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.     

Frameshift Suppression by thyA Mutants of ESCHERICHIA COLI K-12

We have extended our previous study on the suppression of frameshift mutants by Escherichia coli thyA mutants by assaying suppression of 15 rIIB frameshift mutants of bacteriophage T4 on one of our suppressing thyA mutant strains. The majority of insertion mutants were suppressible, whereas none of the deletion mutants tested was suppressible. Frameshift suppression could be inhibited by adding thymidine to the assay medium, but was not affected by the presence of a restrictive rpsL mutation in the host strain. We suggest that the frameshift suppression event occurs at a nonsense codon generated by the frameshift mutation.     

Isolation and Mapping of t Gene Mutants of Bacteriophage T4D

A procedure for selective isolation of T4 t mutants is described. At 120 min after infection of Escherichia coli cells with a low multiplicity of T4 bacteriophage, the mixture was sedimented through a linear sucrose gradient, and infected cells that remained intact were collected as the fastest sedimenting fraction. Ten to 50% of the phage released by chloroform treatment of this fraction were t mutants. Collection of a high proportion of t mutants depended on efficient elimination of cells that would survive because of superinfection lysis inhibition. This was accomplished by early addition of anti-T4 serum and heat-killed cells to inactivate progeny wild-type phage released at the normal burst time. Of 85 t mutants that were isolated and mapped, 23 new mutations were found, 14 of which are suppressible by an rII mutation and 9 of which are suppressible by rII or amber suppressors. Two hot-spot sites for spontaneous mutations were found; 14 mutants at one site, represented by a frameshift mutation, and 12 mutants at a second site were obtained from 39 spontaneous mutants independently isolated from different parental plaques. On our map of the t gene, the distance between the farthest t mutations is 6% recombination. A nonreverting triple t mutant, constructed to contain a frameshift mutation between two amber mutations, exhibited the same t mutant phenotype observed with revertible t mutants.     

Fertility regulation in F-like resistance transfer factors

Mutants of the R factor R100 have been isolated that mediate high-frequency transfer of the R factor during conjugation. Complementation tests revealed two classes of mutants, operator-constitutive and repressor-negative. Some of the latter class were suppressible by amber and ochre suppressors. The results support a simple model of regulation for the control of R-factor-mediated piliation.     

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.     

Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae.

Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1. The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression. Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis. The response of all four proteins is blocked by gln3 mutations. In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants. Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia. We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.     

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.     

Wild-type and mutant alleles of the Aspergillus nidulans developmental regulator gene brlA: correlation of variant sites with protein function

The DNA sequences of two wild-type and eleven mutant alleles of the developmental regulator gene brlA from Aspergillus nidulans, which encodes a zinc-finger protein, were characterized. Variant sites were located on rescued plasmids or PCR products based either on their meiotic map position or the use of denaturing gradient gel electrophoresis. Mutations in three null mutants, one of which is partially suppressible, encode premature stop codons. Two environmentally sensitive mutants were characterised by substitution of leucines required for stabilisation of α-helices in each of the two putative zinc-finger domains. A third zinc-finger substitution is predicted to disrupt recognition of a guanine residue in the DNA target. The mutations in four other leaky mutants map C-terminal to the zinc fingers; one minimally leaky mutant has a premature stop codon, which results in the removal of the last 38 residues of the protein product. Received: 16 February 1999 / Accepted: 22 July 1999     

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.     

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.     

