Yeast amber suppressors corresponding to tRNA3Leu genes

Amber suppressors previously isolated from the yeast Saccharomyces cerevisiae and belonging to the same phenotypic class (Liebman et al., 1976) were assigned to nine different linkage groups named SUP52 through SUP60. One of these suppressors, SUP52, had been shown to cause the insertion of leucine and had been genetically mapped (Liebman et al., 1977). The following additional amber suppressors were mapped: SUP53 maps near the centromere of chromosome III closely linked to leu2; SUP54 maps on chromosome VII, 6 cM distal to trp5; SUP56 maps on chromosome I, 5.4 cM distal to ade1; SUP57 maps on chromosome VI, closely linked to met10; and SUP58 maps on the left arm of chromosome XI, loosely linked to met14. We show by protein analysis that like SUP52, the suppressors SUP53 through SUP56 are leucine-inserters. Furthermore, by hybridization with a cloned tRNA3Leu probe we demonstrate that at least SUP53, SUP54, SUP55 and SUP56 contain mutations in redundant tRNA3Leu genes because they each generate a new XbaI site in a DNA fragment encompassing a tRNA3Leu gene. These new XbaI sites are predicted by the known sequences of tRNA3Leu genes if the CAA anticodon mutates to the amber suppressing anticodon CTA. It is likely that each of the nine suppressors in this phenotypic class contain similar mutations in different tRNA3Leu genes since we find that there are approximately nine unlinked redundant copies of tRNA3Leu genes in haploid strains.     

Genetic and molecular analyses of the SUP201 gene: a tRNA(3Arg) nonsense suppressor of yeast cyrl-2.

The temperature-sensitive cyr1-2 mutant in Saccharomyces cerevisiae produces low levels of adenylate cyclase and cyclic AMP at 25 degrees C and is unable to synthesize repressible acid phosphatase at 25 degrees C. Suppressor mutants of cyr1-2 were isolated by detecting acid phosphatase activity. One of the dominant suppressor mutations isolated was designated SUP201 and characterized. The SUP201 mutant gene was isolated from a gene library made from cyr1-2 SUP201 mutant DNA. Nucleotide sequence analysis of the cloned SUP201 gene revealed that the SUP201 gene was a mutated tRNA gene flanking GCN4, which worked as a UGA suppressor.     

Isolation of a yeast gene involved in species-specific pre-tRNA processing.

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.     

Increased tRNA concentration in yeast containing mutant termination translation factors eRF1 and eRF3

Earlier we have characterized strains bearing mutations in essential genes SUP45 and SUP35 of yeast S. cerevisiae, encoding translation termination factors eRF1 and eRF3 respectively. In the present work nonsense-mutants on genes SUP45 and SUP35 have been compared by a level of eight tRNA: tRNATyr, tRNAGln, tRNATrp, tRNALeu and tRNAArg (previously described as potentially suppressor tRNA), and also tRNAPro, tRNAHis and tRNAGly. We have not revealed preferable increase in amount of natural suppressor tRNA. The majority of the investigated mutations leads to increase in a level of all investigated tRNA. The mechanisms providing viability of nonsense-mutants on essential genes SUP45 and SUP35 are discussed.     

Concerted deletions and inversions are caused by mitotic recombination between delta sequences in Saccharomyces cerevisiae.

Deletions of a tyrosine tRNA suppressor gene, SUP4-o, are mediated by recombination between short repeated delta sequences in Saccharomyces cerevisiae. The arrangement of the five solo delta sequences that surround the SUP4 locus was established by DNA sequence analysis. Seven deletion classes were identified by genomic blotting. DNA sequence analysis also showed that the delta sequences within a 6.5-kilobase region of the SUP4 locus were the endpoints of these events. In three of these classes, an adjacent interval surrounded by delta sequences was inverted in concert with the deletion. The frequency of all deletion classes decreased in strains that contained mutations in the recombination and repair gene RAD52. We present two gene conversion mechanisms by which these rearrangements could have been generated. These models may also explain deletions between repeated sequences in other systems.     

Deletions of a tyrosine tRNA gene in S. cerevisiae.

Genetic fine structure analysis of a tyrosine tRNA in yeast revealed that complete deletions of the gene occurred at an unusually high frequency. Among 56 spontaneous mutations at the SUP4 locus, 16 were classified as deletions as judged by their failure to recombine with any other mutations known to map within the gene. Physical analysis of each deletion confirmed the genetic result. The deletions fall into two size classes: ten are 2100 bp deletions and six are 2800 bp deletions. These results imply that the physical structure of the region surrounding the SUP4 locus, which is known to contain short repeated segments, has a direct role in promoting deletions.     

The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae.

The product of the yeast SUP45 gene (Sup45p) is highly homologous to the Xenopus eukaryote release factor 1 (eRF1), which has release factor activity in vitro. We show, using the two-hybrid system, that in Saccharomyces cerevisiae Sup45p and the product of the SUP35 gene (Sup35p) interact in vivo. The ability of Sup45p C-terminally tagged with (His)6 to specifically precipitate Sup35p from a cell lysate was used to confirm this interaction in vitro. Although overexpression of either the SUP45 or SUP35 genes alone did not reduce the efficiency of codon-specific tRNA nonsense suppression, the simultaneous overexpression of both the SUP35 and SUP45 genes in nonsense suppressor tRNA-containing strains produced an antisuppressor phenotype. These data are consistent with Sup35p and Sup45p forming a complex with release factor properties. Furthermore, overexpression of either Xenopus or human eRF1 (SUP45) genes also resulted in anti-suppression only if that strain was also overexpressing the yeast SUP35 gene. Antisuppression is a characteristic phenotype associated with overexpression of both prokaryote and mitochondrial release factors. We propose that Sup45p and Sup35p interact to form a release factor complex in yeast and that Sup35p, which has GTP binding sequence motifs in its C-terminal domain, provides the GTP hydrolytic activity which is a demonstrated requirement of the eukaryote translation termination reaction.     

The spectrum of spontaneous mutations in a Saccharomyces cerevisiae uracil-DNA-glycosylase mutant limits the function of this enzyme to cytosine deamination repair.

Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, O. N., V. M. Golubovskaya, T. A. Kusmin, and N. V. Tomilin, Mutat. Res. 213:135-140, 1989). This hypothesis was investigated with Saccharomyces cerevisiae. Mutation frequencies and spectra were determined for an ung1 deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme. Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S. cerevisiae strain, and the mutator effect was completely suppressed in the ung1 deletion strain carrying the wild-type UNG1 gene on a multicopy plasmid. Sixty-nine independently derived mutations in the SUP4-o gene were sequenced. All but five of these were due to GC----AT transitions. From this analysis, we conclude that the mutator phenotype of the ung1 deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S. cerevisiae and that the UNG1 gene is not required for strand-specific mismatch repair in S. cerevisiae.