Nonsense mutations in the amylomaltase gene and other loci of Streptococcus pneumoniae

Molecular Genetics and Genomics - Maltose-negative mutations in the amylomaltase gene of Streptococcus pneumoniae were examined for the presence of nonsense mutations. Out of 28 single-site mutants...     

Nonsense mutations in the essential gene<Emphasis Type="Italic"> SUP35</Emphasis> of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> are non-lethal

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.Communicated by C. P. Hollenberg     

Genetic Analysis of the Maltose A Region in Escherichia coli

The genetic map of the maltose A locus of Escherichia coli contains at least three closely linked genes, malT, malP, and malQ. The order of these genes is established by deletion mapping. MalP and malQ, the presumed structural genes for maltodextrin phosphorylase and amylomaltase, belong to the same operon. MalT may be a regulator gene involved in the positive control of this operon.     

Isolation of Lactococcus lactis nonsense suppressors and construction of a food-grade cloning vector

Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNA_gin and the other an amber suppressor with an altered tRNA_ser. The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food-grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following etectroporation of a suppressible purine auxotroph, can be done on purine-free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food-grade plasmid giving a four- to fivefold increase in lysine aminopeptidase activity.     

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.     

Nonsense Mutations in the Maltose A Region of the Genetic Map of Escherichia coli

The isolation of one amber mutation in malQ, one ochre mutation in malP, and seven amber mutations in malT is reported. A study of their phenotypic expressions in the presence of the amber suppressor su(III) and the ochre suppressor su(c) suggests that (i) malQ is the structural gene for amylomaltase; (ii) malQ and the structural gene for maltodextrin phosphorylase, malP, belong to the same operon; (iii) the malT product, which promotes the expression of the malP-malQ operon, is a protein synthesized in limiting amounts by the wild-type strain.     

Overexpression of genes encoding tRNA<Superscript>Tyr</Superscript> and tRNA<Superscript>Gln</Superscript> increases the viability of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains with nonsense mutations in the <Emphasis Type="Italic">SUP45</Emphasis> gene

At present, the machinery supporting the viability of organisms possessing nonsense mutations in essential genes is not entirely understood. Nonsense mutants of Saccharomyces cerevisiae yeast containing a premature translation termination codon in the essential SUP45 gene are known. These strains are viable in the absence of mutant suppressor tRNAs; hence, the existence of alternative mechanisms providing nonsense suppression and mutant viability is conjectured. Analysis of clones obtained by transformation of a strain bearing a nonsense-mutant allele of SUP45 with a multicopy yeast genomic library revealed three genes encoding wild-type tRNA^Tyr and four genes encoding wild-type tRNA^Gln, which increased nonsense mutant viability. Moreover, overexpression of these genes leads to an increase in the amount of the full-length eRF1 protein in cells and compensates for heat sensitivity in the nonsense mutants. Probable ways of tRNA^Tyr and tRNA^Gln influence on the increase in the viability of strains with nonsense mutations in SUP45 are discussed.     

Genetic and Physiological Characterization of met15 Mutants of SACCHAROMYCES CEREVISIAE: a Selective System for Forward and Reverse Mutations

One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus.—It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.     

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.     

Isolation and characterization of context mutations affecting the suppressibility of nonsense mutations

Summary Secondary mutations which increase the efficiency of suppression of nonsense mutations in the rHB cistron of bacteriophage T4 have been isolated. These secondary mutations, called context mutations, map at sites very close to the nonsense codon, possibly on the promotor distal side. In context-nonsense double mutants, the amount of suppressed gene product is increased approximately 10-fold. The context mutations examined can act on the UAA (ochre) nonsense allele as well as on the UAG (amber) nonsense allele at a given site. These context mutations affect all suppression mechanisms analyzed (genetic suppressors. 5-fluorouracil suppression and spontaneous suppression).We suggest that context mutations affect information which is significant to the termination of polypeptide chains. According to our view, context mutations change the immediate neighborhood of nonsense mutations and so reduce the degree of resemblance to the sequences normally used for the termination of translation.     

The paradox of viable <Emphasis Type="Italic">sup45</Emphasis> STOP mutations: a necessary equilibrium between translational readthrough,activity and stability of the protein

The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q → TAA, 242R → TGA, 317L → TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.     

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.     

Relief of polarity in DNA-dependent cell-free synthesis of enzymes of the galactose operon of Escherichia coli

Summary Polar mutations of the galactose operon of both, nonsense and insertion type have been studied in a system for DNA-dependent synthesis of the galactose enzymes of Escherichia coli. In vivo, these mutations reduce to different degrees the level of expression of the gene located on the promoter-distal side of the mutation. No such polar effects are observed in vitro. This relief of polarity is neither due to the action of nonsense suppressors, nor to random initiation of mRNA synthesis.A special aspect of this study concerns those insertion mutations which carry a segment of DNA of foreign origin inserted near the control region of the galactose operon. In vivo, mutants of this type produce only one percent or less of the three galactose enzymes as compared to the wildtype. The residual enzyme synthesis is not or only slightly affected by inducer. In contrast, DNA carrying such insertion mutations is fully active in the cell-free enzyme synthesis and sensitive to the controls exerted by the galactose repressor and by the catabolite gene activator protein.     

