Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER. Xvii. Heat- and Cold-Sensitive Lethals on Chromosome 3

Ethyl methanesulfonate-treated third chromosome of Drosophila melanogaster were tested for the presence of dominant and recessive temperature-sensitive lethal mutations at 17 degrees , 22 degrees and 29 degrees C. Out of 1,176 chromosomes tested, no dominant ts lethals, 21 heat-sensitive, 22 cold-sensitive and 10 heat-cold-sensitive lethals were recovered. Heat-cold sensitivity was produced by a single mutation in all cases. Sixty-two percent of the ts lethals were fertile as homozygotes in both sexes. Surprisingly, 88% of the ts lethals mapped between st and Sb, a region straddling the centromere and estimated to comprise 12.9% of the genetic length and 55% of the physical length of chromosome 3. All but one of the heat- and cold-sensitive lethals complemented with each other at their respective restrictive temperatures.     

Radiosensitivity of Drosophila lines. VII. The effect of the maternal genotype on the yield of recessive and dominant lethal mutations induced by the gamma irradiation of males

The effect of material repair on induction of paternal mutations was tested with radiosensitive rad(2)201G1 mutant. Basc males were irradiated at doses from 0 to 60 Gy of gamma-rays and mated to the radiosensitive mutant or control females. Frequencies of sex-linked recessive lethals and dominant lethals (induced in the paternal genome) were determined. With control females, the rate of recessive lethals increased linearly from 0 to 60 Gy. With rad(2)201G1 mutant, an increase in spontaneous and induced rates of paternal dominant lethals was observed; the rate of sex-linked recessive lethals increased non-linearly from 0 to 60 Gy.     

I−R hybrid dysgenesis in Drosophila melanogaster: nature and site specificity of induced recessive lethals

This paper presents results of the genetic and cytological analysis of 144 sex-linked recessive lethals, plus 1 non-lethal. All of them were induced by I−R hybrid dysgenesis. This collection of mutants was pooled from experiments involving inducer chromosomes that differ in the chrosomal position of their I elements. Our results show that 30% of the recessive lethals are associated with chromosomal rearrangements which depend on the strength of the I−R interaction. These lethals are induced on both inducer- and reactive-origin chromosomes, and their frequency is dependent on the structure of the inducer chromosome used. The I−R-induced lethals occur along the entire length of the X chromosome. These sites probably correspond to specific loci which are more or less homologous with I. The complementation relationshups showed that some specific loci were more frequently involved in all the lethal mutations tested. The most sensitive loci are, in order of observation: l(1)J1, ct, f, ma¹ and m. Among induced recessive lethals considered to be point mutation, complementation tests showed that many of them are in fact multilocius deficiencies which can be detected only at the molecular level.

It seems that the production of I−R rearrangements (cytologically visible or not) may be the most important mechanism leading to lethal mutations. These mutations probably occur during the transposition of I elements, hence their importance from an evolutionary standpoint.     

The mutational spectrum of procarbazine in Drosophila melanogaster.

The antineoplastic agent Procarbazine was tested for the induction of genetic damage in Drosophila melanogaster. The compound was administered to adult males by oral application. The following types of genetic damage were measured: (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. Procarbazine is highly mutagenic in causing recessive lethal mutations in all stages of spermatogenesis. In sperm a clear-cut concentration-effect relationship is not apparent, but in spermatids such a relationship is obtained for mutation induction at low levels of procarbazine exposure, while at high concentrations the induction of recessive lethals is not a function of concentration. A low induction of total sex-chromosome loss (X,Y) and dominant lethals was observed in metabolically active germ cells (spermatids), but procarbazine failed to produce well-defined breakage events, such as partial sex-chromosome loss (YL,YS) and II-III translocations. The results obtained in Drosophila melanogaster are discussed and compared with the mutational pattern reported in the mouse after procarbazine treatment.     

