Influence of streptomycin on UV-induced TRP+ reversions in a streptomycin-resistant strain of E. Coli B/R

UV mutagenesis has been compared in the E. coli B/r trp^? WWP-2 Hcr⁺ and in a mutant (Stm^R 28) resistant to 100 μg streptomycin/ml. In the Stm^R strain it is possible to score UV-induced Trp⁺ reversions, and survival, on media supplemented with, or lacking, streptomycin. Such experiments revealed a marked enhancement of mutagenic effect of UV by streptomycin. On analysis it was shown that in the Stm^R strain a fraction of UV-induced Trp⁺ reversions, dut to ochre suppressor, achieve full phenotypic expression only in the presence of streptomycin. Thus an inhibitory effect of the Stm^R 28 mutation on the activity of some ochre suppressors is relieved by streptomycin.     

Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.     

Deep Genome-Wide Measurement of Meiotic Gene Conversion Using Tetrad Analysis in Arabidopsis thaliana

Gene conversion, the non-reciprocal exchange of genetic information, is one of the potential products of meiotic recombination. It can shape genome structure by acting on repetitive DNA elements, influence allele frequencies at the population level, and is known to be implicated in human disease. But gene conversion is hard to detect directly except in organisms, like fungi, that group their gametes following meiosis. We have developed a novel visual assay that enables us to detect gene conversion events directly in the gametes of the flowering plant Arabidopsis thaliana. Using this assay we measured gene conversion events across the genome of more than one million meioses and determined that the genome-wide average frequency is 3.5×10⁻⁴ conversions per locus per meiosis. We also detected significant locus-to-locus variation in conversion frequency but no intra-locus variation. Significantly, we found one locus on the short arm of chromosome 4 that experienced 3-fold to 6-fold more gene conversions than the other loci tested. Finally, we demonstrated that we could modulate conversion frequency by varying experimental conditions.     

Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae

The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (<10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges. Received: 24 December 1996 / Accepted: 24 July 1997     

Mutationsorte vom oo-Typ in einem Strukturgen

Summary In two o^o-type mutant strains ofE. coli K12 the mutant sites are shown to be located entirely within the structural gene of the decarboxylase component of the pyruvate dehydrogenase complex. One of the two mutant sites (probably of theochre-type) could be converted by mutation to anamber site which also completely inhibits the expression of the closely linked structural genes. The inhibition, therefore, is apparently caused by an inability to initiate, or an early termination of, the synthesis of the decarboxylase protein.

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Mit 3 Textabbildungen     

Gene conversion in nonsense suppressors of Schizosaccharomyces pombe. I. The influence of the genetic background and of three mutant genes (rad2, mut1 and mut2) on the frequency of the post-meiotic segregation.

Summary The frequency of gene conversion was assessed at the anticodon site of the sup3 gene of Schizosaccharomyces pombe. In order to detect a possible influence of the genetic background on the relative frequency of post-meiotic segregations amongst conversions, three similar crosses were analyzed which differed as follows: In cross I the two parents were derived by spontaneous mutation from one and the same strain. The heterogeneity of the genetic background between these two parent strains is assumed to be at a minimum level. In cross II the crossing partners were strains of the Bernese stock collection and differ probably in their genetic background to some extent. Last, in cross III, one of the parent strains was ten times repeatedly mutagenized with nitrosoguanidine in order to introduce cryptic mutations in the genome. A maximum degree of background heterogeneity between parents is expected for this cross. Neither the total conversion frequency nor the frequency of post-meiotic segregations amongst conversions were found to be significantly different in these three crosses.In addition, no effect of the radiation-sensitive mutation, rad2-44, and of two mutator mutations, mut1-4 and mut2-9, on the conversion pattern could be detected.     

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53^R167H mutant allele, orthologous to oncogenic human mutant TP53^R175H and mouse Trp53^R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53^R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53^R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53^R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.     

Restoration of enzyme activity by recessive missense suppressors in the fungus Coprinus.

