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.     

Ribosomal suppressors and antisuppressors in Podospora anserina: Altered susceptibility to paromomycin and relationships between genetic and phenotypic suppression

Informational suppressors and antisuppressors have been previously isolated in Podospora anserina, and their properties suggest that they could be ribosomal mutants involved in the control of translational fidelity. In this paper we present results concerning relationships between these mutants and paromomycin, an aminoglycoside antibiotic known to stimulate translational errors. The mutants were found to manifest an altered growth sensitivity to this drug as compared with the wild-type strain: Most of the suppressors were more sensitive and, in contrast, most of the antisuppressors were more resistant to paromomycin. Moreover, phenotypic suppression of an auxotrophic mutation by paromomycin was observed only if a suppressor and an antisuppressor had been introduced in the strain. These results suggest that ambiguity levels could be altered in the suppressor and antisuppressor strains. In addition, paromomycin was shown to abolish sporulation, which suggests relationships between mistranslation and a step of cellular differentiation.This work was supported by a DGRST grant and by a NATO grant.     

Mutations affecting translational fidelity in the eucaryote Podospora anserina: Characterization of two ribosomal restrictive mutations

Summary Fifty-nine mutations that restrict suppressor efficiency were selected in the fungus Podospra anserina using four different screening methods. Previous genetic analysis has shown that these antisuppressors lie in six loci and that they could be similar to ribosomal restrictive mutations known in Escherichia coli. The present study deals with the response of two of them, AS1-1 and AS6-1, to paromomycin and low temperature both in vivo and in vitro. The data demonstrate that ribosomes of the mutant and double-mutant strains are equally resistant to the ambiguity effect of paromomycin. These data are the first demonstration of mutations that increase translational fidelity in a eucaryotic organism.     

Search for ribosomal mutants in Podospora anserina: Genetic analysis of mutants resistant to paromomycin

It has recently been shown that paromomycin, an antibiotic of the aminoglycoside family, is also active on eukaryotic cytoplasmic ribosomes. In the fungus Podospora anserina, genetic analysis of ten mutants resistant to high doses of paromomycin shows that this resistance is caused by mutations in two different nuclear genes. These mutants display pleiotropic phenotypes (cold sensitivity, mycelium and spore appearance and coloration, cross-resistance to other antibiotics). Double mutants are either lethal or very altered and unstable. Moreover, the cytochrome spectra of these mutants seem to indicate that cytoplasmic protein synthesis is affected. The mutants also display a slight suppressor effect. We can therefore assume that these mutations affect cytoplasmic ribosomes.This work was supported by a C.N.R.S. Grant (ATP Microbiologie No. 3052) and by a NATO Grant.     

Ribosomal recessive suppressors cause a respiratory deficiency in yeast Saccharomyces cerevisiae

Summary Recessive suppressor mutations in yeast Saccharomyces cerevisiae alter a component of the cytoplasmic ribosomes, relaxing the control of translational fidelity. As a consequence ribosomes can misread nonsense codons as amino acids (Surguchov et al. 1980a).The suppressor mutants are often respiratory deficient, being unable to grow on non-fermentable substrates. The study of the cytochrome spectra has revealed that the cytochrome b and aa₃ contents were lower in the mutants than in the parent strains. Furthermore, the suppresor mutations often cause hypersensitivity to paromomycin and neomycin on media with a non-fermentable source of carbon. Some of the suppressor mutants exhibited both erythromycin and chloramphenicol-dependent growth on media containing ethanol or glycerol as a sole carbon source.These results suggest that the mutations altering cytoplasmic ribosomes may simultaneously impair the mitochondrial translation. A coupling of cytoplasmic and mitochondrial protein synthesis in yeast cells is proposed. The existence of a common protein component participating both in mitochondrial and cytoplasmic protein synthesis apparatus is discussed.     

Characterization of an amber suppressor in Pneumococcus.

Summary Partial revertant has been isolated, with resistance to aminopretin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su⁺) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su⁺ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidilate synthetase gene are suppressible. Thymidilate synthetase activity is partially restored by su⁺. Optochin mutants can also be suppressed. Thus su⁺ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycine is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor.The t-RNA extracted from the suppressor strain su⁺ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su⁺ gene is amber specific.Thus su⁺ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su⁺; this is additional evidence that both categories contain point mutations.     

Modulation of allele leakiness and adaptive mutability inEscherichia coli

It is shown that partial phenotypic suppression of two ochre mutations (argE3 andlacZU118) and an amber mutation (inargE) by sublethal concentrations of streptomycin in anrpsL ⁺ (streptomycin-sensitive) derivative of theEscherichia coli strain AB1157 greatly enhances their adaptive mutability under selection. Streptomycin also increases adaptive mutability brought about by theppm mutation described earlier. Inactivation ofrecA affects neither phenotypic suppression by streptomycin nor replication-associated mutagenesis but abolishes adaptive mutagenesis. These results indicate a causal relationship between allele leakiness and adaptive mutability.     

