Isolation and Characterization of Amber Suppressors in Yeast

Nonsense suppressors were obtained in a haploid yeast strain containing eight nutritional mutations, that are assumed to be amber or ochre, and the cyc1-179 amber mutation that has a UAG codon corresponding to position 9 in iso-1-cytochrome c. Previous studies established that the biosynthesis and function of iso-1-cytochrome c is compatible with replacements at position 9 of amino acids having widely different structures (Stewart and Sherman 1972). UV-induced revertants, selected on media requiring the reversion of one or two of the amber nutritional markers, were presumed to contain a suppressor if there was the unselected reversion of at least one other marker. The 1088 suppressors that were isolated could be divided into 78 phenotypic classes. Only 43 suppressors of three classes caused the production of more than 50% of the normal amount of iso-1-cytochrome c in the cyc1-179 strain. Genetic analyses indicated that all of these highly efficient amber suppressors are allelic to one or another of the eight suppressors which cause the insertion of tyrosine at ochre (UAA) codons (Gilmore, Stewart and Sherman 1971). Furthermore, only tyrosine has been identified at position 9 in iso-1-cytochrome c in cyc1-179 strains suppressed with these efficient amber suppressors.     

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.     

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.     

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.     

Demonstration of several independent loci involved in the synthesis of iso-2-cytochrome c in yeast

Summary This study concerns the chromosomal genes controlling the synthesis of cytochrome c in yeast. In the wild type there are two molecular species of cytochrome c : iso-1 (major from) and iso-2 (minor form) which differ in many positions of their amino-acid sequence. A mutation, CY1cy1-1, in the structural gene for iso-1, leads to iso-1 deficiency, while retaining a normal albeit small amount of iso-2-cytochrome c.The cyI-1 mutant does not grow on DL-lactate as sole carbon source, while the wild type does. This property was used for selecting cytochrome c rich revertants (CYT) from cytochrome c deficient strains cy1-1; ca 200 revertants were isolated after extensive nitrous acid mutagenesis from a haploid cy1-1 strain or from a diploid cy1-1/cy1-1 strain and ca 30 of them were analyzed genetically and biochemically. The cytochrome c of seven (CYT) revertants was extracted and characterized; none of them contained iso-1-cytochrome c, but all contained large amount of iso-2-cytochrome csufficient to compensate for the deficiency. It was concluded that none of the revertants resulted from back mutation of cy1-1 and that the cy1-1 mutation is a deletion or some other irreversible aberration. These conclusions were corroborated by genetic analysis. It was shown that every reversion is due to a chromosomal mutation segregating as a single gene. Five unlinked gene loci, CY2A, CY2B, CY2C, CY2D, CY2E, were uncovered in this way. None of them were linked to the CY1 locus. Revertants selected in the diploid strain were dominant or semi-dominant while those selected in the haploid strain were recessive. To the first class belong alleles at loci CY2A, CY2B, CY2C, while to the latter belong alleles at loci CY2D and CY2E.Five unlinked loci are implicated in iso-2-cytochrome c synthesis. Mutations selected at these loci act as suppressors of cytochrome c deficiency caused by a deletion of the CY1 locus. In fact the muations do not restore the synthesis of the deficient protein (iso-1-cytochrome c), but increase the synthesis of an another protein, structurally alike (iso-2-cytochrome c), and having very similar if not identical physiological activity. We propose the term of compensator genes to define this type of mutations. We discuss some possible mechanisms to explain the rarity of compensator mutations and the hypothesis that the locus CY2A could correspond not only to the regulatory gene for iso-2-cytochrome c but also to the structural one.     

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.     

Primary site and second site revertants of missense mutants of the evolutionarily invariant tryptophan 64 in iso-1-cytochrome c from yeast.

The three missense mutants cyc1-132, cyc1-166 and cyc1-189 in the yeast Saccharomyces cerevisiae contain nonfunctional and thermolabile iso-1-cytochromes c and have different replacements of the tryptophan at position 64 which corresponds to the invariant tryptophan residue found in cytochromes c from all eukaryotic species. The cyc1-166 and cyc1-189 mutants contain single replacements of, respectively, serine 64 and cysteine 64, while the cyc1-132 mutant contains a double replacement of glycine 64 and alanine 65 instead of the normal tryptophan 64 and aspartic acid 65. Twenty-three intragenic revertants having at least partially functional iso-1-cytochromes c arose from these three missense mutants by single amino acid replacements of either tryptophan, phenylalanine, tyrosine or leucine at position 64, or by second-site replacements in which the mutant residues at position 64 are retained and the normal serine 45 is replaced by phenylalanine 45. Specific activities of the iso-1-cytochromes c were estimated by growth of strains on lactate medium and are as follows, in terms of the normal, for iso-1-cytochromes c altered specifically in the ways shown: 100% for phenylalanine 64; 25% for tyrosine 64; between 0 and 25% for leucine 64; 100% for phenylalanine 45, cysteine 64; 25% for phenylalanine 45, serine 64; between 0 and 25% for phenylalanine 45, glycine 64, alanine 65; and 0% for serine 64, for cysteine 64, and for glycine 64, alanine 65 iso-1-cytochromes c. The results demonstrate that small residues of glycine, serine, and cysteine at position 64 are incompatible with function; they imply that many of the 10 amino acids accessible by single base-pair substitution but not observed in primary site revertants also are incompatible with function; and they show that large hydrophobic residues of phenylalanine, leucine, and tyrosine at position 64 are capable of restoring at least partial function. The second site revertants indicate that deleterious effects of the three missense mutants can be compensated by the introduction of phenylalanine 45, which may occupy space normally filled by tryptophan 64. Altered shapes of Calpha-band spectra and at least partial instability were characteristics of all iso-1-cytochromes c found lacking tryptophan 64. Apparently, the principal role of the invariant tryptophan is stabilization of the active protein structure, by providing a large hydrophobic group at the proper location.     

