Production of Null Mutants in the Major Intestinal Esterase Gene (Ges-1) of the Nematode Caenorhabditis Elegans

Omnipotent suppressors decrease translational fidelity and cause misreading of nonsense codons. In the presence of the non-Mendelian factor [eta+], some alleles of previously isolated omnipotent suppressors are lethal. Thus the current search was conducted in an [eta+] strain in an effort to identify new suppressor loci. A new omnipotent suppressor, SUP39, and alleles of sup35, sup45, SUP44 and SUP46 were identified. Efficiencies of the dominant suppressors were dramatically reduced in strains that were cured of non-Mendelian factors by growth on guanidine hydrochloride. Wild-type alleles of SUP44 and SUP46 were cloned and these clones were used to facilitate the genetic analyses. SUP44 was shown to be on chromosome VII linked to cyh2, and SUP46 was clearly identified as distinct from the linked sup45.     

Recessive Uaa Suppressors of the Yeast SACCHAROMYCES CEREVISIAE

Recessive lysine-independent revertants were isolated from a ψ⁺ haploid strain of the yeast Saccharomyces cerevisiae containing one of the leucine-inserting UAA suppressors, SUP29, and various UAA mutations including lys1-1. The majority of the revertants were found to have recessive suppressors in addition to the pre-existing SUP29 mutation. The recessive suppressors were able to suppress only a very limited number of UAA mutations, and none of the UAG mutations thus far examined. The recessive inefficient UAA suppressors were assigned to three complementation groups, sup111, sup112, and sup113. A high incidence of gene conversion was observed for an allele of sup111. An antisuppressor acting on sup111, but not detectably on SUP29, was inadvertently obtained during the course of the study. Interactions between SUP29, sup111 and the antisuppressor asu12 were studied.     

The Yeast Omnipotent Suppressor Sup46 Encodes a Ribosomal Protein Which Is a Functional and Structural Homolog of the Escherichia Coli S4 Ram Protein

The accurate synthesis of proteins is crucial to the existence of a cell. In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors). We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13. S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations. Both copies of the S13 genes contain introns. Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns. The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes. S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast. Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins. SUP46 and S4 are most similar in their central amino acid sequences. Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.     

Serine insertion caused by the ribosomal suppressor SUP46 in yeast

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.     

Dominant Suppressors of a Muscle Mutant Define an Essential Gene of CAENORHABDITIS ELEGANS

The sup-11 I locus of C. elegans was defined by rare dominant suppressors of unc-93(e1500) III, a mutation that affects muscle structure. All ten of these dominant suppressors have a recessive "scrawny" phenotype. Two additional classes of sup-11 alleles were identified. One class, null alleles, was obtained by reversion of the dominant suppressor activity. These null alleles are recessive embryonic lethals, indicating that sup-11 is an essential gene. Members of the second class, rare semidominant revertants of the "scrawny" phenotype, are partial suppressors of unc-93(e1500). The genetic properties of the dominant suppressor mutations suggest that they are rare missense mutations that confer a novel activity to the sup-11 protein. We consider some of the ways that sup-11 alleles might suppress unc-93(e1500), including the possibilities that the altered sup-11 proteins restore function to a protein complex or are modified products of a gene that is a member of an unc-93 gene family.     

Altered 40 S ribosomal subunits in omnipotent suppressors of yeast

The five suppressors SUP35, SUP43, SUP44, SUP45 and SUP46, each mapping at a different chromosomal locus in the yeast Saccharomyces cerevisiae, suppress a wide range of mutations, including representatives of all three types of nonsense mutations, UAA, UAG and UGA. We have demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosomes from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin. Furthermore, ribosomal subunit mixing experiments established that the 40 S ribosomal subunit, and this subunit only, is responsible for the higher levels of misreading. Thus, the gene products of SUP35, SUP44, SUP45 and SUP46 are components of the 40 S subunit or are enzymes that modify the subunit. In addition, a protein from the 40 S subunit of the SUP35 suppressor has an altered electrophoretic mobility; this protein is distinct from the altered protein previously uncovered in the 40 S subunit of the SUP46 suppressor. In contrast to the ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46, the ribosomes from the SUP43 suppressor do not significantly misread polyuridylate templates in vitro, suggesting that this locus may not encode a ribosomal component or that the misreading is highly specific.     

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.     

Altered ribosomal protein S11 from the SUP46 suppressor of yeast

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.     

Yeast omnipotent supressor SUP1 (SUP45): nucleotide sequence of the wildtype and a mutant gene.

