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

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.     

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.     

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

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 cyc1 mutants were previously determined from alterations of iso-1-cytochromes c in intragenic revertants. These 14 cyc1 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). NQO² induced a high rate of reversion in the initiation mutant cyc1-131, 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 cyc1 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 cyc1-179 contained the expected replacements of residues of tyrosine, and ten NQO-induced revertants of each of the cyc1-131 and cyc1-133 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.     

Cytochrome oxidase assembly does not require catalytically active cytochrome C

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.     

Substitution of serine caused by a recessive lethal suppressor in yeast.

The SUP-RL1 suppressor in the yeast Saccharomyces cerevisiae causes lethality in haploid strains but not in diploid or aneuploid strains that are heterozygous for the suppressor locus. This recessive lethal suppressor acts on amber (UAG) nutritional markers, and can cause the production of approximately 50% of the normal amount of iso-1-cytochrome c in disomic strains that are heterozygous for the SUP-RL1 suppressor, and that contain the cyc1-179 allele which has an amber codon corresponding to amino acid position 9. The suppressed iso-1-cytochrome c contains a residue of serine at the position that corresponds to the site of the amber codon. SUP-RL1 was found to lie between thr4 and MAL2 on chromosome III, approximately 30 map units from the mating-type locus. It is suggested that the gene product of SUP-RL1 may be a species of serine transfer RNA that normally reads the serine codon UCG, and that is represented only once in the haploid genome.     

Mutants of Yeast Defective in Iso-1-Cytochrome c

A medium containing chlorolactate has been devised to enrich for mutants that are unable to utilize lactate for growth, and therefore that may be defective in cytochrome c. Complementation tests of 6,520 chlorolactate-resistant mutants that were obtained spontaneously or induced with UV, ICR-170, or nitrosoimidazolidone resulted in the identification of 195 mutations at the cyc1 locus, which controls the primary structure of iso-1-cytochrome c. These 195 mutants, with 16 cyc1 mutants previously isolated, were examined for total cytochrome c by spectroscopic methods, growth on lactate medium, suppressibility by defined nonsense suppressors, mutational sites by x-ray-induced recombination, ability to revert, and in 86 cases, whether intragenic revertants contain altered iso-1-cytochrome c. Except for the deletion mutant cyc1-1, all of the mutants appeared to contain single-site mutations that could be assigned to at least 35 different sites within the gene. The cyc1 mutants either completely lacked iso-1-cytochrome c or contained iso-1- cytochromes c that were completely or partially nonfunctional. In spite of the fact that the cyc1 mutants obtained by the chlorolactate procedure were selected on the basis of defective function, 68% appeared to completely lack iso-1-cytochrome c. The remaining cyc1 mutants contained below normal amounts of iso-1-cytochromes c. Studies at several incubation temperatures indicated that these nonfunctional iso-1-cytochromes c were thermolabile. It is suggested that the predominant means for abolishing iso-1-cytochrome c by mutations are either through a complete loss, such as produced by chain terminating codons, or impairments through drastic changes of tertiary structure which lead to instability and thermolability.     

Amino acid replacements of the evolutionarily invariant tryptophan at position 64 in mutant forms of iso-1-cytochrome c from Saccharomyces cerevisiae

Tryptophan located at position 59 in vertebrate cytochromes c and at position 64 in yeast iso-1-cytochrome c is an evolutionarily invariant residue that is believed to be essential to the operation of the cytochrome c molecule. We show that this residue is replaced in at least partially functional iso-1-cytochromes c from cyc1 revertants of the yeast Saccharomyces cerevisiae. Tryptophan, tyrosine and leucine are found at position 64 in the revertants from the cyc1-84 mutant, confirming the genetic evidence (Sherman et al., 1974) that the mutant contains an UAG nonsense codon and establishing that the site of the mutation corresponds to the normal tryptophan 64. In a revertant from the cyc1.189 mutant, position 64 is occupied by a residue of phenylalanine. All three altered proteins are unstable, implying that tryptophan 64 has an essential and unique role for maintaining the normal structure of the cytochrome c molecule. In addition the iso-1-cytochrome c with leucine 64 and tyrosine 64 have greatly reduced biological activities, while iso-1-cytochrome c with the phenylalanine replacement has at least 20% of the wild-type activity or more. It remains uncertain whether the reduced specific activities are due to distorted tertiary structures or due to the specific lack of the tryptophan residue that may also have a direct functional role.     

