of plant sex chromosomes.

    PubMed

    Hobza, Roman; Vyskot, Boris

    2007-01-01

    A recent progress in plant molecular biology has led to enormous available data of DNA sequences, including complete nuclear genomes of Arabidopsis, rice, and poplar. On the other hand, in plant species with more complex genomes, containing widespread repetitive sequences, it is important to establish genomic resources that help us to focus on particular part of genomes. Laser technology enables to handle with specific subcellular structures or even individual chromosomes. Here we present a comprehensive protocol to isolate and characterize DNA sequences derived from the sex chromosomes of white campion (Silene latifolia). This dioecious plant has become the most favorite model to study the structure, function, and evolution of plant sex chromosomes due to a large and distinguishable size of both the X and Y chromosomes. The protocol includes a versatile technique to prepare metaphase chromosomes from either germinating seeds or in vitro cultured hairy roots. Such slides can be used for laser chromosome microdissection, fluorescence in situ-hybridization mapping, and immunostaining. Here we also demonstrate some applications of the laser-dissected chromosome template, especially a modified FAST-FISH technique to paint individual chromosomes, and construction and screening of chromosome-specific DNA libraries.

  13. Artificial gravity.

    PubMed

    Scott, William B

    2005-04-25

    NASA's Artificial Gravity program consists of a team of researchers from Wyle Laboratories, NASA Johnson Space Center, and the University of Texas Medical Branch (UTMB). The short-radius centrifuge (SRC), built by Wyle Laboratories, will be integrated with UTMB's conducted bedrest studies, which mimic the detrimental effects of weightlessness (or microgravity). Bedrest subjects will be spun on the SRC at various accelerations and for various time periods, while being monitored medically. Parameters such as bone loss, muscle atrophy, balance control, and oxygen consumption will then be compared in order to research ways of mitigating the impact on astronauts' physiology. Other potential benefits from these studies extend to population groups on Earth, such as bedridden patients. PMID:15852559

  14. Artificial rheotaxis

    PubMed Central

    Palacci, Jérémie; Sacanna, Stefano; Abramian, Anaïs; Barral, Jérémie; Hanson, Kasey; Grosberg, Alexander Y.; Pine, David J.; Chaikin, Paul M.

    2015-01-01

    Motility is a basic feature of living microorganisms, and how it works is often determined by environmental cues. Recent efforts have focused on developing artificial systems that can mimic microorganisms, in particular their self-propulsion. We report on the design and characterization of synthetic self-propelled particles that migrate upstream, known as positive rheotaxis. This phenomenon results from a purely physical mechanism involving the interplay between the polarity of the particles and their alignment by a viscous torque. We show quantitative agreement between experimental data and a simple model of an overdamped Brownian pendulum. The model notably predicts the existence of a stagnation point in a diverging flow. We take advantage of this property to demonstrate that our active particles can sense and predictably organize in an imposed flow. Our colloidal system represents an important step toward the realization of biomimetic microsystems with the ability to sense and respond to environmental changes. PMID:26601175

  15. Acentric chromosome ends are prone to fusion with functional chromosome ends through a homology-directed rearrangement.

    PubMed

    Ohno, Yuko; Ogiyama, Yuki; Kubota, Yoshino; Kubo, Takuya; Ishii, Kojiro

    2016-01-01

    The centromeres of many eukaryotic chromosomes are established epigenetically on potentially variable tandem repeats; hence, these chromosomes are at risk of being acentric. We reported previously that artificially created acentric chromosomes in the fission yeast Schizosaccharomyces pombe can be rescued by end-to-end fusion with functional chromosomes. Here, we show that most acentric/functional chromosome fusion events in S. pombe cells harbouring an acentric chromosome I differed from the non-homologous end-joining-mediated rearrangements that result in deleterious dicentric fusions in normal cells, and were elicited by a previously unidentified homologous recombination (HR) event between chromosome end-associated sequences. The subtelomere repeats associated with the non-fusogenic ends were also destabilized in the surviving cells, suggesting a causal link between general subtelomere destabilization and acentric/functional chromosome fusion. A mutational analysis indicated that a non-canonical HR pathway was involved in the rearrangement. These findings are indicative of a latent mechanism that conditionally induces general subtelomere instability, presumably in the face of accidental centromere loss events, resulting in rescue of the fatal acentric chromosomes by interchromosomal HR.

  16. Evolution of Chromosome 6 of Solanum Species Revealed by Comparative Fluorescence in Situ Hybridization Mapping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparative genome mapping is an important tool in evolutionary research. Here we demonstrate a comparative fluorescent in situ hybridization (FISH) mapping strategy. A set of 13 bacterial artificial chromosome (BAC) clones derived from potato chromosome 6 was used for FISH mapping in seven differen...

  17. Library Venturing.

    ERIC Educational Resources Information Center

    Wilson, H. Donald

    1986-01-01

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

  18. Moving toward a higher efficiency of microcell-mediated chromosome transfer

    PubMed Central

    Liskovykh, Mikhail; Lee, Nicholas CO; Larionov, Vladimir; Kouprina, Natalay

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine. However, a safe and efficient MMCT technique remains an important challenge. The original MMCT protocol includes treatment of donor cells by Colcemid to induce micronucleation, where each chromosome becomes surrounded with a nuclear membrane, followed by disarrangement of the actin cytoskeleton using Cytochalasin B to help induce microcells formation. In this study, we modified the protocol and demonstrated that replacing Colcemid and Cytochalasin B with TN-16 + Griseofulvin and Latrunculin B in combination with a Collage/Laminin surface coating increases the efficiency of HAC transfer to recipient cells by almost sixfold and is possibly less damaging to HAC than the standard MMCT method. We tested the improved MMCT protocol on four recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells that could facilitate the cell engineering by HACs. PMID:27382603

  19. Moving toward a higher efficiency of microcell-mediated chromosome transfer.

    PubMed

    Liskovykh, Mikhail; Lee, Nicholas Co; Larionov, Vladimir; Kouprina, Natalay

    2016-01-01

    Microcell-mediated chromosome transfer (MMCT) technology enables individual mammalian chromosomes, megabase-sized chromosome fragments, or mammalian artificial chromosomes that include human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) to be transferred from donor to recipient cells. In the past few decades, MMCT has been applied to various studies, including mapping the genes, analysis of chromosome status such as aneuploidy and epigenetics. Recently, MMCT was applied to transfer MACs/HACs carrying entire chromosomal copies of genes for genes function studies and has potential for regenerative medicine. However, a safe and efficient MMCT technique remains an important challenge. The original MMCT protocol includes treatment of donor cells by Colcemid to induce micronucleation, where each chromosome becomes surrounded with a nuclear membrane, followed by disarrangement of the actin cytoskeleton using Cytochalasin B to help induce microcells formation. In this study, we modified the protocol and demonstrated that replacing Colcemid and Cytochalasin B with TN-16 + Griseofulvin and Latrunculin B in combination with a Collage/Laminin surface coating increases the efficiency of HAC transfer to recipient cells by almost sixfold and is possibly less damaging to HAC than the standard MMCT method. We tested the improved MMCT protocol on four recipient cell lines, including human mesenchymal stem cells and mouse embryonic stem cells that could facilitate the cell engineering by HACs. PMID:27382603

  20. Speech Recognition for A Digital Video Library.

    ERIC Educational Resources Information Center

    Witbrock, Michael J.; Hauptmann, Alexander G.

    1998-01-01

    Production of the meta-data supporting the Informedia Digital Video Library interface is automated using techniques derived from artificial intelligence research. Speech recognition and natural-language processing, information retrieval, and image analysis are applied to produce an interface that helps users locate information and navigate more…

  1. Gene order is conserved within the human chromosome 21 linkage group on mouse chromosome 10

    SciTech Connect

    Irving, N.G.; Cabin, D.E.; Swanson, D.A.; Reeves, R.H. )

    1994-05-01

    One hundred progeny from each of two intersubspecific mouse backcrosses were used to construct a comparative genetic map of a region of mouse chromosome 10 (MMU10) that is homologous to the distal tip of the long arm of human chromosome 21 (HSA21). The analysis included five genes and three simple sequence repeat markers, two of which flanked the HSA21-homologous cluster on either side. Analysis of 200 backcross progeny detected at least one crossover between each pair of adjacent genes and demonstrated that the proximal to distal orientation of the cluster was reversed between human and mouse. The order was determined to be Fyn-1-D10Mit20-S100b-Col6a1-Itgb2-Pfk1/D10Mit7-D10Mit11. Comparative mapping supports the order of corresponding markers on HSA21 determined using pulsed-field gel electrophoresis and radiation hybrid line data. However, sequence tagged site content mapping of human yeast artificial chromosomes (YACs) yielded conflicting data on the relative positions of human COL6A1 and S100B on HSA21. This discrepancy was resolved here by demonstrating that several key YACs used in the human contig analysis were mistyped for S100B. The murine map reported here provides a scaffold for construction of physical maps and yeast artificial chromosome contigs that will be useful in the development of mouse models for the study of Down syndrome. 28 refs., 4 figs., 2 tabs.

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

    PubMed

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

    2004-04-01

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

  3. Fifty probands with extra structurally abnormal chromosomes characterized by fluorescence in situ hybridization

    SciTech Connect

    Blennow, E.; Telenius, H.; Nordenskjoeld, M.

    1995-01-02

    Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 (inv dup(15)), including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality. 42 refs., 2 figs., 2 tabs.

  4. Rice chromosome segment substitution line selection utilizing SNP markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chromosome segment substitution lines (CSSLs) are a powerful tool for identifying naturally occurring, favorable alleles in unadapted germplasm. Six CSSL libraries in rice (Oryza sativa) are being developed from crosses between three different accessions of the rice progenitor species, O. rufipogon...

  5. Microdissected double-minute DNA detects variable patterns of chromosomal localizations and multiple abundantly expressed transcripts in normal and leukemic cells

    SciTech Connect

    Sen, S.; Zhou, Hongyi; Stass, S.A.; Sen, P. ); Mulac-Jericevic, B.; Pirrotta, V. )

    1994-02-01

    Double-minute (dm) chromosomes are cytogenetically resolvable DNA amplification-mediating acentric extrachromosomal structures that are commonly seen in primary tumors, tumor cell lines, and drug-resistant cells grown in vitro. Selective isolation of dm DNAs with standard molecular biological techniques is difficult, and thus, detailed studies to elucidate their structure, site of chromosomal origin, and chromosomal reintegration patterns have been limited. In those instances in which a gene has been localized on dms, characterization of the remainder of the DNA, which far exceeds the size of the gene identified, has remained inconclusive. dms seen in the acute myeloid leukemia cell line HL-60 have been shown to harbor the c-myc protooncogene. In this paper, the authors report the successful isolation of the dm-specific DNAs from these cells by the microdissection/polymerase chain reaction technique and demonstrate that the dm DNAs derived from a single discrete normal chromosome segment 8q24.1-q24.2 reintegrate at various specific locations in the leukemic cells. The microdissected dm DNA detects multiple abundantly expressed transcripts distinct from c-myc mRNA on Northern blots. By devising a [open quotes]transcript selection[close quotes] strategy, they cloned the partial genomic sequence of a gene from the microdissected DNA that encodes two of these RNAs. This strategy will be generally applicable for rapid cloning of unknown amplified genes harbored on dms. With DNA from 20 microdissected dms, they constructed a genomic library of about 20,000 recombinant microclones with an average insert size of about 450 bp. The microclones should help in isolating corresponding yeast artificial chromosome clones for high-resolution physical mapping of dms in HL-60 cells. Furthermore, application of the microdissection technique appears to be an extremely feasible approach to characterization of dms in other cell types. 42 refs., 6 figs., 1 tab.

  6. Physical mapping of human chromosome 16. Annual progress report

    SciTech Connect

    Sutherland, G.R.

    1993-08-01

    We aim to isolate cDNAs mapping to human chromosome 16 and localise such cDNAs on the high resolution physical map. In collaboration with LANL, PCR primers will be synthesised from cDNA sequences mapped to chromosome 16 and used as ESTs in the generation of mega-YAC contigs for this chromosome. Probing of high density cosmid grids will enable integration of the ESTs into cosmid contigs and location of the cosmid contigs on the YAC contig. A hn-cDNA library has been constructed from the hybrid CY18 which contains chromosome 16 as the only human chromosome. A modified screening protocol has been successfully developed and 15 hn-cDNA clones have been sequenced and localised on the hybrid map. Sequence analysis of four of these revealed that they were known cDNAs, which are now mapped to chromosome 16. Development of techniques to allow the isolation of longer cDNAs from the identified exons is in progress. This will depend on PCR amplification of cDNAs from a total human CDNA library.

  7. Capturing Chromosome Conformation

    NASA Astrophysics Data System (ADS)

    Dekker, Job; Rippe, Karsten; Dekker, Martijn; Kleckner, Nancy

    2002-02-01

    We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.

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

    PubMed Central

    Wu, Shao-Ying; Wan, Qiu-Hong

    2012-01-01

    The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC. PMID:22403630

  9. Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

    PubMed

    Ye, Qing; He, Ke; Wu, Shao-Ying; Wan, Qiu-Hong

    2012-01-01

    The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC.

  10. Isolation of a 97-kb minimal essential MHC B locus from a new reverse-4D BAC library of the golden pheasant.

    PubMed

    Ye, Qing; He, Ke; Wu, Shao-Ying; Wan, Qiu-Hong

    2012-01-01

    The bacterial artificial chromosome (BAC) system is widely used in isolation of large genomic fragments of interest. Construction of a routine BAC library requires several months for picking clones and arraying BACs into superpools in order to employ 4D-PCR to screen positive BACs, which might be time-consuming and laborious. The major histocompatibility complex (MHC) is a cluster of genes involved in the vertebrate immune system, and the classical avian MHC-B locus is a minimal essential one, occupying a 100-kb genomic region. In this study, we constructed a more effective reverse-4D BAC library for the golden pheasant, which first creates sub-libraries and then only picks clones of positive sub-libraries, and identified several MHC clones within thirty days. The full sequencing of a 97-kb reverse-4D BAC demonstrated that the golden pheasant MHC-B locus contained 20 genes and showed good synteny with that of the chicken. The notable differences between these two species were the numbers of class II B loci and NK genes and the inversions of the TAPBP gene and the TAP1-TAP2 region. Furthermore, the inverse TAP2-TAP1 was unique in the golden pheasant in comparison with that of chicken, turkey, and quail. The newly defined genomic structure of the golden pheasant MHC will give an insight into the evolutionary history of the avian MHC. PMID:22403630

  11. Molecular mapping of chromosomes 17 and X

    SciTech Connect

    Barker, D.F.

    1989-01-01

    The basic aims of this project are the construction of high density genetic maps of chromosomes 17 and X and the utilization of these maps for the subsequent isolation of a set of physically overlapping DNA segment clones. The strategy depends on the utilization of chromosome specific libraries of small (1--15 kb) segments from each of the two chromosomes. Since the time of submission of our previous progress report, we have refined the genetic map of markers which we had previously isolated for chromosome 17. We have completed our genetic mapping in CEPH reference and NF1 families of 15 markers in the pericentric region of chromosome 17. Physical mapping results with three probes, were shown be in very close genetic proximity to the NF1 gene, with respect to two translocation breakpoints which disrupt the activity of the gene. All three of the probes were found to lie between the centromere and the most proximal translocation breakpoint, providing important genetic markers proximal to the NF1 gene. Our primary focus has shifted to the X chromosome. We have isolated an additional 30 polymorphic markers, bringing the total number we have isolated to over 80. We have invested substantial effort in characterizing the polymorphisms at each of these loci and constructed plasmid subclones which reveal the polymorphisms for nearly all of the loci. These subclones are of practical value in that they produce simpler and stronger patterns on human genomic Southern blots, thus improving the efficiency of the genetic mapping experiments. These subclones may also be of value for deriving DNA sequence information at each locus, necessary for establishing polymerase chain reaction primers specific for each locus. Such information would allow the use of each locus as a sequence tagged site.

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

    PubMed Central

    2011-01-01

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

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

    PubMed

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

    2016-02-01

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

  14. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

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

    Papers on art libraries, librarianship, and documentation presented at the 1982 International Federation of Library Associations (IFLA) conference include: (1) "The Tyranny of Distance: Art Libraries in Canada," a description by Mary F. Williamson of Canada's regional art libraries which serve both art students and the general public; (2) "A…

  15. Identification of a novel retrotransposon with sex chromosome-specific distribution in Silene latifolia.

    PubMed

    Kralova, Tereza; Cegan, Radim; Kubat, Zdenek; Vrana, Jan; Vyskot, Boris; Vogel, Ivan; Kejnovsky, Eduard; Hobza, Roman

    2014-01-01

    Silene latifolia is a dioecious plant species with chromosomal sex determination. Although the evolution of sex chromosomes in S. latifolia has been the subject of numerous studies, a global view of X chromosome structure in this species is still missing. Here, we combine X chromosome microdissection and BAC library screening to isolate new X chromosome-linked sequences. Out of 8 identified BAC clones, only BAC 86M14 showed an X-preferential signal after FISH experiments. Further analysis revealed the existence of the Athila retroelement which is enriched in the X chromosome and nearly absent in the Y chromosome. Based on previous data, the Athila retroelement belongs to the CL3 group of most repetitive sequences in the S. latifolia genome. Structural, transcriptomics and phylogenetic analyses revealed that Athila CL3 represents an old clade in the Athila lineage. We propose a mechanism responsible for Athila CL3 distribution in the S. latifolia genome. PMID:24751661

  16. Physical map of mouse Chromosome 17 in the region relevant for positional cloning of the hybrid sterility 1 gene

    SciTech Connect

    Trachtulec, Z.; Vincek, V.; Hamvas, R.M.J.

    1994-09-01

    Hybrid sterility 1 (Hst1) is the major gene responsible for sterility of male hybrids between Mus musculus and certain laboratory strains. Thus, Hst1 is of importance in studying both postreproductive isolation of closely related species and male fertility. It has been mapped to mouse chromosome 17 in the region corresponding to the third inversion of the t haplotypes. The aim of the present study was to construct a physical map of the Hst1 region as the first step in an effort to clone the gene. Three yeast artificial chromosome (YAC) libraries (Princeton, Whitehead, and ICRF) were screened with polymerase chain reaction (PCR) oligonucleotide primers and DNA probes specific for loci previously mapped into the region of the third inversion. The isolated YAC clones were restriction mapped and arranged into contigs. Sixteen YAC clones were arranged into a single contig encompassing a region approximately 2000 kb long based on restriction mapping of highly overlapping but independently derived YAC clones. Five new loci in the region of the third inversion were mappd and the order and approximate physical distances of 12 loci established in this contig. The Hst1 gene maps approximately 0.2 cM proximal to the D17Ph1 locus encompassed by the YAC contig. Since the contig extends at least 1200 kb proximal to D17Ph1, it should contain the Hst1 gene.

  17. Sequential cloning of chromosomes

    DOEpatents

    Lacks, Sanford A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism's chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  18. Sequential cloning of chromosomes

    DOEpatents

    Lacks, S.A.

    1995-07-18

    A method for sequential cloning of chromosomal DNA of a target organism is disclosed. A first DNA segment homologous to the chromosomal DNA to be sequentially cloned is isolated. The first segment has a first restriction enzyme site on either side. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction (class IIS) enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes. 9 figs.

  19. Sequential cloning of chromosomes

    SciTech Connect

    Lacks, S.A.

    1991-12-31

    A method for sequential cloning of chromosomal DNA and chromosomal DNA cloned by this method are disclosed. The method includes the selection of a target organism having a segment of chromosomal DNA to be sequentially cloned. A first DNA segment, having a first restriction enzyme site on either side. homologous to the chromosomal DNA to be sequentially cloned is isolated. A first vector product is formed by ligating the homologous segment into a suitably designed vector. The first vector product is circularly integrated into the target organism`s chromosomal DNA. The resulting integrated chromosomal DNA segment includes the homologous DNA segment at either end of the integrated vector segment. The integrated chromosomal DNA is cleaved with a second restriction enzyme and ligated to form a vector-containing plasmid, which is replicated in a host organism. The replicated plasmid is then cleaved with the first restriction enzyme. Next, a DNA segment containing the vector and a segment of DNA homologous to a distal portion of the previously isolated DNA segment is isolated. This segment is then ligated to form a plasmid which is replicated within a suitable host. This plasmid is then circularly integrated into the target chromosomal DNA. The chromosomal DNA containing the circularly integrated vector is treated with a third, retrorestriction enzyme. The cleaved DNA is ligated to give a plasmid that is used to transform a host permissive for replication of its vector. The sequential cloning process continues by repeated cycles of circular integration and excision. The excision is carried out alternately with the second and third enzymes.

