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     

Reverse mutation inDrosophila and the status of the particulate gene

Summary Following the discussion and examination of relevant experimental observations, it is proposed that the identity between spontaneous phenotypic reversion and reverse (back) mutation is established only after a number of criteria are fulfilled. These criteria include the demonstration that the reverting mutant itself as well as the reversions are not associated with chromosomal rearrangements, that reversion occurrs in the absence of crossing over, that independent suppressor or modifier mutants are not involved, that the phenonomenon of gene conversion does not occur and that the reversion represents and autonomous event.Previously reported instances of partial and complete phenotypic reversions, especially those inDrosophila, are discussed and evaluated in terms of the aforementioned criteria.Detailed, critical data are presented showing that in terms of the listed criteria the sex-linked, recessive bristle mutantsf ¹ andf ³ⁿ inDrosophila melanogaster undergo reverse mutation at characteristic rates.The relationship between reverse mutation and the nature of the gene is discussed. It is concluded that the facts of reverse mutation fit best the concept of a particulate gene.     

Unstable elements at the locusLuteus-1 inPetunias hybrida: Genetic analysis of their mobility

At theLul (Luteus-1) locus from Petunia, semi-dominant mutations occur, characterized by partial chlorophyll deficiencies. They can show phenotypic instability with green spot reversions. Two types of unstable mutations are described. Thelu-ml type presents a low rate of somatic reversions that are regulated by non-linked various activating factors. In contrast thelu-m3 instability shows high reversion rates regulated by a linked specific activating factor (La3), not found otherwise among the genome. Under the effect of therecombination modulator (Rm1), lu-m3 andLa3 were separated inducing a stabilization oflu-m3. This allele,lu-m3rs, however, seems to be able to respond again to a transactivation by reintroduction ofLa3 through crossing. The high transposition potential of the associationlu-m3rs-La3 was implemented to test its capacity to reinster. This experiment, conducted on a large scale, has allowed us to detect and study two unstable mutations concerning the flower pigmentation. Those two genes that control the anthocyanin production and the flavonol synthesis, respectively, are strictly linke toLul. Consequently it can be assumed that those two unstable mutations are due to reinsertion of thelu-m3rs transposons through proximal transpositions.     

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.     

O0 and strong-polar mutations in the gal operon are insertions

Summary Three dg phages carrying strong-polar mutations in the gal operon are denser than the corresponding phages carrying the wildtype gal operon or reversions of the mutations to the Gal ⁺ phenotype. The latter phages have the same density. It is concluded that these strong-polar mutations are insertions of DNA into the gal operon.The amount of inserted DNA is different in the three mutations and is calculated to be 450, 1,080 and 1,800 nucleotide pairs respectively.The strong-polar phenotype is also found in a mutant supplied by A. Taylor which carries a Mu-1 phage integrated into the transferase gene.     

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.     

Insertion mutations in the control region of the Gal operon of E. coli. I. Biological characterization of the mutations

Summary Galactose negative mutations are described which reduce the maximum expression of all three gal genes about 100-fold. The residual enzyme synthesis is not or only slightly inducible.These pleiotropic mutations map in the control region of the gal operon. No recombination is observed between these mutations. All mutants revert spontaneously to a Gal⁺ phenotype. In some mutations wildtype-like as well as constitutive revertants are obtained. The frequency of reversion can be increased by nitrosoguanidine (NG) in all mutants. The revertants, induced by this mutagen, are of a constitutive type.     

Structural analysis of a polysaccharide from<Emphasis Type="Italic">Chlorella kessleri</Emphasis> by means of gas chromatography—mass spectrometry of its saccharide alditols

Interpretation of gas chromatographic-mass spectrometric data of oligosaccharide alditols was used to determine their structures and to derive the structure of a water soluble polysaccharide isolated fromChlorella kessleri.¹H- and¹³C-NMR was employed to assess the configuration of glycosidic bonds and individual monosaccharides were assigned to thel ord series by means of gas chromatography of the acetylated (S)-2-butyl glycosides.     

Mechanism and role of furosemide-sensitive K+ transport in L cells: A genetic approach

