Sinergistic action of genetic and phenotypic suppression of nonsense mutations in yeast Saccharomyces cerevisiae

Summary It was found that the phenotypic suppression induced by the paromamine-containing antibiotic paromomycin could be significantly strengthened by a ribosomal suppressor mutation in yeast Saccharomyces cerevisiae. As a result the suppressor efficient towards ochre mutations in the presence of paromomycin acquired the ability to suppress both amber and opal mutations. It is suggested that phenotypic suppression by paromomycin and genotypic suppression by sup 1 both involve a similar mechanism of misreading.     

A novel method for detection and characterization of ochere suppressors in Escherichia coli

Summary We have found a new method for specifically detecting the occurrence of ochre (UAA) suppression in Escherichia coli. It is based on a procedure we used several years ago to distinguish trpA missense mutants from nonsense mutants, and relies on the generally low efficiency of suppression that seems to be characteristic of ochre suppressors in E. coli. Suppressed ochre mutants are distinguishable from trpA revertants by their inability to grow on glucose minimal medium containing a low concentration (1.5 m/ml) of indole and a high concentration (50 g/ml) of 5-methyl-DL-tryptophan (Ind-5MT). The procedure provides a specific and rapid means for detection of UAA derived from missense codons and has also been exploited to obtain different classes of ochre suppressors derived from the amber suppressor supDam and from a glycine tRNA missense suppressor. The Ind-5MT phenotype seems to depend in some way on the location of the ochre codon within the trpA messenger RNA. The method can be put to many uses and should be generally applicable to all low-efficiency nonsense suppressors, including those specific for UAG and UGA.Preliminary reports of portions of this work were presented at the spring meeting of the Texas Branch of the American Society for Microbiology, College Station, Texas, March, 1975     

Oo mutations in the galactose operon in E. coli

Summary Eleven mutants lacking the three enzymes of galactose fermentation were investigated.Eight of the mutants revert spontaneously to the Gal ⁺ phenotype. These cannot be deletions. Six of these spontaneously reverting mutants do not respond to the mutagens 2-aminopurine, ethyl-methanesulfonate and N-Methyl-N-Nitro-N-nitrosoguanidine. It is concluded that these o ^o mutations cannot be reverted by base substitution.The eleven o ^o mutants are not of the amber or ochre type as shown by their behaviour towards suppressor genes.The possible nature of the mutations is discussed.     

mRNA distal to polar nonsense and insertion mutations in the gal operon of E. coli

Summary mRNA of the galactose operon of E. coli was measured in wildtype E. coli and in gal operon amber and insertion mutants. The mRNA coded by the distal half of the operon is reduced in the mutants. This reduction is more pronounced in the insertion mutants than in the amber mutants. It was compared with the polar effects of the mutations on the enzymes of the operon.     

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors

Summary A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons.     

First identification of an amber nonsense mutation in Schizosaccharomyces pombe: major differences in the efficiency of homologous versus heterologous yeast suppressor tRNA genes

Summary In Saccharomyces cerevisiae ochre and opal, as well as amber mutations are known, whereas in the fission yeast Schizosaccharomyces pombe no amber alleles have been described. We have characterized trp1-566, an amber allele in the trp1 locus of S. pombe. The identification of trp1-566 as an amber allele is based on the following results: (a) The nonsense allele can be converted to an ochre allele by nitrosoguanidine mutagenesis. (b) trp1-566 is suppressed by a bona fide S. pombe amber suppressor tRNA, supSI. The supSI gene was obtained by primer-directed in vitro mutagenesis of a tRNASer from S. pombe. Unexpectedly, an S. cerevisiae amber suppressor tRNASer, supR21, transformed into S. pombe, failed to suppress trp1-566. Northern analysis of S. pombe transformants, containing supRL1 or S. cerevisiae tRNA^Leu or tRNA^Tyr genes reveals that these genes are not transcribed in the fission yeast. As an additional tool for the analysis of nonsense mutations in S. pombe, we obtained by nitrosoguanidine mutagenesis two unlinked amber suppressor alleles, sup13 and sup14, which act on trp1-566.     

An amber mutation in the gene rpsA for ribosomal protein S1 in Escherichia coli

Summary An amber mutation has been induced in the gene rpsA (which codes fo ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression. That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA. The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E. coli.     