Spontaneous mutations in dry spores of Neurospora crassa

Summary Atwood'S method was used for scoring recessive lethals in aNeurospora heterokaryon with an amycelial component. Conidia were stored dry at 30° and 4°C, and samples were tested for lethals every few weeks over a period of 7 months. At 30°, lethals accumulated in strictly linear proportion with time, at a rate of 0.3% lethals per week. At 4°, the rate of accumulation was much slower; the data are not sufficient to decide whether it was linear. When conidia that had spent 24 weeks at 4° were transferred to 30°, the proportion of lethals increased steeply to the level that had meanwhile been reached in the warm series. This points to the possibility of resolving the mutational process into successive steps with different temperature coefficients. Reasons are given for presuming that none of the lethals had been present in the culture of origin and for excluding their occurrence through errors of gene replication during storage or on the plates.When samples of conidia from the warm series were inserted into growth tubes, 80% or more of the accumulated lethals were lost during the initial stages of growth. Subsequently, lethals accumulated again at a rate that remained constant for at least 2 weeks. This, together with similar observations byAtwood andPittenger (1955), opens the possibility of studying mutation rates during growth in growth tubes.Operated by Union Carbide Corporation for the United States Atomic Energy Commission.Work carried out while the author was a Visiting Investigator at Oak Ridge National Laboratory on leave of absence from Edinburgh University.     

Metronidazole (Flagyl): lack of induction of sister-chromatid exchanges in CHO-K1 cells

328 X-linked recessive lethal mutations induced in late spermatids by hycanthone methanesulfonate were tested for coverage by duplications that comprised, in total, about 24% of the euchromatic X chromosome; 78 lethals appeared to be covered. Crossover localization tests of a random sample of 38 non-covered lethals revealed 4 chromosomes carrying a lethal within a duplicated segment. Lethals localized to a particular region were crossed to reference deficiencies and single-locus mutations, and inter se, to ascertain their genetic extent. The proportion of multi-locus deletions among these 78 covered and 4 non-covered lethals was 3/48, 1/10 and 13/24 for the distal, medial and proximal regions, respectively. A storage period of 9 days did not noticeably influence these proportions. In the sample of 38 non-covered lethals, and among 17 of the covered single-site lethals, 4 cases of strong crossover suppression were detected. Comparison of these results with data obtained with other mutagens suggests that induction of multi-locus deletions, and possibly of other types of chromosome rearrangement, could in part depend on other mechanisms than those acting in the formation of translocations and chromosome loss. For the purpose of mutagen testing, these findings imply that, in Drosophila, results in the regular genetic tests for chromosome breakage events do not always accurately predict the capacity of a mutagen to induce multi-locus deletions. This is of importance since transmissible multi-locus deletions have been considered a significant source of genetic damage in man.     

CONTINUING STUDIES ON THE SOUTH AMHERST DROSOPHILA MELANOGASTER NATURAL POPULATION DURING THE 1970'S AND 1980'S

Continuing investigations on the South Amherst Drosophila melanogaster natural population following the significant decline and recovery of lethal (le) and semilethal (sle) frequencies in the late 1960's (Ives, 1970) show that the population has been remarkably stable although it contains MR (male recombination) and/or P DNA elements (Kidwell et al., 1977a; Green, 1980). A 13-year study affirms that the lethals present are nonrandomly distributed along the second chromosome and deficient on the right; they differ significantly in distribution from spontaneous (Ives, 1973) and δ-induced lethals (Minamori and Ito, 1971). Between 1970 and 1977, a total of 4,083 second chromosomes from the Markert subpopulation were analyzed; 28.9% of the chromosomes were lethal and 7.25% were semilethal in homozygous condition. Frequencies are similar for early summer and late fall collections although the rate of allelism among lethals is significantly higher in early summer than in late fall. For the large fall (1970–1979) Porch site population, 2,519 second chromosomes were analyzed; 29.5% were lethal and 8.0% were sublethal as homozygotes; the rate of allelism among lethals was 1.50%. At Hockanum, 1977–1983, lethal and semilethal frequencies were lower; the rate of allelism among lethals was 1.43%. The chromosome map distribution of lethals does not change between summer and late fall at Markert. The overall distributions of lethals at the Markert and Hockanum sites are similar. In tests for male recombination (MR) activity in the population over a 6-year period, a total of 0.47% recombinants were observed; these were uniformly distributed along the second chromosome. Comparisons are made with other long studied D. melanogaster populations.     

