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.     

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.     

Nonsense Mutations in Essential Genes of Saccharomyces Cerevisiae

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.     

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.     

Streptomycin-Induced Phenotypic Suppression of Hydroxylamine-Induced Nonsense Mutants in the rII A Cistron of Bacteriophage T4

Twenty-one hydroxylamine-induced rII A cistron nonsense mutants were tested for streptomycin (SM)-induced phenotypic suppression by exposing Escherichia coli SBO (nonpermissive host) to phage in the presence and absence of SM. All nine amber, four of six ochre, and five of six opal mutants were phenotypically suppressible by SM. For suppressible mutants, the ratio of the average burst size in the presence of SM to size in the absence of SM ranged from 12 to 242 for the ambers, 3 to 33 for the ochres, and 4 to 14 for the opals. Increased susceptibility of the amber mutants to SM-induced phenotypic suppression relative to the susceptibility of the opal and ochre mutants may reflect a neighboring base effect, such that a 3′-terminal adenine inhibits misreading of a 5′-terminal uracil.     

Fine Structure Analysis of the ade3 Locus in SACCHAROMYCES CEREVISIAE

Twenty-six spontaneous mutants at the ade3 locus of Saccharomyces cerevisiae have been mapped and characterized with respect to revertibility, osmotic remediability and temperature sensitivity. Twelve of the twenty-six are temperature sensitive, 25 of 26 are osmotic remedial and 21 of 26 revert. Two of the mutants map as deletions. At least five of the 26 are nonsense mutations but are also, unexpectedly, osmotic remedial. Three nonsense mutations are also temperature sensitive, again an unexpected result. The two multisite mutations are both temperature sensitive and osmotic remedial. For mutants at this locus osmotic remediability and temperature sensitivity cannot be considered diagnostic criteria for missense mutations.     

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.     

Saturated fatty acid mutants in yeast

Summary Thirty-nine saturated fatty acid requiring mutants were isolated after treatment with ethyl methanesulfonate (EMS). These mutants have been assigned to three major complementation classes and from tetrad analysis appear to represent three unlinked genes: at least two loci are not linked to any centromere. Two major complementation categories are delineated by two non-allelic mutnats. Though fully complementary to each other, each fails to complement any mutant in several different complementation subgroups. Characterized by their response to external suppressors, these mutants represent nonsense mutations. One mutant is of the amber variety and the other is ochre. Tetrad analysis of diverse hybrids involving the saturated fatty acid auxotrophs revealed cryptic aneuploidy for chromosomes III, VIII and the as yet unidentified chromosomes that carry the above ochre and amber mutants. Clearly, this represents a minimal estimate. A more precise evaluation requires inclusion of sufficient genetic markers adequate to monitor the entire yeast genome. Gene dosage alterations due to aneuploidy had no apparent effect on the efficiency of interallelic complementation. The implications of these findings on the regulation and assembly of fatty acid synthetase are discussed. A working hypothesis concerning the relation between the effect of the primary lesion and nondisjunction is presented.Supported by PHS Grant No. GM-17317 (S.F.) and an NSF Predoctoral Fellowship (S.A.H.).     

Mutators in SACCHAROMYCES CEREVISIAE: MUT1–1, MUT1–2 and MUT2–1

The purpose of this study was to characterize two mutator stocks of yeast which were induced and selected on the basis of high spontaneous reversion rates of the suppressible "ochre" nonsense allele lys1-1. In the mutator stock VA-3, a single mutation, designated mut1-1, is responsible for the increase in the reversion rate of the ochre alleles lys1-1 and arg4-17. In stock VA-105, there are two separate mutator mutations. Tetrad analysis data showed these two loci are loosely linked. Based on complementation data, one of these mutations is at the same locus as mut1-1 and designated mut1-2. The second mutator of stock VA-105 was designated mut2-1. All three mutators are recessive. Both mut1-1 and mut1-2 give a high mutation rate for ochre nonsense suppressor (SUP) loci, but not for the ochre nonsense alleles. On the contrary, the mutation rates of the ochre alleles are greatly reduced. With the mutant mut2-1 there were mutations at both the lys1-1 site and its suppressors; mut2-1 is as effective as mut1-2 but not as effective as mut1-1 in inducing reversions of a missense mutant, his1-7. Neither mut1-1, mut1-2 nor mut2-1 were effective in inducing reversions of a putative frameshift mutation, hom3-10, or in inducing forward mutations to canavanine resistance.     