  20. CHROMOSOMES OF AMERICAN MARSUPIALS.

    PubMed

    BIGGERS, J D; FRITZ, H I; HARE, W C; MCFEELY, R A

    1965-06-18

    Studies of the chromosomes of four American marsupials demonstrated that Caluromys derbianus and Marmosa mexicana have a diploid number of 14 chromosomes, and that Philander opossum and Didelphis marsupialis have a diploid number of 22. The karyotypes of C. derbianus and M. mexicana are similar, whereas those of P. opossum and D. marsupialis are dissimilar. If the 14-chromosome karyotype represents a reduction from a primitive number of 22, these observations suggest that the change has occurred independently in the American and Australasian forms.

  1. Graph Library

    2007-06-12

    GraphLib is a support library used by other tools to create, manipulate, store, and export graphs. It provides a simple interface to specifS’ arbitrary directed and undirected graphs by adding nodes and edges. Each node and edge can be associated with a set of attributes describing size, color, and shape. Once created, graphs can be manipulated using a set of graph analysis algorithms, including merge, prune, and path coloring operations. GraphLib also has the abilitymore » to export graphs into various open formats such as DOT and GML.« less

  2. Cell Libraries

    NASA Technical Reports Server (NTRS)

    1994-01-01

    A NASA contract led to the development of faster and more energy efficient semiconductor materials for digital integrated circuits. Gallium arsenide (GaAs) conducts electrons 4-6 times faster than silicon and uses less power at frequencies above 100-150 megahertz. However, the material is expensive, brittle, fragile and has lacked computer automated engineering tools to solve this problem. Systems & Processes Engineering Corporation (SPEC) developed a series of GaAs cell libraries for cell layout, design rule checking, logic synthesis, placement and routing, simulation and chip assembly. The system is marketed by Compare Design Automation.

  3. A new chromosome was born: comparative chromosome painting in Boechera.

    PubMed

    Koch, Marcus A

    2015-09-01

    Comparative chromosome painting is a powerful tool to study the evolution of chromosomes and genomes. Analyzing karyotype evolution in cruciferous plants highlights the origin of aberrant chromosomes in apomictic Boechera and further establishes the cruciferous plants as important model system for our understanding of plant chromosome and genome evolution. PMID:26228436

  4. The human glutamate receptor delta 2 gene (GRID2) maps to chromosome 4q22.

    PubMed

    Hu, W; Zuo, J; De Jager, P L; Heintz, N

    1998-01-01

    We isolated the human glutamate receptor delta 2 (GRID2) gene, which has 97.0% identity in amino acid sequence to the mouse glutamate receptor delta 2 (Grid2) gene. We subsequently mapped this gene to human chromosome 4q22 by radiation hybrid mapping and by hybridization to two overlapping human yeast artificial chromosomes that are located in 4q22. The Grid2 gene, which is mutated in lurcher (Lc) mice, maps to mouse chromosome 6. Thus, the mapping of the GRID2 gene to human chromosome 4q22 confirms and refines a region of synteny between mouse and human genomes.

  5. Gene amplification in Rhynchosciara salivary gland chromosomes.

    PubMed Central

    Glover, D M; Zaha, A; Stocker, A J; Santelli, R V; Pueyo, M T; De Toledo, S M; Lara, F J

    1982-01-01

    Late in the fourth larval instar, several regions of the Rhynchosciara americana salivary gland chromosomes undergo "DNA puffing." We have constructed a library of cloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands during the period of development when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puff sites C3 and C8 and encode 1.25-kilobase and 1.95-kilobase RNA molecules, respectively. We estimate from the quantitation of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing. Images PMID:6953439

  6. Direct and inverted reciprocal chromosome insertions between chromosomes 7 and 14 in a woman with recurrent miscarriages

    SciTech Connect

    Ying-Tai Wang; Zhao-Cai Wang; Bajalica, S.; Han, F.Y.; Bui, T.H.; Xie, Y.G.

    1994-09-01

    We present the first case of direct and inverted reciprocal chromosome insertions between human chromosomes 7 and 14, ascertained because of repeated spontaneous abortions. Prometaphase GTG banding analysis showed the karyotype to be 46, XX, inv ins (7;14)(7pter {yields} 7q11.23::14q32.2 {yields} 14q22::7q21.2 {yields} 7qter), dir ins(14;7)(14pter {yields} 14q22::7q11.23 {yields} 7q21.2::14q32.2 {yields} 14qter). Origins of the insertion have been confirmed by chromosome painting with libraries specific for chromosomes 7 and 14 using fluorescence in situ hybridization. 5 refs., 3 figs.

  7. Lithuanian Library History.

    ERIC Educational Resources Information Center

    Gudauskas, Renaldas

    1994-01-01

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

  8. Rural Libraries in Illinois.

    ERIC Educational Resources Information Center

    Crossland, Brent, Ed.; And Others

    1993-01-01

    This theme issue includes 13 articles that discuss rural library concerns in Illinois. Topics addressed include strengthening library services; rural trends and their impact on libraries; partnerships; Cooperative Extension Service revitalization; financing; economic development; resource sharing in school libraries; and public libraries and user…

  9. Marketing the Virtual Library

    ERIC Educational Resources Information Center

    Fagan, Jody Condit

    2009-01-01

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

  10. Deutsches Museum Library.

    ERIC Educational Resources Information Center

    Berninger, Ernst H.; Reineke, Eva

    1986-01-01

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

  11. Library Directions in 1988.

    ERIC Educational Resources Information Center

    DeCandido, GraceAnne A.

    1989-01-01

    Reviews major library issues and events of 1988, including: (1) the Federal Bureau of Investigation's Library Awareness Program; (2) cooperation with USSR libraries; (3) library finance; (4) preservation; and (5) special programing. News about a number of prominent library professionals is included in a sidebar. (MES)

  12. Rural Library Service.

    ERIC Educational Resources Information Center

    Vavrek, Bernard; And Others

    1989-01-01

    Five articles present an overview of trends and issues affecting rural libraries. The areas discussed include the status of rural library services; outreach programs; the role of library cooperation in the support of rural library service; the development of rural information centers; and political marketing of the rural library. (CLB)

  13. Chromosome doubling method

    DOEpatents

    Kato, Akio

    2006-11-14

    The invention provides methods for chromosome doubling in plants. The technique overcomes the low yields of doubled progeny associated with the use of prior techniques for doubling chromosomes in plants such as grasses. The technique can be used in large scale applications and has been demonstrated to be highly effective in maize. Following treatment in accordance with the invention, plants remain amenable to self fertilization, thereby allowing the efficient isolation of doubled progeny plants.

  14. A verified minimal YAC contig for human chromosome 21

    SciTech Connect

    Graw, S.L.; Patterson, D.; Drabkin, H.

    1994-09-01

    The goal of this project is the construction of a verified YAC contig of the complete long arm of human chromosome 21 utilizing YACs from the CEPH and St. Louis libraries. The YACs in this contig have been analyzed for size by PFGE, tested for chimerism by FISH or end-cloning, and verified for STS content by PCR. This last analysis has revealed a number of cases of conflict with the published STS order. To establish correct order, we have utilized STS content analysis of somatic cell hybrids containing portions of chromosome 21. Additional problems being addressed include completeness of coverage and possible deletions or gaps. Questions of completeness of the CEPH 810 YAC set arose after screening with 57 independently derived probes failed to identify clones for 11 (19%). Ten of the 11, however, do detect chromosome 21 cosmids when used to screen Lawrence Livermore library LL21NC02`G,` a cosmid library constructed from flow-sorted chromosomes 21. Remaining gaps in the contig are being closed by several methods. These include YAC fingerprinting and conversion of YACs to cosmids. In addition, we are establishing the overlap between the physical NotI map and the YAC contig by testing YACs for NotI sites and screening the YACs in the contig for the presence of NotI-linking clones.

  15. Micromechanics of human mitotic chromosomes

    NASA Astrophysics Data System (ADS)

    Sun, Mingxuan; Kawamura, Ryo; Marko, John F.

    2011-02-01

    Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.

  16. The Whole Library Handbook 3: Current Data, Professional Advice, and Curiosa about Libraries and Library Services.

    ERIC Educational Resources Information Center

    Eberhart, George M., Comp.

    This handbook contains articles, guidelines, and other information from the field of library science organized into the following chapters: (1) "Libraries," including some basic figures, academic libraries, public libraries, school libraries, special libraries, national libraries, state libraries, small libraries, facilities, the past, and the…

  17. Agricultural Libraries and Information.

    ERIC Educational Resources Information Center

    Russell, Keith W., Ed.; Pisa, Maria G., Ed.

    1990-01-01

    Eleven articles address issues relating to agricultural libraries and information, including background on agricultural libraries and information, trend management, document delivery, reference services, user needs and library services, collection development, technologies for international information management, information sources,…

  18. The mapping of novel genes to human chromosome 19

    SciTech Connect

    Buenaventura, J.M.

    1994-12-01

    The principle goal of our laboratory is the discovery of new genes on human chromosome 19. One of the strategies to achieve this goal is through the use of cDNA clones known as {open_quotes}expressed sequence tags{close_quotes} (ESTs). ESTs, short segments of sequence from a cDNA clone that correspond to the mRNA, occur as unique regions in the genome and, therefore, can be used as markers for specific positions. In collaboration with researchers from Genethon in France, fifteen cDNA clones from a normalized human infant brain cDNA library were tested and determined to map to chromosome 19. A verification procedure is then followed to confirm assignment to chromosome 19. First, primers for each cDNA clone are developed and then amplified by polymerase chain reaction from genomic DNA. Next, a {sup 32}P-radiolabeled probe is made by polymerase chain reaction for each clone and then hybridized against filters containing an LLNL chromosome 19-specific cosmid library to find putative locations on the chromosome. The location is then verified by running a polymerase chain reactions from the positive cosmids. With the Browser database at LLNL, additional information about the positive cosmids can be found. Through use of the BLAST database at the National Library of Medicine, homologous sequences to the clones can be found. Among the fifteen cDNA clones received from Genethon, all have been amplified by polymerase chain reaction. Three have turned out as repetitive elements in the genome. Ten have been mapped to specific locations on chromosome 19. Putative locations have been found for the remaining two clones and thus verification testing will proceed.

  19. Induction of genomic instability and activation of autophagy in artificial human aneuploid cells.

    PubMed

    Ariyoshi, Kentaro; Miura, Tomisato; Kasai, Kosuke; Fujishima, Yohei; Oshimura, Mitsuo; Yoshida, Mitsuaki A

    2016-08-01

    Chromosome missegregation can lead to a change in chromosome number known as aneuploidy. Although aneuploidy is a known hallmark of cancer cells, the various mechanisms by which altered gene and/or DNA copy number facilitate tumorigenesis remain unclear. To understand the effect of aneuploidy occurring in non-tumorigenic human breast epithelial cells, we generated clones harboring artificial aneuploidy using microcell-mediated chromosome transfer. Our results demonstrate that clones with artificial aneuploidy of chromosome 8 or chromosome 22 both show inhibited proliferation and genomic instability. Also, the increased autophagy was observed in the artificially aneuploidy clones, and inhibition of autophagy resulted in increased genomic instability and DNA damage. In addition, the intracellular levels of reactive oxygen species were up-regulated in the artificially aneuploid clones, and inhibition of autophagy further increased the production of reactive oxygen species. Together, these results suggest that even a single extraneous chromosome can induce genomic instability, and that autophagy triggered by aneuploidy-induced stress is a mechanism to protect cells bearing abnormal chromosome number. PMID:27343755

  20. Trends in Artificial Intelligence.

    ERIC Educational Resources Information Center

    Hayes, Patrick

    1978-01-01

    Discusses the foundations of artificial intelligence as a science and the types of answers that may be given to the question, "What is intelligence?" The paradigms of artificial intelligence and general systems theory are compared. (Author/VT)

  1. Major Histocompatibility Complex Genes Map to Two Chromosomes in an Evolutionarily Ancient Reptile, the Tuatara Sphenodon punctatus

    PubMed Central

    Miller, Hilary C.; O’Meally, Denis; Ezaz, Tariq; Amemiya, Chris; Marshall-Graves, Jennifer A.; Edwards, Scott

    2015-01-01

    Major histocompatibility complex (MHC) genes are a central component of the vertebrate immune system and usually exist in a single genomic region. However, considerable differences in MHC organization and size exist between different vertebrate lineages. Reptiles occupy a key evolutionary position for understanding how variation in MHC structure evolved in vertebrates, but information on the structure of the MHC region in reptiles is limited. In this study, we investigate the organization and cytogenetic location of MHC genes in the tuatara (Sphenodon punctatus), the sole extant representative of the early-diverging reptilian order Rhynchocephalia. Sequencing and mapping of 12 clones containing class I and II MHC genes from a bacterial artificial chromosome library indicated that the core MHC region is located on chromosome 13q. However, duplication and translocation of MHC genes outside of the core region was evident, because additional class I MHC genes were located on chromosome 4p. We found a total of seven class I sequences and 11 class II β sequences, with evidence for duplication and pseudogenization of genes within the tuatara lineage. The tuatara MHC is characterized by high repeat content and low gene density compared with other species and we found no antigen processing or MHC framework genes on the MHC gene-containing clones. Our findings indicate substantial differences in MHC organization in tuatara compared with mammalian and avian MHCs and highlight the dynamic nature of the MHC. Further sequencing and annotation of tuatara and other reptile MHCs will determine if the tuatara MHC is representative of nonavian reptiles in general. PMID:25953959

  2. Major Histocompatibility Complex Genes Map to Two Chromosomes in an Evolutionarily Ancient Reptile, the Tuatara Sphenodon punctatus.

    PubMed

    Miller, Hilary C; O'Meally, Denis; Ezaz, Tariq; Amemiya, Chris; Marshall-Graves, Jennifer A; Edwards, Scott

    2015-05-07

    Major histocompatibility complex (MHC) genes are a central component of the vertebrate immune system and usually exist in a single genomic region. However, considerable differences in MHC organization and size exist between different vertebrate lineages. Reptiles occupy a key evolutionary position for understanding how variation in MHC structure evolved in vertebrates, but information on the structure of the MHC region in reptiles is limited. In this study, we investigate the organization and cytogenetic location of MHC genes in the tuatara (Sphenodon punctatus), the sole extant representative of the early-diverging reptilian order Rhynchocephalia. Sequencing and mapping of 12 clones containing class I and II MHC genes from a bacterial artificial chromosome library indicated that the core MHC region is located on chromosome 13q. However, duplication and translocation of MHC genes outside of the core region was evident, because additional class I MHC genes were located on chromosome 4p. We found a total of seven class I sequences and 11 class II β sequences, with evidence for duplication and pseudogenization of genes within the tuatara lineage. The tuatara MHC is characterized by high repeat content and low gene density compared with other species and we found no antigen processing or MHC framework genes on the MHC gene-containing clones. Our findings indicate substantial differences in MHC organization in tuatara compared with mammalian and avian MHCs and highlight the dynamic nature of the MHC. Further sequencing and annotation of tuatara and other reptile MHCs will determine if the tuatara MHC is representative of nonavian reptiles in general.

  3. Artificial life and Piaget.

    PubMed

    Mueller, Ulrich; Grobman, K H.

    2003-04-01

    Artificial life provides important theoretical and methodological tools for the investigation of Piaget's developmental theory. This new method uses artificial neural networks to simulate living phenomena in a computer. A recent study by Parisi and Schlesinger suggests that artificial life might reinvigorate the Piagetian framework. We contrast artificial life with traditional cognitivist approaches, discuss the role of innateness in development, and examine the relation between physiological and psychological explanations of intelligent behaviour.

  4. Karyotype and identification of all homoeologous chromosomes of allopolyploid Brassica napus and its diploid progenitors.

    PubMed

    Xiong, Zhiyong; Pires, J Chris

    2011-01-01

    Investigating recombination of homoeologous chromosomes in allopolyploid species is central to understanding plant breeding and evolution. However, examining chromosome pairing in the allotetraploid Brassica napus has been hampered by the lack of chromosome-specific molecular probes. In this study, we establish the identification of all homoeologous chromosomes of allopolyploid B. napus by using robust molecular cytogenetic karyotypes developed for the progenitor species Brassica rapa (A genome) and Brassica oleracea (C genome). The identification of every chromosome among these three Brassica species utilized genetically mapped bacterial artificial chromosomes (BACs) from B. rapa as probes for fluorescent in situ hybridization (FISH). With this BAC-FISH data, a second karyotype was developed using two BACs that contained repetitive DNA sequences and the ubiquitous ribosomal and pericentromere repeats. Using this diagnostic probe mix and a BAC that contained a C-genome repeat in two successive hybridizations allowed for routine identification of the corresponding homoeologous chromosomes between the A and C genomes of B. napus. When applied to the B. napus cultivar Stellar, we detected one chromosomal rearrangement relative to the parental karyotypes. This robust novel chromosomal painting technique will have biological applications for the understanding of chromosome pairing, homoeologous recombination, and genome evolution in the genus Brassica and will facilitate new applied breeding technologies that rely upon identification of chromosomes.

  5. Alabama Public Library Service Library Directory and 1996 Statistical Report.

    ERIC Educational Resources Information Center

    Alabama Public Library Service, Montgomery.

    This publication presents library contact information and statistics for Alabama public libraries for fiscal year 1996 (October 1, 1995-September 30, 1996). The library directory is arranged by type of library: public libraries, single-county public library systems, multi-county public library systems, and multitype library systems. Entries…

  6. Chromosomal localization of several families of repetitive sequences by in situ hybridization.

    PubMed Central

    Devine, E A; Nolin, S L; Houck, G E; Jenkins, E C; Brown, W T

    1985-01-01

    Four recombinant DNA clones (H1, H7, H12, and H15) carrying low-repetitive human DNA were previously isolated from a human genomic library based on their specificity for chromosome 21 and were studied for their distribution as determined by in situ hybridization. Clone H7 hybridized to the satellite regions of chromosomes 13, 14, 15, 21, and 22 as well as to the centromere region of chromosome 1. Clone H12 hybridized strongly to chromosomes 11 and 17 and the centromere of the X. Clones H1 and H15 had a very widespread distribution throughout the genome. Clone H15 hybridized significantly more to the short arm of chromosome 18 than to any other chromosomal segment. Clone H1 hybridized strongly to the centromere of chromosome 19 and also showed random distribution on all the other human chromosomes. We conclude that these probes appear to represent four repetitive families that demonstrate in situ hybridization patterns that do not correspond with those of any other repetitive family. Further, the in situ hybridization patterns do not show the strong chromosome 21 specificity originally defined by Southern blot analysis. The nature and chromosomal localization of these repetitive families should be useful in regional mapping and evolutionary studies and give additional insight into chromosomal organization. Images Fig. 1 Fig. 3 PMID:3976654

  7. The Library of Virginia's Digital Library Project.

    ERIC Educational Resources Information Center

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

    1997-01-01

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

  8. Development of a novel HAC-based “gain of signal” quantitative assay for measuring chromosome instability (CIN) in cancer cells

    PubMed Central

    Kim, Jung-Hyun; Lee, Hee-Sheung; Lee, Nicholas C. O.; Goncharov, Nikolay V.; Kumeiko, Vadim; Masumoto, Hiroshi; Earnshaw, William C.; Kouprina, Natalay; Larionov, Vladimir

    2016-01-01

    Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene (“loss of signal” assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this “loss of signal” assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this “gain of signal” assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The “gain of signal” assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level. PMID:26943579

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

    PubMed

    Wolny, Elzbieta; Fidyk, Wojciech; Hasterok, Robert

    2013-04-01

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

  10. Vertical integration of cosmid and YAC resources for interval mapping on the X-chromosome

    SciTech Connect

    Holland, J.; Coffey, A.J.; Giannelli, F.; Bentley, D.R. )

    1993-02-01

    The vertical integration of cosmid and yeast artificial chromosome (YAC) resources is of particular importance in the development of high-resolution maps of selected regions of the human genome. A resource of approximately 95,000 cosmids constructed using DNA from primary fibroblasts of karyotype 49,XXXXX was validated by detailed characterization of a 200-kb cosmid contig spanning exons 8-20 of the dystrophin gene. This resource was used to construct contigs in 0.65 Mb of Xq26 by hybridization of gel-purified YAC DNA to high-density gridded arrays of the cosmid library; positive cosmids were overlapped by fingerprinting. Contigs were oriented and ordered relative to existing YACs in the region using cross-hybridization. The overlaps between a representative set of cosmids define 54 intervals of 5-20 kb and were used to construct a high-resolution cosmid interval map of the region, locating markers, dinucleotide repeats, and candidate CpG islands. This approach can be applied rapidly to large regions of the genome and without recourse to subcloning of individual YACs. 49 refs., 5 figs.