The primary structures of the yeast recessive omnipotent suppressor gene SUP1 (SUP45) and one of its mutant alleles (sup1-ts36) was determined. The gene codes for a protein of 49 kD. The mutant protein differs from the wildtype form in one amino acid residue (Ser instead of Leu) in the N-terminal part. The codon usage differs significantly from that of yeast ribosomal protein genes. However, an upstream element resembling a conserved oligonucleotide in the region 5' to ribosomal protein genes in S. cerevisiae has been found. A DNA probe internal to the SUP1 gene does not exhibit detectable homology to genomic DNA neither from higher eucaryotes nor from eu- or archaebacteria. The hypothetical function of this protein in control of translational fidelity is discussed.     

An Antisuppressor That Acts on Omnipotent Suppressors in Yeast

Six partially dominant antisuppressors were obtained that reduce the efficiency of two omnipotent yeast suppressors, sup45 and sup35, thought to be ribosomal ambiguity mutations. Each of these six antisuppressors was shown to fall within a single Mendelian locus, named asu9. The asu9 mutations are specific for omnipotent suppressors; they have no effect on several dominant tRNA-like suppressors. In the absence of suppressors, asu9 causes sensitivity to the aminoglycoside antibiotic, paromomycin. The properties of asu9 are consistent with the hypothesis that asu9 alters yeast ribosomal proteins.     

Dominant and recessive informational suppressors of a missense mutation in Coprinus

Summary Twenty-one suppressor gene mutations which suppress the met-5.1 missense mutation of Coprinus were separated into six groups (A-F) on the basis of dominance or recessiveness, linkage to the met-5 locus, comlementation in heterozygous cells and growth behaviour. The actual number of suppressor loci could not be determined because crosses between suppressed mutants were inviable. The allele specificity of group A, C, D and F suppressors was confirmed by appropriate crosses. Group B and E suppressors were not tested because of close linkage to the met-5 locus. No evidence for functional suppression of met-5 mutations was obtained thus it is likely that all the suppressors cause translational corelation of met-5.1. Suppressors in four groups (C-F) have properties expected of tRNA structural gene mutations: the group C mutation is dominant, the other mutations are recessive but do not complement in heterozygous cells. The relative efficiencies of the tRNA species involved was assessed by comparing the degree to which the different sup ⁺ mutations depressed the growth rate on methionine supplemented medium. The dominant mutation depressed growth to the greatest extent and is, therefore, the most efficient suppressor. The least efficient suppressors did not depress growth at all. When growth was compared on minimal medium it was found that the more efficient the suppressor the less well it restored growth. The mutations in groups A and B depressed growth more than the tRNA mutations but affect some other component in translation because they are recessive and complement normally. It is suggested that they may act to alter tRNA modifying enzymes.     

Isolation and Characterization of Omnipotent Suppressors in the Yeast Saccharomyces Cerevisiae

Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness. Genetic analysis of 12 representative suppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP35 (chromosome IV), SUP45 (chromosome II) and SUP46 (chromosome II), as well as three new loci SUP42 (chromosome IV), SUP43 (chromosome XV) and SUP44 (chromosome VII). Suppressors belonging to the same locus had a wide range of different phenotypes. Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressors encode proteins that effect different functions and that altered forms of each of the proteins can effect the same function.     

Serine-inserting UAA suppression mediated by yeast tRNASer

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 tRNA^Ser from SUP16, SUP17 and SUP19 strains can translate UAA codons. In contrast, tRNA^Ser 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 tRNA^Ser, and that the UAA suppression is mediated by mutationally altered tRNA molecules. The mechanism of SUP22 suppression remains unknown.     

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

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.     

Patterns of Genetic and Phenotypic Suppression of lys2 Mutations in the Yeast SACCHAROMYCES CEREVISIAE

A total of 358 lys2 mutants of Saccharomyces cerevisiae have been characterized for suppressibility by the following suppressors: UAA and UAG suppressors that insert tyrosine, serine or leucine; a putative UGA suppressor; an omnipotent suppressor SUP46; and a frameshift suppressor SUF1–1. In addition, the lys2 mutants were examined for phenotypic suppression by the aminoglycoside antibiotic paromomycin, for osmotic remediability and for temperature sensitivity. The mutants exhibited over 50 different patterns of suppression and most of the nonsense mutants appeared similar to nonsense mutants previously described. A total of 24% were suppressible by one or more of the UAA suppressors, 4% were suppressible by one or more of the UAG suppressors, while only one was suppressible by the UGA suppressor and only one was weakly suppressible by the frameshift suppressor. One mutant responded to both UAA and UAG suppressors, indicating that UAA or UAG mutations at certain rare sites can be exceptions to the specific action of UAA and UAG suppressors. Some of the mutants appeared to require certain types of amino acid replacements at the mutant sites in order to produce a functional gene product, while others appeared to require suppressors that were expressed at high levels. Many of the mutants suppressible by SUP46 and paromomycin were not suppressible by any of the UAA, UAG or UGA suppressors, indicating that omnipotent suppression and phenotypic suppression need not be restricted to nonsense mutations. All of the mutants suppressible by SUP46 were also suppressible by paromomycin, suggesting a common mode of action of omnipotent suppression and phenotypic misreading.     