Sporulation properties of cytochrome a-deficient mutants of Bacillus subtilis

Three classes of cytochrome a-deficient mutants of Bacillus subtilis have been found to be asporogenic or oligosporogenic. All three classes showed declines in adenosine 5'-triphosphate (ATP) concentrations during early sporulation, at a time when ATP levels in wild-type strains are constant. Class III mutants were found to be deficient in aconitase and isocitric dehydrogenase, and showed reduced maximum growth in nutrient sporulation medium. These mutants also suffered the most rapid decline in ATP concentration in early sporulation, and exhibited neither the biphasic oxygen consumption curve nor the increase in pH normally observed at the end of logarithmic growth in nutrient sporulation medium. Nicotinamide adenine dinucleotide oxidase activities of purified membrane preparations were approximately normal for mutants in all classes, except for two of the class II mutants and one class III mutant. Neither cytochrome a nor cytochrome c appears to be an obligatory intermediate in cyanide-sensitive nicotinamide adenine dinucleotide oxidation in B. subtilis.     

Structure and function in a Bacillus subtilis sporulation-specific sigma factor: molecular nature of mutations in spoIIAC

The spoIIAC gene was cloned from chromosomal DNA of seven spoIIAC mutants of Bacillus subtilis, and the complete sequence of the gene was determined for each mutant. Three of the mutants proved to have chain-terminating mutations (one of which, previously shown to be suppressible by sup-3, was identified as amber); these led in every case to complete failure either to manufacture spores or to synthesize two enzymes normally associated with stage II of sporulation. The four remaining mutations were missense, and these corresponded to a phenotype in which a few spores are formed and about half the wild-type quantities of the two enzymes are made. Of the four missense mutations, two were near the promoter-distal end of the gene, in a region believed to correspond to the DNA-binding domain of the sigma factor that spoIIAC encodes. The remaining two mutations were in the region of the gene that is thought to correspond to the domain of the protein that interacts with core RNA polymerase.     

Effects of mutations to streptomycin resistance on the rate of translation of mutant genetic information

Gartner, T. K. (University of California, Santa Barbara), and E. Orias. Effects of mutations to streptomycin resistance on the rate of translation of mutant genetic information. J. Bacteriol. 91:1021-1028. 1966.-The effects of mutations to streptomycin resistance of independent origin upon the translation of suppressible mutant information were studied in an isogenic series of strains of Escherichia coli. The group of suppressible mutants included 1 mutation in the z gene of the lac operon of E. coli (O(0) (2) allele), 12 mutations distributed among the two rII cistrons of T4, and 13 mutations distributed among at least five cistrons of phage T7. It was concluded that the mutations to streptomycin resistance cause a significant decrease in the rate of translation of the suppressible codons, and that this effect is limited to a few types of codons.     

The nucleotide sequence of the first externally suppressible--1 frameshift mutant, and of some nearby leaky frameshift mutants

Nine mutants within a 23 nucleotide sequence of the trpE gene of Salmonella typhimurium have been characterized. trpE91, a mutant which is externally suppressible has a single base deletion. Eight (or nine) nucleotides upstream of this deletion, two independently isolated mutations have the same transversion. In combination with trpE91 these mutations lead to partial restoration of synthesis of anthranilate synthetase in the absence of external suppressors. In the transversion the sequence A CA is changed to A AA and this new sequence may be the site where frameshifting occurs to allow leakiness. Leakiness is displayed by two further mutants of the same sign as trpE91, and one of the opposite sign, in the absence of any base substitution or external suppressors. Specific sequences, e.g., UUUC, may be especially prone to frameshifting and this sequence is created at the site of the +1 frameshift mutant which displays leakiness. In the new reading frame generated by the two -1 frame leaky mutants, a tryptophan codon is encountered. Leakiness is necessarily detected in the absence of tryptophan and under these conditions there will be a shortage of charged tryptophan tRNA. The possibility of such functional imbalance leading to frameshifting in these mutants is discussed.     

Complementation between temperature-sensitive and deletion mutants of reovirus.

