John W. Stewart

Specificity and frequency of ultraviolet-induced reversion of an iso-1-cytochrome c ochre mutant in radiation-sensitive strains of yeast☆

The basis for the specific pattern of ultraviolet-induced reversion of cyc1-9, an ochre allele of the structural gene for iso-1-cytochrome c, has been examined in radiation-sensitive strains of yeast. Previous analysis, using RAD+ strains, showed that 21 out of 23 cyc1-9 revertants induced by ultraviolet light arose by A · T to G · C transition at the first position in the UAA codon, the remaining two occurring by A · T to T · A transversion at the second position (Stewart et al., 1972; Sherman & Stewart, 1974). All possible base-pair substitutions could be obtained with the aid of other mutagens. It has now been shown that this specificity depends largely on the action of the RAD6 locus, since ultraviolet-induced revertants of cyc1-9 arose by a variety of base-pair substitutions in a strain carrying the rad6-1 allele. Induced reversion frequencies in strains carrying this allele are much lower than normal, though significantly higher than the spontaneous frequency, and the strains are more sensitive to the lethal effects of both ultraviolet and X-irradiation. The phenotypically similar rad18-2 mutation, which appears to block the same repair pathway as rad6-1, also has some effect on the reversion specificity, but its action depends on the presence of other, unidentified, mutations. Specificity was, however, completely unaltered in an excision-defective strain carrying the rad1-2 allele. Induced reversion frequency of cyc1-9 was much higher than normal in this strain. Photoreactivation studies indicated that pyrimidine dimers were responsible for most of the revertants in RAD+, rad1 and rad6 strains. These experiments show that the RAD6+ locus is intimately concerned with error-prone repair, and suggest that excision repair is substantially error-free.

Amino acid replacements resulting from super-suppression of nonsense mutants of iso-1-cytochrome c from yeast

Abstract The eight class I, set 1 super-suppressor genes, SUP2, SUP3, SUP4, SUP5, SUP6, SUP7, SUP8 and SUP11 are not closely linked and map at distinct loci throughout the genome of yeast. Each of these suppressors causes the production of 5 to 10% of the normal amount of iso-1-cytochrome c when it is individually coupled to the ochre (UAA) mutant cy1 -2. All eight iso-1-cytochromes c contain a residue of tyrosine at position 20 which corresponds to the site of the ochre codon. Several of these super-suppressors also were shown to act on cy1 -9, but at a much lower efficiency. It was shown that iso-1-cytochrome c from one of the suppressed cy1 -9 strains contains a tyrosine at position 2, which corresponds to the site of the ochre codon in this mutant. It is suggested that the gene product of the eight super-suppressors is tyrosine transfer RNA.

Serine substitutions caused by an ochre suppressor in yeast

Abstract The suppressor SUQ5 in yeast can cause the production of approximately 10 to 20% of the normal amount of iso-l-cytochrome c when coupled to the ochre (UAA) mutants cyc1–2 and cyc1–72. The iso-l-cytochromes c contain residues of serine at positions that correspond to the sites of the ochre codons. SUQ5 is efficient only in strains having the non-Mendelian factor ψ+, although the low amount of suppressed iso-l-cytochrome c from a ψ−SUQ5 cyc1–72 strain was also shown to contain serine at the ochre site. Thus SUQ5 differs from the eight other characterized suppressors of UAA in yeast, which were previously shown to insert residues of tyrosine at ochre sites (Gilmore et al., 1971) and which are only effective in strains haying the non-Mendelian factor ψ−, since they generally cause inviability in the ψ+ state. Like the tyrosine-inserting suppressors, SUQ5 can also act on another ochre allele cyc1–9, but with a very low efficiency of approximately 0.4%, while it does not appear to act at all on amber (UAG) mutants. SUQ5 was found to be 6.4 cM (centiMorgans) from tyr7 on chromosome XVI. It is suggested that the gene product of SUQ5 is serine tRNA.

Demonstration of UAG as a nonsense codon in bakers' yeast by amino-acid replacements in iso-1-cytochrome c

Abstract Lysine 9 was replaced by serine, glutamine, tyrosine, leucine or tryptophan in iso-1-cytochrome c from 39 out of 42 intragenic revertants of cy1-179 . No other sequence changes were found in these 39 proteins. It is concluded that the defect in cy1-179 is UAG coding position 9. Less than 0.5% of the normal amount of iso-1-cytochrome c is present in the cy1-179 strain, but the known replacements at position 9 and at neighboring positions strongly suggest that any amino acid at position 9 would permit the formation of at least 75% of the normal amount of iso-1-cytochrome c , having at least 10% of the normal specific activity. It is concluded that UAG is a termination signal in yeast. Since, in addition, cy1-179 is known to be suppressed by a super-suppressor that does not act on UAA, apparently UAG is a translation termination codon in yeast. It is concluded also that only single base-pair substitutions occurred in 39 of the 42 reversion mutations, which arose spontaneously and which were induced by X-ray and ultraviolet irradiation, and by treatments with diethylsulfate and nitrous acid. For all these mutagens and spontaneous events, the frequencies and proportions of G·C, C·G and T·A replacements of A·T base-pairs strongly depend on the local base sequence. The other 3 intragenic revertants, which were induced by X-rays and nitrous acid, registered multiple base-pair substitutions. The sites of multiple change were restricted to the region coding residues 6 through 10. It is suggested that inaccurate repair processes caused the multiplicity of substitutions.

