Abstract
Techniques for constructing genomic yeast DNA libraries and for isolating yeast genes from them, mainly by complementation, have been known for over 15 yr. The first method (1) involved making the library in an Escherichia coli plasmid vector, but as soon as “shuttle” vectors—capable of stable propagation in both E. coli and Saccharomyces cerevisiae—became available, these were used in preference. The key advantage of shuttle vectors is that they permit selection for expression of yeast genes in a yeast S. cerevisiae host, but the vector containing the desired yeast gene can then be transferred (“shuttled”) back to allow easier plasmid DNA isolations and manipulations. In essence, the usual approach is to isolate pure genomic DNA from the yeast of interest, partially digest the DNA with an appropriate restriction enzyme (so ensuring any particular sequence of interest will occur intact, on a reasonably sized DNA fragment), size-fractionate the cut DNA (to eliminate small, under gene-sized fragments), insert the DNA in an appropriate shuttle vector, and transform it into E. coli, to make an initial, bacterial, clone library. E. coli transformation is very efficient, compared with yeast transformation, and so allows a good-sized clone library to be generated from even relatively small quantities of ligated genomic-vector DNA. Library DNA is then extracted from the pooled E. coli clones and transformed into an appropriate host strain of S. cerevisiae, thus creating a clone library in yeast, which is then used to screen or select for a desired gene.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Struhl, K., Cameron, J. R., and Davis, R. W. (1976) Functional genetic expression of eukaryotic DNA in Escherichia coli. Proc. Natl. Acad. Sci. USA 73, 1471–1475.
Parent, S. A., Fenimore, C. M., and Bastian, K. A. (1985) Vector systems for the expression, analysts and cloning of DNA sequences in Saccharomyces cerevisiae. Yeast 1, 83–138.
Naysmith, K. A. and Reed, S. I. (1980) Isolation of genes by complementation in yeast molecular cloning of a cell-cycle gene. Proc. Natl. Acad. Sci. USA 77, 2119–2123.
Tréton, B. Y., Le Dall, M.-T., and Gaillardin, C. M. (1992) Complementation of acid phosphatase mutation by a genomic sequence from the yeast Yarrowia lipolytica identifies a new phosphatase. Curr. Genet. 22, 345–355.
Kinsella, B. T., Larkin, A., Bolton, M., and Cantwell, B. A. (1991) Molecular cloning and characterisation of a Candida tsukubaensis α-glucosidase gene in Saccharomyces cerevisiae. Curr. Genet. 20, 45–52.
Abarca, D., Fernandez-Lobato, M., and Jiminez, A. (1991) Isolation of new gene (SWA2) encoding an α-amylase from Schwanniomyces occidentalis and its expression in Saccharomyces cerevisiae. FEBS Lett. 279, 41–44.
Stark, M. J. R. and Milner, J. F. (1989) Cloning and analysis of the Kluyveromyces lactis TRP1 gene: a chromosomal locus flanked by genes encoding inorganic pyrophosphatase and histone H3. Yeast 5, 35–50.
Tsay, Y. H. and Robinson, G. W. (1991) Cloning and characterisation of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase. Mol. Cell Biol. 11, 620–631.
Burke, D. T., Carle, G. F., and Olson, M. V. (1987) Cloning large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 2336, 806–812.
Sikorski, R. S. and Hiefer, P. (1989) A system of shuttle vectors and host strains designed to give efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122, 19–27.
Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 412–488.
Brearley, R. D. and Kelly, D. E. (1991) Genetic engineering techniques in yeast, in Genetically-engineered proteins and enzymes from yeasts: production and control (Wiseman, A., ed.), Ellis Horwood, Chichester, pp. 75–95.
Cryer, D. R., Eccleshall, R., and Marmur, J. (1975) Isolation of yeast DNA, in Methods in Cell Biology (Prescott, D. M., ed.), Academic, New York and London, pp. 39–44.
Birnboim, H. C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523.
Ward, A. C. (1990) Single-step purification of shuttle vectors from yeast for high frequency back transformation into E. coli. Nucleic Acids Res. 18, 5319.
Bignell, G. R., Bruce, I. J., and Evans, I. H. Electrophoretic karyotype of the amylolytic yeast Lipomyces starkeyi and cloning and chromosomal localisation of its TRP1 gene, Current Genetics, in press.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Gingold, E. B. (1984) The use of restriction endonucleases, in Methods in Molecular Biology, vol 2, Nucleic Acids. (Walker, J. W., ed.), Humana, Clifton, NJ, pp. 217–223.
McClelland, M., Hanish, J., Nelson, M., and Patel, Y. (1988) KGB: a single buffer for all restriction endonucleases. Nucleic Acids Res. 16, 364.
Gaastra, W. and Jorgensen, P. L. (1984) The extraction and isolation of DNA from gels, in Methods in Molecular Biology, vol 2 (Walker, J. M., ed.), Humana, Clifton, NJ, pp. 67–76.
Dale, J. W. and Greenaway, P. J. (1984) The use of alkaline phosphase to prevent vector regeneration, in Methods in Molecular Biology, vol 2, Nucleic Acids (Walker, J. W., ed.), Humana, Clifton, NJ, pp. 231–236.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Bignell, G.R., Evans, I.H. (1996). Genomic Yeast DNA Clone Banks. In: Evans, I.H. (eds) Yeast Protocols. Methods in Molecular Biology™, vol 53. Humana Press. https://doi.org/10.1385/0-89603-319-8:155
Download citation
DOI: https://doi.org/10.1385/0-89603-319-8:155
Publisher Name: Humana Press
Print ISBN: 978-0-89603-319-1
Online ISBN: 978-1-59259-540-2
eBook Packages: Springer Protocols