Summary As part of a genetic study of the mechanisms for cation transport in cultured mammalian cells, two mouse fibroblastic cell lines have been compared with respect to unidirectional⁴²K⁺ influx. The cell lines areLM(TK ^–) andLTK-5, a mutant selected fromLM(TK ^–) by the ability to grow in medium containing 0.2mm K⁺. In both cell lines, the overall influx can be resolved into three components: (i) a ouabain- and vanadate-sensitive component ( ⁱ M_K ^f), presumably the Na/K pump, which is a saturable function of extracellular K⁺ with aK _1/2 of 1.3mm; (ii) a furosemide-sensitive component ( ⁱ M_k ^fx), also a saturable function of extracellular K⁺, with aK _1/2 of 6mm; and (iii) a diffusional component ( ⁱ M_k ^d); which is a linear function of extracellular K⁺.By several independent criteria, ⁱ M_k ^o and ⁱ M_k ^f appear to be distinct transport processes. First, as indicated above, they can be separated with the use of inhibitors. In addition, they can be separated genetically, since theLTK-5 mutant shows a threefold elevation in ⁱ M_k ^f with no change in ⁱ M_k ^o. And finally, extracellular Na⁺ has no effect on ⁱ M_k ^o, but stimulates ⁱ M_k ^f, a result consistent with the notion that ⁱ M_k ^f influx occurs by Na–K cotransport.Further experiments were directed towards understanding the nature of theLTK-5 mutation and the physiological role of ⁱ M_k ^f. LTK-5 differs from the parental cell line, not only in having an increased ⁱ M_k ^f, but also in having a large cell volume, a slow maximal growth rate, and an ability to grow at 0.2mm K⁺. The most straightforward interpretation — that the increased ⁱ M_k ^f is itself responsible—is unlikely since the addition of furosemide to the growth medium had no effect upon the growth rate or cell volume of the mutant at either normal or low extracellular K⁺ concentrations. It did, however, render the parent capable of growth at 0.2mm K⁺. Possible interpretations are discussed.     

The use of ethyl methanesulfonate for the induction of mutants inMycobacterium phlei PA

The mutagenic effect of ethyl methanesulfonate in a concentration of 0.2m on a prototrophic, acid-fast strainMycobacterium phlei PA was studied by following the induction of changes of three genetic markers: prototrophy to auxotrophy and sensitivity to two antituberculosis drugs (INH and STM) to resistence. Ethylmethanesulfonate was found to be a very effective mutagen in all three cases. Thirty auxotrophic strains were obtained, out of which eight exhibited a low frequency of spontaneous reversions and could hence be used for further studies. Of the phenotypes induced the glycine⁻ (serine⁻) type was most frequently isolated and represented more than half of all auxotrophs obtained. Requirements for lysine and purines were also observed. The EMS treatment (1% survival of the basic suspension) resulted in a 74-fold increase of the frequency of INH-resistant mutants and a frequency of STM-resistant mutants about 1.1/2 to almost 2 orders of magnitude higher than the spontaneous values     

Mitomycin C induces genomic rearrangements involving transposable elements in Drosophila melanogaster

Summary Mitomycin C was injected into the abdomen of male flies of the y ² sc¹ w^aG strain of Drosophila melanogaster. They were mated with females bearing attached-X chromosomes, and the male offspring (F₁) were analysed for the appearance of mutations in the X chromosome. We observed y ¹ and sc ⁺ reversions induced either by excision of mdg4 (gypsy) with retention of one long terminal repeat (LTR) or by insertion of a foreign sequence into mdg4, partial reversion of the w ^aG mutation, w ^aGw ^aGd, and unstable f mutations. The overall mutation frequency was considerably higher than in control flies of the y ² sc¹ w^aG strain. Possible mechanisms of genomic rearrangements induced by Mitomycin C, in particular the role of homologous recombination, are discussed.     

Chloroplast mutations induced by 9-aminoacridine hydrochloride are independent of the<Emphasis Type="Italic"> plastome mutator</Emphasis> in<Emphasis Type="Italic"> Oenothera</Emphasis>

Oenothera plants homozygous for the recessive plastome mutator allele (pm) show chloroplast DNA (cpDNA) mutation frequencies that are about 1,000-fold higher than spontaneous levels. The pm-encoded gene product has been hypothesized to have a function in cpDNA replication, repair and/or mutation avoidance. Previous chemical mutagenesis experiments with the alkylating agent nitroso-methyl urea (NMU) showed a synergistic effect of NMU on the induction of mutations in the pm line, suggesting an interaction between the pm-encoded gene product and one of the repair systems that corrects alkylation damage. The goal of the experiments described here was to examine whether the pm activity extends to the repair of damage caused by non-alkylating mutagens. To this end, the intercalating mutagen, 9-aminoacridine hydrochloride (9AA) was tested for synergism with the plastome mutator. A statistical analysis of the data reported here indicates that the pm-encoded gene product is not involved in the repair of the 9AA-induced mutations. However, the recovery of chlorotic sectors in plants derived from the mutagenized seeds shows that 9AA can act as a mutagen of the chloroplast genome.Communicated by R. Hagemann     

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.     

Pre-steady-state analysis of the turn-on and turn-off of water permeability in the kidney collecting tubule

