Bun-Ichiro Ono

Inheritance of chromosome length polymorphisms in Saccharomyces cerevisiae

SummaryAlthough Saccharomyces cerevisiae strains generally have similar chromosomal band patterns as revealed by pulsed field gel electrophoresis, individual bands often move slightly differently from one strain to the other. Surveying strains from our stock collection, we found that nearly all the bands of a certain pair of strains differed in their mobility. Some of these chromosome length polymorphisms segregated in a 2:2 ratio, indicating that they resulted from single structural alterations (i.e. additions or deletions). One of these was mapped on the right arm of chromosome 1. Others did not segrate in a simple 2:2 ratio. That is, there were progenies which had bands not present in either parent. We suggest that these new bands are the products of recombination between homologous chromosomes having two or more structural alterations.

Misreading of the ribosomal suppressor SUP46 due to an altered 40 S subunit in yeast

Abstract The dominant suppressor, SUP46 , in the yeast Saccharomyces cerevisiae acts on a wide range of different types of mutations. The incorporation of phenylalanine and the misincorporation of leucine in a cell-free system programmed with poly(U) indicated that the ribosomes from a SUP46 strain produce abnormally high rates of translation errors. Furthermore, the cell-free translation system was used to demonstrate that the SUP46 defect resides in the 40 S ribosomal subunit. The growth of SUP46 strains was shown to be unusually sensitive to paromomycin, an aminoglycoside antibiotic that is known to induce translation errors. In addition, paromomycin stimulated mistranslation with SUP46 ribosomes to a greater extent than with normal ribosomes. These results indicate that SUP46 suppression is caused by increased translation errors as a result of the mutationally altered 40 S ribosomal subunit. Paromomycin appears to produce translation errors in SUP46 strains at rates that are too high for cellular growth.

Altered ribosomal protein S11 from the SUP46 suppressor of yeast

Abstract The dominant suppressor SUP46 of the yeast Saccharomyces cerevisiae was shown to act on a wide range of mutations (preceding paper by Ono et al., 1981). Masurekar et al. (1981) demonstrated that ribosomes from the SUP46 strain make an abnormally high rate of errors in a cell-free translation system. These findings indicated that SUP46 suppression was the result of abnormal ribosomes misreading mutant codons. We have used two-dimensional polyacrylamide gel electrophoresis to show that the S11 protein from the 40 S ribosomal subunit has an altered electrophoretic mobility. Thus the gene product of the SUP46 locus is either the S11 ribosomal protein or an enzyme that modifies the S11 protein. These results demonstrate that the altered S11 protein is responsible for the suppression by misreading.

Serine insertion caused by the ribosomal suppressor SUP46 in yeast

Abstract The ribosomal suppressor SUP46 isolated from the yeast Saccharomyces cerevisiae suppresses a broad range of mutations, including at least some UAA, UAG and UGA alleles. The SUP46 suppressor causes the insertion of serine into iso-1-cytochrome c at the site of the UAA mutation in the cyc1-72 allele. It is believed that the altered ribosomes in the SUP46 suppressor allow a serine tRNA to misread UAA codons.

Mutagenicity of 5-bromouracil and N6-hydroxyadenine studied by yeast oligonucleotide transformation assay

The mutagenicity of 5-bromouracil (BrU) and N6-hydroxyadenine (HA) was tested by means of the yeast oligonucleotide transformation procedure. BrU-containing oligonucleotide was not mutagenic; although two mutants (per 200 micrograms oligonucleotide) were obtained, they were attributed to base insertion or base substitution at positions different from BrU. This result supports the view that BrU mutagenesis is dependent on intracellular nucleotide pool imbalance. In contrast, HA-containing oligonucleotide was highly mutagenic; 56 mutants (per 140 micrograms oligonucleotide) were obtained. Of 21 induced mutants examined, 20 had G and one had C at the HA position, a result indicating that HA-->G changes took place. To provide back-up evidence, we carried out a general reversion assay for base HA using a set of yeast tester strains, and the results showed that HA induces exclusively AT-to-GC and GC-to-AT transitions. We conclude that in S. cerevisiae HA is a classic base analog mutagen, causing AT-to-GC and GC-to-AT transitions by ambiguous base pairing. The present work has clearly demonstrated the usefulness of the oligonucleotide transformation procedure for elucidating mutagenicity of modified bases.

