Meiotic Recombination and Sporulation in Repair-Deficient Strains of Yeast

A genetic system designed to monitor recombination and sporulation in various repair-deficient yeast strains was constructed. Variously heterozygous at seven or eight sites distributed across the genome, the system facilitated sensitive detection of changes in frequency or pattern of meiotic recombination. Ten rad mutants sensitive primarily to UV-irradiation and without terminal blocks in the sporulation process were studied. Seven were defective in excision repair (rad1, rad2, rad3, rad4, rad10, rad14 and rad16), and three were defective in mutagenic repair (rad5, rad9 and rad18). Individually, each mutant displayed behavior consistent with an orthodox meiosis including a wild-type meiotic recombination profile with respect to gene conversion, PMS and intergenic map distances. Accordingly, we conclude that these mutants are without major effect on meiotic heteroduplex formation or correction. However, certain combinations of excision-defective mutants with rad18 exhibited marked ascosporal inviability. Tetraploids homozygous for rad1 and rad18 produce a large proportion of diploid spores containing a recessive lethal.     

Panel discussion on submammalian systems

A test of X-ray-induced recessive lethal mutations in mouse spermatogonia (500 rad) was carried out. The test was based on familial analysis, which allowed division on the P pairs into those with lethal heterozygous members and in others assumed to be lethal-free. The F1 males from the latter group, in back-crosses to their daughters, gave an excessive rate of intra-uterine death in comparison with lethal-free males. The excessive death is assumed to reflect the rate of new (induced + spontaneous) recessive lethals or rather lethal equivalents.Three ways of estimating the rate of new recessive lethal mutations gave a mean of 5.5% per genome. From previous tests we can assume that 1% are spontaneous mutations. Thus the data indicate that the mutation rate per rad per gamete is 9 × 10^?5. This value is identical with a previous estimate.The results are discussed in relation to population tests performed in the early 60'2. It is concluded that the lack of observable deterioration in the populations after several consecutive generations of exposure is in accord with the estimates in the present analysis which are more than an order of magnitude lower than assumed at the start of the population tests.It is also stressed that species with different DNA contents show similarities in point estimates of doubling dosages.     

Genetic Effects of Acute Spermatogonial X-Irradiation of the Laboratory Rat

The genetic effects of one generation of spermatogonial X-irradiation in rats, by a single dose of 600r in one experiment and by a fractionated dose of 450r in another, were measured in three generations of their descendants. Estimates of dominant lethal mutation rates—(2 to 3) x 10 ^-4/gamete/r—from litter size differences between irradiated and nonirradiated stock were consistent with previous estimates from rats and mice. Similar consistency was found for estimates of sex-linked recessive mutation rates—(1 to 2) x 10^-4 chromosome/r—from male proportions within strains; however, when measured in crossbreds the proportion of males was higher in the irradiated than in the nonirradiated lines. This inconsistency in results is in keeping with the contradictory results reported for recessive sex-linked lethal mutation rates in mice. The effects used to estimate recessive lethal mutation rates which were unusually high—(2 to 14) x 10^-4/gamete/r—were not significant. Other factors that could have contributed to the observed effects are postulated.     

Mutation Induction in the Male Recombination Strains of DROSOPHILA MELANOGASTER

One group of the second chromosome lines isolated from a southern Texas population of Drosophila melanogaster, which has been known to show relatively high frequencies of male recombinations, was found to increase the frequency of sex-linked recessive lethal mutations from a control frequency of 0.18% to 1.63%. The second group, which showed a very much reduced frequency of male recombinations, was found to cause a slight increase to 0.48%, although it was not statistically significant. The first group was also tested for the recessive lethal mutation frequency in the second chromosome; the frequency increased from a control frequency of 0.28% to 2.82%. Mapping of a portion of the sex-linked lethals indicated a distribution along the entire X chromosome, although there was a tendency of clustering towards the tip of the X chromosome. One sex-linked lethal line so far tested was found to be associated with an inversion (approximate breakpoints, 14A-18A). It was suggested that the element causing male recombination might be similar to the hi mutator gene studied earlier by Ives (1950).     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. I. Recessive Lethal Mutations

The efficiency of the adult feeding method for EMS treatment in Drosophila melanogaster was studied by measuring the frequency of induced recessive lethals on the second chromosome. The treatment was most effective when mature spermatozoa or spermatids were treated and was much less effective on earlier stages. The number of mutations induced was proportional to the concentration except at the highest doses. The recessive lethal rate was estimated to be about 0.012 per second chromosome per 10(-4) M. In addition, about 0.004-0.005 recessive lethals per 10(-4) M were found in a later generation in chromosomes that had not shown the lethal effect in the previous generation. When the experiments are done in a consistent manner and gametes treated as mature sperm or spermatids are sampled, the results are highly reproducible. However, modifications of the procedure, such as starvation before EMS treatment, can considerably alter the effectiveness of the mutagen.     