The CAN1 Locus of SACCHAROMYCES CEREVISIAE: Fine-Structure Analysis and Forward Mutation Rates

A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.     

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.     

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.     

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.     

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.     

Histidinol Dehydrogenase (hisD) Mutants of Salmonella typhimurium

A multidisciplinary analysis has been applied to over 150 hisD mutants of Salmonella typhimurium in a study of gene-enzyme relationship. The mutants were examined for production of immunologically cross-reacting material by using antibody to purified histidinol dehydrogenase, and for genetic complementation by using a set of F' factors bearing Escherichia coli hisD complementing mutants. Classifications as to missense, nonsense, frameshift, or deletion mutant are proposed on the basis of mutagenesis and suppression tests. For the suppression tests the mutants were examined both by a simultaneous suppression technique and by testing for response to E. coli F' factors bearing a recessive lethal amber and a recessive lethal ochre suppressor. The data are interpreted in relation to the position of the mutations in the recombination and complementation maps and in relation to the known composition of histidinol dehydrogenase. The gene hisD appears to be single cistron for the production of a single biosynthetic polypeptide.     

Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene.

Seven xanthine dehydrogenase and cross-reacting material negative Drosophila melanogaster rosy stocks were screened for amber and ochre nonsense mutations. Amber and ochre nonsense suppressors were created by site-directed mutagenesis starting from a wild-type tRNA(Tyr) gene. The suppressor tRNA genes were subcloned into a pUChsneo transformation vector providing heat-shock controlled neomycin resistance. The seven rosy stocks were germline transformed with amber and ochre tDNA(Tyr), and the G1 generation was screened for Geneticin resistance. Surviving rosy516 flies transformed with the amber suppressor showed an eye colour intermediate between the original ry516 stock and the wild-type, suggesting that ry516 is an amber nonsense mutant. This was confirmed by sequencing the relevant part of the ry516 gene; the analysis revealed a C-to-T transition in a CAG glutamine codon at nucleotide 1522 of the wild-type rosy gene.     

Absence of suppressible alleles at the his 1 locus of yeast

Summary No suppressible alleles have been found at the his1 locus of Saccharomyces cerevisiae. Seventeen noncomplementing alleles have been tested against the strong ochre suppressor SUP2. These alleles, plus an additional 34 complementing alleles, were previously tested against a weaker suppressor SUP11. These results, which are in marked contrast to experience with other yeast loci where suppressible alleles are frequent, may be explained if the his1 product is transcribed from a polycistronic message so that nonsense mutations lead to loss of a second nonsupplementable function, or if the his1 protein serves as an essential subunit for an unknown enzyme complex.     

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.     

Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

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

Summary Maltose-negative mutations in the amylomaltase gene of Streptococcus pneumoniae were examined for the presence of nonsense mutations. Out of 28 single-site mutants tested, 3 were shown to be suppressible by an amber suppressor previously found by Gasc et al. (1979). In the presence of the suppressor these mutants manifested 10–30% of wild type amylomaltase activity. In addition to the amylomaltase governed by malM, and the maltosaccharide phosphorylase governed by malP (which maps to the side of malM distal to the regulatory gene, malR), a new maltose-inducible protein, governed by another gene, malX, was observed in gel electrophoretic patterns. The malX gene maps on the side of malM proximal to the malR gene. The approximate molecular weights of the amylomaltase, phosphorylase and malX polypeptides are 62,000, 87,000 and 50,000, respectively. There appear to be no polar effects of the nonsense mutations in the malM gene on synthesis of the gene products of either malP or malX. In a search for nonsense mutants at other loci, one was found in the end gene, which governs the major endonuclease, a membrane enzyme. None were detected among 5 mismatch-repair defective hex mutants analyzed.