  11. Prison Libraries Inside Out.

    ERIC Educational Resources Information Center

    Singer, Glen

    2000-01-01

    Discussion of prison libraries provides an inside look at the correctional institution environment, prison security concerns, inmate patrons and library use, library collections and services, librarians and staff, the day-to-day operation of a prison library, and future possibilities and needs. (Author/LRW)

  12. Inside Prison Libraries.

    ERIC Educational Resources Information Center

    Vogel, Brenda; And Others

    1989-01-01

    Issues related to prison libraries are discussed in six articles. Topics covered include the history of American penitentiary ideology; standards for prison libraries; the controversy as to whether prison libraries should serve prisoners or be used as penological tools; and the lack of knowledge about prison libraries within the general library…

  13. Libraries, Ebooks, and Competition

    ERIC Educational Resources Information Center

    Hellman, Eric

    2010-01-01

    People keep writing articles about how valuable libraries are, even with ebooks and the Internet. What people are overlooking is that the reason libraries are having such fits dealing with a changing environment is not that libraries are unrecognized as fountains of value, it's that libraries are so valuable that they attract voracious new…

  14. Alaska Library Directory, 1996.

    ERIC Educational Resources Information Center

    Jennings, Mary, Ed.

    This directory of Alaska's Libraries lists: members of the Alaska Library Association (AkLA) Executive Council and Committee Chairs; State Board of Education members; members of the Governor's Advisory Council on Libraries; school, academic and public libraries and their addresses, phone and fax numbers, and contact persons; personal,…

  15. School Libraries in Hawaii.

    ERIC Educational Resources Information Center

    Bard, Therese Bissen

    This paper outlines the history, functions, administration, and current focus of school library services in Hawaii, which is the only state in the United States with a library staffed by a trained librarian in every public school. Its first school library was established in 1882. Elementary school libraries developed concurrently with secondary…

  16. California: Library Information Technologies.

    ERIC Educational Resources Information Center

    Will, Barbara, Ed.

    1996-01-01

    Describes six information technology projects in California libraries, including Internet access in public libraries; digital library developments at the University of California, Berkeley; the World Wide Web home page for the state library; Pacific Bell's role in statewide connectivity; state government initiatives; and services of the state…

  17. College and University Libraries.

    ERIC Educational Resources Information Center

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

    1986-01-01

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

  18. AMERICA 2000 Library Partnership.

    ERIC Educational Resources Information Center

    Office of Educational Research and Improvement (ED), Washington, DC.

    The United States Department of Education, the National Endowment for the Humanities, the Library of Congress, the National Commission on Libraries and Information Science, and the National Institute for Literacy have formed the AMERICA 2000 Library Partnership to support libraries in their work toward the six National Education Goals announced by…

  19. Conservation of Library Materials.

    ERIC Educational Resources Information Center

    Illinois Libraries, 1985

    1985-01-01

    Twelve articles cover books as artifacts; workstations for conservation of library materials; care of scrapbooks, albums, and photographs; map preservation; library environment; flood recovery; disaster prevention and preparedness; incorporating preservation into library organization; and bibliography of Chester Public Library (Illinois) First…

  20. Special Library Services.

    ERIC Educational Resources Information Center

    Ensley, Robert F., Ed.

    1975-01-01

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

  1. Growing Competition for Libraries.

    ERIC Educational Resources Information Center

    Gibbons, Susan

    2001-01-01

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

  2. Economics of Academic Libraries.

    ERIC Educational Resources Information Center

    Baumol, William J.; Marcus, Matityahu

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

  3. Facility Focus: Libraries.

    ERIC Educational Resources Information Center

    College Planning & Management, 2002

    2002-01-01

    Describes the design of the Charles V. Park Library at Central Michigan University and the Martha Rivers and E. Bronson Ingram Library, an addition to the Frederick Ferris Thompson Memorial Library at Vassar College. Discusses the libraries as examples of merging tradition with technology. Includes photographs. (EV)

  4. Idaho Library Trustee Manual.

    ERIC Educational Resources Information Center

    Idaho State Library, Boise.

    The public library board of trustees is an example of the great democratic tradition of giving authority for the operation of public agencies to members of the general public. The public library board is legally responsible for overseeing the efficient and effective operation of the public library. The board hires the library's administrator,…

  5. The gene for human glutaredoxin (GLRX) is localized to human chromosome 5q14

    SciTech Connect

    Padilla, C.A.; Holmgren, A.; Bajalica, S.; Lagercrantz, J.

    1996-03-05

    Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescence in situ hybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human-hamster and a human-mouse hybrid panel and using a human glutaredoxin cDNA as a probe. 13 refs., 2 figs.

  6. The Y Chromosome

    ERIC Educational Resources Information Center

    Offner, Susan

    2010-01-01

    The Y chromosome is of great interest to students and can be used to teach about many important biological concepts in addition to sex determination. This paper discusses mutation, recombination, mammalian sex determination, sex determination in general, and the evolution of sex determination in mammals. It includes a student activity that…

  7. Why Chromosome Palindromes?

    PubMed Central

    Betrán, Esther; Demuth, Jeffery P.; Williford, Anna

    2012-01-01

    We look at sex-limited chromosome (Y or W) evolution with particular emphasis on the importance of palindromes. Y chromosome palindromes consist of inverted duplicates that allow for local recombination in an otherwise nonrecombining chromosome. Since palindromes enable intrachromosomal gene conversion that can help eliminate deleterious mutations, they are often highlighted as mechanisms to protect against Y degeneration. However, the adaptive significance of recombination resides in its ability to decouple the evolutionary fates of linked mutations, leading to both a decrease in degeneration rate and an increase in adaptation rate. Our paper emphasizes the latter, that palindromes may exist to accelerate adaptation by increasing the potential targets and fixation rates of incoming beneficial mutations. This hypothesis helps reconcile two enigmatic features of the “palindromes as protectors” view: (1) genes that are not located in palindromes have been retained under purifying selection for tens of millions of years, and (2) under models that only consider deleterious mutations, gene conversion benefits duplicate gene maintenance but not initial fixation. We conclude by looking at ways to test the hypothesis that palindromes enhance the rate of adaptive evolution of Y-linked genes and whether this effect can be extended to palindromes on other chromosomes. PMID:22844637

  8. Integration of genetic, physical, and cytogenetic maps for Brassica rapa chromosome A7.

    PubMed

    Xiong, Z; Kim, J S; Pires, J C

    2010-07-01

    Bacterial artificial chromosome (BAC) contigs have been genetically mapped to the 10 linkage groups of Brassica rapa by BAC end sequences (BES). To integrate the genetic, physical, and cytogenetic maps, fluorescence in situ hybridization (FISH) was used to anchor the assembly of BAC contigs onto Brassica chromosomes using representative BACs. This BAC-FISH approach can be used to identify chromosome arms on separate mitotic metaphase chromosomes or to map multiple BACs to single long pachytene chromosomes. As part of an international consortium that is sequencing the B. rapa genome, we integrated the linkage and physical maps with the B. rapa cytogenetic map for chromosome A7 by hybridizing BACs to mitotic chromosomes and along the length of pachytene chromosome spreads. A total of 31 BACs that were putatively located on A7 were used as probes for FISH analyses; however, only 19 BACs mapped unambiguously to A7 while the remaining BACs either mapped to other chromosomes or hybridized to multiple locations. We then created a multicolor FISH cocktail of 16 BAC probes to simultaneously hybridize the entire length of the A7 chromosome. We successfully applied the 16 A7 BAC probe mix to B. rapa, B. oleracea, and domesticated and resynthesized genotypes of B. napus to demonstrate that this approach can facilitate studies of genome evolution by integrating the genetic, physical, and cytogenetic maps among closely related species of Brassica.

  9. Chromatin Folding, Fragile Sites, and Chromosome Aberrations Induced by Low- and High- LET Radiation

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Cox, Bradley; Asaithamby, Aroumougame; Chen, David J.; Wu, Honglu

    2013-01-01

    We previously demonstrated non-random distributions of breaks involved in chromosome aberrations induced by low- and high-LET radiation. To investigate the factors contributing to the break point distribution in radiation-induced chromosome aberrations, human epithelial cells were fixed in G1 phase. Interphase chromosomes were hybridized with a multicolor banding in situ hybridization (mBAND) probe for chromosome 3 which distinguishes six regions of the chromosome in separate colors. After the images were captured with a laser scanning confocal microscope, the 3-dimensional structure of interphase chromosome 3 was reconstructed at multimega base pair scale. Specific locations of the chromosome, in interphase, were also analyzed with bacterial artificial chromosome (BAC) probes. Both mBAND and BAC studies revealed non-random folding of chromatin in interphase, and suggested association of interphase chromatin folding to the radiation-induced chromosome aberration hotspots. We further investigated the distribution of genes, as well as the distribution of breaks found in tumor cells. Comparisons of these distributions to the radiation hotspots showed that some of the radiation hotspots coincide with the frequent breaks found in solid tumors and with the fragile sites for other environmental toxins. Our results suggest that multiple factors, including the chromatin structure and the gene distribution, can contribute to radiation-induced chromosome aberrations.

  10. Structural and functional partitioning of bread wheat chromosome 3B.

    PubMed

    Choulet, Frédéric; Alberti, Adriana; Theil, Sébastien; Glover, Natasha; Barbe, Valérie; Daron, Josquin; Pingault, Lise; Sourdille, Pierre; Couloux, Arnaud; Paux, Etienne; Leroy, Philippe; Mangenot, Sophie; Guilhot, Nicolas; Le Gouis, Jacques; Balfourier, Francois; Alaux, Michael; Jamilloux, Véronique; Poulain, Julie; Durand, Céline; Bellec, Arnaud; Gaspin, Christine; Safar, Jan; Dolezel, Jaroslav; Rogers, Jane; Vandepoele, Klaas; Aury, Jean-Marc; Mayer, Klaus; Berges, Hélène; Quesneville, Hadi; Wincker, Patrick; Feuillet, Catherine

    2014-07-18

    We produced a reference sequence of the 1-gigabase chromosome 3B of hexaploid bread wheat. By sequencing 8452 bacterial artificial chromosomes in pools, we assembled a sequence of 774 megabases carrying 5326 protein-coding genes, 1938 pseudogenes, and 85% of transposable elements. The distribution of structural and functional features along the chromosome revealed partitioning correlated with meiotic recombination. Comparative analyses indicated high wheat-specific inter- and intrachromosomal gene duplication activities that are potential sources of variability for adaption. In addition to providing a better understanding of the organization, function, and evolution of a large and polyploid genome, the availability of a high-quality sequence anchored to genetic maps will accelerate the identification of genes underlying important agronomic traits. PMID:25035497

  11. Genetic association between chromosome 8 microsatellite (MS8-134) and Werner syndrome (WRN): Chromosome microdissection and homozygosity mapping

    SciTech Connect

    Ye, Lin; Nakura, Jun; Mitsuda, Noriaki; Miki, Tetsuro

    1995-08-10

    Werner syndrome (WRN) is an autosomal recessive disorder characterized by premature aging that has been mapped to the short arm of chromosome 8, 8p11.2-p12. To refine the genetic map around the WRN region, we have isolated eight microsatellites for this region from a microdissection library. We typed members of Japanese families with WRN on the basis of homozygosity mapping analysis. There was no obligate recombination between the WRN locus and microsatellite clone, MS8-134 (D8S1055). The maximum lod score was 20.28 at {theta} = 0.00. Alleles for MS8-134 showed association with WRN in a case-control study (OR = 3.55, 95% CI 1.56-8.07, P < 0.01). Such microsatellites from a microdissection library of the definite chromosome region may be useful for positional cloning of the WRN gene. 23 refs., 1 figs., 1 tab.

  12. Mitotic chromosome structure and condensation.

    PubMed

    Belmont, Andrew S

    2006-12-01

    Mitotic chromosome structure has been the cell biology equivalent of a 'riddle, wrapped in a mystery, inside an enigma'. Observations that genetic knockout or knockdown of condensin subunits or topoisomerase II cause only minimal perturbation in overall chromosome condensation, together with analysis of early stages of chromosome condensation and effects produced by histone H1 depletion, suggest a need to reconsider textbook models of mitotic chromosome condensation and organization. PMID:17046228

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

    ERIC Educational Resources Information Center

    Lyons, Ray; Lance, Keith Curry

    2010-01-01

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

  14. Laser microdissection-based analysis of the Y sex chromosome of the Antarctic fish Chionodracohamatus (Notothenioidei, Channichthyidae).

    PubMed

    Cocca, Ennio; Petraccioli, Agnese; Morescalchi, Maria Alessandra; Odierna, Gaetano; Capriglione, Teresa

    2015-01-01

    Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodracohamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Chionodracohamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Chionodracohamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action.

  15. Laser microdissection-based analysis of the Y sex chromosome of the Antarctic fish Chionodraco hamatus (Notothenioidei, Channichthyidae)

    PubMed Central

    Cocca, Ennio; Petraccioli, Agnese; Morescalchi, Maria Alessandra; Odierna, Gaetano; Capriglione, Teresa

    2015-01-01

    Abstract Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodraco hamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Chionodraco hamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Chionodraco hamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action. PMID:25893071

  16. Genetic mapping of the branchio-oto-renal syndrome and construction of YAC contig spanning the BOR region on chromosome 8q

    SciTech Connect

    Kumar, S.; Kimberling, W.J.; Bumegi, J.

    1994-09-01

    Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder which consists of external, middle and inner ear malformations, branchial cleft sinuses, cervical fistulas, mixed hearing loss and renal anomalies. The prevalence of BOR syndrome is approximately 1:40,000, and it has been reported to occur in about 2% of profoundly deaf children. The BOR syndrome has been localized to chromosome 8q. Initial localization results indicated a distance of about 15 cM between the flanking markers D8S87 and PENK for the BOR gene. This localization has been further refined, using new markers, to a distance of about 7 cM. The multipoint analysis was carried out using markers D8S285, PENK, D8S166, D8S260, D8S510, D8S553, D8S543, D8S530, D8S279, D8S164, D8S286 and D8S275. For cloning the BOR gene, an overlapping Yeast Artificial Chromosome (YAC) contig map of the critical region is being constructed. We have isolated eight YACs from the CEPH Mega YAC library and their size and quality are being characterized by PFGE and FISH analysis. Additional STSs and polymorphic markers developed from the region will be used to further refine the region and close the contig. The availability of this contig will be a useful resource for the systematic search for identifying transcribed sequences from this region.

  17. Physical organization of mixed protease inhibitor gene clusters, coordinated expression and association with resistance to late blight at the StKI locus on potato chromosome III.

    PubMed

    Odeny, Damaris Achieng; Stich, Benjamin; Gebhardt, Christiane

    2010-12-01

    Protease inhibitors (PIs) play a role in plant defence against pests and pathogens as well as in plant development. Potato (Solanum tuberosum) contains abundant levels of diverse PIs. Most potato Kunitz-type inhibitor (KTI) genes map to the StKI locus on potato chromosome III, which is linked to a quantitative trait locus (QTL) for resistance to Phytophthora infestans. To elucidate the physical organization of PIs at the StKI locus, we screened bacterial artificial chromosome (BAC) libraries with KTI probes. Ten different clones were selected, sequenced and annotated. Of 100 putative genes, 22 corresponded to five PI classes. Expression analysis by quantitative real-time PCR (qRT-PCR) using PI class-specific primers in different tissues of the tetraploid potato cultivars 'Nikita' and 'Baltica' revealed different transcript levels, depending on PI type and genotype. During the compatible interaction with a complex race of P. infestans, four PI classes showed coordinated expression over 3 d after infection, a strong decrease in infected leaves and a transient induction in systemic leaves. Basal transcript levels in non-infected leaves differed strongly between the two genotypes examined. Two microsatellite markers located within the PI gene cluster were associated with resistance to P. infestans in a population of potato varieties and breeding clones. PMID:20716067

  18. Chromosomal breakpoints characterization of two supernumerary ring chromosomes 20.

    PubMed

    Guediche, N; Brisset, S; Benichou, J-J; Guérin, N; Mabboux, P; Maurin, M-L; Bas, C; Laroudie, M; Picone, O; Goldszmidt, D; Prévot, S; Labrune, P; Tachdjian, G

    2010-02-01

    The occurrence of an additional ring chromosome 20 is a rare chromosome abnormality, and no common phenotype has been yet described. We report on two new patients presenting with a supernumerary ring chromosome 20 both prenatally diagnosed. The first presented with intrauterine growth retardation and some craniofacial dysmorphism, and the second case had a normal phenotype except for obesity. Conventional cytogenetic studies showed for each patient a small supernumerary marker chromosome (SMC). Using fluorescence in situ hybridization, these SMCs corresponded to ring chromosomes 20 including a part of short and long arms of chromosome 20. Detailed molecular cytogenetic characterization showed different breakpoints (20p11.23 and 20q11.23 for Patient 1 and 20p11.21 and 20q11.21 for Patient 2) and sizes of the two ring chromosomes 20 (13.6 Mb for case 1 and 4.8 Mb for case 2). Review of the 13 case reports of an extra r(20) ascertained postnatally (8 cases) and prenatally (5 cases) showed varying degrees of phenotypic abnormalities. We document a detailed molecular cytogenetic chromosomal breakpoints characterization of two cases of supernumerary ring chromosomes 20. These results emphasize the need to characterize precisely chromosomal breakpoints of supernumerary ring chromosomes 20 in order to establish genotype-phenotype correlation. This report may be helpful for prediction of natural history and outcome, particularly in prenatal diagnosis.

  19. Familial complex chromosomal rearrangement resulting in a recombinant chromosome.

    PubMed

    Berend, Sue Ann; Bodamer, Olaf A F; Shapira, Stuart K; Shaffer, Lisa G; Bacino, Carlos A

    2002-05-15

    Familial complex chromosomal rearrangements (CCRs) are rare and tend to involve fewer breakpoints and fewer chromosomes than CCRs that are de novo in origin. We report on a CCR identified in a child with congenital heart disease and dysmorphic features. Initially, the child's karyotype was thought to involve a straightforward three-way translocation between chromosomes 3, 8, and 16. However, after analyzing the mother's chromosomes, the mother was found to have a more complex rearrangement that resulted in a recombinant chromosome in the child. The mother's karyotype included an inverted chromosome 2 and multiple translocations involving chromosomes 3, 5, 8, and 16. No evidence of deletion or duplication that could account for the clinical findings in the child was identified.

  20. An artificial muscle computer

    NASA Astrophysics Data System (ADS)

    Marc O'Brien, Benjamin; Alexander Anderson, Iain

    2013-03-01

    We have built an artificial muscle computer based on Wolfram's "2, 3" Turing machine architecture, the simplest known universal Turing machine. Our computer uses artificial muscles for its instruction set, output buffers, and memory write and addressing mechanisms. The computer is very slow and large (0.15 Hz, ˜1 m3); however by using only 13 artificial muscle relays, it is capable of solving any computable problem given sufficient memory, time, and reliability. The development of this computer shows that artificial muscles can think—paving the way for soft robots with reflexes like those seen in nature.