Multigeneration exposure test of Drosophila melanogaster to ELF magnetic fields

Mutations, other than dominant lethals, were accumulated on wild type second chromosomes (+) of Drosophila melanogaster during exposure to 50 Hz sinusoidal alternating magnetic fields of 0.5 or 5 mT (rms) for 40 generations by the Curly/Plum(Cy/Pm) accumulation method. We maintained, for 40 generations under continuous exposure, each (+) chromosome as a heterozygote with (Cy) chromosome. Viability of the (+) chromosome was tested by sib-mating of (Cy/+) male and (Cy/+) female in a culture every 10th generation to obtain the homozygote. Viability indices, defined as twice the ratio of number of (+/+) flies to that of (Cy/+) flies plus 1 in the progeny of the test mating, also were calculated, which equaled 1.00 at the starting point. For the control and 0.5 and 5 mT exposed groups, percent frequencies of recessive lethal lines, defined as a line with (+/+) flies less than 0.3% in the test mating, were, respectively, 1.9, 0.9, and 2.9% (10th), 9.0, 4.9, and 9.5% (20th), 30.3, 22.9, and 30.4% (30th), and 39.9, 32.4, and 43.3% (40th generation). For the control and 0.5 and 5 mT groups, average viability indices, excluding lethals and markedly deleterious, were, respectively, 0.778, 0.796, and 0.752 (20th), 0.704, 0.698, and 0.694 (30th), and 0.669, 0.678, and 0.595 (40th generation). Their decreasing rates were 0.0054, 0.0059, and 0.0078 per generation. No significant difference was detected among the exposure levels in either the recessive lethal mutation frequency or the viability index. Bioelectromagnetics 19:335–340, 1998. © 1998 Wiley-Liss, Inc.     

Chromosomal superkiller mutants of Saccharomyces cerevisiae.

Yeast strains carrying a 1.5 X 10(6)-dalton double-stranded RNA in virus-like particles secrete a protein toxin which is lethal to strains not carrying this species of double-stranded RNA. We find that recessive mutations in any of four chromosomal genes result in the superkiller phenotype, i.e., increased secretion of killer toxin activity by strains carrying the killer genome. These genes are designated ski1 through ski4 (for superkiller), ski3 and ski4 are located on chromosome XIV, and ski1 is on chromosome VII. A ski1 mutation results in a decreased rate of cell growth. The kex1 and kex2 mutations are epistatic to each ski mutation.     

Site-Specific Instability in DROSOPHILA MELANOGASTER: The Origin of the Mutation and Cytogenetic Evidence for Site Specificity

During a study of delayed mutations, an unstable X chromosome (Uc) was detected. Spontaneous X-linked recessive lethal mutations were detected in 34 of 993 sperm sampled from 50 males carrying this chromosome. All but three of the 34 lethals originated as clusters in three of the 50 males Cytogenetic and complementation analyses revealed 14 intrachromosomal rearrangements: ten inversions, two reverse repeats, one deficiency and one transposition. Eight of the 14 rearrangements have one break in the 6F1-2 doublet and two rearrangements have a break in 6F1-5 of the X chromosome. The remaining four rearrangements have in addition to the aberrations a lethal point mutation between 6F1 and 6F5. Though each of the lethal lines was established from a single lethal-bearing female, chromosome polymorphism is evident in 17 of the 18 lines having rearrangements, with certain aberrations recurring in several lines. The lethal mutations revert frequently to the nonlethal state, and cytological evidence indicates that more than one mutational event may occur at the unstable locus of the chromosome during one generation. Two lethal lines had more than one type of chromosome rearrangement sharing a common breakpoint. These observations are consistent with the view that the instability of the Uc lines is caused by a transposable element capable of site-specific chromosome breaks and perpetual generation of mutations. The mutagenic and genetic properties of transposable elements can be related to the two-mutation theory of KNUDSON (1971) for cancer initiation.     

Induction of pure and sectored mutant clones in excision-proficient and deficient strains of yeast.