Formation of composite iso-cytochromes c by recombination between non-allelic genes of yeast

We present evidence that two non-allelic genes, located on two non-homologous chromosomes in the yeast Saccharomyces cerevisiae, recombine and in this process generate new composite genes containing portions of both genes. The two genes CYC1 and CYC7 encode, respectively, iso-1-cytochrome c and iso-2-cytochrome c; CYC1 is located on the right arm of chromosome X and CYC7 is located on the left arm of chromosome V. The coding regions of CYC1 and CYC7 and the corresponding iso-1-cytochrome c and iso-2-cytochrome c are approximately 80% homologous. Composite genes were uncovered among revertants of certain but not all cyc1 mutants lacking iso-1-cytochrome c; composite genes were observed in most revertants from the low-reverting strains cyc1-11, cyc1-136 and cyc1-158, and in low proportions of the revertants from the typically reverting strains cyc1-94 and cyc1-156. Protein analysis of 14 composite iso-cytochromes c and DNA sequencing of five composite genes indicated that recombinational events produced replacements of central portions of the cyc1 gene with a corresponding segment from the wild-type CYC7⁺ gene. The replacements varied in length from 13% to 61% of the translated portion of the CYC1 locus. The formation of composite genes occurred spontaneously at very low frequencies and at low but enhanced frequencies after treatments with mutagens including ultraviolet light, ethylmethane sulfonate, methylmethane sulfonate and nitrous acid. Genetic tests indicated that composite genes are formed mitotically by a conversion-like event in which the wild-type CYC1⁺ allele remains intact. Recombination between non-allelic genes can lead to identical sequences at different loci and to diverse composite genes. These results support the indirect evidence from other eukaryotic systems that non-allelic genes with extensive but not complete homology recombine during evolution.     

Substitutions of proline 76 in yeast iso-1-cytochrome c. Analysis of residues compatible and incompatible with folding requirements

Fine-structure genetic mapping previously revealed numerous nonfunctional cyc1 mutations having alterations at or near the site corresponding to amino acid position 76 of iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. DNA sequencing of the alterations in four of these cyc1 mutations indicated that the normal Pro-76 was replaced by Leu-76. Revertants containing at least partially functional iso-1-cytochromes c were isolated, and the alterations were analyzed by DNA sequencing and protein analysis. Specific activities of the altered iso-1-cytochromes c were estimated in vivo by growth of the strains in lactate medium; compared to normal iso-1-cytochrome c with Pro-76, the following activities were associated with the following replacements: approximately 90% for Val-76, approximately 60% for Thr-76, approximately 30% for Ser-76, approximately 20% for Ile-76, and 0% for Leu-76. In order to develop an understanding of the factors that determine whether or not an altered iso-1-cytochrome c will function, we undertook a theoretical analysis which led to the conclusion that the activity of the proteins was dependent on both short- and long-range interactions. Short-range interactions were estimated from studies on known protein structures which gave the likelihood that various amino acids would be found in a local backbone configuration similar to the native protein; long-range interactions with the rest of the molecule were analyzed by considering the size of the side chain. We believe this approach can be used to analyze a wide variety of mutant proteins.     

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.     

A Highly Revertible Cyc1 Mutant of Yeast Contains a Small Tandem Duplication

A mutant, cyc1-96, that reverts spontaneously at an extremely high rate, was uncovered after examining approximately 500 cyc1 mutants which lack or have defective iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Cloning and DNA sequencing of appropriate fragments revealed that the cyc1-96 mutation contained a 19 bp duplication whereas the spontaneously arising revertants contained the normal wild-type sequence. Because the 19 bp segment in the wild-type sequence is flanked by a 5 bp repeat and because the cyc1-96 mutation arose spontaneously, the 19 bp duplication may have arisen by slippage and misalignment during DNA synthesis. The high reversion rate was not diminished in strains containing the rad52 mutation, which generally reduces mitotic recombination, including recombination associated with the elimination of a segment of a long direct repeat. Thus the loss of segments from short and long duplications occur by different mechanisms. We suggest that the high reversion rates of cyc1-96 and other short duplications are due to misalignment errors during replication.