GENETIC ANALYSIS OF STAPHYLOCOCCAL NUCLEASE: IDENTIFICATION OF THREE INTRAGENIC "GLOBAL" SUPPRESSORS OF NUCLEASE-MINUS MUTATIONS

Abstract

A collection of 77 unique missense mutations distributed across the gene encoding staphylococcal nuclease (nuc) has been assembled. These mutations were induced by random gap misrepair mutagenesis of the cloned gene and were identified in E. coli transformants expressing reduced levels of nuclease activity. Four nuc - mutations which alter amino acid residues at positions outside of the active site region of the enzyme were submitted to a second round of mutagenesis, and characterization of several independent NUC+ isolates lead to the identification of three second-site suppressor mutations within the protein-coding sequence of the nuc gene. On separation from the mutation originally suppressed and recombination with a number of other nuc - mutations, all three suppressors displayed the property of "global" suppression, i.e., phenotypic suppression of the nuclease-minus character of multiple different alleles. A simple and generally applicable strategy was used to obtain efficient homologous recombination between plasmids for purposes of mapping nuc - mutations, mapping second-site suppressors and constructing double mutant combinations from pairs of single mutations.