We have analyzed the genes encoding tRNAUCASer of yeast. By hybridization analysis we have established that only three genes encode tRNAUCASer, all of which have been cloned. The DNA sequences of the coding regions of all the three genes are identical, although considerable sequence divergence exists at the 3′ and 5′ flanking regions. The suppressor locus to which each of the three cloned tRNAUCASer genes corresponds was determined by integrative transformation of yeast. A leu2- sup+ yeast strain was transformed to Leu+ with plasmids which carried the LEU2 gene of yeast and one of the DNA fragments containing a tRNAUCASer gene. Integration of the plasmid at the homologous transfer RNA locus on the chromosome introduced the LEU2 marker at the site, which could then be followed in standard genetic crosses. By this method, we have shown that the three cloned fragments correspond to the serine-inserting nonsense suppressors SUP16, SUP17 and SUP19. Finally, the SUP16 allele was recovered directly from yeast by integrating a pBR322-LEU2-sup16+ hybrid plasmid at the SUP16 locus. Appropriate digestion of genomic DNA isolated from the transformed strain permitted recovery of either the wild-type allele or the SUP16 allele on plasmids that were otherwise identical to the original transforming plasmid. This method is generally applicable to the recovery of any mutant allele of yeast for which one has DNA containing the corresponding wildtype gene.
All Science Journal Classification (ASJC) codes
- Structural Biology
- Molecular Biology