A Genetic Analysis of Suppressors of the Pf10 Mutation in Chlamydomonas Reinhardtii

A mutation at the PF10 locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pf10 motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the pf10 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XII and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-1) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another.     

Interaction of UAG suppressors and omnipotent suppressors in Saccharomyces cerevisiae

Haploids bearing the dominant UAG suppressor, SUP7-a, and various alleles of the omnipotent suppressor sup35 were examined. The presence of the UAG suppressor reduced the efficiency of some alleles of sup35, and caused other sup35 alleles to be lethal. A nonclassical interaction of the dominant suppressor tRNA and the ribosome is proposed to explain these observations.     

Suppression of Two Missense Alleles of the TRP5 Locus of SACCHAROMYCES CEREVISIAE

A suppressor SUP101 of alleles trp5-67 and trp5-18 of the trp5 locus of Saccharomyces cerevisiae is described. The two suppressible mutations have been previously classified as missense. The suppression does not result from a physiological bypass of the tryptophan synthetase-catalyzed reaction, since the suppression is allele-specific. IU alleles trp5-70, tryp5-95, and trp5-102; IA alleles trp5-81, trp5-101, and trp5-103; and the ochre alleles trp5-33 and trp5-48 are not suppressed by SUP101. SUP101 does not suppress ochre alleles ade2-1, his5-2, arg4-17, lys1-1, amber alleles trp1-1, tyr7-1, or unclassified alleles at a number of other loci. These results indicate SUP101 is a missense suppressor. Growth on tryptophanless media is dependent upon gene dosage of both the suppressor and the suppressible alleles. Only the diploids homozygous both for the suppressor and suppressible alleles produce growth equivalent to growth of the haploids bearing a suppressible allele and the suppressor. Suppressor-bearing strains grow poorly even on tryptophan-supplemented media. In more than 100 asci analyzed partial growth inhibition on the complete medium always segregated with the suppressor.     

Gene Conversion and Intragenic Recombination at at the SUP6 Locus and the Surrounding Region in SACCHAROMYCES CEREVISIAE

Spontaneous secondary mutations of the ochre suppressor SUP6 were selected in a haploid strain of Saccharomyces cerevisiae . Unselected tetrads were dissected from crosses heterozygous for one of three alleles of SUP6 and for three other loci in this region which span a length of 14 map units (his2, cdc14 and met10). The study showed that all of these markers were characterized by high frequency of meiotic gene conversion and long conversion lengths which frequently extended into adjacent marked loci. Despite the high conversion frequency of SUP6 , recombination between alleles of this locus reached a maximum frequency of only 2 x 10^-3 prototrophs/spore. Although the allelic recombination frequencies were not distance dependent and consequently could not be used to order the alleles, the inequality between the two recombinant outside marker combinations among selected intragenic recombinants produced an internally consistent map of the suppressor locus. Recombination at SUP6 (whether detected as conversion in tetrads or the production of recombinants among random spores) was accompanied by significantly less than 50% outside marker recombination.     

A Novel Dominant Transformer Allele of the Sex-Determining Gene Her-1 of Caenorhabditis Elegans

We have characterized a novel dominant allele of the sex-determining gene her-1 of Caenorhabditis elegans. This allele, called n695, results in the incomplete transformation of XX animals into phenotypic males. Previously characterized recessive her-1 alleles transform XO animals into phenotypic hermaphrodites. We have identified five new recessive her-1 mutations as intragenic suppressors of n695. Three of these suppressors are weak, temperature-sensitive alleles. We show that the recessive her-1 mutations are loss-of-function alleles, and that the her-1(n695) mutation results in a gain-of-function at the her-1 locus. The existence of dominant and recessive alleles that cause opposite phenotypic transformations demonstrates that the her-1 gene acts to control sexual identity in C. elegans.