The yeast cloning vector YEp13 contains a tRNALeu3 gene that can mutate to an amber suppressor

Abstract

We have shown that the yeast-Escherichia coli shuttle vector YEp13 contains, as part of its yeast chromosomal segment, a tRNALeu3 gene. We have also isolated and characterized a variant of YEp13 , namely YEp13 -a, which is capable of suppressing a variety of yeast amber-suppressible alleles in vivo. YEp13 -a differs from YEp13 by a single point mutation, which changes the three-nucleotide, plus-strand sequence corresponding to the tRNALeu3 anticodon from the normal C-A-A to C-T-A. This nucleotide change creates a site for the restriction enzyme XbaI in the suppressor tRNALeu3 gene. We have taken advantage of the correlation between the suppressor mutation and the XbaI site formation, to show that the tRNALeu3 gene on YEp13 corresponds to the genetically characterized yeast chromosomal amber suppressor SUP53 . We have also shown that SUP53 is located just centromere-distal to LEU2 on chromosome III. Finally, comparison of the DNA sequence of SUP53 and its flanking regions with the sequences of other cloned yeast tRNALeu3 genes has revealed considerable sequence homology in the immediate 5'-flanking regions of these genes.