Biosynthesis of sulphur amino acids in Saccharomyces cerevisiae: regulatory roles of methionine and S-adenosylmethionine reassessed

Summarycys4-1, a mutation in the reverse trans-sulphuration pathway, relieves the sulphate assimilation pathway and homocysteine synthase from methionine-mediated repression. Since the mutation blocks the synthesis of cysteine from methionine downstream from homocysteine, this indicates that neither methionine nor S-adenosylmethionine serve as low-molecular-mass effectors in this regulatory system, contradicting earlier hypotheses.

Genetic mapping of leucine-inserting UAA suppressors in Saccharomyces cerevisiae

SummaryOf S. cerevisiae UAA specific suppressors three leucine-insertors have been remaining unmapped. Mapping of them was attempted by using a set of eight strains that contained a total of 60 markers distributing over the entire genome; the markers were about 30 cM apart and they covered about 70% of the genome. Two supressors, SUP28 and SUP33, were localized on the right arm of chromosome XIV and on the left arm of chromosome XI, respectively. The other suppressor, SUP32, was not mapped, but its location was confined to a few regions in the genome. The present result and the previously available informations clearly show that the UAA suppressor loci are dispersedly distributing ovet the entire S. cerevisiae genome.

Serine-inserting UAA suppression mediated by yeast tRNASer☆

Abstract The number of loci that give rise to serine-inserting UAA suppressors in the yeast Saccharomyces cerevisiae was determined by examining over 100 of the revertants that suppressed the two UAA markers his4-1176 and leu2-1: the his4-1176 marker is suppressed by serine-inserting but not by tyrosine- or leueine-inserting suppressors and the leu2-1 marker is suppressed by all UAA suppressors. The suppressors could be assigned to one or other of the four loci: SUP16 and SUP17. which were previously known to yield serine-inserting suppressors, and SUP19 and SUP22. The chromosomal map position of SUP19 suggested that it may be allelic to the previously reported suppressor SUP20, while the SUP22 suppressor has not been described. Representatives of all of the four suppressors were found to insert serine at the UAA site in iso-1-cytochrome c from suppressed cyc1-72 strains. The degree of suppression by the serine-inserting suppressors was SUP16 > SUP17 > SUP19 > SUP22. The efficiency of suppression of each of the four serine suppressors was increased by the chromosomal mutation sal and by the cytoplasmic determinant ψ+. Read-through of the synthetase gene of the RNA bacteriophage Qβ in a cell-free system was used to demonstrate that tRNASer from SUP16, SUP17 and SUP19 strains can translate UAA codons. In contrast, tRNASer or total tRNA from SUP22 strains had no suppressing activity. The results suggest that the three loci SUP16, SUP17 and SUP19 encode iso-accepting species of tRNASer, and that the UAA suppression is mediated by mutationally altered tRNA molecules. The mechanism of SUP22 suppression remains unknown.

Cloning and mapping of the CYS4 gene of Saccharomyces cerevisiae

SummaryA DNA fragment containing the CYS4 gene of Saccharomyces cerevisiae was isolated from a genomic library. The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV. According to the 2 μm DNA chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VII. By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ade3 on the right arm of chromosome VII.

Chromium resistant mutants of the yeast Saccharomyces cerevisiae.

SummaryMany strains of Saccharomyces cerevisiae do not grow on YPD agar containing 750 μg/ml CrO3. Mutants able to grow in the presence of 850 μg/ml CrO3 were obtained from such strains after UV mutagenesis. All of the mutants grew even in the presence of 1,000 μ/ml CrO3. Chromium resistance was dominant or partial dominant over normal response, therefore it was impossible to determine the number of genetic loci by complementation analysis. However, the segregation of representative mutants strongly indicated that resistance was determined by single mutations. In addition, a limited analysis of recombination suggested that the chromium resistant mutations were located on a certain region of the yeast genome. Although it was determined that the mutants had slightly reduced rates of Cr6+ uptake, the exact mechanism of resistance was not discovered. According to the studies of interactions between resistant mutations and sensitive mutations, however, we have proposed a preliminary pathway of Cr6+ detoxification.