  1. The B chromosomes of the African cichlid fish Haplochromis obliquidens harbour 18S rRNA gene copies

    PubMed Central

    2010-01-01

    Background Diverse plant and animal species have B chromosomes, also known as accessory, extra or supernumerary chromosomes. Despite being widely distributed among different taxa, the genomic nature and genetic behavior of B chromosomes are still poorly understood. Results In this study we describe the occurrence of B chromosomes in the African cichlid fish Haplochromis obliquidens. One or two large B chromosome(s) occurring in 39.6% of the analyzed individuals (both male and female) were identified. To better characterize the karyotype and assess the nature of the B chromosomes, fluorescence in situ hybridization (FISH) was performed using probes for telomeric DNA repeats, 18S and 5S rRNA genes, SATA centromeric satellites, and bacterial artificial chromosomes (BACs) enriched in repeated DNA sequences. The B chromosomes are enriched in repeated DNAs, especially non-active 18S rRNA gene-like sequences. Conclusion Our results suggest that the B chromosome could have originated from rDNA bearing subtelo/acrocentric A chromosomes through formation of an isochromosome, or by accumulation of repeated DNAs and rRNA gene-like sequences in a small proto-B chromosome derived from the A complement. PMID:20051104

  2. Degeneration of a Nonrecombining Chromosome

    NASA Astrophysics Data System (ADS)

    Rice, William R.

    1994-01-01

    Comparative studies suggest that sex chromosomes begin as ordinary autosomes that happen to carry a major sex determining locus. Over evolutionary time the Y chromosome is selected to stop recombining with the X chromosome, perhaps in response to accumulation of alleles beneficial to the heterogametic but harmful to the homogametic sex. Population genetic theory predicts that a nonrecombining Y chromosome should degenerate. Here this prediction is tested by application of specific selection pressures to Drosophila melanogaster populations. Results demonstrate the decay of a nonrecombining, nascent Y chromosome and the capacity for recombination to ameliorate such decay.

  3. Summer library reading programs.

    PubMed

    Fiore, Carole D

    2007-01-01

    Virtually all public libraries in the United States provide some type of summer library reading program during the traditional summer vacation period. Summer library reading programs provide opportunities for students of many ages and abilities to practice their reading skills and maintain skills that are developed during the school year. Fiore summarizes some of the research in the field and relates it to library programs and usage by students. Several traditional and innovative programs from U.S. and Canadian libraries are described. She concludes with a call for further research related to summer library reading programs.

  4. The chromosome cycle of prokaryotes.

    PubMed

    Kuzminov, Andrei

    2013-10-01

    In both eukaryotes and prokaryotes, chromosomal DNA undergoes replication, condensation-decondensation and segregation, sequentially, in some fixed order. Other conditions, like sister-chromatid cohesion (SCC), may span several chromosomal events. One set of these chromosomal transactions within a single cell cycle constitutes the 'chromosome cycle'. For many years it was generally assumed that the prokaryotic chromosome cycle follows major phases of the eukaryotic one: -replication-condensation-segregation-(cell division)-decondensation-, with SCC of unspecified length. Eventually it became evident that, in contrast to the strictly consecutive chromosome cycle of eukaryotes, all stages of the prokaryotic chromosome cycle run concurrently. Thus, prokaryotes practice 'progressive' chromosome segregation separated from replication by a brief SCC, and all three transactions move along the chromosome at the same fast rate. In other words, in addition to replication forks, there are 'segregation forks' in prokaryotic chromosomes. Moreover, the bulk of prokaryotic DNA outside the replication-segregation transition stays compacted. I consider possible origins of this concurrent replication-segregation and outline the 'nucleoid administration' system that organizes the dynamic part of the prokaryotic chromosome cycle.

  5. Spatial control of chromosomal location in a live cell with functionalized magnetic particles

    NASA Astrophysics Data System (ADS)

    Hong, Juhee; Purwar, Prashant; Cha, Misun; Lee, Junghoon

    2015-11-01

    Long-range chromosomal travel is a phenomenon unique to cell division. Methods for non-invasive, artificial manipulation of chromosomes, such as optical or magnetic tweezers, have difficulty in producing the motion of whole chromosomes in live cells. Here, we report the spatial control of chromosomes over 10 μm in a live mouse oocyte using magnetic particles driven by an external magnetic field. Selective capture of the chromosomes was achieved using antibodies specific for histone H1 in the chromosome that were conjugated to magnetic particles (H1-BMPs). When an external magnetic field was applied, the chromosomes captured by the H1-BMPs traveled through the cytosol and accumulated near the cell membrane though the movement of the chromosomes captured by H1-BMPs was strongly disturbed by the distribution of the cytoskeleton (e.g. actin filaments). Being non-invasive in nature, our approach will enable new opportunities in the remote manipulation of subcellular elements.Long-range chromosomal travel is a phenomenon unique to cell division. Methods for non-invasive, artificial manipulation of chromosomes, such as optical or magnetic tweezers, have difficulty in producing the motion of whole chromosomes in live cells. Here, we report the spatial control of chromosomes over 10 μm in a live mouse oocyte using magnetic particles driven by an external magnetic field. Selective capture of the chromosomes was achieved using antibodies specific for histone H1 in the chromosome that were conjugated to magnetic particles (H1-BMPs). When an external magnetic field was applied, the chromosomes captured by the H1-BMPs traveled through the cytosol and accumulated near the cell membrane though the movement of the chromosomes captured by H1-BMPs was strongly disturbed by the distribution of the cytoskeleton (e.g. actin filaments). Being non-invasive in nature, our approach will enable new opportunities in the remote manipulation of subcellular elements. Electronic supplementary

  6. Chromosome 19 International Workshop

    SciTech Connect

    Pericak-Vance, M.A. . Medical Center); Ropers, H.H. . Dept. of Human Genetics); Carrano, A.J. )

    1993-01-04

    The Second International Workshop on Human Chromosome 19 was hosted on January 25 and 26, 1992, by the Department of Human Genetics, University Hospital Nijmegen, The Netherlands, at the 'Meerdal Conference Center'. The workshop was supported by a grant from the European Community obtained through HUGO, the Dutch Research Organization (NWO) and the Muscular Dystrophy Association (MDA). Travel support for American participants was provided by the Department of Energy. The goals of this workshop were to produce genetic, physical and integrated maps of chromosome 19, to identify inconsistencies and gaps, and to discuss and exchange resources and techniques available for the completion of these maps. The second day of the meeting was largely devoted to region or disease specific efforts. In particular, the meeting served as a platform for assessing and discussing the recent progress made into the molecular elucidation of myotonic dystrophy.

  7. Chromosomal evolution in Rodentia.

    PubMed

    Romanenko, S A; Perelman, P L; Trifonov, V A; Graphodatsky, A S

    2012-01-01

    Rodentia is the most species-rich mammalian order and includes several important laboratory model species. The amount of new information on karyotypic and phylogenetic relations within and among rodent taxa is rapidly increasing, but a synthesis of these data is currently lacking. Here, we have integrated information drawn from conventional banding studies, recent comparative painting investigations and molecular phylogenetic reconstructions of different rodent taxa. This permitted a revision of several ancestral karyotypic reconstructions, and a more accurate depiction of rodent chromosomal evolution.

  8. Chromosome abnormalities in glioma

    SciTech Connect

    Li, Y.S.; Ramsay, D.A.; Fan, Y.S.

    1994-09-01

    Cytogenetic studies were performed in 25 patients with gliomas. An interesting finding was a seemingly identical abnormality, an extra band on the tip of the short arm of chromosome 1, add(1)(p36), in two cases. The abnormality was present in all cells from a patient with a glioblastoma and in 27% of the tumor cells from a patient with a recurrent irradiated anaplastic astrocytoma; in the latter case, 7 unrelated abnormal clones were identified except 4 of those clones shared a common change, -Y. Three similar cases have been described previously. In a patient with pleomorphic astrocytoma, the band 1q42 in both homologues of chromosome 1 was involved in two different rearrangements. A review of the literature revealed that deletion of the long arm of chromosome 1 including 1q42 often occurs in glioma. This may indicate a possible tumor suppressor gene in this region. Cytogenetic follow-up studies were carried out in two patients and emergence of unrelated clones were noted in both. A total of 124 clonal breakpoints were identified in the 25 patients. The breakpoints which occurred three times or more were: 1p36, 1p22, 1q21, 1q25, 3q21, 7q32, 8q22, 9q22, 16q22, and 22q13.

  9. Interpreting Chromosome Aberration Spectra

    NASA Technical Reports Server (NTRS)

    Levy, Dan; Reeder, Christopher; Loucas, Bradford; Hlatky, Lynn; Chen, Allen; Cornforth, Michael; Sachs, Rainer

    2007-01-01

    Ionizing radiation can damage cells by breaking both strands of DNA in multiple locations, essentially cutting chromosomes into pieces. The cell has enzymatic mechanisms to repair such breaks; however, these mechanisms are imperfect and, in an exchange process, may produce a large-scale rearrangement of the genome, called a chromosome aberration. Chromosome aberrations are important in killing cells, during carcinogenesis, in characterizing repair/misrepair pathways, in retrospective radiation biodosimetry, and in a number of other ways. DNA staining techniques such as mFISH ( multicolor fluorescent in situ hybridization) provide a means for analyzing aberration spectra by examining observed final patterns. Unfortunately, an mFISH observed final pattern often does not uniquely determine the underlying exchange process. Further, resolution limitations in the painting protocol sometimes lead to apparently incomplete final patterns. We here describe an algorithm for systematically finding exchange processes consistent with any observed final pattern. This algorithm uses aberration multigraphs, a mathematical formalism that links the various aspects of aberration formation. By applying a measure to the space of consistent multigraphs, we will show how to generate model-specific distributions of aberration processes from mFISH experimental data. The approach is implemented by software freely available over the internet. As a sample application, we apply these algorithms to an aberration data set, obtaining a distribution of exchange cycle sizes, which serves to measure aberration complexity. Estimating complexity, in turn, helps indicate how damaging the aberrations are and may facilitate identification of radiation type in retrospective biodosimetry.

  10. Correctional Libraries, Library Standards, and Diversity.

    ERIC Educational Resources Information Center

    Shirley, Glennor L.

    2003-01-01

    Respondents to a survey of prison librarians (n=35) regarding how they cope with demands for materials and services related to diversity issues felt that adherence to Library Standards for Adult Correctional Institutions was often impeded by security concerns. Racial differences between library providers and prisoners was not considered relevant.…

  11. Art Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

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

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

  12. Administrative Libraries Section. Special Libraries Division. Papers.

    ERIC Educational Resources Information Center

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

    Six papers on the theme, "Administrative Libraries in a Technological World: Past, Present, and Future--A General Stocktaking and a State of the Art Report" focus on West German telecommunications and information technology and the use of data processing and other technological aids in West German administrative libraries. These papers, which were…

  13. Artificial insemination in poultry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Artificial insemination is a relative simple yet powerful tool geneticists can employ for the propagation of economically important traits in livestock and poultry. In this chapter, we address the fundamental methods of the artificial insemination of poultry, including semen collection, semen evalu...

  14. Artificial intelligence: Recent developments

    SciTech Connect

    Not Available

    1987-01-01

    This book presents the papers given at a conference on artificial intelligence. Topics considered at the conference included knowledge representation for expert systems, the use of robots in underwater vehicles for resource management, precision logic, an expert system for arc welding, data base management, a knowledge based approach to fault trees, and computer-aided manufacturing using simulation combined with artificial intelligence.

  15. Origin of human chromosome 2: An ancestral telomere-telomere fusion

    SciTech Connect

    Ijdo, J.W.; Baldini, A.; Ward, D.C.; Reeders, S.T.; Wells, R.A. )

    1991-10-15

    The authors identified two allelic genomic cosmids from human chromosome 2, c8.1 and c29B, each containing two inverted arrays of the vertebrate telomeric repeat in a head-to-head arrangement, 5{prime}(TTAGGG){sub n}-(CCCTAA){sub m}3{prime}. Sequences flanking this telomeric repeat are characteristic of present-day human pretelomeres. BAL-31 nuclease experiments with yeast artificial chromosome clones of human telomeres and fluorescence in situ hybridization reveal that sequences flanking these inverted repeats hybridize both to band 2q13 and to different, but overlapping, subsets of human chromosome ends. They conclude that the locus cloned in cosmids c8.1 and c29B is the relic of an ancient telomere-telomere fusion and marks the point at which two ancestral ape chromosomes fused to give rise to human chromosome 2.

  16. Assignment of Chinook salmon (Oncorhynchus tshawytscha) linkage groups to specific chromosomes reveals a karyotype with multiple rearrangements of the chromosome arms of rainbow trout (Oncorhynchus mykiss).

    PubMed

    Phillips, Ruth B; Park, Linda K; Naish, Kerry A

    2013-12-09

    The Chinook salmon genetic linkage groups have been assigned to specific chromosomes using fluorescence in situ hybridization with bacterial artificial chromosome probes containing genetic markers mapped to each linkage group in Chinook salmon and rainbow trout. Comparison of the Chinook salmon chromosome map with that of rainbow trout provides strong evidence for conservation of large syntenic blocks in these species, corresponding to entire chromosome arms in the rainbow trout as expected. In almost every case, the markers were found at approximately the same location on the chromosome arm in each species, suggesting conservation of marker order on the chromosome arms of the two species in most cases. Although theoretically a few centric fissions could convert the karyotype of rainbow trout (2N = 58-64) into that of Chinook salmon (2N = 68) or vice versa, our data suggest that chromosome arms underwent multiple centric fissions and subsequent new centric fusions to form the current karyotypes. The morphology of only approximately one-third of the chromosome pairs have been conserved between the two species.

  17. Onion artificial muscles

    NASA Astrophysics Data System (ADS)

    Chen, Chien-Chun; Shih, Wen-Pin; Chang, Pei-Zen; Lai, Hsi-Mei; Chang, Shing-Yun; Huang, Pin-Chun; Jeng, Huai-An

    2015-05-01

    Artificial muscles are soft actuators with the capability of either bending or contraction/elongation subjected to external stimulation. However, there are currently no artificial muscles that can accomplish these actions simultaneously. We found that the single layered, latticed microstructure of onion epidermal cells after acid treatment became elastic and could simultaneously stretch and bend when an electric field was applied. By modulating the magnitude of the voltage, the artificial muscle made of onion epidermal cells would deflect in opposing directions while either contracting or elongating. At voltages of 0-50 V, the artificial muscle elongated and had a maximum deflection of -30 μm; at voltages of 50-1000 V, the artificial muscle contracted and deflected 1.0 mm. The maximum force response is 20 μN at 1000 V.

  18. Israeli Special Libraries

    ERIC Educational Resources Information Center

    Foster, Barbara

    1974-01-01

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

  19. National Library of Medicine

    MedlinePlus

    ... ideas about NLM's third century, the future of big data, and the role of libraries in supporting research ... ideas about NLM's third century, the future of big data, and the role of libraries in supporting research ...

  20. Facility Focus: Libraries.

    ERIC Educational Resources Information Center

    College Planning & Management, 2003

    2003-01-01

    Describes the designs of the Ferris State University Library for Information, Technology and Education (FLITE), and the Meyer Library and Information Technology Center at Southwest Missouri State University. Includes photographs. (EV)

  1. Reutilizing Existing Library Space.

    ERIC Educational Resources Information Center

    Davis, Marlys Cresap

    1987-01-01

    This discussion of the reutilization of existing library space reviews the decision process and considerations for implementation. Two case studies of small public libraries which reassigned space to better use are provided, including floor plans. (1 reference) (MES)

  2. Selecting Library Furniture & Equipment.

    ERIC Educational Resources Information Center

    Media & Methods, 1997

    1997-01-01

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

  3. Mitotic chromosome length scales in response to both cell and nuclear size

    PubMed Central

    Ladouceur, Anne-Marie; Dorn, Jonas F.

    2015-01-01

    Multicellular development requires that cells reduce in size as a result of consecutive cell divisions without increase in embryo volume. To maintain cellular integrity, organelle size adapts to cell size throughout development. During mitosis, the longest chromosome arm must be shorter than half of the mitotic spindle for proper chromosome segregation. Using high-resolution time-lapse microscopy of living Caenorhabditis elegans embryos, we have quantified the relation between cell size and chromosome length. In control embryos, chromosome length scaled to cell size. Artificial reduction of cell size resulted in a shortening of chromosome length, following a trend predicted by measurements from control embryos. Disturbing the RAN (Ras-related nuclear protein)-GTP gradient decoupled nuclear size from cell size and resulted in chromosome scaling to nuclear size rather than cell size; smaller nuclei contained shorter chromosomes independent of cell size. In sum, quantitative analysis relating cell, nuclear, and chromosome size predicts two levels of chromosome length regulation: one through cell size and a second in response to nuclear size. PMID:26033258

  4. Protein engineering with artificial chemical nucleases.

    PubMed

    Larragy, Ruth; Fitzgerald, Jenny; Prisecaru, Andreea; McKee, Vickie; Leonard, Paul; Kellett, Andrew

    2015-08-21

    Herein we report the application of oxidative artificial chemical nucleases as novel agents for protein engineering. The complex ion [Cu(Phen)2(H2O)](2+) (CuPhen; Phen = 1,10-phenanthroline) was applied under Fenton-type conditions against a recombinant antibody fragment specific for prostate-specific antigen (PSA) and compared against traditional DNA shuffling using DNase I for the generation of recombinant mutagenesis libraries. We show that digestion and re-annealment of single chain variable fragment (scFv) coding DNA is possible using CuPhen. Results indicate recombinant library generation in this manner may generate novel clones—not accessible through the use of DNase I—with CuPhen producing highly PSA-specific binding antibodies identified by surface plasmon resonance.

  5. Artificial evolution with adeno-associated viral libraries.

    PubMed

    Perabo, Luca; Huber, Anke; Märsch, Stephan; Hallek, Michael; Büning, Hildegard

    2008-02-01

    After attracting the attention of the scientific community due to a number of favourable characteristics that make it an attractive vector for human gene therapy [1,2], AAV has been thoroughly investigated in the past two decades. Standard technologies for the manipulation of the viral genome and for efficient packaging and purification protocols have paved the road for trial and error manipulation by educated guesses to study viral infectious biology by reverse genetics and to generate improved vectors for human gene transfer. However, despite remarkable progress, our limited knowledge of molecular mechanisms implicated in virus-cell interactions has been a limiting factor. Combinatorial engineering and high-throughput selection techniques hold the potential to boost technological improvement by offering the possibility to screen large numbers of randomly generated clones by appropriate selection protocols. These approaches not only require lesser knowledge of viral biology, but can also be employed as valuable tools to investigate molecular mechanisms that drive the infection process. In this review we recapitulate the rationale for employment of combinatorial methods in AAV vector development and the accomplishments achieved so far, discussing current limitations and interesting developments that are in sight.

  6. Incidental Prenatal Diagnosis of Sex Chromosome Aneuploidies: Health, Behavior, and Fertility

    PubMed Central

    Pieters, J. J. P. M.; Kooper, A. J. A.; van Kessel, A. Geurts; Braat, D. D. M.; Smits, A. P. T.