Summary The acu-1 locus in Coprinus is the structural gene for acetyl-CoA synthetase. Five suppressor gene mutations, which suppress the acu-1,34 missense allele, were induced by mutagen treatment. All five suppressors were shown to have properties expected for tRNA structural gene mutations: they are recessive, they show a gene dosage effect in any doubly heterozygous combination of two sup ⁺ mutations and they are allele specific in action.Crosses between suppressed mutants established that at least four suppressor loci were represented. Doubly suppressed mutants derived from these crosses were used to show that the gene dosage effect is maintained when two sup ⁺ mutations are in cis as well as trans combinations in the two nuclei of the basidiomycete dikaryon.Extracts of the unsuppressed acu-1.34 mutant contained less than 2% of wild type acetyl-CoA synthetase activity whereas extracts of four of the five suppressor strains showed activities ranging from 28 to 37% of wild type. Only a slight increase in activity was detected in the fifth suppressor strain but this was associated with a temperature sensitive sup ⁺ phenotype. All five sup ⁺ mutations restored the ability of the acu-1.34 mutant to induce isocitrate lyase, an enzyme which, under the conditions of growth used, can only be induced when acetyl-CoA synthetase activity is present. Thus all five suppressors act to restore normal acu-1 protein function.     

An Amber Suppressor of Escherichia coli Strain KO1

The suppression characteristics of Escherichia coli strain KO1 have been investigated. The growth patterns of nonsense mutants of RNA (GA and f2) and DNA (λ and T4) phages suggested that KO1 carried an amber, but not ochre or opal suppressors. The comparison of KO1 with previously identified amber suppressors indicated that KO1 differed from su1, su3 and su6 in its suppression pattern. KO1 and su2 shared some properties in common, for instance, their ability to suppress GA amber mutants with one exception (amN20) and the restriction of suppression capacity by the str^r mutation. However, the suppression efficiency of KO1 (48%) was about three times that of su2 (18%). A possibility that KO1 contained a new amber suppressor is discussed.     

Transformation by integration in Podospora anserina

Summary A new transformation system for spheroplasts of Podospora anserina has been developed. The recipient leu1-1 strain is auxotrophic for leucine. The plasmid DNA does not carry the wild-type allele leu ⁺.but a tRNA suppressor: su4-1 or su8-1. The following protocol for genetic analysis has been developed: the [leu ⁺transformants are crossed with another mutant strain, carrying the 193 mutation. This mutation prevents the pigmentation of the spores and is also suppressed by the cloned suppressor. Thus, the genetic analysis of the transformants can be performed directly on ordered tetrads by the observation of pigmentation restoration. The first application of the method is described comparing the integration points when different suppressors are used. Integration of the plasmid DNA in the homologous site was not the rule; in most cases the integration point was located elsewhere.     

Letter: Mutant tyrosine transfer ribonucleic acids of Escherichia coli: construction by recombination of a double mutant A1G82 chargeable with glutamine

A double mutant A1G82 of the su_III⁺ tyrosine gene in Escherichia coli was constructed by genetic cross. This mutant is a stronger glutamine-inserting amber suppressor than either of the single mutants A1 or G82.     

A single nucleotide polymorphism in suppressor of cytokine signalling‐2 is associated with growth and feed conversion efficiency in pigs

Feed efficiency and growth are the most important traits in pig production, and very few genetic markers have been reported to be associated with feed efficiency. The suppressor of cytokine signalling‐2 (encoded by SOCS2) is the main negative regulator of somatic growth, and the knockout of SOCS2 and naturally mutant mice have high‐growth phenotypes. Porcine SOCS2 was selected as a primary positional candidate for feed efficiency, because it is located on chromosome 5q, in the vicinity of a Quantitative Trait Locus (QTL) region for food conversion ratio in pigs. Here, we report five single nucleotide polymorphisms identified by sequencing of the promoter region and exon 1. One PCR–RFLP assay was designed for genotyping the polymorphism c.1667A > G (GenBank Accession No AY312266 ). Association analyses were performed in an Australian mapping resource pedigree population (PRDC‐US43) for food conversion ratio, backfat, IGF1 level and growth traits and showed significant effects on average daily gain on test (ADG2) (P < 0.01) and marginal association with food conversion ratio (FCR) (P < 0.08).     

