Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.     

Rates of forward and reverse mutation inDrosophila after exposure to mustard gas and X-rays

After treatment with mustard gas, reversions of the mutantforked ³ⁿ were observed with a frequency of 1 in 7,500. In the absence of indications for either suppressors or chromosome-rearrangements, these data provide evidence that a chemical mutagen can produce back mutations inDrosophila. Half the number of reversions was characterized by mosaic manifestation. This shows that delayed appearance after chemical treatment also holds for true gene mutations. One partial reversion to nearly normal type was not due to back mutation, but to a rearrangement, presumably involving a duplication of theforked containing region.The study on reversion off ³ⁿ was combined with tests for recessive visibles at 15 selected loci of the X-chromosome. Mutations at theruby locus were most frequently induced by mustard gas (1 in 1,700). About one quarter of the forward mutations were fractionals. After exposure to 5,000 r X-irradiation both reversions off ³ⁿ and forward mutations at the loci under study were observed with frequencies comparable to those induced by mustard gas. Thus, no indication for mutagenspecific differences in mutational response have been obtained. After treatment with mustard gas a higher ratio of visibles to lethals was observed than after exposure to X-irradiation. It is pointed out that comparisons of the mutagenic effect of a chemical mutagen with that of X-radiation, even if restricted to visible mutations, inevitably involve an underestimate for the chemical, due to delayed effects of the latter.The experiments were carried out mainly at the Department of Genetics, State University of Utrecht (Director: Prof. Dr.C. L. Rümke). A. J. M. van Hedel, G. J. O. Jansen, V. Labordus andS. C. M. Schouten collaborated on parts of this project.     

Genetic analysis of instability in Petunia hybrida

Summary In crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles (an1 ^+/+) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1.     

A case of mutagen specificity attributable to a plating medium effect

Summary In anadn ^– met ^– di-auxotrophic strain ofSchizosaccharomyces pombe met ⁺ reversions are several hundred times more frequent thanadn ⁺ reversions after treatment with ultra-violet light. They are only slightly more frequent thanadn ⁺ reversions when HNO₂ is used as a mutagen (mutagen specificity). The poor response of theadn-1,199 allele to the mutagenic action of U.V. can be largely overcome by replacing themet-4,D19 allele with its normalmet ⁺ allele (influence of the genetic background). It was shown that both the mutagen specificity and its dependence on the genetic background are due, largely at least, to the inhibition ofadn ⁺ reversions on a plating medium containingl-methionine. This inhibition is very strong for U.V.-induced reversions but only weak for HNO₂-induced ones. It would be wrong to assume that other mutants at theadenine-1 locus behave in the same manner.With 1 Figure in the Text     

Localization of a non-Mendelian gene in Chlamydomonas reinhardii

Summary Transfer of a non-Mendelian neamine-dependent (nd) mutant to an antibioticfree medium results in neamine-sensitive and neamine-resistent revertants. These reversions are caused by extranuclear mutations.The neamine-sensitive revertants are no more able to split offnd-cells after back-donation to neamine containing medium. Therefore they are different from the streptomycin-sensitive revertants of a streptomycin-dependent (sd) mutant. These mutants were capable ofsd-segregation though their potence ofsd-segregation diminished on antibiotic-free medium with increasing time of cultivation.The different behaviour can be explained by the fact that manysd-genes are present which have to be appointed to the mitochondria. On the other side, thend-gene exists only in few copies and is located therefore in the chloroplast.Several experiments with differing methods are discussed to localize the extranuclear genes.

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Vorgelegt durch G. Melchers     

Random mutant generation and its utility in uncovering structural and functional features of cytochromeb inSaccharomyces cerevisiae

The generation of random mutations in the mitochondrial cytochromeb gene ofSaccharomyces cerevisiae has been used as a most fruitful means of identifying subregions that play a key role in thebc ₁ complex mechanism, best explained by the protonmotive Q cycle originally proposed by Peter Mitchell. Selection for center i and center o inhibitor resistance mutants, in particular, has yielded much information. The combined approaches of genetics and structural predictions have led to a two-dimensional folding model for cytochromeb that is most compatible with current knowledge of the protonmotive Q cycle. A three-dimensional model is emerging from studies of distant reversions of deficient mutants. Finally, interactions between cytochromeb and the other subunits of thebc ₁ complex, such as the iron-sulfur protein, can be affected by a single amino acid change.     