    2011-01-01

    Objective. To assess the diagnostic relevance of incidental prenatal findings of sex chromosome aneuploidies. Methods. We searched with medical subject headings (MeSHs) and keywords in Medline and the Cochrane Library and systematically screened publications on postnatally diagnosed sex chromosomal aneuploidies from 2006 to 2011 as well as publications on incidentally prenatally diagnosed sex chromosomal aneuploidies from 1980 to 2011. Results. Postnatally diagnosed sex chromosomal aneuploidies demonstrated three clinical relevant domains of abnormality: physical (22–100%), behavior (0–56%), and reproductive health (47–100%), while incidentally prenatally diagnosed sex chromosomal aneuploidies demonstrated, respectively, 0–33%, 0–40%, and 0–36%. Conclusion. In the literature incidental prenatal diagnosis of sex chromosomal aneuploidies is associated with normal to mildly affected phenotypes. This contrasts sharply with those of postnatally diagnosed sex chromosomal aneuploidies and highlights the importance of this ascertainment bias towards the prognostic value of diagnosis of fetal sex chromosomal aneuploidies. This observation should be taken into account, especially when considering excluding the sex chromosomes in invasive prenatal testing using Rapid Aneuploidy Detection. PMID:22191050

  7. Metagenomic chromosome conformation capture (meta3C) unveils the diversity of chromosome organization in microorganisms

    PubMed Central

    Marbouty, Martial; Cournac, Axel; Flot, Jean-François; Marie-Nelly, Hervé; Mozziconacci, Julien; Koszul, Romain

    2014-01-01

    Genomic analyses of microbial populations in their natural environment remain limited by the difficulty to assemble full genomes of individual species. Consequently, the chromosome organization of microorganisms has been investigated in a few model species, but the extent to which the features described can be generalized to other taxa remains unknown. Using controlled mixes of bacterial and yeast species, we developed meta3C, a metagenomic chromosome conformation capture approach that allows characterizing individual genomes and their average organization within a mix of organisms. Not only can meta3C be applied to species already sequenced, but a single meta3C library can be used for assembling, scaffolding and characterizing the tridimensional organization of unknown genomes. By applying meta3C to a semi-complex environmental sample, we confirmed its promising potential. Overall, this first meta3C study highlights the remarkable diversity of microorganisms chromosome organization, while providing an elegant and integrated approach to metagenomic analysis. DOI: http://dx.doi.org/10.7554/eLife.03318.001 PMID:25517076

  8. A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution.

    PubMed

    Paesold, Susanne; Borchardt, Dietrich; Schmidt, Thomas; Dechyeva, Daryna

    2012-11-01

    We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North-South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome-specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S-5.8S-25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber-FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.

  9. A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution.

    PubMed

    Paesold, Susanne; Borchardt, Dietrich; Schmidt, Thomas; Dechyeva, Daryna

    2012-11-01

    We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North-South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome-specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S-5.8S-25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber-FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA. PMID:22775355

  10. Public Libraries Going Green

    ERIC Educational Resources Information Center

    Miller, Kathryn

    2010-01-01

    Going green is now a national issue, and patrons expect their library to respond in the same way many corporations have. Libraries are going green with logos on their Web sites, programs for the public, and a host of other initiatives. This is the first book to focus strictly on the library's role in going green, helping you with: (1) Collection…

  11. Reforming Prison Libraries.

    ERIC Educational Resources Information Center

    Coyle, William J.

    1989-01-01

    Discusses the current widespread acceptance of the public library model for prison libraries, in which preferences of the inmates are the chief consideration in programing and collection development. It is argued that this model results in recreational programs and collections that fail to fulfill the prison library's role in education and…

  12. PAL: Positional Astronomy Library

    NASA Astrophysics Data System (ADS)

    Jenness, T.; Berry, D. S.

    2016-06-01

    The PAL library is a partial re-implementation of Pat Wallace's popular SLALIB library written in C using a Gnu GPL license and layered on top of the IAU's SOFA library (or the BSD-licensed ERFA) where appropriate. PAL attempts to stick to the SLA C API where possible.

  13. Creating a Classroom Library.

    ERIC Educational Resources Information Center

    Hepler, Susan; And Others

    1992-01-01

    Presents ideas for creating classroom libraries, noting how to set up a library (create a space, build and organize the collection, and set rules), where to find books at bargain prices (e.g., garage sales, libraries, book clubs, and grants), basic books to include, and information on authors and illustrators. (SM)

  14. Marketing Academic Libraries

    ERIC Educational Resources Information Center

    Mallon, Melissa, Ed.

    2013-01-01

    Ask any academic librarian if marketing their library and its services is an important task, and the answer will most likely be a resounding "yes!" Particularly in economically troubled times, librarians are increasingly called upon to promote their services and defend their library's worth. Since few academic libraries have in-house marketing…

  15. The Library Morphs

    ERIC Educational Resources Information Center

    Waters, John K.

    2008-01-01

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

  16. The Electronic, Eclectic Library.

    ERIC Educational Resources Information Center

    Dowlin, Kenneth E.

    1980-01-01

    Utilization of telecommunications and computer technology to increase access to information is identified as a primary goal for public libraries. To further service in the future, libraries should consider database services, online access to library resources, online community conferencing, community databases, network resource sharing,…

  17. A Truly Bookless Library

    ERIC Educational Resources Information Center

    Kolowich, Steve

    2011-01-01

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

  18. Summer Library Reading Programs

    ERIC Educational Resources Information Center

    Fiore, Carole D.

    2007-01-01

    Virtually all public libraries in the United States provide some type of summer library reading program during the traditional summer vacation period. Summer library reading programs provide opportunities for students of many ages and abilities to practice their reading skills and maintain skills that are developed during the school year. Fiore…

  19. Worthington Libraries, OH

    ERIC Educational Resources Information Center

    Berry, John N., III

    2007-01-01

    Worthington, Ohio, has deep library roots. A library has been part of its history since the planning by settlers before the city's birth in 1803. Among the treasures brought by James Kilbourne and the Scioto Company from Connecticut to the new, planned community they built was a collection of books for their new subscription library. The books…

  20. A Library That Swings

    ERIC Educational Resources Information Center

    Spragens, Thomas A.

    1971-01-01

    The Grace Doherty Library (Centre College, Danville, Kentucky) is a library that "swings," being so designed that a substantial part of its space is used alternately for classroom purposes during the day and for library reading-study space in the peak evening hours. (Author)

  1. Spanish Museum Libraries Network.

    ERIC Educational Resources Information Center

    Lopez de Prado, Rosario

    This paper describes the creation of an automated network of museum libraries in Spain. The only way in which the specialized libraries in the world today can continue to be active and to offer valid information is to automate the service they offer, and create network libraries with cooperative plans. The network can be configured with different…

  2. Place as Library?

    ERIC Educational Resources Information Center

    Davenport, Nancy

    2006-01-01

    Digital technology is redrawing the library's blueprint. Planners are thinking in new ways about how to design libraries as places for learning rather than primarily as storehouses of information. This thinking has given rise to much discussion--and to many publications--about the "library as place." In this article, the author asks why not also…

  3. The Honor System Library

    ERIC Educational Resources Information Center

    Marie, Kristen L.

    2005-01-01

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

  4. Technostress and Library Values.

    ERIC Educational Resources Information Center

    Gorman, Michael

    2001-01-01

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

  5. Library Journal Classics.

    ERIC Educational Resources Information Center

    Crunden, Frederick; And Others

    1993-01-01

    Includes four articles originally published in the 1890s. Highlights include a discussion of freedom of speech, political opinions, and the role of the public library in social reform; the establishment of a congressional or national library; censorship in libraries; and a criticism of a librarian by his assistant. (LRW)

  6. School Libraries and Innovation

    ERIC Educational Resources Information Center

    McGrath, Kevin G.

    2015-01-01

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

  7. Simple Library Bookkeeping.

    ERIC Educational Resources Information Center

    Hoffman, Herbert H.

    A simple and cheap manual double entry continuous transaction posting system with running balances is developed for bookkeeping by small libraries. A very small library may operate without any system of fiscal control but when a library's budget approaches three figures, some kind of bookkeeping must be introduced. To maintain control over his…

  8. Changing State Digital Libraries

    ERIC Educational Resources Information Center

    Pappas, Marjorie L.

    2006-01-01

    Research has shown that state virtual or digital libraries are evolving into websites that are loaded with free resources, subscription databases, and instructional tools. In this article, the author explores these evolving libraries based on the following questions: (1) How user-friendly are the state digital libraries?; (2) How do state digital…

  9. The Library Lobby

    ERIC Educational Resources Information Center

    Ralston, Anthony

    1971-01-01

    The library is examined as a university-wide resource and as a research facility. The use of the library relative to computing facilities is also considered as is the cost and value of library usage in relation to computer usage. (Author)

  10. Merchandising Your Library.

    ERIC Educational Resources Information Center

    Sivulich, Kenneth G.

    1989-01-01

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

  11. Library Building and Design.

    ERIC Educational Resources Information Center

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

    1997-01-01

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

  12. Public Library Finance.

    ERIC Educational Resources Information Center

    Mason, Marilyn Gell

    This study reviews trends in public library finance; examines recent political, economic, and technological changes; and assesses the impact of these changes on public library services. A history of the public library in America is presented, as well as an analysis of the principles of economics and public finance which reveals that current…

  13. Library Studies I Workbook.

    ERIC Educational Resources Information Center

    Frost, William J.

    Developed for use in the Library Studies I component of the Library Studies Program at Bloomsburg University (Pennsylvania), this self-paced workbook is intended to acquaint students with the Harvey A. Andruss Library and help them develop information-seeking skills. The workbook is designed to be used in conjunction with an exercise book, and…

  14. Supervision in Libraries.

    ERIC Educational Resources Information Center

    Bailey, Martha J.

    Although the literature of library administration draws extensively on that of business management, it is difficult to compare library supervision to business or industrial supervision. Library supervisors often do not have managerial training and may consider their management role as secondary. The educational level of the staff they supervise…

  15. Libraries and the Environment.

    ERIC Educational Resources Information Center

    LaRue, James; And Others

    1991-01-01

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

  16. Imagining the Digital Library in a Commercialized Internet.

    ERIC Educational Resources Information Center

    Heckart, Ronald J.

    1999-01-01

    Discusses digital library planning in light of Internet commerce and technological innovation in marketing and customer relations that are transforming user expectations about Web sites that offer products and services. Topics include user self-sufficiency; personalized service; artificial intelligence; collaborative filtering; and electronic…

  17. A FISH-based chromosome map for the European corn borer yields insights into ancient chromosomal fusions in the silkworm.

    PubMed

    Yasukochi, Y; Ohno, M; Shibata, F; Jouraku, A; Nakano, R; Ishikawa, Y; Sahara, K

    2016-01-01

    A significant feature of the genomes of Lepidoptera, butterflies and moths, is the high conservation of chromosome organization. Recent remarkable progress in genome sequencing of Lepidoptera has revealed that syntenic gene order is extensively conserved across phylogenetically distant species. The ancestral karyotype of Lepidoptera is thought to be n=31; however, that of the most well-studied moth, Bombyx mori, is n=28, and diverse studies suggest that three chromosomal fusion events occurred in this lineage. To identify the boundaries between predicted ancient fusions involving B. mori chromosomes 11, 23 and 24, we constructed fluorescence in situ hybridization (FISH)-based chromosome maps of the European corn borer, Ostrinia nubilalis (n=31). We first determined a 511 Mb genomic sequence of the Asian corn borer, O. furnacalis, a congener of O. nubilalis, and isolated bacterial artificial chromosomes and fosmid clones that were expected to localize in candidate regions for the boundaries using these sequences. Combined with FISH and genetic analysis, we narrowed down the candidate regions to 40 kb-1.5 Mb, in strong agreement with a previous estimate based on the genome of a butterfly, Melitaea cinxia. The significant difference in the lengths of the candidate regions where no functional genes were observed may reflect the evolutionary time after fusion events.

  18. A FISH-based chromosome map for the European corn borer yields insights into ancient chromosomal fusions in the silkworm.

    PubMed

    Yasukochi, Y; Ohno, M; Shibata, F; Jouraku, A; Nakano, R; Ishikawa, Y; Sahara, K

    2016-01-01

    A significant feature of the genomes of Lepidoptera, butterflies and moths, is the high conservation of chromosome organization. Recent remarkable progress in genome sequencing of Lepidoptera has revealed that syntenic gene order is extensively conserved across phylogenetically distant species. The ancestral karyotype of Lepidoptera is thought to be n=31; however, that of the most well-studied moth, Bombyx mori, is n=28, and diverse studies suggest that three chromosomal fusion events occurred in this lineage. To identify the boundaries between predicted ancient fusions involving B. mori chromosomes 11, 23 and 24, we constructed fluorescence in situ hybridization (FISH)-based chromosome maps of the European corn borer, Ostrinia nubilalis (n=31). We first determined a 511 Mb genomic sequence of the Asian corn borer, O. furnacalis, a congener of O. nubilalis, and isolated bacterial artificial chromosomes and fosmid clones that were expected to localize in candidate regions for the boundaries using these sequences. Combined with FISH and genetic analysis, we narrowed down the candidate regions to 40 kb-1.5 Mb, in strong agreement with a previous estimate based on the genome of a butterfly, Melitaea cinxia. The significant difference in the lengths of the candidate regions where no functional genes were observed may reflect the evolutionary time after fusion events. PMID:26264548

  19. Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences.

    PubMed

    Deng, Chuan-Liang; Zhang, Wei-Li; Cao, Ying; Wang, Shao-Jing; Li, Shu-Fen; Gao, Wu-Jun; Lu, Long-Dou

    2015-12-01

    The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X,X(m) and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However,there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.

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

    PubMed Central

    Wang, Congli; Ulloa, Mauricio; Shi, Xinyi; Yuan, Xiaohui; Saski, Christopher; Yu, John Z.; Roberts, Philip A.

    2015-01-01

    Genetic and physical framework mapping in cotton (Gossypium spp.) were used to discover putative gene sequences involved in resistance to common soil-borne pathogens. Chromosome (Chr) 11 and its homoeologous Chr 21 of Upland cotton (G. hirsutum) are foci for discovery of resistance (R) or pathogen-induced R (PR) genes underlying QTLs involved in response to root-knot nematode (Meloidogyne incognita), reniform nematode (Rotylenchulus reniformis), Fusarium wilt (Fusarium oxysporum f.sp. vasinfectum), Verticillium wilt (Verticillium dahliae), and black root rot (Thielaviopsis basicola). Simple sequence repeat (SSR) markers and bacterial artificial chromosome (BAC) clones from a BAC library developed from the Upland cotton Acala Maxxa were mapped on Chr 11 and Chr 21. DNA sequence through Gene Ontology (GO) of 99 of 256 Chr 11 and 109 of 239 Chr 21 previously mapped SSRs revealed response elements to internal and external stimulus, stress, signaling process, and cell death. The reconciliation between genetic and physical mapping of gene annotations from new DNA sequences of 20 BAC clones revealed 467 (Chr 11) and 285 (Chr 21) G. hirsutum putative coding sequences, plus 146 (Chr 11) and 98 (Chr 21) predicted genes. GO functional profiling of Unigenes uncovered genes involved in different metabolic functions and stress response elements (SRE). Our results revealed that Chrs 11 and 21 harbor resistance gene rich genomic regions. Sequence comparisons with the ancestral diploid D5 (G. raimondii), A2 (G. arboreum) and domesticated tetraploid TM-1 AD1 (G. hirsutum) genomes revealed abundance of transposable elements and confirmed the richness of resistance gene motifs in these chromosomes. The sequence information of SSR markers and BAC clones and the genetic mapping of BAC clones provide enhanced genetic and physical frameworks of resistance gene-rich regions of the cotton genome, thereby aiding discovery of R and PR genes and breeding for resistance to cotton diseases. PMID

  1. Artificial intelligence in medicine.

    PubMed Central

    Ramesh, A. N.; Kambhampati, C.; Monson, J. R. T.; Drew, P. J.

    2004-01-01

    INTRODUCTION: Artificial intelligence is a branch of computer science capable of analysing complex medical data. Their potential to exploit meaningful relationship with in a data set can be used in the diagnosis, treatment and predicting outcome in many clinical scenarios. METHODS: Medline and internet searches were carried out using the keywords 'artificial intelligence' and 'neural networks (computer)'. Further references were obtained by cross-referencing from key articles. An overview of different artificial intelligent techniques is presented in this paper along with the review of important clinical applications. RESULTS: The proficiency of artificial intelligent techniques has been explored in almost every field of medicine. Artificial neural network was the most commonly used analytical tool whilst other artificial intelligent techniques such as fuzzy expert systems, evolutionary computation and hybrid intelligent systems have all been used in different clinical settings. DISCUSSION: Artificial intelligence techniques have the potential to be applied in almost every field of medicine. There is need for further clinical trials which are appropriately designed before these emergent techniques find application in the real clinical setting. PMID:15333167

  2. Planning & Urban Affairs Library Manual.

    ERIC Educational Resources Information Center

    Knobbe, Mary L., Ed.; Lessel, Janice W., Ed.

    Written especially for persons without a library degree who are operating a small urban study or planning agency library on a part-time basis. Subjects covered are: (1) library function and staff function, duties and training; (2) physical layout and equipment of library; (3) establishing and maintaining the library; (4) library administration;…

  3. Chromosome assortment in Saccharum.

    PubMed

    Al-Janabi, S M; Honeycutt, R J; Sobral, B W

    1994-12-01

    Recent work has revealed random chromosome pairing and assortment in Saccharum spontaneum L., the most widely distributed, and morphologically and cytologically variable of the species of Saccharum. This conclusion was based on the analysis of a segregating population from across between S. spontaneum 'SES 208' and a spontaneously-doubled haploid of itself, derived from anther culture. To determine whether polysomic inheritance is common in Saccharum and whether it is observed in a typical biparental cross, we studied chromosome pairing and assortment in 44 progeny of a cross between euploid, meiotically regular, 2n=80 forms of Saccharum officinarum 'LA Purple' and Saccharum robustum ' Mol 5829'. Papuan 2n=80 forms of S. robustum have been suggested as the immediate progenitor species for cultivated sugarcane (S. officinarum). A total of 738 loci in LA Purple and 720 loci in Mol 5829 were amplified and typed in the progeny by arbitrarily primed PCR using 45 primers. Fifty and 33 single-dose polymorphisms were identified in the S. officinarum and S. robustum genomes, respectively (χ 2 at 98%). Linkage analysis of single-dose polymorphisms in both genomes revealed linkages in repulsion and coupling phases. In the S. officinarum genome, a map hypothesis gave 7 linkage groups with 17 linked and 33 unlinked markers. Four of 13 pairwise linkages were in repulsion phase and 9 were in coupling phase. In the S. robustum genome, a map hypothesis gave 5 linkage groups, defined by 12 markers, with 21 markers unlinked, and 2 of 9 pairwise linkages were in repulsion phase. Therefore, complete polysomic inheritance was not observed in either species, suggesting that chromosomal behavior is different from that observed by linkage analysis of over 500 markers in the S. spontaneum map. Implications of this finding for evolution and breeding are discussed.

  4. The Isolation of Polygenic Factors Controlling Bristle Score in Drosophila Melanogaster. I. Allocation of Third Chromosome Sternopleural Bristle Effects to Chromosome Sections

    PubMed Central

    Shrimpton, A. E.; Robertson, A.

    1988-01-01

    A single third chromosome C, with a high sternopleural bristle score, had been extracted from an artificial selection line. C was divided into five chromosomal sections by recombination with a multiply marked third chromosome ruseca, which had a low sternopleural bristle score. A nonuniform distribution of sternopleural bristle effect with physical length of chromosome was observed. The second section (26-44 cM) of C carried the most sternopleural bristle effect (10 bristles when homozygous), the first (0-26 cM) and third (44-62 cM) also carried significant sternopleural bristle effects (six and five bristles, respectively). The fourth section (62-71 cM) carried a small but significant effect (less than one bristle) while the fifth section (71-101 cM) carried little effect when alone (less than one bristle), though it did carry effects which had an epistatic interaction with those of the first and second sections. PMID:17246416

  5. Identification of an antibacterial protein by functional screening of a human oral metagenomic library.

    PubMed

    Arivaradarajan, Preeti; Warburton, Philip J; Paramasamy, Gunasekaran; Nair, Sean P; Allan, Elaine; Mullany, Peter

    2015-09-01

    Screening of a bacterial artificial chromosome (BAC) library containing metagenomic DNA from human plaque and saliva allowed the isolation of four clones producing antimicrobial activity. Three of these were pigmented and encoded homologues of glutamyl-tRNA reductase (GluTR), an enzyme involved in the C5 pathway leading to tetrapyrole synthesis, and one clone had antibacterial activity with no pigmentation. The latter contained a BAC with an insert of 15.6 kb. Initial attempts to localize the gene(s) responsible for antimicrobial activity by subcloning into pUC-based vectors failed. A new plasmid for toxic gene expression (pTGEX) was designed enabling localization of the antibacterial activity to a 4.7-kb HindIII fragment. Transposon mutagenesis localized the gene to an open reading frame of 483 bp designated antibacterial protein1 (abp1). Abp1 was 94% identical to a hypothetical protein of Neisseria subflava (accession number WP_004519448.1). An Escherichia coli clone expressing Abp1 exhibited antibacterial activity against Bacillus subtilis BS78H, Staphylococcus epidermidis NCTC 11964 and B4268, and S. aureus NCTC 12493,ATCC 35696 and NCTC 11561. However, no antibacterial activity was observed against Pseudomonas aeruginosa ATCC 9027, N. subflava ATCC A1078, E. coli K12 JM109 and BL21(DE3) Fusobacterium nucleatum ATCC 25586 and NCTC 11326, Prevotella intermedia ATCC 25611, Veillonella parvula ATCC 10790 or Lactobacillus casei NCTC 6375. PMID:26347298

  6. Library and Information Science's Ontological Position in the Networked Society: Using New Technology to Get Back to an Old Practice

    ERIC Educational Resources Information Center

    Kåhre, Peter

    2013-01-01

    Introduction: This paper concerns the ontological position of library and informations science in the networked society. The aim of the study is to understand library use and library functions in the age of Internet and artificial intelligent programmed search engines. Theoretical approach: The approach discusses so called sociocognitive tools in…

  7. A YAC contig map of plasmodium falciparum chromosome 4: Characterization of a DNA amplification between two recently separated isolates

    SciTech Connect

    Rubio, J.P.; Triglia, T.; Cowman, A.F.

