Expression of mutant alanine tRNAs increases spontaneous mutagenesis in Escherichia coli

The expression of mutA, an allele of the glycine tRNA gene glyV, can confer a novel mutator phenotype that correlates with its ability to promote Asp-->Gly mistranslation. Both activities are mediated by a single base change within the anticodon such that the mutant tRNA can decode aspartate codons (GAC/U) instead of the normal glycine codons (GCC/U). Here, we investigate whether specific Asp-->Gly mistranslation is required for the unexpected mutator phenotype. To address this question, we created and expressed 18 individual alleles of alaV, the gene encoding an alanine tRNA, in which the alanine anticodon was replaced with those specifying other amino acids such that the mutant (alaVX) tRNAs are expected to potentiate X-->Ala mistranslation, where X is one of the other amino acids. Almost all alaVX alleles proved to be mutators in an assay that measured the frequency of rifampicin-resistant mutants, with one allele (alaVGlu) being a stronger mutator than mutA. The alaVGlu mutator phenotype resembles that of mutA in mutational specificity (predominantly transversions), as well as SOS independence, but in a puzzling twist differs from mutA in that it does not require a functional recA gene. Our results suggest that general mistranslation (as opposed to Asp-->Gly alone) can induce a mutator phenotype. Furthermore, these findings predict that a large number of conditions that increase translational errors, such as genetic defects in the translational apparatus, as well as environmental and physiological stimuli (such as amino acid starvation or exposure to antibiotics) are likely to activate a mutator response. Thus, both genetic and epigenetic mechanisms can accelerate the acquisition of mutations.     

Effects of Escherichia coli dnaE antimutator alleles in a proofreading-deficient mutD5 strain.

We have previously isolated seven mutants of Escherichia coli which replicate their DNA with increased fidelity. These mutants were isolated as suppressors of the elevated mutability of a mismatch-repair-defective mutL strain. Each mutant was shown to contain a single amino acid substitution in the dnaE gene product, the alpha (i.e., polymerase) subunit of DNA polymerase III holoenzyme responsible for replicating the E. coli chromosome. The mechanism(s) by which these antimutators exert their effect is of interest. Here, we have examined the effects of the antimutator alleles in a mutD5 mutator strain. This strain carries a mutation in the dnaQ gene, which results in defective exonucleolytic proofreading. Our results show that dnaE mutations also confer a strong antimutator phenotype in this background, the effects being generally much greater than those observed previously in the mutL background. The results suggest that the dnaE antimutator alleles can exert their effect independently of exonucleolytic proofreading activity. The large magnitude of the antimutator effects in the mutD5 background can be ascribed, at least in part, to the (additional) restoration of DNA mismatch repair, which is generally impaired in mutD5 strains because of error saturation. The high mutability of mutD5 strains was exploited to isolate a strong new dnaE antimutator allele on the basis of its ability to suppress the high reversion rate of an A.T-->T.A transversion in this background. A model suggesting how the dnaE antimutator alleles might exert their effects in proofreading-proficient and -deficient backgrounds is presented.     

Replication fidelity in E. coli: Differential leading and lagging strand effects for dnaE antimutator alleles

DNA Pol III holoenzyme (HE) is the major DNA replicase of Escherichia coli. It is a highly accurate enzyme responsible for simultaneously replicating the leading- and lagging DNA strands. Interestingly, the fidelity of replication for the two DNA strands is unequal, with a higher accuracy for lagging-strand replication. We have previously proposed this higher lagging-strand fidelity results from the more dissociative character of the lagging-strand polymerase. In support of this hypothesis, an E. coli mutant carrying a catalytic DNA polymerase subunit (DnaE915) characterized by decreased processivity yielded an antimutator phenotype (higher fidelity). The present work was undertaken to gain deeper insight into the factors that influence the fidelity of chromosomal DNA replication in E. coli. We used three different dnaE alleles (dnaE915, dnaE911, and dnaE941) that had previously been isolated as antimutators. We confirmed that each of the three dnaE alleles produced significant antimutator effects, but in addition showed that these antimutator effects proved largest for the normally less accurate leading strand. Additionally, in the presence of error-prone DNA polymerases, each of the three dnaE antimutator strains turned into mutators. The combined observations are fully supportive of our model in which the dissociative character of the DNA polymerase is an important determinant of in vivo replication fidelity. In this model, increased dissociation from terminal mismatches (i.e., potential mutations) leads to removal of the mismatches (antimutator effect), but in the presence of error-prone (or translesion) DNA polymerases the abandoned terminal mismatches become targets for error-prone extension (mutator effect). We also propose that these dnaE alleles are promising tools for studying polymerase exchanges at the replication fork.     

Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles

Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species. Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection. We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures. The analysis involved screening for mutators among revertants of S. typhimurium histidine auxotrophs selected for the His⁺ phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn. The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts. Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%). the his⁺ reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds. The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype. We calculate that the frequency of mutators in the unselected population of S. typhimurium is 1–4×10⁻⁶, an incidence of 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli.     

Cidofovir resistance in vaccinia virus is linked to diminished virulence in mice

Cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC)] is recognized as a promising drug for the treatment of poxvirus infections, but drug resistance can arise by a mechanism that is poorly understood. We show here that in vitro selection for high levels of resistance to HPMPC produces viruses encoding two substitution mutations in the virus DNA polymerase (E9L) gene. These mutations are located within the regions of the gene encoding the 3'-5' exonuclease (A314T) and polymerase (A684V) catalytic domains. These mutant viruses exhibited cross-resistance to other nucleoside phosphonate drugs, while they remained sensitive to other unrelated DNA polymerase inhibitors. Marker rescue experiments were used to transfer A314T and/or A684V alleles into a vaccinia virus Western Reserve strain. Either mutation alone could confer a drug resistance phenotype, although the degree of resistance was significantly lower than when virus encoded both mutations. The A684V substitution, but not the A314T change, also conferred a spontaneous mutator phenotype. All of the HPMPC-resistant recombinant viruses exhibited reduced virulence in mice, demonstrating that these E9L mutations are inextricably linked to reduced fitness in vivo. HPMPC, at a dose of 50 mg/kg of body weight/day for 5 days, still protected mice against intranasal challenge with the drug-resistant virus with A314T and A684V mutations. Our studies show that proposed drug therapies offer a reasonable likelihood of controlling orthopoxvirus infections, even if the viruses encode drug resistance markers.     

Molecular and physiological characterization of Arabidopsis GAI alleles obtained in targeted Ds-tagging experiments

Bioactive gibberellin (GA) is an essential regulator of vascular plant development. The GAI gene of Arabidopsis thaliana (L.) Heynh. encodes a product (GAI) that is involved in GA signalling. The dominant mutant gai allele encodes an altered product (gai) that confers reduced GA responses, dwarfism, and elevated endogenous GA levels. Recessive, presumed loss-of-function alleles of GAI confer normal height and resistance to the GA biosynthesis inhibitor paclobutrazol. One explanation for these observations is that GAI is a growth repressor whose activity is opposed by GA, whilst gai retains a constitutive repressor activity that is less affected by GA. Previously, we described gai-t6, a mutant allele which contains an insertion of a maize Ds transposable element into gai. Here we describe the molecular and physiological characterization of two further alleles (gai-t5, gai-t7) identified during the Ds mutagenesis experiment. These alleles confer paclobutrazol resistance and normal endogenous GA levels. Thus the phenotype conferred by gai-t5, gai-t6 and gai-t7 is not due to elevated GA levels, but is due to loss of gai, a constitutively active plant growth repressor.     

Mutators, population size, adaptive landscape and the adaptation of asexual populations of bacteria.

Selection of mutator alleles, increasing the mutation rate up to 10, 000-fold, has been observed during in vitro experimental evolution. This spread is ascribed to the hitchhiking of mutator alleles with favorable mutations, as demonstrated by a theoretical model using selective parameters corresponding to such experiments. Observations of unexpectedly high frequencies of mutators in natural isolates suggest that the same phenomenon could occur in the wild. But it remains questionable whether realistic in natura parameter values could also result in selection of mutators. In particular, the main parameters of adaptation, the size of the adapting population and the height and steepness of the adaptive peak characterizing adaptation, are very variable in nature. By simulation approach, we studied the effect of these parameters on the selection of mutators in asexual populations, assuming additive fitness. We show that the larger the population size, the more likely the fixation of mutator alleles. At a large population size, at least four adaptive mutations are needed for mutator fixation; moreover, under stronger selection stronger mutators are selected. We propose a model based on multiple mutations to illustrate how second-order selection can optimize population fitness when few favorable mutations are required for adaptation.     

Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains

mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.     

Temperature-Sensitive Lethal Pseudorevertants of ste Mutations in Saccharomyces cerevisiae

A procedure was devised to isolate mutations that could restore conjugational competence to temperature sensitive ste mutants and simultaneously confer temperature-sensitive lethal growth phenotypes. Three such mutations, falling into two complementation groups, were identified on the basis of suppression of ste5 alleles. These same mutations were later shown to be capable of suppressing ste4 and ste7 alleles. Five mutations in a single complementation group were isolated as suppressors of ste2 alleles. None of the mutations described in this study conferred a homogeneous cell cycle arrest phenotype, and all were shown to define complementation groups distinct from those previously identified in studies of cell division cycle (cdc) mutations. In no instance did pseudoreversion appear to be achieved by mutational G1 arrest of ste mutant cells. Instead, it is proposed that the mutations restore conjugation by reestablishing the normal pheromone response.     

A novel mutation in ribosomal protein S4 that affects the function of a mutated RF1

Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination. RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA. RF1 and RF2 have been shown to bind to several ribosomal proteins. To study this interaction in vivo, prfA1, a mutant form of RF1 has been used. A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 degrees C and shows an increased misreading of UAG and UAA. In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts(+); on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1. The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive. The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16. These three mutations all confer both a Ts (44 degrees C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not. The three alleles were sequenced and shown to be truncations of the S4 protein. None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation. Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants. Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough.     

Gain-of-Function Mutations of fem-3, a Sex-Determination Gene in Caenorhabditis elegans

We have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild-type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3(gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3(gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All nine fem-3(gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3(gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal.     

Derivative Alleles of the Arabidopsis Gibberellin-Insensitive (gai) Mutation Confer a Wild-Type Phenotype

The gai mutation of Arabidopsis confers a dwarf phenotype resembling that of mutants defective in gibberellin (GA) biosynthesis. However, gai mutant plants differ from GA biosynthesis mutants because they fail to respond to exogenous GAs and accumulate endogenous GA species to higher (rather than lower) levels than found in wild-type controls. The gai mutation, therefore, identifies a gene that modulates the response of plant cells to GA. We have mapped gai with respect to visible and restriction fragment length polymorphism (RFLP) markers from chromosome 1. To observe the phenotype exhibited by individuals potentially lacking wild-type (GAI) function, we have also isolated novel irradiation-induced derivative alleles of gai. When homozygous, these alleles confer a revertant phenotype that is indistinguishable from the wild type. gai is a semidominant mutation that exerts its effects either because it is a gain-of-function mutation or because it is a loss-of-function or reduced-function mutation. The genetic and physiological properties of the derivative alleles are considered with reference to these alternative modes of dominance of gai. Because these alleles are potential deletion or rearrangement mutations, together with the closely linked RFLP markers identified in the linkage mapping experiments, they provide useful resources for the isolation of the gai locus via a map-based cloning approach.     

The balance between mutators and nonmutators in asexual populations

Mutator alleles, which elevate an individual's mutation rate from 10 to 10,000-fold, have been found at high frequencies in many natural and experimental populations. Mutators are continually produced from nonmutators, often due to mutations in mismatch-repair genes. These mutators gradually accumulate deleterious mutations, limiting their spread. However, they can occasionally hitchhike to high frequencies with beneficial mutations. We study the interplay between these effects. We first analyze the dynamics of the balance between the production of mutator alleles and their elimination due to deleterious mutations. We find that when deleterious mutation rates are high in mutators, there will often be many "young," recently produced mutators in the population, and the fact that deleterious mutations only gradually eliminate individuals from a population is important. We then consider how this mutator-nonmutator balance can be disrupted by beneficial mutations and analyze the circumstances in which fixation of mutator alleles is likely. We find that dynamics is crucial: even in situations where selection on average acts against mutators, so they cannot stably invade, the mutators can still occasionally generate beneficial mutations and hence be important to the evolution of the population.     