Mutagenicity of hycanthone in Drosophila melanogaster

The schistosomicidal agent hycanthone was tested for mutagenicity in Drosophila melanogaster. The compound was administered either by injection into adult males or by larval feeding. The following types of genetic damage were measured:(1) complete and mosaic sex-linked recessive lethal mutations; (2) II–III translocations; and (3) dominant lethals.In postmeiotic germ cells, especially in late spermatids, a pronounced increase was found in the frequency of sex-linked recessive lethals, both completes and mosaics. By contrast, translocations and dominant lethals were not induced.     

Mechanism of hycanthone mutagenesis: the induction of temperature-sensitive mutations in Drosophila melanogaster.

A total of 358 sex-linked recessive lethals induced by hycanthone methane sulphonate (HMS) were checked for temperature sensitivity, along with 239 EMS-induced lethals as a positive control. Only about 1-2% of the HMS-induced lethals were temperature-sensitive, in contrast to about 7-8% for EMS-induced lethals. This can be reasonably explained by assuming that in Drosophila, HMS mainly acts by inducing frame-shift mutations or deletions.     

Radioprotective effect of six chemicals against X-ray-induced genetic damage in D. melanogaster

In Drosophila melanogater six chemicals were tested for radioprotectiveeffect against X-ray-induced genetic damage such as sex-linked recessive lethals and autosomal translocations using Oster's ring-X chromosome stock. A 2-day brood pattern was followed to score the damage induced at different spermatogenic stages separately. In all cases the chemicals were injected before X-irradiation. 10-mM solution of reduced glutathione (GSH) provided statistically significant protection against sex-linked recessive lethals in all broods. In translocation tests this chemical reduced the frequency in all broods but the result is not statistically significant. Cysteamine (MEA) did not show any protective effect but the frequency of lethals was slightly reduced in the first and fourth broods. 2-Aminoethyl isothiuronium Br·HBr (AET) showed a statistically significant protective effect when the data of the replicate experiments were pooled. Negative results were obtained for 5-hydroxytryptamine (5-HT) in sex-linked lethal tests. Aminoethyl phosphorothioate (AEPT) reduced the frequencies of both sex-linked lethals and autosomal translocations in all broods consistently but the results are not statistically significant. In tests for both lethals and translocations the reduction was largest in the stages with highest radiosensitivity. N(3-Aminopropyl)aminoethyl phosphorothioate (3AP-AEPT) gave no protection.     

Analysis of lethal events induced by ultraviolet in a heterokaryon of Neurospora.

Summary The relative frequencies of heterokaryons and the two kinds of homokaryons have been scored among colonies from conidia harvested from a heterokaryon and treated with UV, in order to determine which kinds of lethal mutations were induced. Recessive lethal mutations were scored directly. The pattern of surviving types indicated that recessive lethals and mitotic lethals (events destroying whole nuclei) occurred with similar frequencies. But the absolute frequency of these mutations was not sufficient to account for the observed kill, suggesting that dominant lethals and/or cytoplasmic lethals were also induced at a similar rate.     

Effect of storage and dose on MMS-induced deletions. Complementation analysis of X-chromosomal recessive lethals in the zeste-white and maroon-like regions of Drosophila melanogaster