Functional interactions in vivo between suppressor tRNA and mutationally altered ribosomal protein S4

Summary Ribosomal mutants (rpsD) which are associated with a generally increased translational ambiguity were investigated for their effects in vivo on individual tRNA species using suppressor tRNAs as models. It was found that nonsense suppression is either increased, unaffected or decreased depending on the codon context and the rpsD allele involved as well as the nature of the suppressor tRNA. Missense suppression of AGA and AGG by glyT(SuAGA/G) tRNA as well as UGG by glyT(SuUGG-8) tRNA is unaffected whereas suppression of UGG by glyT(SuUGA/G) or glyV(SuUGA/G) tRNA is decreased in the presence of an rpsD mutation. The effects on suppressor tRNA are thus not correlated with the ribosomal ambiguity (Ram) phenotype of the rpsD mutants used in this study. It is suggested that the mutationally altered ribosomes are changed in functional interactions with the suppressor tRNA itself rather than with the competing translational release factor(s) or cognate aminoacyl tRNA. The structure of suppressor tRNA, particularly the anticodon loop, and the suppressed codon as well as the codon context determine the allele specific functional interactions with these ribosomal mutations.     

Suppression of temperature-sensitive aminoacyl-tRNA synthetase mutations by ribosomal mutations: A possible mechanism

Summary The biochemical basis of suppression of a temperature-sensitive alanyl-tRNA synthetase (alaS) mutation by mutational alterations of the ribosome has been investigated. Measurement of the polyU-dependent polyphenylalanine synthesis showed that ribosomes from the suppressor strains are less active than ribosomes from the unsuppressed aminoacyl-tRNA synthetase mutant. In this system no increased translational ambiguity could be detected for the suppressor ribosomes. This fact and also the findings that the ram-1 mutation is not able to suppress the aminoacyl-tRNA synthetase mutation and that presence of the suppressor allele is not accompanied by a measureably improved alanyl-tRNA synthetase activity argue against the possibility that suppression might be due to increased translational misreading rates of the alanyl-tRNA synthetase mRNA.It has been further found that partial suppression of temperature sensitive growth of the alaS mutation can be achieved by independent ribosomal mutations leading to reduced growth rates because of a mutation to antibiotic resistance. Addition of low concentrations of a variety of antibiotics acting at the ribosomal level can also partially revert the temperature-sensitive phenotype of the alaS mutant. Although the possibility cannot be excluded that suppression is due to the stabilisation or activation of the mutant enzyme by some indirect effect of the suppressor ribosomal mutations, the following working hypothesis is favoured at the moment: It is assumed that limitation of the aminoacyl-tRNA synthetase activity in a certain range of the restrictive temperature causes growth inhibition by the premature termination of polypeptide synthesis at the ribosome or by the unbalanced synthesis of the individual cellular proteins under this condition. The mechanism of suppression by ribosomal mutations is proposed to consist of the release of this growth inhibition by the reduction of the rate of polypeptide synthesis, which would keep amino acid incorporation from exceeding the slow charging of tRNA and thus exhausting the pool of charged tRNA. In the suppressor strains, therefore, growth at the semi-restrictive temperature is no longer limited by the aminoacylation of tRNA but by the translational process at the mutated ribosome. This influence of the ribosomal mutation on the speed of translation could be directly or indirectly coupled with an effect on translational fidelity resulting in the prevention of the binding of uncharged or non-cognate charged tRNA or in the tighter binding of peptidyl-tRNA when cognate aminoacyl-tRNA is limiting.     

Suppression of mitochondrially-determined resistance to chloramphenicol and paromomycin by nuclear genes in Saccharomyces cerevisiae.

Summary Phenotypic revertants of a drug resistant strain of Saccharomyces cerevisiae were induced by mutgenesis with manganese. Several of these drug sensitive mutants have been shown to result from mutations in the nuclear genome that cause phenotypic modification (suppression) of the mitochondrially-determined drug resistant genotype.Four mutants carrying a single recessive nuclear gene capable of modifying mitochondrial chloramphenicol resistance are described; these may be assigned to three complementation groups. Chloramphenicol resistant mutants mapping at five separate mitochondrial loci are described. At least two of the nuclear genes cause modification of mitochondrial chloramphenicol resistance determined by mutations at three of these loci, but the other two loci are apparently non-suppressible by these nuclear alleles. This indicates that these modifiers do not act by causing a general decrease in cellular or mitochondrial permeability to the drug.A single dominant nuclear modifier of mitochondrial paromomycin resistance has been identified. It is non-allelic to and does not interact with the genes modifying mitochondrial chloramphenicol resistance.     

