Cloning Genes by Complementation in Yeast

Mark D. Rose, James Broach

Research output: Contribution to journalArticle

208 Citations (Scopus)

Abstract

This chapter describes the use of genomic and cDNA banks to isolate specific genes by complementation in Saccharomyces cerevisiae. The most straightforward approach to cloning genes from plasmidborne banks is complementation of a recessive marker. A recipient strain is constructed that carries a recessive mutation in the gene of interest as well as a nonreverting null allele of the chromosomal cognate of the selectable marker carried on the plasmid vector, This strain is then transformed with pools of plasmids from a bank constructed from wild-type genomic DNA. Transformants are recovered by selecting for eomplementation by the vector-borne selectable marker. Cloning genes that are defined by dominant alleles is a straightforward extension of cloning by complementation of recessive alleles. The only difference is that the clone bank has to be constructed de novo from genomic or cDNA prepared from the strain carrying the dominant mutation. In the absence of any direct information about the identity of a gene or its gene product, one recourse is to isolate a set of genes whose regulation fulfills some interesting set of criteria. One approach to achieving this end has been to clone random genomic fragments into a plasmid carrying an enhancerless promoter that drives expression of a readily scored gene, such as lacZ. Random transformants are then examined for conditional expression of lacZ in response to the desired signal.

Original languageEnglish (US)
Pages (from-to)195-230
Number of pages36
JournalMethods in Enzymology
Volume194
Issue numberC
DOIs
StatePublished - Jan 1 1991

Fingerprint

Cloning
Yeast
Organism Cloning
Genes
Yeasts
Plasmids
Alleles
Complementary DNA
Clone Cells
Dominant Genes
Mutation
Gene expression
Saccharomyces cerevisiae
DNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

Rose, Mark D. ; Broach, James. / Cloning Genes by Complementation in Yeast. In: Methods in Enzymology. 1991 ; Vol. 194, No. C. pp. 195-230.
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Cloning Genes by Complementation in Yeast. / Rose, Mark D.; Broach, James.

In: Methods in Enzymology, Vol. 194, No. C, 01.01.1991, p. 195-230.

Research output: Contribution to journalArticle

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