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

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 14 and alkaxyotes. The screening, isolation and mapping of the nrbB intron mutations was conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase. We have isolated 159 hydroxylamine-induced nrdB mutants, determined which mutations are in nrdB by marker rescue with clones of the nrdB gene and have mapped these mutations by marker rescue using subclones of the nrdB intron. Thirty out of the 159 nrdB mutations are in or near the intron. These mutations cluster towards the ends, mainly the 3′ end. We have performed deletion mapping to further map mutations in the 3′ end of the intron. The mutations map in regions of conserved structural elements, thus supporting secondary structure predictions similar to those of the well studied td intron in the T4 gene coding for thymidylate synthase.