Temperature-Sensitive Pleiotropic Revertants from a Mutant in the AROM Gene Cluster of NEUROSPORA CRASSA

Genetical and biochemical studies have been performed with revertants induced in a polyaromatic mutant (No. 58) in the arom gene cluster of Neurospora crassa. In addition to complete and partial revertants able to grow on minimal at both 25 degrees and 35 degrees , temperature-sensitive revertants capable of growth on minimal at 25 degrees but not at 35 degrees have been recovered. One of these revertants has been shown to lack biosynthetic dehydroquinase activity at both temperatures (utilizing the inducible catabolic isozyme for growth at 25 degrees ), to have dehydroshikimate reductase activity only at 25 degrees , and to form an arom aggregate having a molecular weight approximately one-half that of wild type. These results are interpreted as indicating that pleiotropic mutants in the arom gene cluster can result from missense mutations, as well as from nonsense mutations as indicated in previous studies.     

Variation of Mutagenic Action on Nonsense Mutants at Different Sites in the Iso-1-Cytochrome c Gene of Yeast

Three ochre and two amber mutants in yeast have been definitively identified by the amino acid replacements in iso-1-cytochromes c from intragenic revertants. Except for rare and sometimes unusual changes, all of the replacements were single amino acids whose codons differed from UAA or UAG by one base. These assignments, which were based on the absence of tryptophan replacements in ochre revertants, could be corroborated from the studies of two groups of suppressors that were shown to act on either the ochre or amber mutants. All five nonsense mutants are located at different sites in the cyc1 gene and all are at sites that can be occupied by amino acids having a wide range of structures. The relative frequencies of the amino acid replacements indicate that identical codons located at different sites may respond differently to a mutagenic agent. Notably glutamine replacements occurred almost exclusively in UV-induced revertants of only one ochre mutant cyc1–9, but not at all or at reduced proportions in the others. Similarly, lysine replacements occurred almost exclusively in the NA-induced revertants of only the ochre mutant cyc1–72, but not at all in the others. These and other results reveal that mutation of A·T base pairs by UV and nitrous acid are dependent upon the location of the codon within the gene as well as the location of the base pair within the codon. From these findings, it appears as if the type of base-pair changes induced by UV and nitrous acid are strongly influenced by adjacent nucleotide sequences.     

Reversion of trpA nonsense mutations by deletion of the chain-termination codons

This paper describes a novel mechanism for reversion of nonsense mutations in the trpA gene of Escherichia coli. This mechanism, deletion of the nonsense codon, was discovered in the course of selecting for missense revertants of trpA(UGA211) and for catalytically active tryptophan synthetase alpha chain revertants of trpA(UAA234) and trpA(UAG234). Each type of revertant trpA was cloned and its DNA sequence determined. trpA(UGA211) gave rise to two previously unidentified types of missense revertant. The first type was expected, namely trpA(CGA211), the result of a base substitution event. The other type, representing approximately 1% of the missense revertants, was unexpected on the basis of single base substitutions and an understanding of which amino acids are functional at alpha chain position 211. It was found to be the result of a 21 base-pair deletion of a region containing codon 211. The tryptophan-independent revertants of both position 234 nonsense mutants occurred at a frequency of approximately 2 per 10(9) viable cells. They were identical in that they both resulted from a 3 base-pair deletion, namely deletion of the chain-terminating codon at position 234. One of them, however, also displayed an A instead of the normal G in the third position of codon 235. The revertants were characterized according to growth in different media and tryptophan synthetase assays performed on crude extracts. These types of mutants should prove interesting and important for the elucidation of alpha chain structure-function relationships, for insight into the assembly and interaction of subunits in this model multienzyme complex, and for the study of mechanisms by which deletions can be generated.     

Super Suppressors in NEUROSPORA CRASSA I. Induction, Genetic Localization and Relationship to a Missense Suppressor

Genetic analyses have been made to test the feasibility of using coincident reversions to prototrophy of multiple mutants to select super suppressors (ssu) in Neurospora crassa. Of five double-mutant strains examined, only those mutant combinations in which both members had the properties of nonsense mutations did revert coincidently. Forty-eight genetically purified coincident revertants were crossed to the wild type, and each was shown to contain a suppressor mutation. Five super suppressors were examined more thoroughly. Tetrad and random spore analysis was used to demonstrate that each behaved as a single gene in crosses. Two super suppressors, ssu-1 and ssu-4 were localized respectively on the right and left arm of linkage group 7. Two others, ssu-2 and ssu-3, appear to map on the right arm of linkage group 1. The fifth super suppressor mapped, ssu-7, lies between ad-8 and ylo-1 on linkage group 6. One super suppressor, ssu-1, was interesting because it mapped near the location reported for the suppressor of the missense mutant tryp-3(td201) (Yourno and Suskind 1964a). However, no overlap was found in action spectrum of the two suppressors. Tetrad analysis showed the two suppressors were located about 10 map units apart, the missense suppressor being the more distal to the centromere.     

