The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Maynard V Olson - One of the best experts on this subject based on the ideXlab platform.
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physical calibration of Yeast Artificial Chromosome contig maps by reca assisted restriction endonuclease rare cleavage
Genomics, 1994Co-Authors: Andreas Gnirke, Pui-yan Kwok, Shawn P Iadonato, Maynard V OlsonAbstract:Clone-based genome maps can be constructed by determining the presence or absence of sequence-tagged sites (STSs) in a redundant collection of Yeast Artificial Chromosome clones (YACs). While STS-content mapping has proven to be an effective means of ordering clone ends and STSs along Chromosomes, the exact physical map positions of these landmarks are not determined. This fundamental weakness can be overcome by RecA-assisted restriction endonuclease (RARE) cleavage, a method that exploits the binding specificity on duplex DNA of a RecA-protein-oligodeoxynucleotide complex to enhance the cleavage specificity of a restriction endonuclease. This technique allows selective cleavage at individual members of a large set of restriction sites. RARE-cleavage mapping was applied to a contig comprising 5 overlapping YACs spanning 580 kb on human Chromosome 14. An STS-content map comprising 10 YAC-end specific STSs and one internal STS was constructed. RARE cleavage was performed on 2 YACs that span the entire contig at the EcoRI sites defining the vector-insert junctions of all 5 YACs, as well as at a HhaI site within the STS that was initially used to screen the YAC library for the clones in the contig. The sizes of the RARE-cleavage fragments were measured by pulsed-field gel electrophoresis and used to convert the STS-content map into a true physical map that indicates precise positions of clone ends and STSs.
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Automatable screening of Yeast Artificial-Chromosome libraries based on the oligonucleotide-ligation assay
Genomics, 1992Co-Authors: Pui-yan Kwok, Maureen F. Gremaud, Deborah A. Nickerson, Leroy Hood, Maynard V OlsonAbstract:The systematic screening of Yeast Artificial-Chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughput for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.
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detection and characterization of chimeric Yeast Artificial Chromosome clones
Genomics, 1991Co-Authors: Eric D Green, Harold Riethman, James E Dutchik, Maynard V OlsonAbstract:Abstract Methods for the construction of Yeast Artificial-Chromosome (YAC) clones have been designed to isolate single, large (100–1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such “chimeric” YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in Yeast, between two Alu sequences located on unrelated segments of human DNA.
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the human hprt gene on a Yeast Artificial Chromosome is functional when transferred to mouse cells by cell fusion
Genomics, 1991Co-Authors: Clare Huxley, David Schlessinger, Yoshiaki Hagino, Maynard V OlsonAbstract:Abstract A 680-kb Yeast Artificial Chromosome (YAC) that contains a functional copyof the human hypoxanthine phosphoribosyltransferase (HPRT) gene has been isolated. This YAC, yHPRT, and another YAC, yXY837, which contains the 3′ end of the HPRT gene, have been mapped with restriction enzymes that cleave human DNA infrequently. The HPRT gene lies near the center of yHPRT. Fusion of yHPRT-containing Yeast spheroplasts with mouse L A-9 cells, which are HPRT-negative, gives rise to HPRT-positive colonies. These colonies contain the human HPRT gene and express human HPRT mRNA. Fusion of Yeast with mammalian cells is an efficient way of testing the integrity and functionality of human DNA contained in YACs.
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Preparation of clone libraries in Yeast Artificial-Chromosome vectors.
Methods in enzymology, 1991Co-Authors: David T Burke, Maynard V OlsonAbstract:Publisher Summary This chapter describes the use of pYAC4 for cloning large fragments of human DNA prepared by partial digestion with EcoRI. Straightforward modifications of these protocols allow the cloning of other types of fragments with alternative Yeast Artificial Chromosome (YAC) vectors. Using the protocols described in the chapter, stable ligation mixtures are obtained in which titers are sufficiently high to prepare a library of 100,000 clones with an average insert size of 275 kb from a few hundred micrograms of high molecular weight human DNA. The expected sampling redundancy for single-copy sequences in such a library is nearly 10. Methods are also described in the chapter for storing large collections of YAC clones picked individually into the wells of microtiter plates in a form suitable for library screening. Sucrose density gradients are used for size fractionation either before ligation, after ligation, or at both steps of the procedure. Illustrative data are shown in the chapter indicating the effects of these choices on the size distribution of the YACs obtained.