The effect of storage on MMS-induced recessive lethals in the zeste-white (3A1-3C2) and the maroon-like (18F4-20F) regions was studied by complementation analysis. (1) Without any exception, all 52 mutants (from unstored spermatozoa) mapped in the zeste-white region were restricted to single complementation units. Furthermore, none of an additional 15 lethals, sampled from sperm that had been stored in females for 9-12 days, was associated with a deletion. (2) Of 34 mutations induced by 8.5 X 10(-2) mM MMS in the maroon-like (mal) region, 4 spanned 2 or more complementation units, and thus are considered to be deletions. A high dose of 2.5 mM MMS provided 55 lethals for analysis of which 4 were deletions. There was no evidence for any difference in the frequency of deletions as the MMS concentration was enhanced from 8.5 X 10(-2) mM to 2.5 mM. However, with storage, 47.1% lethals (16 of 34 mutants induced by 2.5 mM MMS) mapped in the mal region were found to involve large structural changes. (3) A high proportion of double mutants in both the zeste-white (z w) and the maroon-like regions was found among the chromosomes analyzed. These double mutants have one lethal positioned within the region studied and the other outside it. Clearly, the proportion of double mutants increased with dose, from 6.3 to 41.7% in z w and from 14.7 to 61.8% in the mal section. Apurinic sites in DNA reacted with MMS are considered as the likely primary lesions responsible for the storage effect on MMS-induced recessive lethals in the mal region. Thus, the ability of MMS to produce delayed deletion lethals seems to correlate with preference for alkylation of base nitrogens. An interesting aspect for further analysis is the apparent infrequency in the zeste-white region of alkylation-induced chromosomal breakage, as observed by various investigators for MMS, EMS and MNNG.     

Distribution of MR-induced sex-linked recessive lethal mutations in Drosophila melanogaster

In the ‘doubling-dose’ method currently used in genetic risk evaluation, two principle assumptions are made and these are: (1) there is proportionality between spontaneous and induced mutations and (2) the lesions that lead to spontaneous and induced mutations are essentially similar. The studies reported in this paper were directed at examining the validity of these two assumptions in Drosophila. An analysis was made of the distribution of sex-linked recessive lethals induced by MR, one of the well-studied mutator systems in Drosophila.

Appropriate genetic complementation tests with 15 defined X-chromosome duplications showed that MR-induced lethals occurred at many sites along the X-chromosome (in contrast to the known locus specificity of MR-induced visible-mutations); some, but not all these sites at which recessive lethals arose in the MR-system are the same as those known to be hot-spots for X-ray-induced lethals. With in situ hybridization we were able to demonstrate that a majority of MR-induced lethals is associated with a particular mobile DNA sequence, the P-element, i.e. they arose as a result of transposition.

The differences between the profiles of MR-induced and X-ray-induced recessive lethals, and the nature of MR-induced and X-ray-induced mutations, thus raise questions about the validity of the assumptions involved in the use of the ‘doubling-dose’ method.     

HIGH INBREEDING DEPRESSION,SELECTIVE INTERFERENCE AMONG LOCI,AND THE THRESHOLD SELFING RATE FOR PURGING RECESSIVE LETHAL MUTATIONS

The evolutionary dynamics of recessive or slightly dominant lethal mutations in partially self-fertilizing plants are analyzed using two models. In the identity-equilibrium model, lethals occur at a finite number of unlinked loci among which genotype frequencies are independent in mature plants. In the Kondrashov model, lethals occur at an infinite number of unlinked loci with identity disequilibrium produced by partial selfing. If the genomic mutation rate to (nearly) recessive lethal alleles is sufficiently high, such that the mean number of lethals (or lethal equivalents) per mature plant maintained at equilibrium under complete outcrossing exceeds 10, selective interference among loci creates a sharp discontinuity in the mean number of lethals maintained as a function of the selfing rate. Virtually no purging of the lethals occurs unless the selfing rate closely approaches or exceeds a threshold selfing rate, at which there is a precipitous drop in the mean number of lethals maintained. Identity disequilibrium lowers the threshold selfing rate by increasing the ratio of variance to mean number of lethals per plant, increasing the opportunity for selection. This theory helps to explain observations on plant species that display very high inbreeding depression despite intermediate selfing rates.     