Segmental Polarity in Drosophila Melanogaster: Genetic Dissection of Fused in a Suppressor of Fused Background Reveals Interaction with Costal-2

fused (fu) is a segment polarity gene that encodes a putative serine/threonine kinase. A complete suppressor of the embryonic and adult phenotypes of fu mutants, Suppressor of fused (Su(fu)), was previously described. The amorphic Su(fu) mutation is viable and displays no phenotype by itself. We have used this suppressor as a tool to perform a genetic dissection of the fu gene. Analysis of the interaction between Su(fu) and 33 fu alleles shows that they belong to three different classes. Defects due to class I fu alleles are fully suppressed by Su(fu). Class II fu alleles lead to a new segment polarity phenotype in interaction with Su(fu). This phenotype corresponds to embryonic and adult anomalies similar to those displayed by the segment polarity mutant costal-2 (cos-2). Class II alleles are recessive to class I alleles in a fu[I]/fu[II];Su(fu)/Su(fu) combination. Class 0 alleles, like class I alleles, confer a normal segmentation phenotype in interaction with Su(fu). However class II alleles are dominant over class 0 alleles in a fu[0]/fu[II];Su(fu)/Su(fu) combination. Alleles of class I and II correspond to small molecular events, which may leave part of the Fu protein intact. On the contrary, class 0 alleles correspond to large deletions. Several class I and class II fu mutations have been mapped, and three mutant alleles were sequenced. These data suggest that class I mutations affect the catalytic domain of the putative Fu kinase and leave the carboxy terminal domain intact, whereas predicted class II proteins have an abnormal carboxy terminal domain. Su(fu) enhances the cos-2 phenotype and cos-2 mutations interact with fu in a way similar to Su(fu). All together these results suggest that a close relationship might exist between fu, Su(fu) and cos-2 throughout development. We thus propose a model where the Fu(+) kinase is a posterior inhibitor of Costal-2(+) while Su(fu)(+) is an activator of Costal-2(+). The expression pattern of wingless and engrailed in fu and fu;Su(fu) embryos is in accordance with this interpretation.     

A frameshift mutation in exon 2 of the phenylalanine hydroxylase gene linked to RFLP haplotype 1

Summary A deletion of a single base in codon 55 (exon 2) of the phenylalanine hydroxylase (PAH) gene has been identified by direct DNA sequencing of 94 phenyl-ketonuria (PKU) chromosomes. This mutation alters the reading frame so that a stop signal (TAA) is generated in codon 60 of the PAH gene. Haplotype analysis revealed that all PKU alleles showing the codon 55 frameshift mutation exhibited haplotype 1. In our panel of DNA probes 13% of all mutant haplotype 1 alleles carry this particular mutation. Patients who were compound heterozygotes for this deletion and R408W in exon 12, or the splice mutation in intron 12, were affected by severe PKU. Thus, the clinical data provide additional evidence that haplotype 1 PKU alleles carry molecular defects which confer a null phenotype. In addition, we were able to show that the newly detected mutation occurs on alleles of different ethnic background.     

Construction and Preliminary Characterization of a Library of “Lethal” Preterminal Protein Mutant Adenoviruses

Adenoviruses containing lethal in-frame insertion mutant alleles of the preterminal protein (pTP) gene were constructed with cell lines that express pTP. Thirty in-frame insertion mutant alleles, including 26 alleles previously characterized as lethal and 4 newly constructed mutant alleles, were introduced into the viral chromosome in place of the wild-type pTP gene. The viruses were tested for ability to form plaques at 37 degrees C in HeLa-pTP cells and at 32 degrees C and 39.5 degrees C in HeLa cells. Two of the newly constructed viruses exhibited temperature sensitivity for plaque formation, one virus did not form plaques in the absence of complementation, seven additional mutants exhibited a greater than 10-fold reduction in plaque formation in the absence of complementation, and another eight mutants exhibited stronger phenotypes than did previously characterized in-frame insertion mutants in the plaque assay. These mutant viruses offer promise for analysis of pTP functions.     

