Cloning plant genes by complementation of yeast mutants.

Publisher Summary This chapter presents a procedure to construct an A. thaliana complementary DNA (cDNA) expression library and transformation strategies that can be used to introduce the cDNA library into mutant Saccharomyces cerevisiae strains and to recover complementing plasmids. To construct the cDNA library, a multicopy S. cerevisiae expression vector pFL61 is used. pFL61 allows the cloning of cDNAs at two incompatible BstXI sites, thus avoiding its recircularization during the ligation step. cDNAs are cloned downstream of the S. cerevisiae phosphoglycerate kinase (PGKI) promoter and upstream of the PGKl terminator. Cloning in this locus, together with the high copy number of the vector, allows high-level constitutive expression in S. cerevisiae when the cDNA is cloned in the proper orientation. This method is highly efficient for identifying plant genes with functional yeast homologs and does not depend on any sequence homology of the encoded proteins. Although it has been used to complement S. cerevisiae mutations, there are no technical limitations to the adaptation of this strategy to cloning genes by complementation of mutations obtained in other genetically amenable lower eukaryotes, such as S. pombe , Aspergillus nidulans , or Neurospora crassa .