Characterization of strong polar mutations in a region immediately adjacent to the L-arabinose operator in Escherichia coli B-r

Seven l-arabinose-negative mutations are described that map in three genetically distinct regions immediately adjacent to the araO (operator) region of the l-arabinose operon. All seven mutants revert spontaneously, exhibit a cis-dominant, trans-recessive polarity effect upon the expression of l-arabinose isomerase (gene araA), and fail to respond to amber, ochre, or UGA suppressors. Three of these mutants exhibit absolute polarity and are not reverted by the mutangens 2-aminopurine, diethyl sulfate, and ICR-191. These may have arisen as a consequence of an insertion mutation in gene araB or in the initiator region of the l-arabinose operon. The four remaining mutants exhibit strong but not absolute polarity on gene araA and respond to the mutagens diethyl sulfate and ICR-191. Three of these mutants are suppressible by two independently isolated suppressors that fail to suppress known nonsense codons. Partially polar Ara(+) revertants with lesions linked to ara are obtained from three of the same four mutants. These polar mutants, their external suppressors, and their partially polar revertants are discussed in terms of the mechanism of initiation of expression of the l-arabinose operon.     

Mutants of Yeast Defective in Iso-1-Cytochrome c

A medium containing chlorolactate has been devised to enrich for mutants that are unable to utilize lactate for growth, and therefore that may be defective in cytochrome c. Complementation tests of 6,520 chlorolactate-resistant mutants that were obtained spontaneously or induced with UV, ICR-170, or nitrosoimidazolidone resulted in the identification of 195 mutations at the cyc1 locus, which controls the primary structure of iso-1-cytochrome c. These 195 mutants, with 16 cyc1 mutants previously isolated, were examined for total cytochrome c by spectroscopic methods, growth on lactate medium, suppressibility by defined nonsense suppressors, mutational sites by x-ray-induced recombination, ability to revert, and in 86 cases, whether intragenic revertants contain altered iso-1-cytochrome c. Except for the deletion mutant cyc1-1, all of the mutants appeared to contain single-site mutations that could be assigned to at least 35 different sites within the gene. The cyc1 mutants either completely lacked iso-1-cytochrome c or contained iso-1- cytochromes c that were completely or partially nonfunctional. In spite of the fact that the cyc1 mutants obtained by the chlorolactate procedure were selected on the basis of defective function, 68% appeared to completely lack iso-1-cytochrome c. The remaining cyc1 mutants contained below normal amounts of iso-1-cytochromes c. Studies at several incubation temperatures indicated that these nonfunctional iso-1-cytochromes c were thermolabile. It is suggested that the predominant means for abolishing iso-1-cytochrome c by mutations are either through a complete loss, such as produced by chain terminating codons, or impairments through drastic changes of tertiary structure which lead to instability and thermolability.     

Nonsense Motility Mutants in SALMONELLA TYPHIMURIUM

Of 313 motility-deficient mutants isolated from an LT2 his(amber) strain fixed in phase 1 by gene vh2(-), 25 regained motility when amber or ochre suppressors were introduced, in F' factors or by transduction. The fla mutants (23 amber, 1 ochre) fell in complementation groups A, B, C, F, K, a new group, M, and at least one further new group; the hypothesis of a fla gene which specifies only an RNA structural component of a flagellum-synthesizing basal apparatus is disproven for the corresponding genes. Hfr and transductional crosses confirmed gene assignments from complementation and indicated that flaM and another new fla locus map near H1. A small minority of motile bacteria were detectable in many of the amber fla mutants. In groups A and F some pairs of amber fla mutants complemented each other, and perhaps each of these groups corresponds to more than one structural gene. The suppressed derivatives of a mutant with an amber mutation in H1 made flagella morphologically and serologically indistinguishable from wild-type flagella. A slow-spreading but flagellate mutant showed mainly non-translational motility in broth, and in a viscous medium the bacteria reversed very frequently; its amber mutation, probably near H1, is inferred to cause a defect in chemotaxis, so that the bacteria give the avoidance reaction continuously.     