Philip Hieter - One of the best experts on this subject based on the ideXlab platform.
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efficient manipulation of the human adenovirus genome as an infectious Yeast Artificial Chromosome clone
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Gary Ketner, Forrest Spencer, Stuart Tugendreich, Carla Connelly, Philip HieterAbstract:Abstract A Yeast Artificial Chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the Yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the YAC. The application of powerful Yeast genetic techniques to an infectious adenovirus clone promises to significantly enhance the genetic analysis of adenovirus and to simplify the construction of adenovirus-based vectors for vaccines or for gene transfer to mammalian cells or whole animals. The adenovirus YAC was produced by homologous recombination in vivo between adenovirus 2 virion DNA and YAC vector plasmids carrying segments of the viral left and right genomic termini. This recombinational cloning strategy is generally applicable to the construction of YACs containing other DNA segments, such as the genomes of other viruses. Further, it is very efficient and may permit the targeted cloning of segments of the genomes of higher organisms directly from genomic DNA.
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Yeast Artificial Chromosome modification and manipulation.
Methods in enzymology, 1992Co-Authors: Roger H. Reeves, William J. Pavan, Philip HieterAbstract:Publisher Summary This chapter describes Yeast Artificial Chromosome (YAC) modification and manipulation. Two types of YAC modification systems have been developed that take advantage of this property to target recombination to highly repetitive DNA elements within YAC DNA inserts. Vectors that integrate into YACs can be used to produce Chromosome-specific libraries from somatic cell hybrids or to introduce deletions, mutations, or mammalian selectable markers into YAC cloned segments. A second vector system facilitates analysis of the large inserts in YACs by fragmenting them, generating nested deletion derivatives. A polyethylene glycol-mediated fusion procedure has been described that can be used to introduce appropriately modified YACs into cultured mammalian cells. This combination of vectors and procedures provides a system for realizing the full potential of YACs in structural and biological analysis of the genome.
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Yeast Artificial Chromosome vectors for efficient clone manipulation and mapping
Genomics, 1991Co-Authors: James H. Shero, Mary Kay Mccormick, Stylianos E. Antonarakis, Philip HieterAbstract:The Yeast Artificial Chromosome (YAC) cloning system allows the cloning of exogenous DNA several hundred kilobases in length. To enhance the usefulness of this technology, Yeast Artificial Chromosome vectors have been designed for efficient clone characterization, manipulation, and mapping. The vectors contain a polylinker with unique EcoRI, BglII, NotI, EagI, SacII, SalI, NruI, NheI, and ClaI cloning sites and T7 bacteriophage promoters positioned to allow the generation of riboprobes from the exogenous DNA ends. Centric and acentric vector arms were constructed as separate plasmids to allow the recovery of both ends of the YAC insert DNA directly in Escherichia coli. In addition, YACs generated using this vector system contain a Yeast gene (SUP 11) that allows visual monitoring of YAC stability and copy number.
M Le M Beau - One of the best experts on this subject based on the ideXlab platform.
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a Yeast Artificial Chromosome based map of the region of Chromosome 20 containing the diabetes susceptibility gene mody1 and a myeloid leukemia related gene
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Markus Stoffel, M Le M Beau, R Espinosa, Stefan K Bohlander, Le D Paslier, David J Cohen, K Xiang, Nancy J Cox, Stefan S Fajans, C I BellAbstract:Abstract We have generated a physical map of human Chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The Yeast Artificial Chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene.
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construction of a 2 8 megabase Yeast Artificial Chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Olufunmilayo I Olopade, M Le M Beau, Helen Pomykala, Fitsum Hagos, Lise Sveen, R Espinosa, Martin Dreyling, Susan Gursky, Walter M Stadler, Stefan K BohlanderAbstract:Abstract Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of Chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping Yeast Artificial Chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the Yeast Artificial Chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.