A very low level of complementation has been found in conventional crosses between various classes of temperature-sensitive (ts) mutants of reovirus. A more definitive test for complementation was devised through a plaque assay on cell monolayers mixedly infected with defective reovirions lacking the L1 segment and prototype ts mutants from one or other of the known classes of reovirus mutants. An increase in the number of plaques on the mixedly infected plates over that on control plates infected with defective virions or ts mutants alone indicated that the ts mutant had been complemented by the defective virus. Class A, B, D, F, and G mutants were complemented at 39 C by the defective viruses, whereas class C and E mutants were not. In tests to determine whether complementation was reciprocal it was found that the defective virions were complemented by a class G mutant but not by the class C mutant. This and previous work (D.A. Spandidos and A. F. Graham, 1975) has therefore shown that of the seven known classes of ts mutants the class C mutant is the only one that neither complements nor is complemented by the defective virions. For this reason the class C ts mutation has been assigned to the L1 segment of the viral genome.     

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.     

Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae

Cellular vanadium metabolism was studied in Saccharomyces cerevisiae by isolating and characterizing vanadate [VO4(3-), V(V)]-resistant mutants. Vanadate growth inhibition was reversed by the removal of the vanadate from the medium, and vanadate resistance was found to be a recessive trait. Vanadate-resistant mutants isolated from glucose-grown cells were divided into five complementation classes containing more than one mutant. Among the vanadate-resistant mutants isolated in maltose medium, the majority of mutants were found in only two complementation groups. Three of the classes of vanadate-resistant mutants were resistant to 2.5 mM vanadate but sensitive to 5.0 mM vanadate in liquid media. Two classes of vanadate-resistant mutants were resistant to growth in media containing up to 5.0 mM vanadate. Electron spin resonance studies showed that representative strains of the vanadate-resistant complementation classes contained more cell-associated vanadyl [VO2+, V(IV)] than the parental strains. 51 Vanadium nuclear magnetic resonance studies showed that one of the vanadate resonances previously associated with cell toxicity (G. R. Willsky, D. A. White, and B. C. McCabe, J. Biol. Chem. 259:13273-132812, 1984) did not accumulate in the resistant strains compared with the sensitive strain. The amount of vanadate remaining in the media after growth was larger for the sensitive strain than for the vanadate-resistant strains. All of the strains were able to accumulate phosphate, vanadate, and vanadyl.     

The molecular basis for rRNA-dependent spectinomycin resistance in Nicotiana chloroplasts

The chloroplast genes coding for the 16S ribosomal RNA from several spectinomycin-resistant Nicotiana mutants were analyzed. Two classes of mutants were identified. In one class, a G to A base transition is found at position 1140 of the tobacco-chloroplast 16S rRNA gene, which eliminates an AatII restriction endonuclease site. This base transition is proximal to a mutation previously described for spectinomycin resistance in Escherichia coli. In the other class, a novel G to A transition is found at position 1012 of the 16S rRNA gene. Although the mutations in the two classes are 128 nucleotides apart, the secondary structure model for 16S rRNA suggests that the two mutated nucleotides are in spatial proximity on opposite sides of a conserved stem structure in the 3' region of the molecule. Phylogenetic evidence is presented linking this conserved stem with spectinomycin resistance in chloroplasts. Perturbation of the stem is proposed to be the molecular-genetic basis for rRNA-dependent spectinomycin resistance.     

Formal Relations between Om Mutants and Their Suppressors in Drosophila Ananassae

Optic morphology (Om) mutants associated with insertions of the tom transposable element at each of three tested loci are neomorphs as defined by the phenotypic equivalence of +/+/Om with +/Om and of +/Om/Om with Om/Om. Mutants behaving as suppressors of Om mutants and mapping to at least six loci are recovered from the same source and in similar frequency as Om mutants. The semidominant and nonpleiotropic suppressors at four of the six loci display defective eye phenes themselves, and the phenotypically normal mutants at a fifth locus are suspected alleles of a gene represented by recessive furrowed eye mutants. These and other properties imply that the suppressors, like suppressible Om mutants, are neomorphic due to insertion of the tom element into a hypothetical sequence they share with other members of a set of genes involved in development of the eye. Concurrently premature expression of both the suppressor and suppressed mutants would allow interaction of their products just as in normal development.     

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.