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.     

Allelspezifische suppressormutationen vonSchizosaccharomyces pombe

By comparing the intragenic distribution of suppressor sensitive mutants in fine structure maps, 13 allele specific suppressor mutations (isolated from revertants in adenine dependent mutants of constitutionad ₇) have been analyzed for their allele specific patterns of action in three different groups of mutants blocked in adenine biosynthesis. The 13 suppressor mutations, which have resulted from mutations at seven different suppressor loci, are characterized by four different suppression patterns. Three of these patterns, which partially overlap, are not locus specific since they include sensitive mutants at each of the three lociad ₇, ad₆ andad ₁ studied. The relative frequency of mutants sensitive to one or the other of the suppressors of this type, the absence of osmotic-remedial strains among the suppressor sensitive mutants, and the polarized complementation behaviour of one suppressiblead ₆ mutant and two suppressiblead ₁ mutants capable of interallelic complementation, suggest that the suppression mechanism involves misreading of a mutant triplet of the nonsense type.     

Neomycin is more efficient than streptomycin in suppressing frameshift mutations

The effects of streptomycin and neomycin on the phenotypic suppression of frameshift mutations in the lacZ gene of Escherichia coli and on the efficiency of suppression of amber mutations in T4 phage by the informational supE tRNA nonsense suppressor were compared. Neomycin stimulated much more efficiently than streptomycin the phenotypic suppression of frameshift mutations. Because neomycin favors mismatches of the central codon base whereas streptomycin favors mismatches of the first codon base, this result suggests that mismatching of the central codon base pair and shifting of the reading frame are two correlated phenomena. In contrast, both streptomycin and neomycin stimulated about equally the efficiency of the tRNA nonsense suppressor, an effect probably related to their interference with the proofreading control in tRNA selection.     

Analysis of revertants of a ribosomal mutation in Podospora anserina: Evidence for new ribosomal mutations which confer hypersensitivity to paromomycin

This paper describes the analysis of cold-resistant revertants of a cold-sensitive mutant. Pm1-1 is a ribosomal mutation screened for its paromomycin resistance. Suppression of its cold sensitivity occurs with two kinds of external mutations localized in two different loci. One of them, PmB, is assumed to be a ribosomal gene. PmB mutations confer hypersensitivity to paromomycin in vivo as well as in vitro in a cell-free protein synthesis system.This work was supported by DGRST Grant MRM/P240 and NATO Grant 1637.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997     

The induction and characterization of recessive suppressors of arg-2 in Schizophyllum commune

Summary Genetic analyses have been made to detect recessive suppressor mutations in eight prototrophic strains derived by treating an arginine dependent strain with hydroxylamine. The results indicate that one strain possesses a recessive suppressor, su-1, which maps outside the arg-2 locus and is capable of suppressing auxotrophy conferred by the arg-2 mutation. This suppressor is incapable of suppressing auxotrophy conferred by eight other loci. Prototrophy in the remaining seven strains resulted from either intragenic suppression, reversion, or from a suppressor mutation that is closely linked to the arg-2 locus. The results of heterokaryotic allelic tests with the seven strains indicate that the mutation to prototrophy is recessive.     

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.     

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells

Summary We have compared the suppression of nonsense mutations by aminoglycoside antibiotics inEscherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules inE. coli and human cells.     

Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli

Summary Extragenic suppressor mutations for dnaA(Ts) mutations mapping in the rpoB gene (-subunit of RNA polymerase) were isolated by selection of spontaneous rifampicin resistant mutants and screening for temperature resistance. Six rpoB mutations were analysed for suppression of 12 different dnaA(Ts) mutations. The analysis showed that all dnaA(Ts) mutations could be suppressed by some rpoB mutation. All six rpoB mutations showed allele specificity when tested for suppression of 12 dnaA (Ts) mutant strains. The allele specificity was found to correlate with the map position of the dnaA (Ts) alleles.     

Joined suppression of rough phenotype and of red colour ofade2-1 mutants inSaccharomyces cerevisiae

A reciprocal suppression of rough phenotype was found in rough mutants of two prototrophic strains ofSaccharomyces cerevisiae on medium containing 1 % glucose. Suppression affects at least three independent rough loci and is strongly influenced by the genetic background. It is accompanied by suppression of red pigmentation inade2-1 mutants, and this too responds similarly to the genetic background. There is evidence for at least two independent loci that modify the action of the suppressor of both rough and red. The suppressor segregates regularly and is linked to one of the tested rough loci. It is not a super-suppressor and probably is not involved in the regulatory mechanism of purine biosynthesis either. An involvement of glucose metabolism in the suppression can be assumed from the lifting of the suppression of rough phenotype on lactate medium.