Temperature sensitive allosuppressor mutants of the fission yeast S. pombe influence cell cycle control over mitosis

Summary A collection of Schizosaccharomyces pombe mutants has been obtained which restore activity to a nonsense suppressing tRNA sup3–5 whose suppressing function has been inactivated by second site mutations within the sup3–5 gene. These mutants were screened for those that were temperature sensitive in suppressing the opal nonsense allele ade6-704. Some of these map within or close to sup3 itself and others define two allosuppressor genes sal2 and sal3. The temperature sensitive mutants fail to efficiently suppress any other opal nonsense alleles although one mutant, sup3–5, r57, rr2, weakly does so at the low temperature. sal2 and sal3 mutants have a pleiotropic effect on the cell cycle causing a transient or complete blockage of mitosis. This blockage and the allosuppressor phenotypes are both eliminated by the presence of wee mutations in wee1 or cdc2. Mutants in sal2 are allelic with cdc25, a gene required for successful completion of mitosis. It is suggested that sal3 and cdc25 influence the mechanism that links the growth rate of the cell with the initiation of mitosis. Mutants in these genes may disturb tRNA biosynthesis or protein synthesis and this disruption may have an effect on both nonsense suppression and the growth rate control over mitosis.     

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.     

Nonsense Suppression of the Major Rhodopsin Gene of Drosophila

We placed UAA, UAG and UGA nonsense mutations at two leucine codons, Leu205 and Leu309, in Drosophila's major rhodopsin gene, ninaE, by site-directed mutagenesis, and then created the corresponding mutants by P element-mediated transformation of a ninaE deficiency strain. In the absence of a genetic suppressor, flies harboring any of the nonsense mutations at the 309 site, but not the 205 site, show increased rhodopsin activity. Additionally, all flies with nonsense mutations at either site have better rhabdomere structure than does the ninaE deficiency strain. Construction and analysis of a 3'-deletion mutant of ninaE indicates that translational readthrough accounts for the extra photoreceptor activity of the ninaE309 alleles and that truncated opsins are responsible for the improved rhabdomere structure. The presence of leucine-inserting tRNA nonsense suppressors DtLa Su+ and DtLb Su+ in the mutant strains produced a small increase (less than 0.04%) in functional rhodopsin. The opal (UGA) suppressor derived from the DtLa tRNA gene is more efficient than the amber (UAG) or opal suppressor derived from the DtLb gene, and both DtLa and DtLb derived suppressors are more efficient at site 205 than 309.     

Establishment of mammalian cell lines containing multiple nonsense mutations and functional suppressor tRNA genes

We describe the generation of mammalian cell lines carrying amber suppressor genes. Nonsense mutants in the herpes simplex virus thymidine kinase (HSV tk) gene, the Escherichia coli xanthine-guanine phosphoribosyl transferase (Eco-gpt) gene and the aminoglycoside 3′ phosphotransferase gene of the Tn5 transposon (NPT-II) were isolated and characterized. Each gene was engineered with the appropriate control signals to allow expression in both E. coli and mammalian cells. Expression in E. coli made possible the use of well developed bacterial and phage genetic manipulations to isolate and characterize the nonsense mutants. Once characterized, the nonsense mutants were transferred into mammalian cells by microinjection and used, in turn, to select for amber suppressor genes. Xenopus laevis amber suppressor genes, prepared by site-specific mutagenesis of a normal X. laevis tRNA gene, were microinjected into the above cell lines and selected for the expression of one or more of the amber mutant gene products. The resulting cell lines, containing functional amber suppressor genes, are stable and exhibit normal growth rates.     

Mutants of Schizosaccharomyces pombe which sporulate in the haploid state

Summary Suppressor mutants of mei1–102, a mutation in one of the mating type cassette genes (mat2-P) which blocks the progression into meiosis, were isolated and characterized in Schizosaccharomyces pombe. These suppressor mutations conferred either temperature-sensitivity or cold-sensitivity. The growth of these strains is halted and sporulation initiated at the restrictive temperatures, regardless of other conditions usually required for the initiation of meiosis i.e. they sporulate in the presence of a nitrogen source and mating type homozygosity. Their most striking feature is that they can sporulate from the haploid state. The haploidy of these mutants was confirmed by genetical analysis and by measurement of the DNA content of the cells. The mutants are all recessive and define a single gene pat1. The pat1 gene maps very close to the centromere of chromosome II. A meiosis defective mutation in mei5 can suppress the temperature-sensitivity caused by pat1, indicating some interaction between them. Spores produced from a haploid cell have poor viability and appear to contain only 1/2C DNA on average.