Isolation and characterization of EMS induced splicing defective point mutations within the intron of the nrdB gene of bacteriophage T4

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. The screening, isolation, and mapping of 31 nrdB intron mutations were conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase. These intron mutations cluster towards the ends, mainly the 3' end, and show a defect in self-splicing. These mutations map in regions of conserved structural elements, thus supporting secondary structure predictions. A distinct pattern of clustering is observed with the highest number of mutations mapping within three of the smaller regions (A, C, and D) of the nrdB intron and no mutations mapping in the largest (B) region. The highest density of mutations mapped in the smallest region (C) of the intron, containing only 96 bases, thus showing a distinct pattern of clustering within the catalytic core.