Summary Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent water channel insertion into and retrieval from the cell apical membrane. The time course of osmotic water permeability (P _f ) following addition and removal of vasopressin (VP) and 8-Br-cAMP was measured continuously by quantitative fluorescence microscopy using an impermeant fluorophore perfused in the lumen. Cortical collecting tubules were subjected to a 120 mOsm bath-to-lumen osmotic gradient at 37°C with 10–15 nl/min lumen perfusion and 10–20 ml/min bath exchange rate. With addition of VP (250 U/ml), there was a 23±3 sec (sem,n=16) lag in whichP _f did not change, followed by a rise inP _f (initial rate 1.4±0.2×10^–4 cm/sec²) to a maximum of 265±10×10^–4 cm/sec. With addition of 8-Br-cAMP (0.01–1mm) there was an 11±2 sec lag. For [8-Br-cAMP]=0.01, 0.1 and 1mm, the initial rate ofP _f increase following the lag was (units 10^–4 cm/sec²): 1.1±0.1, 1.2±0.1 and 1.7±0.3. MaximumP _f was (units 10^–4 cm/sec): 64±4, 199±9 and 285±11. With removal of VP,P _f decreased to baseline (12×10^–4 cm/sec) with aT _1/2 of 18 min; removal of 0.1 and 1mm 8-Br-cAMP gaveT _1/2 of 4 and 8.5 min. These results demonstrate (i) a brief lag in theP _f response, longer for stimulation by VP than by 8-Br-cAMP, representing the transient build-up of biochemical intermediates proximal to the water channel insertion step, (ii) similar initialdP _f /dt (water channel insertion) over a wide range of [8-Br-cAMP] and steady-stateP _f values, and (iii) more rapidP _f decrease with removal of 8-Br-cAMP than with VP. These pre-steady-state results define the detailed kinetics of the turn-on and turn-off of tubuleP _f and provide kinetic evidence that the rate-limiting step for turn-on ofP _f is not the step at which VP regulates steady-stateP _f . If water channel insertion is assumed to be the rate-limiting step in the turn-on ofP _f , these results raise the possibility that water channels must be activated following insertion into the apical membrane.     

The nature of reverse mutations in Drosophila melanogaster

A selection system for the screening of reversions has been constructed and used to test reversions of lethals located in the proximal region of the X chromosome of Drosophila and of Kpn mutations.Spontaneous and induced reversions have been screened, X-rays and ethyl methanesulphonate (EMS) being the mutagens used in the induction experiments.No genuine back-mutation was found in 6·10⁵ gametes scored. Sterile reversions of all four lethals tested were obtained. Their frequency suggested that at least in three of the lethals the sterile reversions represented “escapers” of the lethal effect rather than true revertants.Three fertile reversions of l^x4 were found and analyzed. All three were autosomal suppressors, located on the second chromosome, allelic to each other, dominant in males and recessive in females.One fertile reversion of l^3DES was found to be an X-linked suppressor. It is suggested that this suppressor is a Y-suppressed lethal, showing a V-type position effect, resulting from an aberration included in the proximal heterochromatin of the X chromosome.Reversions of Kpn were obtained at a similar rate to that found in previous reports²².The absence of true back-mutants in our experiments, in contrast to findings in previous reports, is discussed. From the existing literature on spontaneous and induced back-mutations in Drosophila melanogaster it appears that for several mutations the rates of forward and back-mutation are of the same order of magnitude. It is suggested that reported cases of back-mutations represent mainly inter- and intrachromosomal recombination in duplicated regions rather than mutational events and that the frequency of true back-mutation in Drosophila is usually of an order of magnitude, similar to that known for microorganisms and fungi.     

Evidence for Mitotic Recombination in W(ei)/+ Heterozygous Mice

A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the Wei allele at the W locus were studied Mice heterozygous in repulsion for both Wei and buff (bf) [i.e. Wei+/+bf] were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; Wei+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of Wei/+ were enhanced significantly following X-irradiation of 9.25-day-old Wei/+ embryos (P less than 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results rise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse.     

Developmental genetics of a P element induced allele of suppressor-of-forked in Drosophila melanogaster

Summary In this paper we describe a new allele of suppressor of forked, su(f) ^hd37, referred to as hd37, which was isolated in a hybrid dysgenesis mutation screen and is shown to be P induced by its high frequency of reversion in hybrid dysgenic crosses, and by in situ hybridization. hd37 suppresses forked and fails to complement the forked suppression of known su(f) alleles. However, it complements the recessive lethality of alleles in both of the su(f) lethal complementation groups. We also describe a new phenotypic effect of su(f) alleles, the enhancement of Minute(3)i ⁵⁵. Recessive lethal alleles enhance the lethal effects of this Minute, but hd37 does not. The temperature sensitive period for forked bristle suppression by hd37 was found to be very narrow, consisting of a short interval (12–18 h) immediately before bristle formation. These results suggest that the several genetic functions associated with this locus may be genetically separable.Journal paper No. J-12137 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2746     

X-ray-induced reversion of thewhite-ivory mutant ofDrosophila melanogaster

Following X irradiation,w ⁱ reverts in oogonia and in spermatogonia. following treatment of adult females,w ⁱ reverts equally frequently in homozygotes and deficiency heterozygotes. Induced reversions are commonly recovered as clusters, indicating that they are of gonial origin. In contrast tow ⁱ , two partial reversions recombine normally withw ^ch . One of these has been tested for X-ray-induced reversion and found to be stable.A part of this investigation began while the senior author was a predoctoral trainee under Public Health Training Grant GM 701.05 at the University of California, Davis. It was completed under Utah State University Research Council Grant U-302.