Isolation of dominant suppressor mutations for position-effect variegation in Drosophila melanogaster

Summary Dominant suppressor mutations for position-effect variegation have been isolated by using a strongly variegated line carrying the w ^m4 chromosome (w ^m4h) and the dominant enhancer mutant En(var)c ¹⁰¹. The use of an effective genetic test system made it possible to isolate more than 100 strongly dominant suppressor mutations for position-effect variegation. This suggests that the phenomenon of position-effect variegation is characterised by a complex genetic basis. The significance of the isolated mutants to genetic dissection of structural and regulatory functions of the eukaryotic chromosome is discussed.     

The detection of post-meiotic segregation without tetrad analysis in Ustilago maydis

Summary Post-meiotic segregation (PMS) results in the formation of mixed genotypes from single meiotic products. A method is described in which single members of tetrads are selected, and these are then tested for their genetic homogeneity. The method is applied to Ustilago maydis using crosses which are heteroallelic for nar 1, the structural gene for nitrate reductase. In the absence of PMS, meiotic products containing a nar ⁺ recombinant are genetically pure (the equivalent of a 6 mutant: 2 wild-type octad). With PMS, a nar ⁺ recombinant clone arises in association with a nar ^- clone and these are otherwise genetically identical (the equivalent of a 7 mutant: 1 wild type octad). The procedure will make it possible to search for mutant strains which are defective in the correction of mismatched bases in hybrid DNA formed during recombination. Among 26 nar ⁺ recombinants from a control cross, PMS was detected on 3 occasions. In an equivalent cross, both parents were uvs 3, a mutant defective in the excision of pyrimidine dimers from DNA. Among 43 nar ⁺ recombinants, 7 arose from PMS. Thus the frequency of PMS for the nar alleles is about 15% and the excision of pyrimidine dimers is probably unrelated to the repair of mismatched bases in hybrid DNA.     

Cis-dominant regulatory mutations affecting the formation of glucose-repressible alcohol dehydrogenase (ADHII) in Saccharomyces cerevisiae

Summary The formation of ADHII in Saccharomyces cerevisiae is regulated by carbon catabolite repression. There are two genes involved in the formation of ADHII: ADR2, the structural gene as identified by electrophoretic variants and ADR1, possibly a regulatory gene. A new genetic element involved in the regulation of ADHII was identified by three allelic mutants insensitive to strong glucose repression. They were called ADR3 ^c (wild type designation ADR3) and found to be tightly linked to the structural gene, ADR2. The alcohol dehydrogenase found in ADR3 ^c mutants could not be distinguished electrophoretically from the ADHII of the glucose-sensitive wild type, ADR3.Dominance relations between ADR3 ^c and ADR3 were established in diploids heterozygous for ADR3 and the two alleles of ADR2 (ADR2-S: slow ADHII, ADR2-F: fast ADHII). During growth on 10% glucose, an ADR3 ^c ADR2-F/ADR3 ADR2-S heterozygous diploid formed only the fast ADHII variant whereas an ADR3 ^c ADR2-S/ADR3 ADR2-F heterozygote produced only the slow form. This was taken as evidence of the cis-dominance of all ADR3 ^c alleles. The cis-effect of ADR3 ^c was also demonstrated in glucose-derepressed diploids. The ADR3 ^c mutations do not only cause glucose-insensitive ADHII formation, but also reduce the activity of the adjacent structural gene during derepression. Thus ADR3 ^c alleles were considered to be controlling site mutations. No pleiotropic effects were observed on the formation of enzymes related to the function of ADHII.An adr1 ADR2 ADR3 single mutant did not form ADHII. In contrast to this, an adr1 ADR2 ADR3 ^c double mutant formed ADHII at a similar level as an ADR1 ADR2 ADR3 ^c mutant. This showed that ADR3 ^c was epistatic over adr1 (previously suggested as a positive regulatory gene). From this it was concluded that ADR1 is in fact a positive regulatory gene the function of which is required for the expression of the structural gene for ADHII, ADR2. ADR3 is the controlling site for the structural gene ADR2. Mutations at this site, ADR3 ^c , alleviate the requirement for the ADR2 gene product. ADR3 ^c is discussed as a promotor or operator site.     