The relationship between radiation-induced and transposon-induced genetic damage during Drosophila oogenesis

The combined effect of X-irradiation and transposon mobility on the frequencies of X-linked recessive lethals and dominant lethals was investigated in female hybrids in the P-M system of hybrid dysgenesis. X-linked lethals were measured in G2 hybrid dysgenic females whose X chromosome was derived from the M X P cross. To test for additivity or synergism, the mutation rate in irradiated dysgenic females was compared to that of unirradiated females as well as to irradiated nondysgenic hybrid females derived from M X M crosses. Eggs collected for 2 days after irradiation, were represented by the more radiation-sensitive A and B oocytes (about 75%) and the least sensitive C oocytes (about 25%). The production of X-linked lethal events in X-irradiated dysgenic females was 8.1%, as compared to 4.5% in dysgenic controls and 3.4% in irradiated, nondysgenic controls, demonstrating an additive effect of radiation and dysgenesis-induced genetic damage. The effect of irradiation on sterility of dysgenic hybrid females was a negative one, resulting in 20% less sterility than expected from an additive effect. The combined effect of radiation and dysgenesis on dominant lethality tested in A, B and C oocytes of the same hybrid females was synergistic. Egg broods collected for 3.5 days after irradiation showed that significantly more damage was induced in the presence of ionizing radiation in dysgenic females than in their nondysgenic counterparts. This effect was most obvious in B and C oocytes. The synergism observed may be related to the inability of cells to repair the increased number of chromosome breaks induced both by radiation and transposon mobility.     

EMS-induced dominant lethal dose response curve in DBA/1J male mice.

An ethyl methanesulfonate (EMS) induced dominant lethal dose response experiment was conducted in strain DBA/1J male mice. Two methods of scoring for dominant lethals, the classic method (dissecting females at mid-pregnancy) and an alternative method (inspection of uterine scars after litters were weaned) were compared. Results indicate that strain DBA/1J has a similar sensitivity to EMS-induced dominant lethals as has been previously reported for other mouse genotypes. Of the two methods used to score dominant lethals, the classic method is more sensitive.     

Lethals, Steriles and Deficiencies in a Region of the X Chromosome of CAENORHABDITIS ELEGANS

Twenty-one X-linked recessive lethal and sterile mutations balanced by an unlinked X-chromosome duplication have been identified following EMS treatment of the small nematode, Caenorhabditis elegans. The mutations have been assigned by complementation analysis to 14 genes, four of which have more than one mutant allele. Four mutants, all alleles, are temperature-sensitive embryonic lethals. Twelve mutants, in ten genes, are early larval lethals. Two mutants are late larval lethals, and the expression of one of these is influenced by the number of X chromosomes in the genotype. Two mutants are maternal-effect lethals; for both, oocytes made by mutant hermaphrodites are rescuable by wild-type sperm. One of the maternal-effect lethals and two larval lethals are allelic. One mutant makes defective sperm. The lethals and steriles have been mapped by recombination and by complementation testing against 19 deficiencies identified after X-ray treatment. The deficiencies divide the region, about 15% of the X-chromosome linkage map, into at least nine segments. The deficiencies have also been used to check the phenotypes of hemizygous lethal and sterile hermaphrodites.     

Induction of sex-linked recessive lethals and autosomal translocations by beta-propiolactone in Drosophila: influence of the route of administration on mutagenic activity.

Beta-propiolactone (BPL) was tested for the induction of sex-linked recessive lethals and autosomal translocations in Drosophila melanogaster. The compound was administered to adult males either by oral application or by abdominal injection. When injected, BPL was a potent inducer of sex-linked recessive lethals. When BPL was given by feeding, its mutagenic activity was detectable only when the flies were starved and when the BPL-containing solutions were renewed several times. Nevertheless, the recessive-lethal frequency was one order of magnitude higher with injection. This difference in effects is attributed to (1) rapid decomposition of the compound in aqueous feeding solutions, and to (2) rapid degradation in vivo which restricts the activity of BPL mainly to the site of application. These data are compared with other studies in which both routes of application were applied. BPL induced translocations in stored spermatozoa when injected, but not when fed. This finding seems a logical consequence of (1) the difference in effectiveness of the two routes of application for BPL, and (2) the existence of different LECs for mutation induction (recessive lethals) and for chromosome breakage (translocations). In Drosophila, the breakage capacity of BPL was one order of magnitude lower than that of MMS, when a comparison was made on the basis of equal recessive-lethal frequencies.     