Specific induction of transitions and transversions of G · C base pairs by 4-nitroquinoline-1-oxide in iso-1-cytochrome c mutants of yeast☆

Abstract The base-pair changes induced by the highly carcinogenic agent, 4-nitroquinoline-1-oxide, have been determined from the reversion rates of defined tester strains and from the amino acid replacements of revertant iso-1-cytochromes c . The mutant codons and the base-pair changes of reverse mutations of 14 cyc 1 mutants were previously determined from alterations of iso-1-cytochromes c in intragenic revertants. These 14 cyc 1 mutants, which were used as tester strains, included nine mutants with altered AUG initiation codons, an ochre (UAA) mutant, an amber (UAG) mutant and three frameshift mutants (Stewart et al. , 1971,1972; Stewart & Sherman, 1972,1974; Sherman & Stewart, 1973). NQO † induced a high rate of reversion in the initiation mutant cyc 1-131, the only mutant in the group which reverts to normal iso-1-cytochrome c by a G · C → A · T transition. In addition, NQO produces a significant rate of reversion of all cyc 1 mutants which revert by G · C transversions, e.g. the amber (UAG) mutant and the initiation mutants containing AGG, and probably CUG mutant codons. It did not revert the ochre mutant which contains no G · C base pairs. Ten NQO-induced revertants of the amber mutant cyc 1-179 contained the expected replacements of residues of tyrosine, and ten NQO-induced revertants of each of the cyc 1-131 and cyc 1-133 initiation mutants all contained the expected normal iso-1-cytochrome c . The structures of these iso-1-cytochromes c and the pattern of reversion of the tester strains indicate that base-pair substitutions arise at G · C base pairs which are the site of NQO attack. Thus NQO induces G · C → A · T transitions, G · C → T · A transversions and possibly G · C → C · G transversions. Because of its mode of action, NQO may be useful in less-defined systems for identifying G · C base pairs in mutant codons.

Structural gene for yeast iso-2-cytochrome c☆

Abstract Protein analysis and genetic studies have led to the identification of the structural genes of iso-1-cytochrome c and iso-2-cytochrome c, which constitute, respectively, 95% and 5% of the total amount of cytochrome c in the yeast Saccharomyces cerevisiae. The structural gene CYC1 for iso-1-cytochrome c was previously identified by Sherman et al. (1966) and the structural gene CYC7 for iso-2-cytochrome c is identified in this investigation. A series of the following mutations were selected by appropriate procedures and shown by genetic tests to be allelic: CYC7+ →CYC7-1 →cyc7-1-1 →CYC7-1-1-A, etc., where CYC7 + denotes the wild-type allele determining iso-2-cytochrome c; CYC7-1 denotes a dominant mutant allele causing an approximately 30-fold increase of iso-2-cytochrome c with a normal sequence, and was used as an aid in selecting deficient mutants; cyc7-1-1 denotes a recessive mutant allele causing complete deficiency of iso-2-cytochrome c; and CYC7-1-1-A denotes an intragenic revertant having an altered iso-2-cytochrome c at the same level as iso-2-cytochrome c in the CYC7-1 strains. The suppression of cyc7-1-1 with the known amber suppressor SUP7-a indicated that the defect in cyc7-1-1 was an amber (UAG) nonsense codon. Sequencing revealed a single amino acid replacement of a tyrosine residue for the normal glutamine residue at position 24 in iso-2-cytochrome c from the suppressed cyc7-1-1 strain and also in five revertants of cyc7-1-1, of which three were due to extragenic suppression and two to intragenic reversion. The nature of the mutation that elevated the level of normal iso-2-cytochrome c in the CYC7-1 strain was not identified, although it occurred at or very near the CYC7 locus but outside the translated portion of the gene and it may be associated with a chromosomal aberration. Genetic studies demonstrated that CYC7 is not linked to CYC1, the structural gene for iso-1-cytochrome c.