We have found that UV-induced mutation frequency in a forward non-selective assay system (scoring white adex ade2 double auxotroph mutants among the red pigmented ade2 clones) increases linearly with dose up to a maximum frequency of about 3 X 10(-3) mutants per survivor and then declines in both RAD wild-type and rad2 excision deficient strains of Saccharomyces cerevisiae. Mutation frequencies of the RAD and the rad2 strains plotted against survival are nearly identical over the entire survival range. On this basis we conclude that unexcised pyrimidine dimers are the predominant type of pre-mutational lesions in both strains. In the RAD wild-type strain pure mutant clones outnumber sectors in a 10:1 ratio at all doses used; in rad2 this ratio varies from 1:1 at low doses up to 10:1 at high doses. As others have concluded for wild-type strains we find also in the rad2 strain that pure clone formation cannot be accounted for quantitatively by lethal sectoring events alone. We conclude that heteroduplex repair is a crucial step in pure mutant clone formation and we examine the plausibility of certain macromolecular mechanisms according to which heteroduplex repair may be coupled with replication, repair and sister strand exchange in yeast mutagenesis.     

Dominant cataract and recessive specific locus mutations in offspring of X-irradiated male mice

Male mice were X-irradiated with 3.0 + 3.0 Gy or 5.1 + 5.1 Gy (fractionation interval 24 h). The offspring were screened for dominant cataract and recessive specific locus mutations. In the 3.0 + 3.0-Gy spermatogonial treatment group, 3 dominant cataract mutations were confirmed in 15 551 offspring examined and 29 specific locus mutations were recovered in 18 139 offspring. In the post-spermatogonial treatment group, 1 dominant cataract mutation was obtained in 1120 offspring and 1 recessive specific locus mutation was recovered in 1127 offspring. The induced mutation rate per locus, per gamete, per Gy calculated for recessive specific locus mutations is 2.0 X 10(-5) in post-spermatogonial stages and 3.7 X 10(-5) in spermatogonia. For dominant cataract mutations, assuming 30 loci, the induced mutation rate is 5.0 X 10(-6) in the post-spermatogonial stages and 1.1 X 10(-6) in spermatogonia. In the 5.1 + 5.1-Gy spermatogonial treatment group, 3 dominant cataract mutations were obtained in 11 205 offspring, whereas in 13 201 offspring 27 recessive specific locus mutations were detected in the spermatogonial group. In the post-spermatogonial treatment group no dominant cataract mutation was observed in 425 offspring and 2 recessive specific locus mutations were detected in 445 offspring. The induced mutation rate per locus, gamete and Gy in spermatogonia for recessive specific locus mutations is 2.8 X 10(-5) and for dominant cataract mutations 0.9 X 10(-6). In post-spermatogonial stages, the mutation rate for recessive specific locus alleles is 6.2 X 10(-5). In the concurrent untreated control group, in 11 036 offspring no dominant cataract mutation and in 23 518 offspring no recessive specific locus mutation was observed. Litter size and the number of carriers at weaning have been determined in the confirmation crosses of the obtained dominant cataract mutants as indicators of viability and penetrance effects. Two mutants had a statistically significantly reduced litter size and one mutant had a statistically significantly reduced penetrance.     

X-ray- and chemically induced germ-line mutation causing phenotypical anomalies in mice

A large and significant increase of phenotypical anomalies was observed in the progeny of ICR parent mice treated before mating with X-rays, urethane, 7,12-dimethylbenz[a]anthracene, ethylnitrosourea (ENU), and 4-nitroquinoline 1-oxide, but the increase was not significant with furylfuramide. Major types of induced anomalies were cleft palate, dwarf, open eyelid, tail anomalies, and exencephalus. Dwarf, open eyelid and tail anomalies were predominant types of viable anomalies and were inherited as if they were dominant mutations with varying expressivity or penetrance. Incidence of prenatal anomalies increased with treated doses of X-rays, urethan, or ENU for both spermatozoa and spermatogonia. Spermatogonia were less sensitive to X-rays and urethane than spermatozoa, while ENU induced a very high incidence of prenatal anomalies by the spermatogonial treatment. In contrast to the previous works with X-rays, there was a clear, almost linear increase of anomalies in the dose range from 0 to 216 rad after spermatogonial exposure. For treatment of oocytes, there was also a clear increase with doses of X-rays and urethane. Doubling doses of X-rays for prenatal anomalies were 12 rad for spermatozoa, 27 rad for spermatogonia, and 19 rad for mature oocytes. These values are similar to those for ordinary mouse mutations. However, the mean rate of prenatal anomalies per rad (1.2 X 10(-4), 6.6 X 10(-5) and 9.1 X 10(-5) for spermatozoa, spermatogonia and mature oocytes, respectively) and that for 1 micrograms/g of ENU (3.4 X 10(-4) for spermatogonia) were 4-40 times higher than that of ordinary mutation in mice, because overall phenotypical abnormalities were scored in this study. Information obtained from the work on phenotypical anomalies is valuable to assess genetic risk of radiation and chemicals, because a majority of human genetic diseases show this kind of irregular and uncertain inheritance and most of the induced anomalies are similar to those found in humans.     