Allele specific and locus non-specific suppressors in Aspergillus nidulans.

Using N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, ethyl methanesulphonate or 4-nitroquinoline-1-oxide mutagenesis and an enrichment method for the isolation of auxotrophs, 25 mutants with defects in the adA locus were obtained after screening 41,376 colonies. One of these, adA24, did not complement with any of the other adA mutants, had a very high reversion rate and had some other properties which usually characterize strains carrying nonsense mutations. All revertants of adA24 carried dominant suppressor mutations. A group of adA24 suppressors was tested for allele and locus specificity. They were found to suppress only some adA alleles, and at the same time, some mutations in the methG, methH, argB and proA loci. It is proposed that the allele specific and locus non-specific adenine suppressors are suppressors of nonsense mutations.     

Nonsense Mutation in the Regulatory Gene ETH2 Involved in Methionine Biosynthesis in SACCHAROMYCES CEREVISIAE

Ethionine-resistant mutants, mapping at the locus eth2-the product of which is involved in pleiotropic regulation of methionine biosynthesis-have been isolated in a strain carrying five ochre nonsense mutations. Selection for nonsense suppressors in such a strain led to characterization of several allele-specific but gene non-specific suppressors which are active on the recessive heteroallele eth2-2 (resulting in partial recovery of sensitivity toward ethionine) as well as on the five other suppressible alleles. Two of these suppressors are unlinked to the eth2 gene and either dominant or semi-dominant. It is concluded that the mutation eth2-2 resulted in a nonsense codon. Enzyme studies indicate that this mutation results in a complete absence of an active product of gene eth2, in contrast with the effect of a former mutation eth2-1 which was interpreted as leading to a modified product of this gene (Cherest, Surdin-Kerjan and de Robichon-Szulmajster 1971). This conclusion is based on the absence of repressibility of methionine group I enzymes and the observation that in a heteroallelic diploid, eth2-1 expression is not masked by eth2-2. The nonsense suppressors studied lead to at least partial recovery of repressibility of methionine group I enzymes. All these results support the idea that the product of gene ETH2 is an aporepressor protein.     

Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs containing premature translation termination codons. In mammalian cells, a termination codon is ordinarily recognized as "premature" if it is located greater than 50-54 nucleotides 5' to the final exon-exon junction. We have described a set of naturally occurring human beta-globin gene mutations that apparently contradict this rule. The corresponding beta-thalassemia genes contain nonsense mutations within exon 1, and yet their encoded mRNAs accumulate to levels approaching wild-type beta-globin (beta(WT)) mRNA. In the present report we demonstrate that the stabilities of these mRNAs with nonsense mutations in exon 1 are intermediate between beta(WT) mRNA and beta-globin mRNA carrying a prototype NMD-sensitive mutation in exon 2 (codon 39 nonsense; beta 39). Functional analyses of these mRNAs with 5'-proximal nonsense mutations demonstrate that their relative resistance to NMD does not reflect abnormal RNA splicing or translation re-initiation and is independent of promoter identity and erythroid specificity. Instead, the proximity of the nonsense codon to the translation initiation AUG constitutes a major determinant of NMD. Positioning a termination mutation at the 5' terminus of the coding region blunts mRNA destabilization, and this effect is dominant to the "50-54 nt boundary rule." These observations impact on current models of NMD.     

The Suppression of AD-3B Mutants by Supersuppressors in NEUROSPORA CRASSA

The action of eight suppersuppressors has been tested on 76 ad-3B mutants which were induced by base-pair transition mutagens. Thirteen mutants were found to be suppressible by at least one ssu gene. Most of the suppressible mutants were found to belong to a class in which one would expect to find nonsense mutants (polar complementing or noncomplementing classes, with AT at the mutant site). However, suppression was observed in other classes, including those containing presumed missense mutants. The specificity of the suppressors and the criteria for molecular classification of the mutants are discussed.     

Procedure for Identifying Nonsense Mutations

A method has been devised for the rapid identification of nonsense mutations (UAG, UAA, UGA codons) in Salmonella. The mutations to be tested are reverted, and the revertants are replica-printed onto lactose plates spread with lawns of tester strains. These tester strains contain F' lac episomes with nonsense mutations in the episomal Z gene. The revertants are infected with the episome from the tester strain lawn. Because S. typhimurium is unable to ferment lactose, only those revertants which have nonsense suppressors are able to grow on lactose. If colonies appear on the lactose plate, it may be concluded that the original strain carries a nonsense mutation, since nonsense suppressors suppress the mutant phenotype.     