Mutagenesis in CAENORHABDITIS ELEGANS. II. a Spectrum of Mutational Events Induced with 1500 R of γ-RADIATION

We previously established a gamma-ray dose-response curve for recessive lethal events (lethals) captured over the eT1 balancer. In this paper we analyze the nature of lethal events produced, with a frequency of 0.04 per eT1 region, at a dose of 1500 r. To do so, we developed a protocol that, in the absence of cytogenetics, allows balanced lethals to be analyzed for associated chromosomal rearrangements. A set of 35 lethal strains was chosen for the analysis. Although the dosage was relatively low, a large number of multiple-break events were observed. The fraction of lethals associated with rearrangements was found to be 0.76. Currently most X- and gamma-ray dosages used for mutagenesis in C. elegans are 6000-8000 r. From our data we conservatively estimated that 43% of rearrangements induced with 8000 r would be accompanied by additional chromosome breaks in the genome. With 1500 r the value was 5%. The 35 lethals studied were derived from 875 screened F1's. Among these lethals there were (1) at least two unc-36 duplications, (2) at least four translocations, (3) at least six deficiencies of chromosome V (these delete about 90% of the unc-60 to unc-42 region) and (4) several unanalyzed rearrangements. Thus, it is possible to recover desired rearrangements at reasonable rates with a dose of only 1500 r. We suggest that the levels of ionizing radiation employed in most published C. elegans studies are excessive and efforts should be made to use reduced levels in the future.     

Maternal age-specific rates of 47,+21 and other cytogenetic abnormalities diagnosed in the first trimester of pregnancy in chorionic villus biopsy specimens: comparison with rates expected from observations at amniocentesis.

Results are presented on chromosome analyses made on 4,481 embryos or fetuses studied through chorionic villus sampling (CVS) in whom there was no known bias to presence of a chromosome abnormality except advanced parental age. We excluded from the analysis most cases in which mosaicism was diagnosed or in which there were cytogenetic discrepancies among samples obtained from the conceptus. There remain 48 cases of 47,+21, 39 cases of other nonlethal abnormalities, and 12 lethal abnormalities diagnosed in 4,481 studied. A regression analysis (restricted to the 3,848 cases diagnosed in the 35-49-year maternal age interval) was done on rates of (1) 47,+21, (2) other abnormalities excluding lethals or (3) including them, and (4) all abnormalities excluding lethals or (5) including them. The model used was y = exp(bx + c), where y is the rate of abnormality, x is maternal age at time of CVS (the modal age of the procedure was 10 gestational weeks from the last menstrual period), and b and c were, respectively, (1) 0.288 and -15.527; (2) 0.272 and -15.173; (3) 0.253 and -14.141; (4) 0.282 and -14.753; and (5) 0.271 and -14.195. We also derived rates of abnormalities at the time of CVS that would be predicted from rates (of nonmosaics) at amniocentesis after adjustment for the difference in gestational age between the usual times that these two procedures are done. The difference between the numbers of abnormalities predicted on the basis of these adjusted amniocentesis rates and the numbers observed at CVS provides an estimate of the spontaneous loss of embryos and fetuses between the usual gestational ages of these procedures. In these data, for 47,+21 the estimated proportion lost is 21% but the result is not significant at the .05 level. For other abnormalities excluding lethals the estimated spontaneous loss is 29% (P approximately .05); including lethals it is 44%. For all abnormalities, excluding lethals, pooled together, the estimate is 24%; including lethals it is 33%. The last three values are all significant at the .05 level or lower. The observed rates of abnormalities at CVS would be approximately 10% to 15% higher if one pooled diagnosed mosaics with the nonmosaics, but the estimated proportion of spontaneous fetal loss would be lower.