Mutants of yeast initiating translation of Iso-1-cytochrome c within a region spanning 37 nucleotides

We used a specially constructed strain, cyc1–345, of the yeast Saccharomyces cerevisiae to isolate revertants that initiated translation of iso-1-cytochrome c at various sites along an extended region of the mRNA. Normal amounts of iso-1-cytochrome c occurred when translation initiated at the abnormal sites corresponding to amino acid positions ?3, ?2, 3 and 5, as well as the normal position ?1; 20% of the normal amounts occurred when translation initiated at the abnormal position 9. These results with cyc1–345 revertants indicate that translation of iso-1-cytochrome c can initiate with the normal efficiency at any site within the region spanning 25 nucleotides. Furthermore, because the lower amount of the short iso-1-cytochrome c in the mutant initiating at position 9 may not necessarily reflect an inefficiency of translation, we believe that translation can initiate with normal or near-normal efficiencies at any site within a 37 nucleotide region, and presumably at any site preceding and following that of the normal initiation codon. These results establish that there is no absolute requirement for a particular sequence 5′ to the initiation codon, and are consistent with our previous suggestion that translation starts at the AUG codon closest to the 5′ end of the mRNA.     

Differentiation between Amber and Ochre Mutants of Yeast by Reversion with 4-Nitroquinoline-1-Oxide

4-nitroquinoline-1-oxide (NQO) induces high frequencies of intragenic revertants of amber (UAG) but not ochre (UAA) mutants of yeast. Distinction of the amber and ochre codons was made with well-characterized nonsense mutants of the iso-1-cytochrome c gene (cyc1 mutants) as well as with nonsense mutants having nutritional requirements. Thus the NQO-induced reversion frequencies corroborated the assignments that were based on the pattern of amino acid replacements in intragenic revertants and on the speficity of suppression. It was concluded from these results and from the results of a previous investigation with other cyc1 mutants (Prakash, Stewart and Sherman 1974) that NQO induces transversions of G:C base pairs at many sites and that the specificity is not strongly influenced by neighboring base pairs in at least the strains examined in these studies. NQO was previously shown to induce G:C → A:T transitions at least at one site and this and the previous study established that it does not significantly mutate A:T base pairs at numerous sites. Thus NQO can be used to selectively mutate G:C base pairs and to determine if the pathways of reverse mutations involve G:C base pairs. Suppressors that act on either amber or ochre mutants were induced with NQO, indicating that they can arise by mutations of G:C base pairs.     

Genes Affecting the Expression of Cytochrome c in Yeast: Genetic Mapping and Genetic Interactions

The four mutant genes, cyc2, cyc3, cyc8 and cyc9, that affect the levels of the two iso-cytochromes c in the yeast Saccharomyces cerevisiae have been characterized and mapped. Both cyc2 and cyc3 lower the amount of iso-1-cytochrome c and iso-2-cytochrome c; whereas, cyc8 and cyc9 increase the amount of iso-2-cytochrome c. The cyc2, cyc3, cyc8 and cyc9 genes are located, respectively, on chromosomes XV, I, II and III, and are, therefore, unlinked to each other and unlinked to CYC1, the structural gene of iso-1-cytochrome c and to CYC7, the structural gene of iso-2-cytochrome c. While some cyc3 mutants are completely or almost completely deficient in cyotchromes c, none of the cyc2 mutants contained less than 10% of parental level of cytochrome c even though over one-half of the mutants contain UAA or UAG nonsense mutations. Thus, it appears as if a complete block of the cyc2 gene product still allows the formation of a residual fraction of cytochrome c. The cyc2 and cyc3 mutant genes cause deficiencies even in the presence of CYC7, cyc8 and cyc9, which normally cause overproduction of iso-2-cytochrome c. We suggest that cyc2 and cyc3 may be involved with the regulation or maturation of the iso-cytochromes c. In addition to having high levels of iso-2-cytochromes c, the cyc8 and cyc9 mutants are associated with flocculent cells and other abnormal phenotypes. The cyc9 mutant was shown to be allelic with the tup1 mutant and to share its properties, which include the ability to utilize exogenous dTMP, a characteristic flocculent morphology, the lack of sporulation of homozygous diploids and low frequency of mating and abnormally shaped cells of alpha strains. The diverse abnormalities suggest that cyc8 and cyc9 are not simple regulatory mutants controlling iso-2-cytochrome c.     

Yeast UAA suppressors effective in psi+ strains: leucine-inserting suppressors.