A naturally thermolabile activity compromises genetic analysis of telomere function in Saccharomyces cerevisiae

The core assumption driving the use of conditional loss-of-function reagents such as temperature-sensitive mutations is that the resulting phenotype(s) are solely due to depletion of the mutant protein under nonpermissive conditions. However, prior published data, combined with observations presented here, challenge the generality of this assumption at least for telomere biology: for both wild-type yeast and strains bearing null mutations in telomere protein complexes, there is an additional phenotypic consequence when cells are grown above 34°. We propose that this synthetic phenotype is due to a naturally thermolabile activity that confers a telomere-specific defect, which we call the Tmp(-) phenotype. This prompted a re-examination of commonly used cdc13-ts and stn1-ts mutations, which indicates that these alleles are instead hypomorphic mutations that behave as apparent temperature-sensitive mutations due to the additive effects of the Tmp(-) phenotype. We therefore generated new cdc13-ts reagents, which are nonpermissive below 34°, to allow examination of cdc13-depleted phenotypes in the absence of this temperature-dependent defect. A return-to-viability experiment following prolonged incubation at 32°, 34°, and 36° with one of these new cdc13-ts alleles argues that the accelerated inviability previously observed at 36° in cdc13-1 rad9-Δ mutant strains is a consequence of the Tmp(-) phenotype. Although this study focused on telomere biology, viable null mutations that confer inviability at 36° have been identified for multiple cellular pathways. Thus, phenotypic analysis of other aspects of yeast biology may similarly be compromised at high temperatures by pathway-specific versions of the Tmp(-) phenotype.     

A Major Role for Bacteriophage T4 DNA Polymerase in Frameshift Mutagenesis

T4 DNA polymerase strongly influences the frequency and specificity of frameshift mutagenesis. Fifteen of 19 temperature-sensitive alleles of the DNA polymerase gene substantially influenced the reversion frequencies of frameshift mutations measured in the T4 rII genes. Most polymerase mutants increased frameshift frequencies, but a few alleles (previously noted as antimutators for base substitution mutations) decreased the frequencies of certain frameshifts while increasing the frequencies of others. The various patterns of enhanced or decreased frameshift mutation frequencies suggest that T4 DNA polymerase is likely to play a variety of roles in the metabolic events leading to frameshift mutation. A detailed genetic study of the specificity of the mutator properties of three DNA polymerase alleles (tsL56, tsL98 and tsL88) demonstrated that each produces a distinctive frameshift spectrum. Differences in frameshift frequencies at similar DNA sequences within the rII genes, the influence of mutant polymerase alleles on these frequencies, and the presence or absence of the dinucleotide sequence associated with initiation of Okazaki pieces at the frameshift site has led us to suggest that the discontinuities associated with discontinuous DNA replication may contribute to spontaneous frameshift mutation frequencies in T4.     

The Origin of Spontaneous Mutation in SACCHAROMYCES CEREVISIAE

Characterization of two antimutator loci in yeast shows that both are members of the same mutagenic repair system known to be responsible for almost all induced mutation (Lawrence and Christensen 1976, 1979a,b; Prakash 1976). One of the these newly isolated antimutator mutations is an allele of rev3 (Lemontt 1971b). Two other alleles of rev3 were tested and were also found to be antimutators. Double mutants carrying rev3 and mutator mutations of rad3, rad51 or rad18 are like rev3 single mutants with respect to spontaneous mutation rate, supporting the hypothesis (Hastings, Quah and von Borstel 1976) that many mutators in yeast act by channelling spontaneous lesions from accurate to mutagenic repair. However, the enhanced mutation rate seen in a radiation-resistant mutator mutant mut1 is not dependent on REV3, but is dependent on another gene designated ANT1. An additive effect on the reduction in spontaneous mutation, seen in the ant1 rev3 double-mutant strain, leads to the conclusion that at least 90% of spontaneous mutations seen in the wild type are caused by mutagenic repair of spontaneous lesions.     

Identification of a region of the poliovirus genome involved in persistent infection of HEp-2 cells.

Poliovirus mutants were selected during the persistent infection of human neuroblastoma cells. These viruses could establish secondary persistent infections in HEp-2 nonneural cells. We report the identification of a region of the genome of a persistent virus (S11) that was sufficient to confer to a recombinant virus the phenotype that causes persistent infection in HEp-2 cells. This region, between nucleotides 1148 and 3481, contained 11 missense mutations mapping exclusively in the genes of capsid proteins VP1 and VP2. Because recombinant viruses carrying only one of these two mutated genes were not able to cause persistent infection, it seems very probable that two or more mutations in these genes are required for expression of the phenotype that causes persistent infection.