Restoration of mRNA splicing by a second-site intragenic suppressor in theT4 ribonucleotide reductase (small subunit) self-splicing intron

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleo tide reductase and contains a 598-base self-splicing intron which is closel y related to other group I introns of T4 and eukaryotes. Thirty-one mutants causing splicing defects in the nrdB intron were isolated. Twenty-three EM S-induced revertants for these 31 primary mutants were isolated by the stra tegic usage of the white halo plaque phenotype. We mapped these revertants by marker rescue using subclones of the nrdB gene. Some of these second-sit e mutations mapped to regions currently predicted by the secondary structur e model of the nrdB intron. One of these suppressor mutants (nrdB753R) was found to be intragenic by marker rescue with the whole nrdB gene. However, this mutation failed to map within the nrdB intron. Splicing assays showed that this pseudorevertant restored splicing proficiency of the nrdB primary mutation to almost wild-type conditions. This is the first example of a mu tation within the exons of a gene containing a self-splicing intron that is capable of restoring a self-splicing defect caused by a primary mutation w ithin the intron. In addition, two other suppressor mutations are of intere st (nrdB429R and nrdB399R). These suppressors were able to restore their pr imary 5' defect but in turn create a 3' splicing defect. Both of these reve rtants mapped in different regions of the intron with respect to their prim ary mutations. (C) 2000 Academic Press.