Mutagenicity of lead chromate in Drosophila melanogaster in the presence of nitrilotriacetic acid (NTA)

By using the sex-linked recessive lethal mutation test in Drosophila melanogaster (standard Basc scheme) we analysed the mutagenic effects of treatments by feeding with nitrilotriacetic acid (NTA: 5 X 10(-2) M), with the insoluble Cr(VI) compound lead chromate, PbCrO4 (supernatant of 4.6 X 10(-4)-M suspension in which the actual concentration was 0.06 gamma/ml as Cr(VI)) and with both compounds preincubated at 3 relative ratios (NTA: 5 X 10(-2) M; PbCrO4: 4.6 X 10(-4), 4.6 X 10(-5) and 9.2 X 10(-6) M, respectively). The estimation of mutation frequencies was done at different developmental stages of the germ cells, namely spermatozoa, spermatids and spermatocytes. Ethyl methanesulphonate (EMS: 5 X 10(-3) M) was used as the reference positive control, with clearly mutagenic results. Treatments with NTA or with PbCrO4 alone did not induce any significant increase of the mutation frequency. PbCrO4 at the 3 concentrations tested was completely soluble in the 5 X 10(-2)-M NTA solution, and the mixture of NTA and PbCrO4 induced significant increases of the frequency of sex-linked lethal mutations, with a significant dose-effect relationship with respect to PbCrO4, apparently as a result of the interaction of the compounds and subsequent release of the genotoxic heavy-metal Cr(VI) ions. This result indicates an important synergistic action of NTA with PbCrO4 under the conditions described.     

X-ray-induced mutants resistant to 8-azaguanine. II. Cell cycle dose response.

Synchronous Chinese hamster ovary cells were irradiated in G1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G1 and S phase were practically identical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 X 10(-7) (75-100 rad) to 8 X 10(-7) (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 X 10(-7) per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency (PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant (PE greater than 0.1) to 7.5 mug/ml AG, 85% to 5.0 mug/ml, and 91% to 3.5 mug/ml. Only 42% showed possible hypoxanthine-phosphoribosyltransferase (hprtase) deficiency as evidenced by HAT sensitivity (PE less than 0.1). Wild type controls exhibited PE's in 3.5 mug/ml AG of less than 0.001 and in HAT of greater than 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild type cells (D0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occurring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10-60 rad for chronic irradiation, similar to that often reported for in vivo studies.     

Mutagenic effect of different types of irradiation on the sex cells of male mice. X. Frequency of recessive lethal mutations and reciprocal translocations in the spermatogonia of mice subjected to fractional gamma-irradiation]

The frequency of recessive lethal mutations and reciprocal translocations was investigated in spermatogonia of CBA male mice which were thrice gamma-irradiated at doses of 300 r with 28 days intervals. The rate of induced recessive lethals was estimated 1) by comparison of embryos survival between the irradiated and control groups in mating of the F1 males with their daughters, and 2) by estimation the frequency of males heterozygotes for recessive lethals in the first generation. In the first case the frequency of recessive lethals was 2,8 +/- 0,8-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete (for the pre- and post-implantation death) and 1,6 +/- 0,1-10(-4) per r per gamete in the second case. The frequency of heterozygotes for reciprocal translocations in the first generations of males was 3,1 +/- 0,9-10(-5) per r per gamete.     

