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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
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
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
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
Browse J, Somerville CR, Slack R (1988) Changes in lipid composition during protoplast isolation. Plant Sci 56:15–20
Burgers PMJ, Percival KJ (1987) Transformation of yeast spheroplasts without cell fusion. Anal Biochem 163:391–397
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
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
Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81:1991–1995
Coulson A, Waterston R, Kiff J, Sulston J, Kohara Y (1988) Genome linking with yeast artificial chromosomes. Nature 335:184–186
Garza D, Ajioka JW, Burke DT, Hard DL (1989) Mapping the Drosophila genome with yeast artificial chromosomes. Science 246:641–646
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
Meyerowitz EM (1989) Arabidopsis, a useful weed. Cell 56:263–269
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
Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120:621–623
Pruitt RE, Meyerowitz EM (1986) Characterization of the genome of Arabidopsis thaliana. J Mol Biol 187:169–183
Sherman F, Fink GR, Hicks JB (1986) Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
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
Somerville CR (1989) Arabidopsis blooms. Plant Cell 1:1131–1135
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
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
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
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
Author information
Authors and Affiliations
Additional information
Communicated by E. Meyerowitz
Rights and permissions
About this article
Cite this article
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
Issue Date:
DOI: https://doi.org/10.1007/BF00260662