Amber mutants of Salmonella-phage P22 in genes engaged in the establishment of lysogeny

Summary Phage mutants were isolated with amber mutations in genes necessary for establishment of lysogeny. These mutants form turbid plaques on su ⁺ strain 527R1 and clear plaques of different types on LT2. According to complementation tests, fourteen mutants fall in the c ₂ gene, four in the c ₃ gene but no amber mutants were found belonging to the c ₁ gene. Pulse labelling experiments to follow DNA synthesis after phage infection were done with the mutants classified by complementation tests. Furthermore the labelling experiments demonstrated that the nonleaky c ₃ amber mutants displayed the same DNA synthesis pattern as c ₁ missense mutants. Since these c ₃ amber mutants complement missense c ₁ mutants it is concluded that the c ₃ and c ₁ genes must act together for the first transient repression of DNA synthesis, i.e., seven minutes after infection. It is suggested that clear plaque forming c ₁ amber mutants cannot be isolated because of polarity leading to defectivity of lysogenic as well as of lytic functions.The majority of the experiments presented are a part of the dissertation of H. D. Dopatka at the University of Göttingen.     

Marker Effects on Reversion of T4rII Mutants

The frequencies of 2-aminopurine- and 5-bromouracil-induced A:T leads to G:C transitions were compared at nonsense sites throughout the rII region of bacteriophage T4. These frequencies are influenced both by adjacent base pairs within the nonsense codons and by extracodonic factors. Following 2AP treatment, they are high in amber (UAG) and lower in opal (UGA) codons than in allelic ochre (UAA) codons. In general, 5BU-induced transitions are more frequent in both amber and opal codons than in the allelic ochre codons. 2AP- and 5BU-induced transition frequencies in the first and third positions of opal codons are correlated with those in the corresponding positions of the allelic ochre codons. Similarly, the frequencies of 2AP-induced transition in the first and second positions of amber codons and their ochre alleles are correlated. However, there is little correlation between the frequencies of 5BU-induced transitions in the first and second positions of allelic amber and ochre codons.     

Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.Translation termination is mediated by one of the three stop codons (UAA, UAG, or UGA). When such stop codons arise in coding sequences due to mutations, referred to as nonsense mutations, they lead to abrupt arrest of the translation process. However, the termination efficiency of such nonsense codons is not 100%, as certain tRNAs have the ability to read these nonsense codons. Genetic code ambiguity is seen in several organisms. Stop codons have been shown to have alternate roles apart from translation termination. In organisms from all three domains of life, UGA encodes selenocysteine through a specialized mechanism. In Methanosarcinaceae, UAG encodes pyrrolysine (3). UAA and UAG are read as glutamine codons in some green algae and ciliates such as Tetrahymena and Diplomonads (24), and UAG alone encodes glutamine in Moloney murine leukemia virus (32). UGA encodes cysteine in Euplotes; tryptophan in some ciliates, Mycoplasma species, Spiroplasma citri, Bacillus, and tobacco rattle virus; and an unidentified amino acid in Pseudomicrothorax dubius and Nyctotherus ovalis (30). In certain cases the context of the stop codon in translational readthrough has been shown to play a role; for example, it has been reported that in vitro in tobacco mosaic virus, UAG and UAA are misread by tRNA^Tyr in a highly context-dependent manner (34, 9).Termination suppressors are of three types, i.e., amber, ochre, and opal suppressors, which are named based on their ability to suppress the three stop codons. Amber suppressors can suppress only amber codons, whereas ochre suppressors can suppress ochre codons (by normal base pairing) as well as amber codons (by wobbling) and opal suppressors can read opal and UGG tryptophan codon in certain cases. As described by Sambrook et al. (27), a few amber suppressors can also suppress ochre mutations by wobbling. The suppression efficiency varies among these suppressors, with amber suppressors generally showing increased efficiency over ochre and opal suppressors. supE44, an amber suppressor tRNA, is an allele of and is found in many commonly used strains of Escherichia coli K-12. Earlier studies have shown that supE44 is a weak amber suppressor and that its efficiency varies up to 35-fold depending on the reading context of the stop codon (8).Translational accuracy depends on several factors, which include charging of tRNAs with specific amino acids, mRNA decoding, and the presence of antibiotics such as streptomycin and mutations in ribosomal proteins which modulate the fidelity of the translational machinery. Among these, mRNA decoding errors have been reported to occur at a frequency ranging from about 10⁻³ to 10⁻⁴ per codon. Translational misreading errors also largely depend on the competition between cognate and near-cognate tRNA species. Poor availability of cognate tRNAs increases misreading (18).Several studies with E. coli and Saccharomyces cerevisiae have shown the readthrough of nonsense codons in suppressor-free cells. In a suppressor-free E. coli strain, it has been shown in vitro that glutamine is incorporated at the nonsense codons UAG and UAA (26). It has been reported that overexpression of wild-type tRNA^Gln in yeast suppresses amber as well as ochre mutations (25). In this study, we have confirmed the presence of an amber suppressor mutation in the glnX gene in a supE44 strain by sequence analysis. This was done essentially because we observed that supE44 could also suppress lacZ ochre mutations, albeit inefficiently. On further investigation using an in vivo luciferase reporter assay system for tRNA-mediated nonsense suppression (28), we found that the efficiency of suppression of amber lesion by supE44 is significantly higher than that reported previously in the literature. An increased ability to suppress ochre and opal nonsense mutations was observed in cells bearing supE44 compared to in the wild type. Such an effect was observed only in the stationary phase and was abolished in logarithmic phase.     

Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin

Orias, E. (University of California, Santa Barbara), and T. K. Gartner. Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin. J. Bacteriol. 91:2210-2215. 1966.-Streptomycin-induced suppression of amber and ochre rII mutants of phage T4 was studied in a streptomycin-sensitive strain of Escherichia coli and four nearly isogenic streptomycin-resistant derivatives of this strain, in the presence and in the absence of an ochre suppressor. Most of the 12 rII mutants tested were suppressed by streptomycin in the streptomycin-sensitive su(-) strain. This streptomycin-induced suppression in the su(-) strain was eliminated by the independent action of at least two of the four nonidentical mutations to streptomycin resistance. In two of the su(+)str-r strains, streptomycin markedly augmented the suppression caused by the ochre suppressor. In those su(-)str-r hosts in which significant streptomycin-induced suppression could be measured, the amber mutants were more suppressible than the ochre mutants.     

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.     

Intragenic localization of chain-terminating triplets arising by transitions and transversions

Mutational changes involving transitions can convert only one sense codon to ochre, two codons to amber, and two codons to UGA. One codon, UGG for tryptophan, can be converted by transitions to either amber or UGA. By transversion changes 15 other codons can be converted to ochre and/or amber and/or UGA. Ten amino acids can never be replaced by chain termination as a result of transition and transversion mutagenesis of single base-pairs. For two systems (bacteriophage T4 lysozyme and Escherichia coli K12 tryptophan synthetase A protein) in which the poly-peptide gene product has been completely sequenced one can construct predictive intra-genic distribution maps for the location of all possible chain-terminating mutations arising as a result of transitions and transversions.     

cur-1, a mutation affecting the phenotype of sup+ strains of Escherichia coli

Summary cur-1, a mutation found in many strains of the Y10 line of Escherichia coli K-12, but not previously described, maps at 27 min, close to galU. cur-1 causes mucoidity or uracil requirement at 29°C depending on the genetic background. These phenotypes are suppressed by amber codon suppressors.     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The pheno-type of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

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.     

Nonsense-missense suppression in yeast

Summary A dominant suppressor has been discovered which suppresses polarity type mutations, noncomplementing ones and those belonging to short nonpolar complementation groups at the ad ₂ locus of S. cerevisiae. The suppressor translates two types of nonsense previously established at ad ₂. These data together with theoretical consideration of relationships between nonsense-translating anticodons and different codons make it possible to suggest a mechanism for the observed suppression. According to this mechanism two nonsense types of ad ₂ are identified as amber and ochre codons.     

Mutagenesis in Escherichia coli. V. Attempted interconversion of ochre and amber suppressors and mutational instability due to an ochre suppressor

Summary A possible quantitative system for the interconversion of ochre and amber suppressors was studied in Escherichia coli WU36-10, a strain in which a leucine requirement is suppressed by amber suppressors and a tyrosine requirement is suppressed by ochre suppressors. The conversion of am Sup-2⁺ to oc Sup-2⁺ occurred at rates similar to those for the de novo induction of such suppressors, both spontaneously and after ultraviolet or gamma irradiation. Both induction and conversion of suppressors showed the phenomenon of mutation frequency decline after ultraviolet light. Conversions in the opposite direction from oc Sup-2⁺ to am Sup-2⁺ were, however, not detected in unmutagenised populations of oc Sup-2⁺ strains derived either by conversion from an am Sup-2⁺ strain or de novo from the parental WU36-10, nor were they detected after treatment with ultraviolet light, gamma radiation or 2-aminopurine. If the conversion of oc Sup-2⁺ to am Sup-2⁺ occurs at all, it is at a rate very considerably lower than that for the conversion of am Sup-2⁺ to oc Sup-2⁺. Some Tyr⁺ oc Sup-2⁺ mutants demonstrated mutation rates c. 100 times greater than those of WU36-10 for mutation to Leu⁺ spontaneously and after ultraviolet or gamma radiation. Possible explanations of this are discussed.     

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.