Ultraviolet mutagenesis of radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans

A mutational tester strain (JP10) of the nematode C. elegans was used to capture recessive lethal mutations in a balanced 300 essential gene autosomal region. The probability of converting a radiation interaction into a lethal mutation was measured in young gravid adults after exposure to fluences of 254-nm ultraviolet radiation (UV) ranging from 0 to 300 Jm-2. Mutation frequencies as high as 5% were observed. In addition, three different radiation-hypersensitive mutations, rad-1, rad-3 and rad-7 were incorporated into the JP10 background genotype, which allowed us to measure mutation frequencies in radiation-sensitive animals. The strain homozygous for rad-3 was hypermutable to UV while strains homozygous for rad-1 and rad-7 were hypomutable. Data showing the effects of UV on larval development and fertility for the rad mutants is also shown and compared for wild-type and JP10 backgrounds.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Genetic Damage at Specific Gene Loci in Drosophila with Betatron X-Rays

The mutation rate was determined for mature sperm at eight specific gene loci on the third chromosome of Drosophila melanogaster using the low ion density radiations of 22 Mev betatron X-rays. A dose of 3000 rads of betatron X-rays produced a mutation rate of 4.36 x 10^-8 per rad/locus. Among the mutations observed, 66% were recessive lethals and 34% viable when homozygous. Only one of the 24 viable mutations was associated with a chromosome aberration. Among the 47 recessive lethals, no two-break aberrations were detected in 48.9% of the lethals, deletions were associated with 42.2%, inversions with 6.7% and translocations with 2.2%.—When these genetic results are compared to those for 250 KV X-rays, the mutation rate for betatron treatments was slightly lower (.76), the recessive lethal rate among induced mutations was higher, and the chromosome aberrations among lethal mutations were slightly lower than with 250 KV X-rays. Although the two types of irradiations differ by an ion density of approximately ten, the amount and types of inheritable genetic damage induced by the two radiations in mature sperm were not significantly different.     

Specific locus mutation rates after repeated small radiation doses to mouse oocytes

When female mice were given a dose of 20 × 10 rad X-rays, the specific locus mutation rate among offspring conceived up to 7 weeks after the end of treatment was 1/39887 or 0.18·10⁻⁷/rad/locus, whereas when the same total dose of 200 rad was given in a single exposure the mutation rate was 9/34813 or 1.85·10¹⁰⁻⁷/rad/locus. The lower mutation rate after the 20 × 10 rad dose was obtained whether the total of 200 rad was given over a period of 5 days or 4 weeks, and if only young conceived in the first 20 days, rather than 7 weeks, were considered. It is suggested that each 10 rad fraction had the same small effect, and hence that these results confirm and extend Russell's previous finding that the dose-response relationship for specific locus mutations in females is curved.     

Saccharomyces cerevisiae MGS1 is essential in strains deficient in the RAD6-dependent DNA damage tolerance pathway

Saccharomyces cerevisiae Mgs1 protein, which possesses DNA-dependent ATPase and single strand DNA annealing activities, plays a role in maintaining genomic stability. We found that mgs1 is synthetic lethal with rad6 and exhibits a synergistic growth defect with rad18 and rad5, which are members of the RAD6 epistasis group important for tolerance of DNA damage during DNA replication. The mgs1 mutant is not sensitive to DNA-damaging agents, but the mgs1 rad5 double mutant has increased sensitivity to hydroxyurea and a greatly increased spontaneous mutation rate. Growth defects of mgs1 rad18 double mutants are suppressed by a mutation in SRS2, encoding a DNA helicase, or by overexpression of Rad52. More over, mgs1 mutation suppresses the temperature sensitivity of mutants in POL3, encoding DNA polymerase delta. mgs1 also suppresses the growth defect of a pol3 mutant caused by expression of Escherichia coli RuvC, a bacterial Holliday junction resolvase. These findings suggest that Mgs1 is essential for preventing genome instability caused by replication fork arrest in cells deficient in the RAD6 pathway and may modulate replication fork movement catalyzed by yeast polymerase delta.     

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells.

The frequency of ethyl methanesulfonate (EMS)-induced mutations to 6-thioguanine resistance in a Chinese hamster ovary cells clone K1-BH4 was studied at many EMS doses including the minimally lethal range (0-100 microng/ml) as well as the exponential killing portion (100-800 microng/ml) of the survival curve. The mutation frequency increases approximately in proportion with increasing EMS concentration at a fixed treatment time. The pooled data for the observed mutation frequency, f(X), as a function of EMS dose X, is adequately described by a linear function f(X)=10(-6)(8.73+3.45 X), where 0 less than or equal to X less than or equal to 800 microng/ml. One interpretation of the linear dose-response is that, as a result of EMS treatment, ethylation of cellular constituents occurs, which is directly responsible for the mutation. Biochemical analyses demonstrate that most of the randomly isolated 6-thioguanine-resistant variants possess a highly reduced or undetectable level of HGPRT activity suggesting that the EMS-induced mutations to 6-thioguanine resistance affect primarily, if not exclusively, the HGPRT locus.