Extragenic Suppression and Synthetic Lethality among Chlamydomonas Reinhardtii Mutants Resistant to anti-Microtubule Drugs

The antimicrotubule agents oryzalin (ORY), colchicine (COL) and taxol (TAX) were used to select three recessive, conditional lethal (ts-) mutants which defined two new essential loci, ory1 and cor1. The two ory1 mutants conferred resistance to ORY, TAX, and COL; the cor1 mutant conferred resistance only to COL. Each of the mutants displayed wild-type sensitivity to a number of unrelated inhibitors. Assembly and disassembly of flagellar microtubules in the ory1 mutants displayed wild-type sensitivity to ORY and COL, suggesting that the ory1 gene product either does not participate in these processes or the ory1 gene product alone is not sufficient to confer resistance. The ory1 locus mapped to linkage group X; cor1 was mapped to the left arm of linkage group XII. A synthetic lethal interaction was observed between ory1 and cor1 mutations, i.e., inferred ory1 cor1 double mutants were inviable at the permissive temperature. The conditional lethal phenotype of ory1-1 was used to select many spontaneous TS+ revertants, which arose at high frequencies. Genetic and phenotypic characterization of the revertants demonstrated that (1) the revertants fell into four phenotypic classes, including some which conferred supersensitivity to ORY and others which conferred cold-sensitive lethality, (2) reversion was caused in most or all cases by extragenic suppressors, (3) suppressor mutations displayed complex behavior in heterozygous (sup/+) diploids, (4) many different loci may be capable of suppressing ory1 mutants, and (5) suppressors of ory1-1 efficiently suppressed an independently isolated allele, ory1-2. Taken together the ory1, cor1, and suppressor mutations identify a number of interacting loci involved in essential cellular processes which are specifically susceptible to antimicrotubule agents.     

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     

Isolation of cell size mutants of a fission yeast by a new selective method: characterization of mutants and implications for division control mechanisms.

Previously known cell size (wee) mutations of fission yeast suppress the mitotic block caused by a defective cdc25 allele. Some 700 revertants of cdc25-22 were obtained after ultraviolet mutagenesis and selection at the restrictive temperature. Most revertants carried the original cdc25 lesion plus a mutation in or very close to the wee1 gene. Two partial wee1 mutations of a new type were found among the revertants. Two new wee mutations mapping at the cdc2 gene (cdc2-w mutants) were also obtained. The various mutations were examined for their effects on cell division size, their efficiency as cdc25 suppressors, and their dominance relations. Full wee1 mutations were found to suppress cdc25 lesions very efficiently, whereas partial wee1 mutations were poor suppressors. The cdc25 suppression ability of cdc2-w mutations was allele specific for cdc2, suggesting bifunctionality of the gene product. The wee1 mutations were recessive for cdc25 suppression; cdc2-w mutations were dominant. A model is proposed for the genetic control of mitotic timing and cell division size, in which the cdc2+ product is needed and is rate limiting for mitosis. The cdc2+ activity is inhibited by the wee1+ product, whereas the cdc25+ product relieves this inhibition.     

Dominant active alleles of RIM101 (PRR2) bypass the pH restriction on filamentation of Candida albicans

Morphological development of the fungal pathogen Candida albicans is profoundly affected by ambient pH. Acidic pH restricts growth to the yeast form, whereas neutral pH permits development of the filamentous form. Superimposed on the pH restriction is a temperature requirement of approximately 37 degrees C for filamentation. The role of pH in development was investigated by selecting revertants of phr2Delta mutants that had gained the ability to grow at acid pH. The extragenic suppressors in two independent revertants were identified as nonsense mutations in the pH response regulator RIM101 (PRR2) that resulted in a carboxy-terminal truncation of the open reading frame. These dominant active alleles conferred the ability to filament at acidic pH, to express PHR1, an alkaline-expressed gene, at acidic pH, and to repress the acid-expressed gene PHR2. It was also observed that both the wild-type and mutant alleles could act as multicopy suppressors of the temperature restriction on filamentation, allowing extensive filamentation at 29 degrees C. The ability of the activated alleles to promote filamentation was dependent upon the developmental regulator EFG1. The results suggest that RIM101 is responsible for the pH dependence of hyphal development.     

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.