    1995-03-20

    We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen. 42 refs., 6 figs., 1 tab.

  8. Chimera-free, high copy number YAC libraries and efficient methods of analysis. Final technical progress report

    SciTech Connect

    1995-10-01

    The first experiment involved a low chimera YAC library in recombination deficient host strains. To determine if the genetic background of the yeast host strain contributes to the formation of chimeric YACs the same YAC ligation mixture was introduced into three isogenic yeast hosts differing only in their recombination abilities. To prepare YACs, human genomic DNA was partially digested with EcoR1 and then ligated to YAC vector pCGS966 arms. DNA was size fractionated before and after ligation by preparative pulsed field gel electrophoresis (CHEF), selecting for fragments greater than 400 kb, and introduced into competent spheroplasts. CHEF gel Southern blots of resulting colony-purified YACs were probed with human DNA to determine if multiple YACs or YAC fragments were present in the same cell. The frequency of chimeric YACs was measured by fluorescence in situ hybridization (FISH) of YACs to human prometaphase spreads. YACs that hybridized to more than one location were assumed to be chimeric. In the second experiment new YAC vectors featuring tags for capture of YACs and YAC inserts were constructed. Yeast Artificial Chromosomes (YACs) have been of tremendous value in the physical mapping of the human genome. Because they can carry very large inserts, YACs are likely not only to contain entire genes but also their control elements. However, the only mode of purification of YAC DNA from current commonly used YAC libraries such as the CEPH library is by pulsed field gel electrophoresis. This is an inefficient, time consuming process and due to the single copy nature of these YACs, often result in poor yields. The vector pCGS1000 was designed to test new efficient ways of YAC DNA purification.

  9. Flight Software Math Library

    NASA Technical Reports Server (NTRS)

    McComas, David

    2013-01-01

    The flight software (FSW) math library is a collection of reusable math components that provides typical math utilities required by spacecraft flight software. These utilities are intended to increase flight software quality reusability and maintainability by providing a set of consistent, well-documented, and tested math utilities. This library only has dependencies on ANSI C, so it is easily ported. Prior to this library, each mission typically created its own math utilities using ideas/code from previous missions. Part of the reason for this is that math libraries can be written with different strategies in areas like error handling, parameters orders, naming conventions, etc. Changing the utilities for each mission introduces risks and costs. The obvious risks and costs are that the utilities must be coded and revalidated. The hidden risks and costs arise in miscommunication between engineers. These utilities must be understood by both the flight software engineers and other subsystem engineers (primarily guidance navigation and control). The FSW math library is part of a larger goal to produce a library of reusable Guidance Navigation and Control (GN&C) FSW components. A GN&C FSW library cannot be created unless a standardized math basis is created. This library solves the standardization problem by defining a common feature set and establishing policies for the library s design. This allows the libraries to be maintained with the same strategy used in its initial development, which supports a library of reusable GN&C FSW components. The FSW math library is written for an embedded software environment in C. This places restrictions on the language features that can be used by the library. Another advantage of the FSW math library is that it can be used in the FSW as well as other environments like the GN&C analyst s simulators. This helps communication between the teams because they can use the same utilities with the same feature set and syntax.

  10. Assessing Library Automation and Virtual Library Development in Four Academic Libraries in Oyo, Oyo State, Nigeria

    ERIC Educational Resources Information Center

    Gbadamosi, Belau Olatunde

    2011-01-01

    The paper examines the level of library automation and virtual library development in four academic libraries. A validated questionnaire was used to capture the responses from academic librarians of the libraries under study. The paper discovers that none of the four academic libraries is fully automated. The libraries make use of librarians with…

  11. Artificial Sweeteners and Cancer

    MedlinePlus

    ... artificial sweeteners and cancer? Saccharin Studies in laboratory rats during the early 1970s linked saccharin with the ... cause cancer in laboratory animals .” Subsequent studies in rats showed an increased incidence of urinary bladder cancer ...

  12. Intelligence: Real or artificial?

    PubMed Central

    Schlinger, Henry D.

    1992-01-01

    Throughout the history of the artificial intelligence movement, researchers have strived to create computers that could simulate general human intelligence. This paper argues that workers in artificial intelligence have failed to achieve this goal because they adopted the wrong model of human behavior and intelligence, namely a cognitive essentialist model with origins in the traditional philosophies of natural intelligence. An analysis of the word “intelligence” suggests that it originally referred to behavior-environment relations and not to inferred internal structures and processes. It is concluded that if workers in artificial intelligence are to succeed in their general goal, then they must design machines that are adaptive, that is, that can learn. Thus, artificial intelligence researchers must discard their essentialist model of natural intelligence and adopt a selectionist model instead. Such a strategic change should lead them to the science of behavior analysis. PMID:22477051

  13. Introduction to artificial intelligence

    SciTech Connect

    Charniak, E.; McDermott, D.

    1985-01-01

    This book is an introduction on artificial intelligence. Topics include reasoning under uncertainty, robot plans, language understanding, and learning. The history of the field as well as intellectual ties to related disciplines are presented.

  14. Chromosome Connections: Compelling Clues to Common Ancestry

    ERIC Educational Resources Information Center

    Flammer, Larry

    2013-01-01

    Students compare banding patterns on hominid chromosomes and see striking evidence of their common ancestry. To test this, human chromosome no. 2 is matched with two shorter chimpanzee chromosomes, leading to the hypothesis that human chromosome 2 resulted from the fusion of the two shorter chromosomes. Students test that hypothesis by looking for…

  15. X chromosome and suicide.

    PubMed

    Fiori, L M; Zouk, H; Himmelman, C; Turecki, G

    2011-02-01

    Suicide completion rates are significantly higher in males than females in most societies. Although gender differences in suicide rates have been partially explained by environmental and behavioral factors, it is possible that genetic factors, through differential expression between genders, may also help explain gender moderation of suicide risk. This study investigated X-linked genes in suicide completers using a two-step strategy. We first took advantage of the genetic structure of the French-Canadian population and genotyped 722 unrelated French-Canadian male subjects, of whom 333 were suicide completers and 389 were non-suicide controls, using a panel of 37 microsatellite markers spanning the entire X chromosome. Nine haplotype windows and several individual markers were associated with suicide. Significant results aggregated primarily in two regions, one in the long arm and another in the short arm of chromosome X, limited by markers DXS8051 and DXS8102, and DXS1001 and DXS8106, respectively. The second stage of the study investigated differential brain expression of genes mapping to associated regions in Brodmann areas 8/9, 11, 44 and 46, in an independent sample of suicide completers and controls. Six genes within these regions, Rho GTPase-activating protein 6, adaptor-related protein complex 1 sigma 2 subunit, glycoprotein M6B, ribosomal protein S6 kinase 90  kDa polypeptide 3, spermidine/spermine N(1)-acetyltransferase 1 and THO complex 2, were found to be differentially expressed in suicide completers. PMID:20010893

  16. Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene.

    PubMed

    Mollereau, C; Simons, M J; Soularue, P; Liners, F; Vassart, G; Meunier, J C; Parmentier, M

    1996-08-01

    Nociceptin (orphanin FQ), the newly discovered natural agonist of opioid receptor-like (ORL1) receptor, is a neuropeptide that is endowed with pronociceptive activity in vivo. Nociceptin is derived from a larger precursor, prepronociceptin (PPNOC), whose human, mouse, and rat genes we have now isolated. The PPNOC gene is highly conserved in the three species and displays organizational features that are strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides, suggesting the four genes belong to the same family-i.e., have a common evolutionary origin. The PPNOC gene encodes a single copy of nociceptin as well as of other peptides whose sequence is strictly conserved across murine and human species; hence it is likely to be neurophysiologically significant. Northern blot analysis shows that the PPNOC gene is predominantly transcribed in the central nervous system (brain and spinal cord) and, albeit weakly, in the ovary, the sole peripheral organ expressing the gene. By using a radiation hybrid cell line panel, the PPNOC gene was mapped to the short arm of human chromosome 8 (8p21), between sequence-tagged site markers WI-5833 and WI-1172, in close proximity of the locus encoding the neurofilament light chain NEFL. Analysis of yeast artificial chromosome clones belonging to the WC8.4 contig covering the 8p21 region did not allow to detect the presence of the gene on these yeast artificial chromosomes, suggesting a gap in the coverage within this contig.

  17. 30526 artificial lift

    SciTech Connect

    Not Available

    1989-01-01

    This book focuses on the four major methods of artificial lift: sucker-rod pumping, gas lift, electrical submersible pumping (ESP) and hydraulic pumping. Though more than 80% of artificially lifted wells worldwide are rod-pumped, the large majority of these wells are low-volume, stripper-type producers. For this reason, sucker-rod pumping papers comprise less than 40% of the 26 SPE papers selected.

  18. Physics of Artificial Gravity

    NASA Technical Reports Server (NTRS)

    Bukley, Angie; Paloski, William; Clement, Gilles

    2006-01-01

    This chapter discusses potential technologies for achieving artificial gravity in a space vehicle. We begin with a series of definitions and a general description of the rotational dynamics behind the forces ultimately exerted on the human body during centrifugation, such as gravity level, gravity gradient, and Coriolis force. Human factors considerations and comfort limits associated with a rotating environment are then discussed. Finally, engineering options for designing space vehicles with artificial gravity are presented.

  19. Transfer of stem cells carrying engineered chromosomes with XY clone laser system.

    PubMed

    Sinko, Ildiko; Katona, Robert L

    2011-01-01

    Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are limited by the amount of foreign DNA they can carry. Mammalian artificial chromosomes have large DNA-carrying capacity and ability to replicate in parallel with, but without integration into, the host genome. Hence they are attractive vectors for transgenesis, cellular protein production, and gene therapy applications as well. ES cells mediated chromosome transfer by conventional blastocyst injection has a limitation in unpredictable germline transmission. The demonstrated protocol of laser-assisted microinjection of artificial chromosome containing ES cells into eight-cell mouse embryos protocol described here can solve the problem for faster production of germline transchromosomic mice.

  20. Heidegger and artificial intelligence

    SciTech Connect

    Diaz, G.

    1987-01-01

    The discipline of Artificial Intelligence, in its quest for machine intelligence, showed great promise as long as its areas of application were limited to problems of a scientific and situation neutral nature. The attempts to move beyond these problems to a full simulation of man's intelligence has faltered and slowed it progress, largely because of the inability of Artificial Intelligence to deal with human characteristic, such as feelings, goals, and desires. This dissertation takes the position that an impasse has resulted because Artificial Intelligence has never been properly defined as a science: its objects and methods have never been identified. The following study undertakes to provide such a definition, i.e., the required ground for Artificial Intelligence. The procedure and methods employed in this study are based on Heidegger's philosophy and techniques of analysis as developed in Being and Time. Results of this study show that both the discipline of Artificial Intelligence and the concerns of Heidegger in Being and Time have the same object; fundamental ontology. The application of Heidegger's conclusions concerning fundamental ontology unites the various aspects of Artificial Intelligence and provides the articulation which shows the parts of this discipline and how they are related.

  1. Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system

    PubMed Central

    Morishita, Maki; Muramatsu, Tomoki; Suto, Yumiko; Hirai, Momoki; Konishi, Teruaki; Hayashi, Shin; Shigemizu, Daichi; Tsunoda, Tatsuhiko; Moriyama, Keiji; Inazawa, Johji

    2016-01-01

    Chromothripsis is the massive but highly localized chromosomal rearrangement in response to a one-step catastrophic event, rather than an accumulation of a series of subsequent and random alterations. Chromothripsis occurs commonly in various human cancers and is thought to be associated with increased malignancy and carcinogenesis. However, the causes and consequences of chromothripsis remain unclear. Therefore, to identify the mechanism underlying the generation of chromothripsis, we investigated whether chromothripsis could be artificially induced by ionizing radiation. We first elicited DNA double-strand breaks in an oral squamous cell carcinoma cell line HOC313-P and its highly metastatic subline HOC313-LM, using Single Particle Irradiation system to Cell (SPICE), a focused vertical microbeam system designed to irradiate a spot within the nuclei of adhesive cells, and then established irradiated monoclonal sublines from them, respectively. SNP array analysis detected a number of chromosomal copy number alterations (CNAs) in these sublines, and one HOC313-LM-derived monoclonal subline irradiated with 200 protons by the microbeam displayed multiple CNAs involved locally in chromosome 7. Multi-color FISH showed a complex translocation of chromosome 7 involving chromosomes 11 and 12. Furthermore, whole genome sequencing analysis revealed multiple de novo complex chromosomal rearrangements localized in chromosomes 2, 5, 7, and 20, resembling chromothripsis. These findings suggested that localized ionizing irradiation within the nucleus may induce chromothripsis-like complex chromosomal alterations via local DNA damage in the nucleus. PMID:26862731

  2. Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system.

    PubMed

    Morishita, Maki; Muramatsu, Tomoki; Suto, Yumiko; Hirai, Momoki; Konishi, Teruaki; Hayashi, Shin; Shigemizu, Daichi; Tsunoda, Tatsuhiko; Moriyama, Keiji; Inazawa, Johji

    2016-03-01

    Chromothripsis is the massive but highly localized chromosomal rearrangement in response to a one-step catastrophic event, rather than an accumulation of a series of subsequent and random alterations. Chromothripsis occurs commonly in various human cancers and is thought to be associated with increased malignancy and carcinogenesis. However, the causes and consequences of chromothripsis remain unclear. Therefore, to identify the mechanism underlying the generation of chromothripsis, we investigated whether chromothripsis could be artificially induced by ionizing radiation. We first elicited DNA double-strand breaks in an oral squamous cell carcinoma cell line HOC313-P and its highly metastatic subline HOC313-LM, using Single Particle Irradiation system to Cell (SPICE), a focused vertical microbeam system designed to irradiate a spot within the nuclei of adhesive cells, and then established irradiated monoclonal sublines from them, respectively. SNP array analysis detected a number of chromosomal copy number alterations (CNAs) in these sublines, and one HOC313-LM-derived monoclonal subline irradiated with 200 protons by the microbeam displayed multiple CNAs involved locally in chromosome 7. Multi-color FISH showed a complex translocation of chromosome 7 involving chromosomes 11 and 12. Furthermore, whole genome sequencing analysis revealed multiple de novo complex chromosomal rearrangements localized in chromosomes 2, 5, 7, and 20, resembling chromothripsis. These findings suggested that localized ionizing irradiation within the nucleus may induce chromothripsis-like complex chromosomal alterations via local DNA damage in the nucleus. PMID:26862731

  3. Advanced comparative cytogenetic analysis of X chromosomes in river buffalo, cattle, sheep, and human.

    PubMed

    Perucatti, A; Genualdo, V; Iannuzzi, A; Rebl, A; Di Berardino, D; Goldammer, T; Iannuzzi, Leopoldo

    2012-05-01

    Based on a recently generated comprehensive gene map for Ovis aries chromosome X (OARX) with an approximately even locus distribution, we assigned selected bacterial artificial chromosome (BAC) probes corresponding to these OARX loci to Bubalus bubalis (BBU) and Bos taurus (BTA) by comparative fluorescence in-situ hybridization (FISH) to improve cytogenetically the X chromosome maps in these species. Twenty-five added loci in BBUX and BTAX, respectively, contribute to a more detailed description of the cytogenetic organization of these chromosomes. Further seven loci were identified in OARX and two DNA probes were assigned to X and Y chromosomes in river buffalo, cattle, and sheep, respectively, and thus identified loci in the pseudoautosomal region. The additional assignments double the number of cytogenetic loci in BBUX and increase their number in BTAX and OARX. The larger quantity of cytogenetic anchors allows a more precise morphological comparison of bovid X chromosomes among each other and with the Homo sapiens (HSA) X chromosome. The anchor loci confirm and refine syntenic fragments in HSAX and identify several evolutionary breakpoints between the compared chromosomes. The cytogenetic assignments in BBUX, BTAX, and OARX represent useable anchors for the ongoing genome sequence assembly in Bovidae. PMID:22669522

  4. Polymerase Chain Reaction-based Suppression of Repetitive Sequences in Whole Chromosome Painting Probes for FISH

    SciTech Connect

    Dugan, L C; Pattee, M; Williams, J; Eklund, M; Bedford, J S; Christian, A T

    2004-04-21

    We have developed a method to suppress the PCR amplification of repetitive sequences in whole chromosome painting probes by adding Cot-1 DNA to the amplification mixture. The repetitive sequences in the Cot-1 DNA bind to their homologous sequences in the probe library, prevent the binding of primers, and interfere with extension of the probe sequences, greatly decreasing PCR efficiency selectively across these blocked regions. A second labeling reaction is then done and this product is resuspended in FISH hybridization mixture without further addition of blocking DNA. The hybridization produces little if any non-specific binding on any other chromosomes. We have been able to successfully use this procedure with both human and rat chromosome probes. This technique should be applicable in producing probes for CGH, M-FISH and SKY, as well as reducing the presence of repetitive DNA in genomic libraries.

  5. Organization of the bacterial chromosome.

    PubMed Central

    Krawiec, S; Riley, M

    1990-01-01

    Recent progress in studies on the bacterial chromosome is summarized. Although the greatest amount of information comes from studies on Escherichia coli, reports on studies of many other bacteria are also included. A compilation of the sizes of chromosomal DNAs as determined by pulsed-field electrophoresis is given, as well as a discussion of factors that affect gene dosage, including redundancy of chromosomes on the one hand and inactivation of chromosomes on the other hand. The distinction between a large plasmid and a second chromosome is discussed. Recent information on repeated sequences and chromosomal rearrangements is presented. The growing understanding of limitations on the rearrangements that can be tolerated by bacteria and those that cannot is summarized, and the sensitive region flanking the terminator loci is described. Sources and types of genetic variation in bacteria are listed, from simple single nucleotide mutations to intragenic and intergenic recombinations. A model depicting the dynamics of the evolution and genetic activity of the bacterial chromosome is described which entails acquisition by recombination of clonal segments within the chromosome. The model is consistent with the existence of only a few genetic types of E. coli worldwide. Finally, there is a summary of recent reports on lateral genetic exchange across great taxonomic distances, yet another source of genetic variation and innovation. PMID:2087223

  6. Cohesin in determining chromosome architecture

    SciTech Connect

    Haering, Christian H.; Jessberger, Rolf

    2012-07-15

    Cells use ring-like structured protein complexes for various tasks in DNA dynamics. The tripartite cohesin ring is particularly suited to determine chromosome architecture, for it is large and dynamic, may acquire different forms, and is involved in several distinct nuclear processes. This review focuses on cohesin's role in structuring chromosomes during mitotic and meiotic cell divisions and during interphase.