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isolation of a Yeast Artificial Chromosome spanning the 8 21 translocation breakpoint t 8 21 q22 q22 3 in acute myelogenous leukemia
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Paul F Erickson, Katheleen Gardiner, M Le M Beau, Manuel O Diaz, David A Patterson, Janet D Rowley, Harry A DrabkinAbstract:The 8;21 translocation is one of the most common specific rearrangements in acute myelogenous leukemia. We have identified markers (D21S65 and a Not I boundary clone, Not-42, referred to as probe B) flanking the Chromosome 21 translocation breakpoint (21q22.3) that demonstrate physical linkage in normal genomic DNA, by using at least three restriction endonucleases (Not I, Sac II, and BssHII), and that are located not more than 250-280 kilobases apart. Pulsed-field gel analysis of DNA from somatic cell hybrids containing the 8;21 translocation Chromosomes demonstrates rearrangement of these markers. A 470-kilobase Yeast Artificial Chromosome, YAC-Not-42, has been isolated that contains both probes. Mapping of lambda subclones constructed from YAC-Not-42 suggests that greater than 95% (25/26 probes tested) of the Yeast Artificial Chromosome DNA is located on the proximal (D21S65) side of the breakpoint. In situ hybridization studies using metaphase Chromosomes from five acute myelogenous leukemia patients with the 8;21 translocation confirmed these results and demonstrated the translocation of probe B to the derivative Chromosome 8. A Chromosome walk of approximately 39 kilobases from probe B has allowed identification of the breakpoint in DNA from a somatic cell hybrid containing the derivative Chromosome 8. Since probe B contains conserved DNA sequences and is in close proximity to the translocation breakpoint, it may represent a portion of the involved gene on Chromosome 21.
Gregory B. Martin - One of the best experts on this subject based on the ideXlab platform.
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Construction of plant Yeast Artificial Chromosome libraries
Plant Molecular Biology Manual, 1994Co-Authors: Gregory B. MartinAbstract:Yeast Artificial Chromosome (YAC) libraries are becoming widely used for the construction of long-range physical maps and for map-based cloning [ 1–6]. In plants, YAC libraries are now available for Arabidopsis, barley, carrot, maize, rice and tomato [7–14]. Recently, map-based cloning projects using YAC libraries have been successful in isolating several plant genes that were known only by their phenotype [15–18]. Detailed analysis of individual YACs is likely to provide new insights into the way repeated sequences and transcribed regions are organized in plant genomes.
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construction of a Yeast Artificial Chromosome library of tomato and identification of cloned segments linked to two disease resistance loci
Molecular Genetics and Genomics, 1992Co-Authors: Gregory B. Martin, Martin W Ganal, Steven D TanksleyAbstract:We have constructed a Yeast Artificial Chromosome (YAC) library of tomato for Chromosome walking that contains the equivalent of three haploid genomes (22 000 clones). The source of high molecular weight DNA was leaf protoplasts from the tomato cultivars VFNT cherry and Rio Grande-PtoR, which together contain loci encoding resistance to six pathogens of tomato. Approximately 11 000 YACs have been screened with RFLP markers that cosegregate withTm-2a andPto — loci conferring resistance to tobacco mosaic virus andPseudomonas syringae pv.tomato, respectively. Five YACs were identified that hybridized to the markers and are therefore starting points for Chromosome walks to these genes. A subset of the library was characterized for the presence of various repetitive sequences and YACs were identified that carried TGRI, a repeat clustered near the telomeres of most tomato Chromosomes, TGRII, an interspersed repeat, and TGRIIl, a repeat that occurs primarily at centromeric sites. Evaluation of the library for organellar sequences revealed that approximately 10% of the clones contain chloroplast sequences. Many of these YAC clones appear to contain the entire 155 kb tomato chloroplast genome. The tomato cultivars used in the library construction, in addition to carrying various disease resistance genes, also contain the wild-type alleles corresponding to most recessive mutations that have been mapped by classical linkage analysis. Thus, in addition to its utility for physical mapping and genome studies, this library should be useful for Chromosome walking to genes corresponding to virtually any phenotype that can be scored in a segregating population.
