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Construction and characterization of a yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking - Molecular Genetics and Genomics

  • ️Somerville, Chris
  • ️Wed May 01 1991

Summary

A yeast artificial chromosome (YAC) genomic library of Arabidopsis thaliana was constructed in a derivative of the vector pYAC4 which was modified to facilitate the production of end-specific probes for chromosome walking. Experiments in which a subset of 2300 clones from the library were probed with 30 restriction fragment length polymorphism (RFLP) markers indicated that, on the average, the entire genome is represented once in each 800 YAC clones. Thus, the complete library of more than 20000 YACs is expected to contain most or all of the Arabidopsis genome with a high probability. The YAC clones examined in the sample had an average insert size of approximately 150 kb±10 and represented more than 5% of the Arabidopsis genome. Based on the properties of the library and the currently available RFLP maps for Arabidopsis, only one or two steps from flanking RFLPs should be sufficient to isolate an average gene in Arabidopsis by chromosome walking with the YACs. In order to facilitate chromosome walking, a method for the production of hybridization probes from the ends of the inserts was employed that is based on a combination of pre-amplification of the vector/insert junction sequences via the polymerase chain reaction and specific transcription from T3 or T7 RNA polymerase promoters flanking the cloning site in the YAC vector.

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References

  • Albertsen HM, Abderrahim H, Cann H, Dansset J, Le Pasher D, Cohen D (1990) Construction and characterization of a yeast artificial chromosome library containing seven haploid human genome equivalents. Proc Natl Acad Sci USA 87:4256–4260

    Google Scholar 

  • Anand R, Villasante A, Tyler-Smith C (Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis. Nucleic Acids Res 17:3425–3433

  • Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA, Seidman JG, Struhl K (1987) Protocols in Molecular Biology. John Wiley and Sons, New York

    Google Scholar 

  • Brownstein BH, Silverman GA, Little RD, Burke DT, Korsmeyer SJ, Schlessinger D, Olson MV (1989) Isolation of single-copy human genes from a library of yeast artificial chromosome clones. Science 244:1348–1351

    Google Scholar 

  • Browse J, Somerville CR, Slack R (1988) Changes in lipid composition during protoplast isolation. Plant Sci 56:15–20

    Google Scholar 

  • Burgers PMJ, Percival KJ (1987) Transformation of yeast spheroplasts without cell fusion. Anal Biochem 163:391–397

    Google Scholar 

  • Burke DT, Carle GF, Olson MV (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806–812

    Google Scholar 

  • Chang C, Bowman JL, DeJohn AW, Lander ES, Meyerowitz EM (1988) Restriction fragment length polymorphism map for Arabidopsis thaliana. Proc Natl Acad Sci USA 85:6856–6860

    Google Scholar 

  • Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995

    CAS  PubMed  Google Scholar 

  • Coulson A, Waterston R, Kiff J, Sulston J, Kohara Y (1988) Genome linking with yeast artificial chromosomes. Nature 335:184–186

    Google Scholar 

  • Garza D, Ajioka JW, Burke DT, Hard DL (1989) Mapping the Drosophila genome with yeast artificial chromosomes. Science 246:641–646

    Google Scholar 

  • Guzman P, Ecker J (1988) Development of large DNA methods for plants: molecular cloning of large segments of Arabidopsis and carrot DNA into yeast. Nucleic Acid Res 16:11091–11105

    Google Scholar 

  • Meyerowitz EM (1989) Arabidopsis, a useful weed. Cell 56:263–269

    Google Scholar 

  • Nam HG, Giraudat J, den Boer B, Moonan F, Loos W, Hauge B, Goodman HM (1989) Restriction fragment length polymorphism linkage map of Arabidopsis thaliana. Plant Cell 1:699–705

    Google Scholar 

  • Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120:621–623

    CAS  PubMed  Google Scholar 

  • Pruitt RE, Meyerowitz EM (1986) Characterization of the genome of Arabidopsis thaliana. J Mol Biol 187:169–183

    Google Scholar 

  • Sherman F, Fink GR, Hicks JB (1986) Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Silverman GA, Te RD, Pollock KM, Sadler JE, Korsmeyer SJ (1989) Use of yeast artificial chromosome clones for mapping and walking within human chromosome segments 19q21.3. Proc Nail Acad Sci USA 86:7485–7489

    CAS  PubMed  Google Scholar 

  • Somerville CR (1989) Arabidopsis blooms. Plant Cell 1:1131–1135

    Google Scholar 

  • Traver CN, Klapholz S, Hyman RW, Davis RW (1989) Rapid screening of a human genomic library in yeast artificial chromosomes for single copy sequences. Proc Natl Acad Sci USA 86:5898–5902

    Google Scholar 

  • Vollrath D, Davis RW (1987) Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res 15:7865–7876

    Google Scholar 

  • Wahl GM, Lewis KA, Ruiz JC, Rothenberg B, Zhao J, Evans GA (1987) Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. Proc Nail Acad Sci USA 84:2160–2164

    Google Scholar 

  • Ward ER, Jen GC (1990) Isolation of single-copy-sequence clones from a yeast artificial chromosome library of randomly sheared Arabidopsis thaliana DNA. Plant Mol Biol 14:561–568

    Google Scholar 

Download references

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Author notes

  1. Erwin Grill

    Present address: Institut für Pflanzenwissenschaften Biochemie und Physiologie der Pflanzen, ETH-Zentrum, Sonneggstrasse 5, CH-8092, Zurich, Switzerland

Authors and Affiliations

  1. MSU-DOE Plant Research Laboratory, Michigan State University, 48824, East Lansing, MI, USA

    Erwin Grill & Chris Somerville

Authors

  1. Erwin Grill

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  2. Chris Somerville

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Additional information

Communicated by E. Meyerowitz

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Grill, E., Somerville, C. Construction and characterization of a yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking. Molec. Gen. Genet. 226, 484–490 (1991). https://doi.org/10.1007/BF00260662

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  • Issue Date: May 1991

  • DOI: https://doi.org/10.1007/BF00260662

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