  7. Tetrasomy 15q: Two marker chromosomes with no detectable alpha-satellite DNA

    SciTech Connect

    Blennow, E.; Telenius, K.; Larsson, C.; Nordenskjoeld, M. ); Vos, D. de; Carter, N.P. ); Henriksson, P.; Johansson, O. )

    1994-05-01

    Two patients with specific and similar phenotypes were both found to have an unusual marker chromosome present in 70%-80% of their lymphocytes at routine cytogenetic examination. The marker chromosomes were isolated by flow sorting and were amplified by degenerate oligonucleotide-primed PCR. These libraries and a cosmid probe located at 15q26 were used to characterize the marker chromosomes by FISH. Both marker chromosomes were found to consist of duplicated chromosome material from the distal part of chromosome 15q and were identified as inv dup(15) (qter[yields]q23::q23[yields]qter) and inv dup(15) (qter[yields]q24[yields]qter), respectively. Hence, the markers did not include any known centromere region, and no alpha-satellite DNA could be detected at the site of the primary construction. Tetrasomy 15q may be a new syndrome, associated with a specific type of marker chromosome. In addition, further analysis of this type of marker chromosome might give new insight into the structure and function of the mammalian centromere. 29 refs., 4 figs., 1 tab.

  8. Region-specific cosmids and STRPs identified by chromosome microdissection and FISH

    SciTech Connect

    Flejter, W.L.; Bennett-Baker, P.; Barcroft, C.L.

    1995-01-20

    A strategy for identifying short tandem repeat (STR)-containing cosmid clones from a specific chromosomal region is described. The approach is based an the use of uncloned, PCR-amplified DNA derived from chromosome microdissection and pooled groups of STR sequences as hybridization probes to screen a cosmid library. Cosmid clones that display a positive signal common to both hybridizations are then characterized for repeat length polymorphisms. This method has been applied to chromosome bands 17q12-q21, a region that includes a gene (BRCA1) involved in early onset familial breast and ovarian cancer. Of 1536 chromosome 17-specific cosmid clones tested, 38 were identified by the dual screening procedure. Fluorescence in situ hybridization revealed that 19 cosmids originated from the microdissected target region. Thirteen of the 19 cosmids were mapped between markers flanking the BRCA1 region and selected for further characterization. Tetranucleotide repeats were identified in 10 of these 13 cosmids. Primers designed for each marker were tested on a panel of 80 CEPH parents for allele sizes, frequencies, and observed heterozygosities. From these studies six polymorphic and one nonpolymorphic STRs were identified. A similar approach should be applicable for screening whole genomic or chromosome-specific cosmid libraries in efforts to isolate new polymorphic markers from any chromosomal region of interest. 32 refs., 3 figs., 2 tabs.

  9. Chromosome choreography: the meiotic ballet.

    PubMed

    Page, Scott L; Hawley, R Scott

    2003-08-01

    The separation of homologous chromosomes during meiosis in eukaryotes is the physical basis of Mendelian inheritance. The core of the meiotic process is a specialized nuclear division (meiosis I) in which homologs pair with each other, recombine, and then segregate from each other. The processes of chromosome alignment and pairing allow for homolog recognition. Reciprocal meiotic recombination ensures meiotic chromosome segregation by converting sister chromatid cohesion into mechanisms that hold homologous chromosomes together. Finally, the ability of sister kinetochores to orient to a single pole at metaphase I allows the separation of homologs to two different daughter cells. Failures to properly accomplish this elegant chromosome dance result in aneuploidy, a major cause of miscarriage and birth defects in human beings. PMID:12907787

  10. Higher order structure of chromosomes.

    PubMed

    Okada, T A; Comings, D E

    1979-04-01

    Isolated Chinese hamster metaphase chromosomes were resuspended in 4 M ammonium acetate and spread on a surface of distilled water or 0.15 to 0.5 M ammonium acetate. The DNA was released in the form of a regular series of rosettes connected by interrossette DNA. The mean length of the rosette DNA was 14 micron, similar to the mean length of 10 micron for chromomere DNA of Drosophila polytene chromosomes. The mean interrosette DNA was 4.2 micron. SDS gel electrophoresis of the chromosomal nonhistone proteins showed them to be very similar to nuclear nonhistone proteins except for the presence of more actin and tubulin. Nuclear matrix proteins were present in the chromosomes and may play a role in forming the rosettes. Evidence that the rosette pattern is artifactual versus the possibility that it represents a real organizational substructure of the chromosomes is reviewed.

  11. Schizophrenia and chromosomal deletions

    SciTech Connect

    Lindsay, E.A.; Baldini, A.; Morris, M. A.

    1995-06-01

    Recent genetic linkage analysis studies have suggested the presence of a schizophrenia locus on the chromosomal region 22q11-q13. Schizophrenia has also been frequently observed in patients affected with velo-cardio-facial syndrome (VCFS), a disorder frequently associated with deletions within 22q11.1. It has been hypothesized that psychosis in VCFS may be due to deletion of the catechol-o-methyl transferase gene. Prompted by these observations, we screened for 22q11 deletions in a population of 100 schizophrenics selected from the Maryland Epidemiological Sample. Our results show that there are schizophrenic patients carrying a deletion of 22q11.1 and a mild VCFS phenotype that might remain unrecognized. These findings should encourage a search for a schizophrenia-susceptibility gene within the deleted region and alert those in clinical practice to the possible presence of a mild VCFS phenotype associated with schizophrenia. 9 refs.

  12. I.D. Weeks Library: Guide to Library Resources.

    ERIC Educational Resources Information Center

    South Dakota Univ., Vermillion. I.D. Weeks Library.

    This document is a guide to library resources in the I. D. Weeks Library at the University of South Dakota, Vermillion. The guide was designed to accompany an academic library instruction course, "Use of Library Resources, A & S 111." The purpose of the course is to familiarize students with the facilities of the I. D. Weeks Library; to acquaint…

  13. Libraries are for People! But Are the People for Libraries?

    ERIC Educational Resources Information Center

    Ohio State Library, Columbus.

    The 1974 Ohio Governor's Conference on Library and Information Services was the first in a series of meetings mandated by the Long Range Program for the Improvement of Library Services developed by the State Library Board to give citizens a voice in the future of their libraries. Information was gathered on the kinds of libraries--school,…

  14. [Evolution of differential chromosome banding].

    PubMed

    Rodionov, A V

    1999-03-01

    Specific chromosome banding patterns in different eukaryotic taxons are reviewed. In all eukaryotes, chromosomes are composed of alternating bands, each differing from the adjacent material by the molecular composition and structural characteristics. In minute chromosomes of fungi and Protozoa, these bands are represented by kinetochores (Kt- (Cd-)bands), nucleolus organizers (N-bands), and telomeres as well as the euchromatin. In genomes of most fungi and protists, long clusters of tandem repeats and, consequently, C-bands were not revealed but they are likely to be found out in species with chromosomes visible under a light microscope, which are several tens of million bp in size. Chromosomes of Metazoa are usually larger. Even in Cnidaria, they contain C-bands, which are replicated late in the S phase. In Deuterostomia, chromosome euchromatin regions differ by replication time: bands replicating at the first half of the S phase alternate with bands replicating at the second half of the S phase. Longitudinal differentiation in the replication pattern of euchromatic regions is observed in all classes of Vertebrata beginning with the bony fish although the time when it developed in Deuterostomia is unknown. Apparently, the evolution of early and late replicating subdomains in Vertebrata euchromatin promoted fast accumulation of differences in the molecular composition of nucleoproteid complexes characteristic of early and late replicating bands. As a result, the more contrasting G/R and Q-banding patterns of chromosomes developed especially in Eutheria. The evolution of Protostomia and Plantae followed another path. An increase in chromosome size was not accompanied by the appearance of wide RBE and RBL euchromatin bands. The G/R-like banding within the interstitial chromosome regions observed in some representatives of Invertebrates and higher plants arose independently in different phylogenetic lineages. This banding pattern seems to be closer to that of C

  15. X-Chromosome dosage compensation.

    PubMed

    Meyer, Barbara J

    2005-01-01

    In mammals, flies, and worms, sex is determined by distinctive regulatory mechanisms that cause males (XO or XY) and females (XX) to differ in their dose of X chromosomes. In each species, an essential X chromosome-wide process called dosage compensation ensures that somatic cells of either sex express equal levels of X-linked gene products. The strategies used to achieve dosage compensation are diverse, but in all cases, specialized complexes are targeted specifically to the X chromosome(s) of only one sex to regulate transcript levels. In C. elegans, this sex-specific targeting of the dosage compensation complex (DCC) is controlled by the same developmental signal that establishes sex, the ratio of X chromosomes to sets of autosomes (X:A signal). Molecular components of this chromosome counting process have been defined. Following a common step of regulation, sex determination and dosage compensation are controlled by distinct genetic pathways. C. elegans dosage compensation is implemented by a protein complex that binds both X chromosomes of hermaphrodites to reduce transcript levels by one-half. The dosage compensation complex resembles the conserved 13S condensin complex required for both mitotic and meiotic chromosome resolution and condensation, implying the recruitment of ancient proteins to the new task of regulating gene expression. Within each C. elegans somatic cell, one of the DCC components also participates in the separate mitotic/meiotic condensin complex. Other DCC components play pivotal roles in regulating the number and distribution of crossovers during meiosis. The strategy by which C. elegans X chromosomes attract the condensin-like DCC is known. Small, well-dispersed X-recognition elements act as entry sites to recruit the dosage compensation complex and to nucleate spreading of the complex to X regions that lack recruitment sites. In this manner, a repressed chromatin state is spread in cis over short or long distances, thus establishing the

  16. Molecular mapping of chromosomes 17 and X. Progress report

    SciTech Connect

    Barker, D.F.

    1989-12-31

    The basic aims of this project are the construction of high density genetic maps of chromosomes 17 and X and the utilization of these maps for the subsequent isolation of a set of physically overlapping DNA segment clones. The strategy depends on the utilization of chromosome specific libraries of small (1--15 kb) segments from each of the two chromosomes. Since the time of submission of our previous progress report, we have refined the genetic map of markers which we had previously isolated for chromosome 17. We have completed our genetic mapping in CEPH reference and NF1 families of 15 markers in the pericentric region of chromosome 17. Physical mapping results with three probes, were shown be in very close genetic proximity to the NF1 gene, with respect to two translocation breakpoints which disrupt the activity of the gene. All three of the probes were found to lie between the centromere and the most proximal translocation breakpoint, providing important genetic markers proximal to the NF1 gene. Our primary focus has shifted to the X chromosome. We have isolated an additional 30 polymorphic markers, bringing the total number we have isolated to over 80. We have invested substantial effort in characterizing the polymorphisms at each of these loci and constructed plasmid subclones which reveal the polymorphisms for nearly all of the loci. These subclones are of practical value in that they produce simpler and stronger patterns on human genomic Southern blots, thus improving the efficiency of the genetic mapping experiments. These subclones may also be of value for deriving DNA sequence information at each locus, necessary for establishing polymerase chain reaction primers specific for each locus. Such information would allow the use of each locus as a sequence tagged site.

  17. The map-based genome sequence of Spirodela polyrhiza aligned with its chromosomes, a reference for karyotype evolution.

    PubMed

    Cao, Hieu Xuan; Vu, Giang Thi Ha; Wang, Wenqin; Appenroth, Klaus J; Messing, Joachim; Schubert, Ingo

    2016-01-01

    Duckweeds are aquatic monocotyledonous plants of potential economic interest with fast vegetative propagation, comprising 37 species with variable genome sizes (0.158-1.88 Gbp). The genomic sequence of Spirodela polyrhiza, the smallest and the most ancient duckweed genome, needs to be aligned to its chromosomes as a reference and prerequisite to study the genome and karyotype evolution of other duckweed species. We selected physically mapped bacterial artificial chromosomes (BACs) containing Spirodela DNA inserts with little or no repetitive elements as probes for multicolor fluorescence in situ hybridization (mcFISH), using an optimized BAC pooling strategy, to validate its physical map and correlate it with its chromosome complement. By consecutive mcFISH analyses, we assigned the originally assembled 32 pseudomolecules (supercontigs) of the genomic sequences to the 20 chromosomes of S. polyrhiza. A Spirodela cytogenetic map containing 96 BAC markers with an average distance of 0.89 Mbp was constructed. Using a cocktail of 41 BACs in three colors, all chromosome pairs could be individualized simultaneously. Seven ancestral blocks emerged from duplicated chromosome segments of 19 Spirodela chromosomes. The chromosomally integrated genome of S. polyrhiza and the established prerequisites for comparative chromosome painting enable future studies on the chromosome homoeology and karyotype evolution of duckweed species.

  18. Transcription of a protein-coding gene on B chromosomes of the Siberian roe deer (Capreolus pygargus)

    PubMed Central

    2013-01-01

    Background Most eukaryotic species represent stable karyotypes with a particular diploid number. B chromosomes are additional to standard karyotypes and may vary in size, number and morphology even between cells of the same individual. For many years it was generally believed that B chromosomes found in some plant, animal and fungi species lacked active genes. Recently, molecular cytogenetic studies showed the presence of additional copies of protein-coding genes on B chromosomes. However, the transcriptional activity of these genes remained elusive. We studied karyotypes of the Siberian roe deer (Capreolus pygargus) that possess up to 14 B chromosomes to investigate the presence and expression of genes on supernumerary chromosomes. Results Here, we describe a 2 Mbp region homologous to cattle chromosome 3 and containing TNNI3K (partial), FPGT, LRRIQ3 and a large gene-sparse segment on B chromosomes of the Siberian roe deer. The presence of the copy of the autosomal region was demonstrated by B-specific cDNA analysis, PCR assisted mapping, cattle bacterial artificial chromosome (BAC) clone localization and quantitative polymerase chain reaction (qPCR). By comparative analysis of B-specific and non-B chromosomal sequences we discovered some B chromosome-specific mutations in protein-coding genes, which further enabled the detection of a FPGT-TNNI3K transcript expressed from duplicated genes located on B chromosomes in roe deer fibroblasts. Conclusions Discovery of a large autosomal segment in all B chromosomes of the Siberian roe deer further corroborates the view of an autosomal origin for these elements. Detection of a B-derived transcript in fibroblasts implies that the protein coding sequences located on Bs are not fully inactivated. The origin, evolution and effect on host of B chromosomal genes seem to be similar to autosomal segmental duplications, which reinforces the view that supernumerary chromosomal elements might play an important role in genome

  19. Field-flow fractionation of chromosomes

    SciTech Connect

    Giddings, J.C.

    1990-09-01

    Research continued on field flow fractionation of chromosomes. Progress in the past year can be organized into three main categories: (1) chromosome sample preparation; (2) preliminary chromosome fractionation; (3) fractionation of a polystyrene aggregate model which approximates the chromosome shape. We have been successful in isolating metaphase chromosomes from the Chinese hamster. We also received a human chromosome sample from Dr. Carolyn Bell-Prince of Los Alamos National Laboratory. Results are discussed. 2 figs.

  20. Integrated circuit cell library

    NASA Technical Reports Server (NTRS)

    Whitaker, Sterling R. (Inventor); Miles, Lowell H. (Inventor)

    2005-01-01

    According to the invention, an ASIC cell library for use in creation of custom integrated circuits is disclosed. The ASIC cell library includes some first cells and some second cells. Each of the second cells includes two or more kernel cells. The ASIC cell library is at least 5% comprised of second cells. In various embodiments, the ASIC cell library could be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more comprised of second cells.

  1. Evaluation of an automated karyotyping system for chromosome aberration analysis

    NASA Technical Reports Server (NTRS)

    Prichard, Howard M.

    1987-01-01

    Chromosome aberration analysis is a promising complement to conventional radiation dosimetry, particularly in the complex radiation fields encountered in the space environment. The capabilities of a recently developed automated karyotyping system were evaluated both to determine current capabilities and limitations and to suggest areas where future development should be emphasized. Cells exposed to radiometric chemicals and to photon and particulate radiation were evaluated by manual inspection and by automated karyotyping. It was demonstrated that the evaluated programs were appropriate for image digitization, storage, and transmission. However, automated and semi-automated scoring techniques must be advanced significantly if in-flight chromosome aberration analysis is to be practical. A degree of artificial intelligence may be necessary to realize this goal.

  2. [Consequences of abnormalities of chromosome structure in domestic animals].

    PubMed

    Popescu, C P

    1990-01-01

    Abnormalities in chromosome structure generally have no phenotypic expression but are very often associated with reproductive disorders. In cattle, sheep and goats, the robertsonian translocation seems to be the most frequent abnormality of chromosome structure. In the pig, reciprocal translocations are very common. The accumulation of data on the frequency of such abnormalities and their effects on reproductive performance prompted an evaluation of their economic consequences in cattle and pigs. In cattle, because of the negative effect of 1/29 translocation, an eradication program, based on the removal of carrier bulls from artificial insemination centers was established. In pig, the main effect of the reciprocal translocations was a reduction in the number of offspring, up to 50%, thus representing a considerable economic loss. PMID:2206288

  3. Mitotic chromosome condensation in vertebrates

    SciTech Connect

    Vagnarelli, Paola

    2012-07-15

    Work from several laboratories over the past 10-15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292-301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories-a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307-316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119-1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579-589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different classes of

  4. The Jordanian Library Scene, 1973

    ERIC Educational Resources Information Center

    International Library Review, 1974

    1974-01-01

    This discussion is made up of several individual papers by different authors: Introduction; Libraries in Jordan; The Royal Scientific Society and its Library; The Royal Scientific Society Course in Librarianship; Jordan Library Association Course in Librarianship; Secondary School Libraries in Jordan; and Notes on West Bank Libraries, 1972. (JB)

  5. Public Relations in Special Libraries.

    ERIC Educational Resources Information Center

    Rutkowski, Hollace Ann; And Others

    1991-01-01

    This theme issue includes 11 articles on public relations (PR) in special libraries. Highlights include PR at the Special Libraries Association (SLA); sources for marketing research for libraries; developing a library image; sample PR releases; brand strategies for libraries; case studies; publicizing a consortium; and a bibliography of pertinent…

  6. Standards for Utah Public Libraries.

    ERIC Educational Resources Information Center

    Utah State Library Div., Salt Lake City. Dept. of Community and Economic Development.

    This document presents standards for Utah public libraries. The benefits of standards for public libraries and for the state library are listed, and responsibilities, benefits, and characteristics of three types of public libraries (i.e., volunteer, transitional, and certified public libraries) are summarized. Specific standards in the following…

  7. Library Latchkey Children. ERIC Digest.

    ERIC Educational Resources Information Center

    Dowd, Frances Smardo

    This digest discusses ways in which public libraries deal with latchkey children who spend their after-school hours at public libraries while their parents are at work. Research conducted in 1990 of 110 public libraries revealed that almost all libraries encountered unattended children after school hours, and that most libraries surveyed were not…

  8. Ohio: Library and Information Networks.

    ERIC Educational Resources Information Center

    Byerly, Greg

    1996-01-01

    Describes the development, current status, and future goals and plans of library and information networks in Ohio. Highlights include OCLC; OhioLINK, incorporating state-assisted universities, two-year colleges, the state library, and private university libraries; a school library network; a public library information network; a telecommunications…

  9. Resources of North Carolina Libraries.

    ERIC Educational Resources Information Center

    Downs, Robert B., Ed.

    The conclusion of the survey is that North Carolina libraries do not have sufficient resources, physical facilities or staff to provide adequate library service for the state. The survey covers the present and potential roles of the State Library, the State Department of Archives and History, public libraries, university libraries, senior and…

  10. FCLib: The Feature Characterization Library.

    SciTech Connect

    Gentile, Ann C.; Doyle, Wendy S. K.; Kegelmeyer, W. Philip,; Ulmer, Craig D.

    2008-11-01

    The Feature Characterization Library (FCLib) is a software library that simplifies the process of interrogating, analyzing, and understanding complex data sets generated by finite element applications. This document provides an overview of the library, a description of both the design philosophy and implementation of the library, and examples of how the library can be utilized to extract understanding from raw datasets.

  11. Optical Disc Applications in Libraries.

    ERIC Educational Resources Information Center

    Andre, Pamela Q. J.

    1989-01-01

    Discusses a variety of library applications of optical disc storage technology, including CD-ROM, digital videodisc, and WORM. Research and development projects at the Library of Congress, National Library of Medicine, and National Agricultural Library are described, products offered by library networks are reviewed, and activities in academic and…

  12. A YAC contig and an EST map in the pericentromeric region of chromosome 13 surrounding the loci for neurosensory nonsyndromic deafness (DFNB1 and DFNA3) and Limb-Girdle muscular dystrophy type 2C (LGMD2C)

    SciTech Connect

    Guilford, P.; Crozet, F.; Blanchard, S.