Anthony P. Monaco - One of the best experts on this subject based on the ideXlab platform.
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Construction of Yeast Artificial Chromosome libraries by pulsed-field gel electrophoresis.
Molecular Biotechnology, 1994Co-Authors: Anthony P. Monaco, Zoia Larin, Hans LehrachAbstract:Yeast Artificial Chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.
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characterization of a Yeast Artificial Chromosome contig spanning the huntington s disease gene candidate region
Nature Genetics, 1992Co-Authors: Gillian P. Bates, Anthony P. Monaco, Marcy E. Macdonald, Sarah Baxendale, John Valdes, Holger Hummerich, Le D Paslier, Danilo A Tagle, Michael R Altherr, Mark RossAbstract:The Huntington's disease (HD) gene has been localized by recombination events to a region covering 2.2 megabases (Mb) DNA within Chromosome 4p16.3. We have screened three Yeast Artificial Chromosome (YAC) libraries in order to isolate and characterize 44 YAC clones mapping to this region. Approximately 50% of the YACs were chimaeric. Unstable YACs were identified across the whole region, but were particularly prevalent around the D4S183 and D4S43 loci. The YACs have been assembled into a contig extending from D4S126 to D4S98 covering roughly 2 Mb DNA, except for a gap of about 250 kilobases (kb). The establishment of a YAC contig which spans the region most likely to contain the HD mutation is an essential step in the isolation of the HD gene.
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a Yeast Artificial Chromosome contig containing the complete duchenne muscular dystrophy gene
Genomics, 1992Co-Authors: Anthony P. Monaco, Zoia Larin, Ann P Walker, Iona Y Millwood, Hans LehrachAbstract:A contig of 36 overlapping Yeast Artificial Chromosome (YAC) clones has been constructed for the complete Duchenne muscular dystrophy (DMD) gene in Xp21. The YACs were isolated from a human 48,XXXX YAC library using the DMD cDNA and brain promoter fragments as hybridization probes. The YAC clones were characterized for exon content using HindIII or EcoRI digests, hybridization of individual DMD cDNA probes, and polymerase chain reaction (PCR) amplification of specific exons near the 5' end of the gene. For comparison to the known long-range restriction map of the DMD gene, YAC clones were digested with SfiI and hybridized with DMD cDNA probes. The combined analysis of the exon content and the SfiI map allowed an approximately 3.2-Mb YAC contig to be constructed. The complete 2.4-Mb DMD gene could be represented in a minimum set of 7 overlapping YAC clones.
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mapping irradiation hybrids to cosmid and Yeast Artificial Chromosome libraries by direct hybridization of alu pcr products
Nucleic Acids Research, 1991Co-Authors: Anthony P. Monaco, Gunther Zehetner, Gregory G Lennon, Christal Douglas, Dean Nizetic, P N GoodfellowAbstract:Abstract A direct hybridization protocol is described for screening cosmid and Yeast Artificial Chromosome libraries with pools of Alu-PCR products from somatic cell or irradiation hybrids. This method eliminates purification, cloning and analysis of each individual Alu-PCR product before library screening. A series of human X Chromosome irradiation hybrids were mapped by this method, using a cosmid reference library for comparisons between overlapping hybrids to identify interesting clones for further analysis.
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Yeast Artificial Chromosome libraries containing large inserts from mouse and human dna
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Zoia Larin, Anthony P. MonacoAbstract:Abstract Yeast Artificial Chromosome (YAC) libraries have been difficult to construct with average insert sizes greater than 400 kilobase pairs when DNA is size-fractionated in low-melting-point agarose. By using Yeast Chromosome in mock cloning experiments, we found that polyamines should be present whenever agarose containing high molecular weight DNA is melted to protect DNA from degradation. By incorporating polyamines during the cloning procedure, we constructed YAC libraries from mouse and human DNA with average insert sizes of 700 and 620 kilobase pairs, respectively. Several genome equivalents of these YAC libraries were replicated onto the surface of many duplicate agar plates using a 40,000 multipin transfer device. High-density filter replicas were screened by hybridization, and 70 mouse YAC clones from 31 loci and 132 human YAC clones from 49 loci were isolated.
