A Novel Approach for Isolation and Mapping of Second-Site Revertants of Intron Mutations in a Ribontjcleotide Reductase Encoding Gene (nrdB) of Bacteriophage T4 Using the White Halo Plaque Phenotype

Abstract

Abstract The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598 base pair self splicing intron which is closely related to other group I introns of T4 and eukaryotes. Previously, the nrdB intron mutations, presumably causing splicing defects of the nrdB transcript, were isolated and mapped in or near the nrdB intron. In this communication, we have isolated 181 hydroxylamine-induced revertants for the above primary nrdB intron mutations by strategic usage of the white halo phenotype. Also, we mapped these revertant mutations by marker rescue with subclones of the nrdB gene. Some of the second site mutations were mapped to regions predicted by the secondary structure model of the nrdB intron. To investigate the involvement of protein factors facilitating splicing of the nrdB transcript, we attempted to isolate extragenic revertants of td-nrdB double mutants by utilizing hydroxylamine mutagenesis. Mapping and sequencing of the suppressor mutations in the extragenic revertants revealed that the second-site mutations are in the frd gene, coding for dihydrofolate reductase. Splicing assays showed that these suppressor mutations do not reverse the splicing defects of td-nrdB mutants, but only affect the halo phenotype, most likely by altering the pyrimidine nucleotide metabolism.