    1995-09-01

    Two forms of inherited childhood nonsyndromic deafness (DFNB1 and DFNA3) and a Duchenne-like form of progressive muscular dystrophy (LGMD2C) have been mapped to the pericentromeric region of chromosome 13. To clone the genes responsible for these diseases we constructed a yeast artificial chromosome (YAC) contig spanning an 8-cM region between the polymorphic markers D13S221. The contig comprises 24 sequence-tagged sites, among which 15 were newly obtained. This contig allowed us to order the polymorphic markers centromere- D13S175-D13S141-D13S143-D13S115-AFM128yc1-D13S292-D13S283-AFM323vh5-D13S221-telomere. Eight expressed sequence tags, previously assigned to 13q11-q12 (D13S182E, D13S183E, D13S502E, D13S504E, D13S505E, D13S837E, TUBA2, ATP1AL1), were localized on the YAC contig. YAC screening of a cDNA library derived from mouse cochlea allowed us to identify an {alpha}-tubulin gene (TUBA2) that was subsequently precisely mapped within the candidate region. 36 refs., 2 figs., 2 tabs.

  13. X-chromosome tiling path array detection of copy number variants in patients with chromosome X-linked mental retardation

    PubMed Central

    Madrigal, I; Rodríguez-Revenga, L; Armengol, L; González, E; Rodriguez, B; Badenas, C; Sánchez, A; Martínez, F; Guitart, M; Fernández, I; Arranz, JA; Tejada, MI; Pérez-Jurado, LA; Estivill, X; Milà, M

    2007-01-01

    Background Aproximately 5–10% of cases of mental retardation in males are due to copy number variations (CNV) on the X chromosome. Novel technologies, such as array comparative genomic hybridization (aCGH), may help to uncover cryptic rearrangements in X-linked mental retardation (XLMR) patients. We have constructed an X-chromosome tiling path array using bacterial artificial chromosomes (BACs) and validated it using samples with cytogenetically defined copy number changes. We have studied 54 patients with idiopathic mental retardation and 20 controls subjects. Results Known genomic aberrations were reliably detected on the array and eight novel submicroscopic imbalances, likely causative for the mental retardation (MR) phenotype, were detected. Putatively pathogenic rearrangements included three deletions and five duplications (ranging between 82 kb to one Mb), all but two affecting genes previously known to be responsible for XLMR. Additionally, we describe different CNV regions with significant different frequencies in XLMR and control subjects (44% vs. 20%). Conclusion This tiling path array of the human X chromosome has proven successful for the detection and characterization of known rearrangements and novel CNVs in XLMR patients. PMID:18047645

  14. Chromosomal localization and structure of the human type II IMP dehydrogenase gene

    SciTech Connect

    Glesne, D.; Huberman, E. |; Collart, F.; Varkony, T.; Drabkin, H.

    1994-05-01

    We determined the chromosomal localization and structure of the gene encoding human type II inosine 5{prime}-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205), an enzyme associated with cellular proliferation, malignant transformation, and differentiation. Using polymerase chain reaction (PCR) primers specific for type II IMPDH, we screened a panel of human-Chinese hamster cell somatic hybrids and a separate deletion panel of chromosome 3 hybrids and localized the gene to 3p21.1{yields}p24.2. Two overlapping yeast artificial chromosome clones containing the full gene for type II IMPDH were isolated and a physical map of 117 kb of human genomic DNA in this region of chromosome 3 was constructed. The gene for type II IMPDH was localized and oriented on this map and found to span no more than 12.5 kb.

  15. Artificial muscles on heat

    NASA Astrophysics Data System (ADS)

    McKay, Thomas G.; Shin, Dong Ki; Percy, Steven; Knight, Chris; McGarry, Scott; Anderson, Iain A.

    2014-03-01

    Many devices and processes produce low grade waste heat. Some of these include combustion engines, electrical circuits, biological processes and industrial processes. To harvest this heat energy thermoelectric devices, using the Seebeck effect, are commonly used. However, these devices have limitations in efficiency, and usable voltage. This paper investigates the viability of a Stirling engine coupled to an artificial muscle energy harvester to efficiently convert heat energy into electrical energy. The results present the testing of the prototype generator which produced 200 μW when operating at 75°C. Pathways for improved performance are discussed which include optimising the electronic control of the artificial muscle, adjusting the mechanical properties of the artificial muscle to work optimally with the remainder of the system, good sealing, and tuning the resonance of the displacer to minimise the power required to drive it.

  16. Artificial intelligence in nanotechnology.

    PubMed

    Sacha, G M; Varona, P

    2013-11-15

    During the last decade there has been increasing use of artificial intelligence tools in nanotechnology research. In this paper we review some of these efforts in the context of interpreting scanning probe microscopy, the study of biological nanosystems, the classification of material properties at the nanoscale, theoretical approaches and simulations in nanoscience, and generally in the design of nanodevices. Current trends and future perspectives in the development of nanocomputing hardware that can boost artificial-intelligence-based applications are also discussed. Convergence between artificial intelligence and nanotechnology can shape the path for many technological developments in the field of information sciences that will rely on new computer architectures and data representations, hybrid technologies that use biological entities and nanotechnological devices, bioengineering, neuroscience and a large variety of related disciplines.

  17. Artificial gravity experiment satellites

    NASA Astrophysics Data System (ADS)

    Harada, Tadashi

    1992-07-01

    An overview of the conceptual study of an artificial gravity experiment satellite based on the assumption of a launch by the H-2 launch vehicle with a target launch date in the Year 2000 is presented. While many satellites provided with artificial gravity have been reported in relation to a manned Mars exploration spacecraft mission, the review has been conducted on missions and test subjects only for experimental purposes. Mission requirements were determined based on the results of reviews on the mission, test subjects, and model missions. The system baseline and development plan were based on the results of a study on conceptual structure and scale of the system, including measures to generate artificial gravity. Approximate scale of the system and arm length, mission orbit, visibility of the operation orbit from ground stations in Japan, and satellite attitude on the mission orbit are outlined.

  18. Artificial vision workbench.

    PubMed

    Frenger, P

    1997-01-01

    Machine vision is an important component of medical systems engineering. Inexpensive miniature solid state cameras are now available. This paper describes how these devices can be used as artificial retinas, to take snapshots and moving pictures in monochrome or color. Used in pairs, they produce a stereoscopic field of vision and enable depth perception. Macular and peripheral vision can be simulated electronically. This paper also presents the author's design of an artificial orbit for this synthetic eye. The orbit supports the eye, protects it, and provides attachment points for the ocular motion control system. Convergence and image fusion can be produced, and saccades simulated, along with the other ocular motions. The use of lenses, filters, irises and focusing mechanisms are also discussed. Typical camera-computer interfaces are described, including the use of "frame grabbers" and analog-to-digital image conversion. Software programs for eye positioning, image manipulation, feature extraction and object recognition are discussed, including the application of artificial neural networks.

  19. Artificial intelligence in nanotechnology

    NASA Astrophysics Data System (ADS)

    Sacha, G. M.; Varona, P.

    2013-11-01

    During the last decade there has been increasing use of artificial intelligence tools in nanotechnology research. In this paper we review some of these efforts in the context of interpreting scanning probe microscopy, the study of biological nanosystems, the classification of material properties at the nanoscale, theoretical approaches and simulations in nanoscience, and generally in the design of nanodevices. Current trends and future perspectives in the development of nanocomputing hardware that can boost artificial-intelligence-based applications are also discussed. Convergence between artificial intelligence and nanotechnology can shape the path for many technological developments in the field of information sciences that will rely on new computer architectures and data representations, hybrid technologies that use biological entities and nanotechnological devices, bioengineering, neuroscience and a large variety of related disciplines.

  20. Specific features in linear and spatial organizations of pericentromeric heterochromatin regions in polytene chromosomes of the closely related species Drosophila virilis and D. kanekoi (Diptera: Drosophilidae).

    PubMed

    Wasserlauf, Irina; Usov, Konstantin; Artemov, Gleb; Anan'ina, Tatyana; Stegniy, Vladimir

    2015-06-01

    Heterochromatin plays an important role in the spatial arrangement and evolution of the eukaryotic genetic apparatus. The closely related species Drosophila virilis (phyla virilis) and D. kanekoi (phyla montana) differ in the amount of heterochromatin along the chromosomes as well as by the presence of the metacentric chromosome 2, which emerged as a result of a pericentric inversion during speciation, in the D. kanekoi karyotype. The purpose of this study was to establish if chromosome rearrangements have any influence on the linear redistribution of centromeric heterochromatin in polytene chromosomes and the spatial organization of chromosomes in the nuclei of nurse cell. We have microdissected the chromocenter of D. virilis salivary gland polytene chromosomes; obtained a DNA library of this region (DvirIII); and hybridized (FISH) DvirIII to the salivary gland and nurse cell polytene chromosomes of D. virilis and D. kanekoi. We demonstrated that DvirIII localizes to the pericentromeric heterochromatin regions of all chromosomes and peritelomeric region of chromosome 5 in both species. Unlike D. virilis, the DvirIII signal in D. kanekoi chromosomes is detectable in the telomeric region of chromosome 2. We have also conducted a 3D FISH of DvirIII probe to the D. virilis and D. kanekoi nurse cell chromosomes. In particular, the DvirIII signal in D. virilis was observed in the local chromocenter at one pole of the nucleus, while the signal belonging to the telomeric region of chromosome 5 was detectable at the other pole. In contrast, in D. kanekoi there exist two separate DvirIII-positive regions. One of these regions belongs to the pericentromeric region of chromosome 2 and the other, to pericentromeric regions of the remaining chromosomes. These results suggest that chromosome rearrangements play an important role in the redistribution of heterochromatin DNA sequences in the genome, representing a speciation mechanism, which, in general, could also affect the

  1. Artificial human vision.

    PubMed

    Dowling, Jason

    2005-01-01

    Can vision be restored to the blind? As early as 1929 it was discovered that stimulating the visual cortex of an individual led to the perception of spots of light, known as phosphenes [1] . The aim of artificial human vision systems is to attempt to utilize the perception of phosphenes to provide a useful substitute for normal vision. Currently, four locations for electrical stimulation are being investigated; behind the retina (subretinal), in front of the retina (epiretinal), the optic nerve and the visual cortex (using intra- and surface electrodes). This review discusses artificial human vision technology and requirements, and reviews the current development projects.

  2. Stable Chromosome Condensation Revealed by Chromosome Conformation Capture.

    PubMed

    Eagen, Kyle P; Hartl, Tom A; Kornberg, Roger D

    2015-11-01

    Chemical cross-linking and DNA sequencing have revealed regions of intra-chromosomal interaction, referred to as topologically associating domains (TADs), interspersed with regions of little or no interaction, in interphase nuclei. We find that TADs and the regions between them correspond with the bands and interbands of polytene chromosomes of Drosophila. We further establish the conservation of TADs between polytene and diploid cells of Drosophila. From direct measurements on light micrographs of polytene chromosomes, we then deduce the states of chromatin folding in the diploid cell nucleus. Two states of folding, fully extended fibers containing regulatory regions and promoters, and fibers condensed up to 10-fold containing coding regions of active genes, constitute the euchromatin of the nuclear interior. Chromatin fibers condensed up to 30-fold, containing coding regions of inactive genes, represent the heterochromatin of the nuclear periphery. A convergence of molecular analysis with direct observation thus reveals the architecture of interphase chromosomes. PMID:26544940

  3. Molecular biology of chromosome function

    SciTech Connect

    Adolph, K.W. )

    1989-01-01

    The structure and function of chromosomes are closely linked since chromosome organization profoundly influences the activity of the genome in replication and transcription. Many fundamental results originated from studies of bacterial and viral systems chosen for their less-complex cycles. However, the processes of replication and transcription show differences between the higher and simpler systems. Three important subjects are covered in this volume: DNA replication and recombination, gene transcription, and chromosome organization. Eukaryotic, prokaryotic, and viral systems are discussed. The information presented is derived from techniques of structural biology and biophysics, including computer graphics and X-ray crystallography, as well as biochemistry, molecular and cell biology.

  4. Numerous transitions of sex chromosomes in Diptera.

    PubMed

    Vicoso, Beatriz; Bachtrog, Doris

    2015-04-01

    Many species groups, including mammals and many insects, determine sex using heteromorphic sex chromosomes. Diptera flies, which include the model Drosophila melanogaster, generally have XY sex chromosomes and a conserved karyotype consisting of six chromosomal arms (five large rods and a small dot), but superficially similar karyotypes may conceal the true extent of sex chromosome variation. Here, we use whole-genome analysis in 37 fly species belonging to 22 different families of Diptera and uncover tremendous hidden diversity in sex chromosome karyotypes among flies. We identify over a dozen different sex chromosome configurations, and the small dot chromosome is repeatedly used as the sex chromosome, which presumably reflects the ancestral karyotype of higher Diptera. However, we identify species with undifferentiated sex chromosomes, others in which a different chromosome replaced the dot as a sex chromosome or in which up to three chromosomal elements became incorporated into the sex chromosomes, and others yet with female heterogamety (ZW sex chromosomes). Transcriptome analysis shows that dosage compensation has evolved multiple times in flies, consistently through up-regulation of the single X in males. However, X chromosomes generally show a deficiency of genes with male-biased expression, possibly reflecting sex-specific selective pressures. These species thus provide a rich resource to study sex chromosome biology in a comparative manner and show that similar selective forces have shaped the unique evolution of sex chromosomes in diverse fly taxa.

  5. Numerous Transitions of Sex Chromosomes in Diptera

    PubMed Central

    Vicoso, Beatriz; Bachtrog, Doris

    2015-01-01

    Many species groups, including mammals and many insects, determine sex using heteromorphic sex chromosomes. Diptera flies, which include the model Drosophila melanogaster, generally have XY sex chromosomes and a conserved karyotype consisting of six chromosomal arms (five large rods and a small dot), but superficially similar karyotypes may conceal the true extent of sex chromosome variation. Here, we use whole-genome analysis in 37 fly species belonging to 22 different families of Diptera and uncover tremendous hidden diversity in sex chromosome karyotypes among flies. We identify over a dozen different sex chromosome configurations, and the small dot chromosome is repeatedly used as the sex chromosome, which presumably reflects the ancestral karyotype of higher Diptera. However, we identify species with undifferentiated sex chromosomes, others in which a different chromosome replaced the dot as a sex chromosome or in which up to three chromosomal elements became incorporated into the sex chromosomes, and others yet with female heterogamety (ZW sex chromosomes). Transcriptome analysis shows that dosage compensation has evolved multiple times in flies, consistently through up-regulation of the single X in males. However, X chromosomes generally show a deficiency of genes with male-biased expression, possibly reflecting sex-specific selective pressures. These species thus provide a rich resource to study sex chromosome biology in a comparative manner and show that similar selective forces have shaped the unique evolution of sex chromosomes in diverse fly taxa. PMID:25879221

  6. Revolution and the Library.

    ERIC Educational Resources Information Center

    Gorniak-Kocikowska, Krystyna

    2001-01-01

    Considers the impact that the computer revolution has had on college and university libraries. Discusses the historical background of changes that resulted from the invention of the printing press; the development of national languages; knowledge acquisition; the historical role of religion; content control of libraries; and changes in students.…

  7. Corporations and Library Fundraising.

    ERIC Educational Resources Information Center

    DiMattia, Susan S.

    1984-01-01

    Examination of corporate donations of cash, products, service, and expertise to libraries highlights industry contributions in 1980; why corporations give; examples of corporate donations to various libraries (Brooklyn Public, New York Public, Altoona Area Public, Boston Public); planning fund-raising compaigns; and seven strategic planning…

  8. Guidelines for National Libraries.

    ERIC Educational Resources Information Center

    Sylvestre, Guy

    This report is designed to provide practical assistance to government officials and librarians responsible for the planning, creation, and development of national library services. Based on a number of authoritative studies and a broad consensus among experts, including directors of national libraries, these guidelines give special attention to…

  9. Health Science Libraries Today.

    ERIC Educational Resources Information Center

    Kenney, Brigitte L.

    This study explores means by which biomedical information might be distributed over a network to physicians and other personnel in the health sciences. Health Science libraries of all types are surveyed in terms of location, facilities, collection, staff, budget, and services. The library's user group is presented, and cooperative agreements among…

  10. Prison Libraries: Bibliography.

    ERIC Educational Resources Information Center

    Gillespie, David M.

    An Alphabetically arranged bibliography, listing 485 entries representing 518 citations taken from "Poole's Index to Periodical Literature" 1802-1906, "Cannon's Bibliography of Library Economy," and "Library Literature" 1921 to mid-1970. Other sources used include: The Index to the Journal of Correctional Education; ten bibliographies taken from…

  11. "Greening" up the Library

    ERIC Educational Resources Information Center

    Woodland, Jean

    2010-01-01

    This paper looks at the library's ability to spread environmental conservation efforts throughout the community. The paper suggests various ways library staff can explore environmentally friendly procedures within the context of the media center as well as ways to promote community interest and support. It identifies areas for improvement, ideas…

  12. Small Public Library Management

    ERIC Educational Resources Information Center

    Pearlmutter, Jane; Nelson, Paul

    2012-01-01

    Anyone at the helm of a small public library knows that every little detail counts. But juggling the responsibilities that are part and parcel of the job is far from easy. Finally, here's a handbook that includes everything administrators need to keep a handle on library operations, freeing them up to streamline and improve how the organization…

  13. Attribution of Library Costs

    ERIC Educational Resources Information Center

    Drake, Miriam A.

    1977-01-01

    Universities conduct a variety of cost-allocation studies that require the collection and analysis of the library cost-data. Cost accounting methods are used in most studies; however, costs are attributed to library user groups in a variety of ways. Cost accounting studies are reviewed and allocation methods are discussed. (Author)

  14. Guidelines on Library Twinning.

    ERIC Educational Resources Information Center

    Doyle, Robert P., Comp.; Scarry, Patricia, Comp.

    This document serves as an overview of the many forms of library twinning, or formal exchanges of materials and/or staff between two institutions. Twinning can be reciprocal, but often involves a one-way arrangement whereby a library in a developing country receives a visit from a librarian of a developed country. Either way, both institutions can…

  15. Library Technology International

    ERIC Educational Resources Information Center

    Breeding, Marshall

    2008-01-01

    The author has been extremely fortunate over the last few years to have had the opportunity to travel to many different parts of the world and speak and work with librarians in many countries and to have the chance to see first-hand some incredible libraries that demonstrate creative approaches to library services, innovative uses of technology,…

  16. Library Laws of Texas.

    ERIC Educational Resources Information Center

    Seidenberg, Ed, Ed.

    Compiled to provide a central reference point for all legislative information pertaining to libraries in the state of Texas, this publication includes all pertinent legislation as amended through the 66th Legislature, Regular Session, 1979. It contains articles dealing specifically with archives, buildings and property, city libraries, non-profit…

  17. Library Automation in Australia.

    ERIC Educational Resources Information Center

    Blank, Karen L.

    1984-01-01

    Discussion of Australia's move toward library automation highlights development of a national bibliographic network, local and regional cooperation, integrated library systems, telecommunications, and online systems, as well as microcomputer usage, ergonomics, copyright issues, and national information policy. Information technology plans of the…

  18. Hospital Library Administration.

    ERIC Educational Resources Information Center

    Cramer, Anne

    The objectives of a hospital are to improve patient care, while the objectives of a hospital library are to improve services to the staff which will support their efforts. This handbook dealing with hospital administration is designed to aid the librarian in either implementing a hospital library, or improving services in an existing medical…

  19. Library Consortia in Hungary

    ERIC Educational Resources Information Center

    Csajbok, Edit; Szluka, Peter; Vasas, Livia

    2012-01-01

    During the last two decades many Hungarian libraries have developed considerably, beyond what was considered possible prior to 1989 and the beginning of events signaling the end of Communism in the country. Some of the modernization of library services has been realized through participation in cooperative agreements. Many smaller and larger…

  20. Dumping the "Library."

    ERIC Educational Resources Information Center

    Crowley, Bill

    1998-01-01

    Discusses an alternative to abandoning the word "library" for "information" in graduate education. Recommends patient, consistent effort by library- and information-science educators to convince academic librarians that if they accept the standards of the teaching/research faculty, including the need to earn a Ph.D., it will raise the prestige of…