Kate Loughney

Identification of the yeast DNA sequences that correspond to specific tyrosine-inserting nonsense suppressor loci☆

Abstract There are eight unlinked genes for yeast tyrosine transfer RNA. In previous work, nonsense suppressors have been isolated at each of the eight loci, and these loci have been genetically mapped (Hawthorne & Leupold, 1974). It has also been demonstrated by RNA-DNA hybridization that the genes are physically located on eight different Eco RI restriction fragments (Olson et al. , 1977). The purpose of the present report is to cross-correlate the set of tyrosine-inserting suppressor loci with the set of tRNA Tyr -hybridizing restriction fragments. This cross-correlation was achieved for six of the eight loci by analyzing the meiotic and mitotic linkage between the tyrosine-inserting suppressors and the genetic determinants of naturally occurring size variants of the tRNA Tyr -hybridizing restriction fragments. Now that individual suppressor loci have been identified with specific DNA fragments, it should be possible to analyze the phenotypes of these mutant genes in terms of their DNA sequences. The method by which these assignments were made also offers a new approach to the general problem of correlating genes with restriction fragments; it is particularly suited to organisms with powerful genetic systems in which hybridization to chromosome spreads in situ is impractical.

Physical analysis of the CYC1-sup4 interval in Saccharomyces cerevisiae.

CYC1 and sup4 are part of a tightly linked cluster of genes on chromosome X in the yeast Saccharomyces cerevisiae. Using as probes previously cloned fragments containing the CYC1 and sup4 genes, we have identified and cloned the deoxyribonucleic acid (DNA) present between these genes in one strain of yeast. We find that the CYC1 and sup4 genes are approximately 21 kilobases apart. In the same strain, the meiotic map distance is approximately 3.7 centimorgans, for a ratio of 5.6 kilobases per centimorgan in this interval. The physical mapping has allowed unambiguous determination of the orientation of CYC1 and sup4 relative to each other, the centromere, and a nearby transfer ribonucleic acid (tRNA(2Ser)) gene. The spontaneous mutation cyc1-1 inactivates the CYC1 gene as well as the neighboring loci OSM1 and RAD7. We have determined that a cyc1-1-bearing strain lacks approximately 13 kilobases of single-copy DNA from the CYC1-sup4 region, including all of the CYC1 coding information. There is a sequence homologous to the middle-repetitive element Ty1 at or near the breakpoint of the cyc1-1 deletion. We discuss the possibility that Ty elements play a role in the formation of such large, spontaneous deletions, which occur frequently in this region of chromosome X in certain yeast strains.

Yeast Suppressor tRNA Genes

The advent of rapid techniques for isolating and characterizing specific eukaryotic DNA sequences offers the promise of studying the structure and function of eukaryotic tRNA genes at the level of individual nucleotides. tRNA genes in any eukaryotic organism are readily accessible by these techniques; both their small size and the availability of purified tRNAs as RNA-DNA hybridization probes facilitate applications of molecular cloning and DNA sequencing to these genes. It has been our view, however, that the complex series of steps by which the coding sequence of a tRNA gene is expressed as a mature, functional tRNA molecule is unlikely to be decipherable by structural studies alone. We have chosen, therefore, to focus our efforts on the molecular cloning and physical analysis of a small set of yeast tRNA genes that have been genetically identified as nonsense suppressors (Hawthorne and Leupold 1974). Twelve genetically mapped loci in yeast correspond to nonsense suppressors of known amino-acid-insertion specificity (Table 1). In only two cases, SUP5 UAG and SUPRL1 UAG (Piper et al. 1976; Piper 1978), have these nonsense suppressor mutations been shown to affect the primary structure of a tRNA, but there is now little doubt that all the loci in Table 1 correspond to tRNA genes. The tyrosine-inserting loci have been particularly intensively studied at the DNA level and a SUP4 UAA gene was shown to be mutant in the DNA sequence specifying a tRNA Tyr anticodon (Goodman et al. 1977; M. V. Olson et al. 1977, 1979, in prep.). HYBRIDIZATION TO RESTRICTION DIGESTS We...