A NOVEL-APPROACH FOR ISOLATION AND MAPPING OF 2ND-SITE REVERTANTS OF INTRON MUTATIONS IN A RIBONUCLEOTIDE REDUCTASE-ENCODING GENE (NRDB) OFBACTERIOPHAGE-T4 USING THE WHITE HALO PLAQUE PHENOTYPE

The nrdB gene of bacteriophage T4 codes for the small subunit of ribon ucleotide 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 d efects of the nrdB transcript, were isolated and mapped in or near the nrdB intron. In this communication, we have isolated 181 hydroxylamin e-induced revertants for the above primary nrdB intron mutations by st rategic usage of the white halo phenotype. Also, we mapped these rever tant mutations by marker rescue with subclones of the nrdB gene. Some of the second site mutations were mapped to regions predicted by the s econdary structure model of the nrdB intron. To investigate the involv ement of protein factors facilitating splicing of the nrdB transcript, we attempted to isolate extragenic revertants of td-nrdB double mutan ts by utilizing hydroxylamine mutagenesis. Mapping and sequencing of t he suppressor mutations in the extragenic revertants revealed that the second-site mutations are in the frd gene, coding for dihydrofolate r eductase. Splicing assays showed that these suppressor mutations do no t reverse the splicing defects of td-nrdB mutants, but only affect the halo phenotype, most likely by altering the pyrimidine nucleotide met abolism.