The Saccharomyces cerevisiae haploid genome includes six copies of the gene encoding tRNATrp which are scattered on five chromosomes. Other, non-functional tDNATrp fragments also occur in the genome. The segments of all six genes which encode the 72-nucleotide mature tRNATrp, as well as a 34-nucleotide intervening sequence, are identical. However, the 5' and 3' flanking sequences diverge virtually at the boundaries of the coding region. We have used an assay based on suppression of UGA mutations by multi-copy clones of tDNATrp to search for functional differences among these genes. Previous studies with one tDNATrp had demonstrated that moderate suppression of a UGA mutation, leu2-2, resulted from introduction of a multi-copy clone of the gene. Attempts to use this assay to select tDNATrp clones from a yeast genomic library yielded only four of the six different clones. The other two genes were amplified by PCR and cloned in pRS202, a 2 mu vector also used for the genomic library. Plasmids bearing the six tRNA genes were transformed into S. cerevisiae strain JG369.3B and scored for their ability to suppress the leu2-2 mutation as well as his4-260, another UGA marker. Two of the six tRNATrp clones were unable to suppress either marker, two evidenced weak suppression of the Leu auxotrophy, and two were able to suppress both markers. Growth rates in liquid media requiring suppression were measured for cell lines carrying each of the clones. Differences greater than 50-fold were observed in media lacking histidine. An evolutionary tree based on 5'-flanking sequence corresponds reasonably well with suppressor activity, while a similar analysis of 3'-flanking sequence does not. This suggests that the functional differences are based on divergence in the 5'-flanking sequences of the tRNATrp genes.

• PMID: 9392080
• DOI full text
• PubMed

Reference Type

Comparative Study | Journal Article | Research Support, U.S. Gov't, Non-P.H.S.

Authors

Ong WC, Ibrahim M, Town M, Johnson JD

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