Accumulation of Ade+ reversions in isoauxotrophic strains ofSaccharomyces cerevisiœ allelic inRAD6 during adenine starvation

A comparative method based on an analysis of accumulation of starvation-induced Ade⁺ reversions and cell death during adenine starvation was developed and exploited for estimating the role ofRAD6 in the starvation-induced reversions. It was shown that inactivation ofRAD6 function inSaccharomyces cerevisiœ markedly enhances the accumulation of Ade⁺ reversions, and therefore it is likely that this gene is taking part in maintaining the low level of starvation-induced mutations in yeast cells. This work was supported by a grant 204-1080-1993 from GACR to the last author andCharles University grant 274/1996 to the first author.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Nonsense mutations in the essential gene<Emphasis Type="Italic"> SUP35</Emphasis> of<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> are non-lethal

In the present work we have characterized for the first time non-lethal nonsense mutations in the essential gene SUP35, which codes for the translation termination factor eRF3 in Saccharomyces cerevisiae. The screen used was based on selection for simultaneous suppression of two auxotrophic nonsense mutations. Among 48 mutants obtained, sixteen were distinguished by the production of a reduced amount of eRF3, suggesting the appearance of nonsense mutations. Fifteen of the total mutants were sequenced, and the presence of nonsense mutations was confirmed for nine of them. Thus a substantial fraction of the sup35 mutations recovered are nonsense mutations located in different regions of SUP35, and such mutants are easily identified by the fact that they express reduced amounts of eRF3. Nonsense mutations in the SUP35 gene do not lead to a decrease in levels of SUP35 mRNA and do not influence the steady-state level of eRF1. The ability of these mutations to complement SUP35 gene disruption mutations in different genetic backgrounds and in the absence of any tRNA suppressor mutation was demonstrated. The missense mutations studied, unlike nonsense mutations, do not decrease steady-state amounts of eRF3.Communicated by C. P. Hollenberg     

Simultaneous transposition of different mobile elements: Relation to multiple mutagenesis in Drosophila melanogaster

Summary Several different transposition events occur simultaneously in one and the same germ cell, as we have found by analyzing different genetic systems in Drosophila melanogaster. (i) In unstable ct ^MR2 strains, stable reversions to ct ⁺ and changes in the type of ct mutation, which depend on an excision or transposition of the mobile element mdg4 (Gerasimova 1981; Gerasimova et al. 1984), are frequently accompanied by the appearance of novel mutations in different loci of the X chromosome. Some of these (sn, w, g) seem to be induced by the P-element and copia. (ii) A stable ct ^MR2 reversion to the wild type frequently coexists with an insertion of one to five copies of the P-element in the X-chromosome. Thus, the number of independent transposition events registered by genetic analysis and in situ hybridization may be as great as six. (iii) In two strains with double unstable mutations (cm, ct, and ct, r), double reversions to the wild type occurred at a high rate (80%–97% of total revertants). They frequently coexisted with novel strain-specific mutations. (iv) The stable strain ct ⁶ g²is destabilized by crossing with the MRh12/Cy strain (which contains a number of P-element copies). Both mutations begin to revert to the wild type. Of the revertants 50% have double reversions. Our experiments revealed a high specificity of insertion sites depending on the nature of transposon and the strain genotype. A possible role played by the burst of transposition in the evolution and possible mechanisms of transposition specificity are discussed.     

Unstable visible mutations induced in Drosophila melanogaster by injections of oncogenic virus DNA into the polar plasm of early embryos

Summary When RSV DNA cloned in pBR 322 or DNA of simian adenovirus Sa7 (C8) is injected into the pole plasm of embryos of various Drosophila stocks, the progeny of 1–70% of the surviving flies display visible mutations. The mutagenesis is partially directed: the loci mutating due to retrovirus and adenovirus DNA do not everlap. The majority of resulting mutants are characterised by high instability: reversions and new mutations occur in them, which sometimes spread over the whole population(explosive instability). The injected sequences are revealed by dot-hybridization in the DNA of many mutant strains, but only rarely by Southern blotting procedures. The results show that the microinjection of oncovirus DNA into embryos is an approach for obtaining highly unstable strains even from wildtype stable Drosophila stocks without crosses with MR lines or the introduction of P elements. The sets of unstable mutations induced by oncovirus DNA is different from those in hybrid dysgenesis.     

The influence of amino acid starvation on the UV reversibility of the strains ofEscherichia coli B/rthy − trp − Hcr + andEscherichia coli B/rthy − trp − Hcr −

The fraction of inducedtrp ⁺ reversions in the strains ofEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁺ andEscherichia coli B/rthy ⁻ trp ⁻ Hcr ⁻ was studied in the course of starvation for an essential amino acid. UV light as a mutagenic factor was used. It was found that there is a decrease in the proportion of inducedtrp ⁺ reversions in the strain ofHcr ⁺ type during starvation. Such a decrease was however observed only with that fraction oftrp ⁺ reversions which is expressed in selective plates where several divisions of irradiated cells are caused. The proportion oftrp ⁺ reversions expressed on minimal plates does not change during starvation. With the strain ofHcr ⁻ type the proportion of inducedtrp ⁺ mutations remains unaltered irrespective of the nature of the selective plates.     