We have previously reported the isolation and characterization of UAA suppressors from a haploid strain of yeast Saccharomyces cerevisiae containing the ψ⁺ non-Mendelian determinant which increases the efficiency of action of certain suppressors (Ono et al., 1979). Most of the suppressors caused the insertion of either tyrosine or serine. In contrast, the pattern of suppression of nutritional markers suggested that the rare suppressor, SUP26, inserted in an amino acid other than tyrosine or serine. In this investigation we report the characterization of additional suppressors, similar to SUP26, that were isolated on a medium lacking uracil and containing canavanine; this medium is expected to exclude serine-inserting suppressors because they do not suppress the ura4-1 marker, and to exclude tyrosine-inserting suppressors because they suppress the can1-100 marker. The total of 155 revertants similar to the SUP26 suppressor were analyzed genetically and these could be assigned to one or another of the six distinct loci SUP26, SUP27, SUP28, SUP29, SUP32 and SUP33. The SUP26, SUP27 and SUP29 loci mapped on chromosomes XII, IV and X, respectively. The detailed map position of the SUP29 suppressor suggests that it may be allelic to the SUP30 suppressor reported by Hawthorne &; Mortimer (1968). These six suppressors had the same pattern of suppression of UAA nutritional markers and all of them had a similar low efficiency of action on the iso-1-cytochrome c mutation cyc1-72. The efficiency of each of these suppressors was increased by a chromosomal allo-suppressor, sal. Each of the six suppressors caused the insertion of leucine in iso-1-cytochrome c at the UAA site of the cyc1-72 mutation. It is suggested that the gene products of these suppressors are redundant forms of the same leucine transfer RNA.     

Variation of Mutagenic Action on Nonsense Mutants at Different Sites in the Iso-1-Cytochrome c Gene of Yeast

Three ochre and two amber mutants in yeast have been definitively identified by the amino acid replacements in iso-1-cytochromes c from intragenic revertants. Except for rare and sometimes unusual changes, all of the replacements were single amino acids whose codons differed from UAA or UAG by one base. These assignments, which were based on the absence of tryptophan replacements in ochre revertants, could be corroborated from the studies of two groups of suppressors that were shown to act on either the ochre or amber mutants. All five nonsense mutants are located at different sites in the cyc1 gene and all are at sites that can be occupied by amino acids having a wide range of structures. The relative frequencies of the amino acid replacements indicate that identical codons located at different sites may respond differently to a mutagenic agent. Notably glutamine replacements occurred almost exclusively in UV-induced revertants of only one ochre mutant cyc1–9, but not at all or at reduced proportions in the others. Similarly, lysine replacements occurred almost exclusively in the NA-induced revertants of only the ochre mutant cyc1–72, but not at all in the others. These and other results reveal that mutation of A·T base pairs by UV and nitrous acid are dependent upon the location of the codon within the gene as well as the location of the base pair within the codon. From these findings, it appears as if the type of base-pair changes induced by UV and nitrous acid are strongly influenced by adjacent nucleotide sequences.     

Structural gene for yeast iso-2-cytochrome c.

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.     

Characterization of amber and ochre suppressors in Salmonella typhimurium.

Amber and ochre suppressor mutations in Salmonella typhimurium were selected. The amino acid insertions directed by the suppressors were inferred from suppression patterns of Escherichia coli lacI amber mutations. These amber mutations only respond to nonsense suppressors that direct the insertion of particular amino acids. Four Salmonella amber suppressors characterized insert serine, glutamine, tyrosine, and (probably) leucine. Of the three ochre suppressors characterized, two direct the insertion of tyrosine and one directs that of lysine. Of the three amber and two ochre suppressors which have been mapped by phage P22 cotransduction, all are located in the same relative position on the Salmonella map as the analogous E. coli suppressors are on the E. coli map.     

Mutagenic specificity: reversion of iso-1-cytochrome c mutants of yeast

In previous studies the nucleotide sequences of numerous mutant codons in the cy1 gene have been identified from altered iso-1-cytochromes c. These studies not only revealed the mutant codons that caused the deficiencies but also experimentally determined which of the base pair changes allowed the formation of functional iso-1-cytochromes c. In this investigation we have quantitatively measured the reversion frequencies of eleven cy1 mutants which were treated with 12 mutagens. The cy1 mutants comprised nine mutants having single-base changes of the AUG initiation codon (Stewart et al., 1971), an ochre mutant cy1–9 (Stewart et al., 1972), and an amber mutant cy1–179 (Stewart &; Sherman, 1972). In some cases the types of induced base changes could be inferred unambiguously from the pattern of reversion. Selective G.C to A.T transitions were induced by ethyl methanesulfonate, diethyl sulfate, N-methyl-N′-nitro-N-nitrosoguanidine, 1-nitrosoimidazolidone-2, nitrous acid, [5-³H]uridine and β-propiolactone. There was no apparent specificity with methyl methanesulfonate, dimethyl sulfate, nitrogen mustard and γ-rays. Ultraviolet light induced high rates of reversion of the ochre and amber mutants, but in these instances it appears as if the selective action is due to particular nucleotide sequences and not due to simple types of base pair changes.     

Serine substitutions caused by an ochre suppressor in yeast.

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.     

Yeast UAA suppressors effective in ψ+ strains serine-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.