A method for rapid mapping of mutations by plasmid rescue strategy inSaccharomyces cerevisiae

Abstract

The products of PRP17 and PRP18 genes are required for the second step of pre-mRNA splicing reactions in Saccharomyces cerevisiae. Temperature-sensitive mutants at either of these loci accumulate products of the first splicing reaction at nonpermissive temperature. To characterize functional regions in these proteins the mutations in three temperature-sensitive alleles of PRP17 and two temperature-sensitive alleles of PRP18 were mapped by the plasmid rescue strategy. One of the procedures adopted in the past is plasmid rescue of the mutant allele followed by sequencing of the entire gene. In this work we describe an adaptation of the above procedure that allows, first, rapid mapping of chromosomal segments bearing the mutations, followed by sequence characterization of the minimal segment. The strategy adopted was to integrate a wild-type copy of the gene at the homologous mutant chromosomal locus, followed by recovery of the chromosomal fragments from these integrants as plasmids in E. coli. The recovered plasmids were screened by a complementation assay for those that contained in them the chromosomal mutation. The mutations in ail the three alleles of PRP17 map to a small region in the N-terminal half of the protein, whereas the temperature-sensitive mutations in the two alleles of PRP18 map to different regions of the PRP18 protein. The recovered mutant plasmids from all five alleles at the two loci were sequenced and the nucleotide changes were found to result in missense mutations in each case. Our strategy is therefore a rapid method to map chromosomal mutations and is of general use in structure-function analysis of cloned genes.