Mobilization of the transposable element mdg4 by hybrid dysgenesis generates a family of unstable cut mutations in Drosophila melanogaster

Summary A family of unstable mutations at the cut locus in Drosophila melanogaster was obtained under the conditions of hybrid dysgenesis (Gerasimova 1981, 1982). The in situ hybridization experiments have shown that, in the original unstable ct ^MR2 mutation, the 7B region of the X chromosome (where cut is located) contains a mobile dispersed genetic element, mdg4. All other unstable ct mutations derived from ct ^MR2 including visible and lethal alleles and unstable ct ⁺ reversions, also contain mdg4 in the 7B region. The X chromosomes of the parent strain (wild type) do not contain mdg4 at all. All stable revertants derived from ct ^MR2, from other unstable ct mutations, or from ct lethals lost mdg4 from the 7B region. The ct ^MR2 X chromosome does not contain P-elements, although a few copies are present in the autosomes. The instability of the ct ^MR2./ct ^MR2 strain remained at a high level for 50 generations (1.5 years) and then rapidly decreased. A new cross with an MRh12/Cy strain (originally used for dysgenesis induction and containing a number of P-elements) increased the instability to a level exceeding the original one. The data strongly suggest that unstable ct mutations in our system are induced by transpositions of mdg4, possibly activated by P-elements.     

Identification of novel RPGR (retinitis pigmentosa GTPase regulator) mutations in a subset of X-linked retinitis pigmentosa families segregating with the RP3 locus

The X-linked form of retinitis pigmentosa (XLRP) is a severe disease of the retina, characterised by night blindness and visual field constriction in a degenerative process, culminating with complete loss of sight within the third decade of life. Genetic mapping studies have identified two major loci for XLRP: RP3 (70%–75% of XLRP) and RP2 (20%–25% of XLRP). The RPGR (retinitis pigmentosa GTPase regulator) gene has been cloned within the RP3 genomic interval and it has been shown that 10%–20% of XLRP families have mutations in this gene. Here, we describe a single-strand conformational polymorphism-based mutation screening of RPGR in a pool of 29 XLRP families for which the disease segregates with the RP3 locus, in order to investigate the proportion of RP3 families with RPGR mutations and to relate the results to previous reports. Five different new mutations have been identified: two splice site mutations for exon 1 and three frameshift mutations in exons 7, 10 and 11. The percentage of RPGR mutations identified is 17% (5/29) in our genetically well-defined population. This figure is comparable to the percentage of RP2 gene mutations that we have detected in our entire XLRP patient pool (10%–15%). A correlation of RPGR mutations with phenotype in the families described in this study and the biochemical characterisation of reported mutations may provide insights into the function of the protein. Electronic Publication     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

Genetic Control of Purine Biosynthesis in Yeast Pichia methanolica. The ADE5 (PUR4) Gene Controlling 5"-Phosphoribosylformyl Glycinamidine Synthetase

By comparing published and experimental data on spontaneous mutability of early genes controlling biosynthesis of purine nucleotides (BPN) in different yeast species in the system from red to white, it was shown that the PUR4 gene encoding 5"-phosphoribosylformyl glycinamidine synthetase (FGAM-synthetase) (EC 6.3.5.3) is the most mutable gene in yeastSaccharomyces cerevisiae (the ADE6 gene), Schizosaccharomyces pombe(the ade3 gene), andPichia methanolica (theADE5 gene). This correlates with a considerably large size of the FGAM-synthetase polypeptide, as compared to the products of other genes belonging to this group. Study of characteristics of spontaneous mutations in early BPN genes of P. methanolica demonstrated that the vast majority of unstable ade5s ^U alleles (mutations with a high reversion frequency ranging from 0.2 × 10^–6 to 2 × 10^–6) appeared solely among mutants for the ADE5 gene. Based on these results, it was assumed that there are two independent mechanisms responsible for reversions of spontaneous mutations in this gene. The DNA sequence that can compensate for theP. methanolica ade5mutation and probably is the structural P-ADE5gene, was cloned from a genomic library of P. methanolicaby the ade6 mutation complementation in the recipient S. cerevisiae strain.     

Induction of unstable mutations in Drosophila melanogaster by microinjection of DNA from oncogenic viruses into the embryonic polar plasma. III. Genetic instability in the absence of an intact P-element

DNA of simian adenovirus Sa7 injected into polar plasma of early Drosophila melanogaster y1snw*; bw; st stock embryos induced one to three unstable visible X-linked mutations in the absence of intact P-element. Numerous mutational events (reversions, new mutations) occur only in four precisely destabilized by the Sa7 DNA loci of X-chromosome and take place during 4-5 generations; in the next generations the level of instability decreased. At the same time, Sa7 DNA induced reversions and new allelic mutations in the snw mutant locus, without exogenous intact P-element.