Yeast UAA suppressors effective in ψ+ strains: Leucine-inserting suppressors☆

Over 200 revertants that suppressed three or more UAA markers were isolated in a haploid strain of yeast, Saccharomyces cerevisiae, containing the ψ+ cytoplasmic determinant which increases the efficiency of action of certain suppressors. These revertants were grouped into classes on the basis of suppression of four nutritional markers and the canavanine-resistant marker can1–100, and on the basis of the efficiency of suppression of the cyc1–72 marker which contains a defined UAA mutant codon corresponding to position 06 in iso-1-cytochrome c. Genetic analysis and other tests indicated that 40% of the suppressors were highly efficient and were allelic to one or another of the known tyrosine-inserting suppressors, that 59% of the suppressors were moderately efficient and were allelic to either the previously known serine-inserting suppressor SUP16 or to the newly discovered serine-inserting suppressor SUP17, and that 1% of the suppressors were inefficient and were allelic to the newly discovered SUP26 suppressor. The SUP16 suppressors were shown to be allelic to the previously characterized suppressor SUQ5 whose locus is on the right arm of chromosome XVI. This location and the pattern of suppression suggests that the SUP16 locus may be identical to the previously described SUP15 locus. Genetic analysis established that the newly discovered SUP17 locus is on the left arm of chromosome IX, between the his6 and lys11 markers. The examination of four different strains revealed that the SUP16 and SUP17 suppressors cause insertion of serine in iso-1-cytochrome c at the UAA site of the cyc1–72 mutant. It is suggested that the gene products of the SUP16 and SUP17 loci are redundant forms of the same serine transfer RNA. Because viable haploid strains containing both suppressors were obtainable, it was concluded that SUP16 and SUP17 could not be the sole genes coding for the only UCA-decoding species of serine tRNA.

Mutations Altering Initiation of Translation of Yeast Iso-1-cytochrome c ; Contrasts between the Eukaryotic and Prokaryotic Initiation Process

INTRODUCTION Although the yeast Saccharomyces cerevisiae is a eukaryotic organism that has a greater genetic complexity than bacteria, it shares many of the technical advantages that permit rapid progress in determining fundamental mechanisms of biological processes. One of the major impacts of yeast in molecular biology is its use for investigating processes that are particularly amenable to genetic analysis and that are different in prokaryotes and eukaryotes. A systematic analysis of mutational alterations at the end of the CYC1 gene in the yeast S. cerevisiae has revealed the nucleotide sequences that effect initiation of translation. The analysis of these findings led us to suggest that certain essential features of the translational process may be fundamentally different from the analogous features in Escherichia coli. Furthermore, we have suggested that these differences between yeast and E. coli reflect differences between eukaryotes and prokaryotes. These findings and conclusions concerning the initiation process are briefly reviewed in this paper. MUTATIONS OF THE AUG INITIATION CODON Protein analysis of mutationally altered forms of iso-1-cytochrome c , along with genetic analysis of the corresponding mutant strains, established that the primary structure of this mitochondrial protein is determined by the CYC1 gene (Sherman et al. 1966), which is located on the right arm of chromosome X (Lawrence et al. 1975). Over 400 independently derived cyc1 mutants that lack iso-1-cytochrome c or that contain nonfunctional iso-1-cytochrome c have been isolated by one of three following procedures: (1) a spectroscopic scanning procedure (Sherman 1964), (2) a benzidine staining procedure (Sherman...

Tyrosine substitutions resulting from suppression of amber mutants of iso-1-cytochrome c in yeast☆

The suppressors SUP6-2 and SUP7-2 can cause the production of approxi- mately 25 to 60% of the normal amount of iso-1-cytochrome c when they are coupled to the amber (UAG) mutants cy1–179 and cy1–76. The iso-1-cytochromes c contain residues of tyrosine at the positions which correspond to the sites of the amber codons. SUP6-2 and SUP7-2 do not suppress ochre (UAA) mutants. The SUP6-2 and the SUP7-2 genes are apparently alleles of the SUP6-1 and SUP7-1 genes, respectively, which cause the insertion of tyrosine at ochre (UAA) codons (ochre-specific suppressors). It is suggested that the gene products of the allelic amber suppressors and ochre-specific suppressors (the SUP6-1 and SUP6-2 suppressors and theSUP7-1 andSUP7-2 suppressors) are two differently altered forms of the same tyrosine tRNA.

Confirmation of UAG as a nonsense codon in bakers' yeast by amino acid replacements of glutamic acid 71 in iso-1-cytochrome c.

Abstract The yeast mutant cy1–76 is more than 99% deficient in iso-1-cytochrome c. Twelve intragenic revertants of cy1–76 have approximately normal amounts of iso-1-cytochromes c, which are altered by replacement of glutamic acid 71 with either tryptophan, leucine, tyrosine, serine, glutamine or lysine. It is concluded that position 71 in functioning iso-1-cytochrome c can be radically varied, and that the defect in cy1–76 is a nonsense codon, UAG, corresponding to position 71. Tryptophan is the replacement in 4 of the 12 revertants of cy1–76. Tryptophan is similarly abundant as a replacement of lysine 9 in the previously studied 42 revertants ofcy1–179, but is not a replacement in the 45 previously studied revertants of cyl-9. Since amino acid replacements indicate that either UAA or UAG nonsense mutations occur in all three mutants, these new results confirm the previously recognized distinction between the two nonsense codons: one, evidently UAG, can be reverted to a tryptophan codon, while the other, apparently UAA, cannot; apparently UGA does not encode tryptophan in yeast.