Mutant analysis by rescue gene excision: New tools for mosaic studies in Drosophila

A host of classical and molecular genetic tools make Drosophila a tremendous model for the dissection of gene activity. In particular, the FLP‐FRT technique for mitotic recombination has greatly enhanced gene loss‐of‐function analysis. This technique efficiently induces formation of homozygous mutant clones in tissues of heterozygous organisms. However, the dependence of the FLP‐FRT method on cell division, and other constraints, also impose limits on its effectiveness. We describe here the generation and testing of tools for Mutant Analysis by Rescue Gene Excision (MARGE), an approach whereby mutant cells are formed by loss of a rescue transgene in a homozygous mutant organism. Rescue‐transgene loss can be induced in any tissue or cell‐type and at any time during development or in the adult using available heat‐shock‐induced or tissue‐specific flippases, or combinations of UAS‐FLP with Gal4 and Gal80^ts reagents. The simultaneous loss of a constitutive fluorescence marker (GFP or RFP) identifies the mutant cells. We demonstrate the efficacy of the MARGE technique by flip‐out (clonal and disc‐wide) of a Ubi‐GFP‐carrying construct in imaginal discs, and by inducing a known yki mutant phenotype in the Drosophila ovary.     

Studies of opal mutations in bacteriophage T4B

Hydroxylamine-induced amber and opal mutants are localized on the map of gene 47 of bacteriophage T4B. The matched map of amber and opal mutations showed the presence of four paired sites which seemed to have arisen in the triplet coding for tryptophan.In growth studies o opal mutants in gene 47 in a series of Su⁺ strains the number of strains bearing a gene-suppressor for amber or ochre mutations also had a weak suppressor activity for some opal mutants. This suppressor acitivity is supposedly due to a second mutation in gene Su_uga.A comparative study of the phage yield with amber and opal mutations located in the same (paired) triplet in gene 47 has shown that the suppressor activity depends on the location of the mutant site along the gene.Experiments dealing with the induction of reversions by nitrous acid in amber and opal mutants with mutational sites located in the same triplet of gene 47 (mutant pairs) have shown the essential influence of the nucleotide sequence in the triplet on the frequency of induced reverse mutations at the given site.     

Polarity of localised conversion in Streptococcus pneumoniae transformation

Summary Localised conversion in pneumococcal transformation is a process that spans a few nucleotides when the 5-ATTAAT/3-TAAGTA configuration occurs at the pairing step. It was first observed in two-point crosses between an amiA mutation (amiA36) carrying this sequence and other closely linked mutants of the locus. The yield of the amiA resistance allele conversion to wild type is 20%. In order to characterize this process, which differs from longpatch conversion by the length of DNA repair, gene requirements and sequence specificity, we devised expreriments to detect the reciprocal conversion, AmiA⁺ to AmiA^r. For this purpose we examined the suppressibility by a pneumococcal informational suppressor of several nonsense mutations at the locus. Amber (UAG) and ochre (UAA) mutations are suppressed whereas UGA is not suppressed. In this genetic background, where amiA36 is partly suppressed, it was possible to select for double mutants in a cross between amiA36 and a closely linked non-suppressible marker. Direct isolation of such double mutants was also performed without any screening in crosses between amiA36 and the same linked marker in cloned DNA. The frequency of double mutants was very low (1/175) suggesting that there is no conversion of wild-type to mutant alleles. Thus conversion is a polarized process changing specifically A to C.     

The Preparation of Congenic Strains of Tetrahymena1

SYNOPSIS. Congenic strains of syngen 1, Tetrahymena pyriformis, were produced by backcrossing the F¹ hybrid between inbred strains C2 and D to strain D in 12 consecutive backcrosses, with selection for certain C2 genes, and then using genomic exclusion to induce homozygosity. Six congenic strains of high breeding performance are available. Five differ from strain D in single genes at 5 different loci. The 6th strain differs at all 5 loci. Assuming the size of the Drosophila gene (4 × 10⁴ nucleotide pairs), we can calculate that strains differing from D by single genes have a heterozygous segment 3 genes long while The strain which differs from D by 5 genes has 5 heterozygous segments and 15 genes contributed from strain C2. A 40-fold increase in heterozygosity would be found with a gene size of 10³ nucleotide pairs. This means that in using these strains for biochemical work we must be aware that some genetic noise still remains.