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.     

Non-random distribution of spontaneous and high temperature-induced recessive lethal mutations in the X-chromosome of Drosophila]

Frequency and localization of spontaneous and induced by high temperature (37 degrees C) recessive lethal mutations in X-chromosome of females belonging to the 1(1) ts 403 strain defective in synthesis of heat-shock proteins (HSP) were studied. No differences in frequencies of both spontaneous and induced lethals between 1(1) ts 403 and control strain were found, thus implying that the disturbances in HSP synthesis have no effect on this process in oocytes of Drosophila melanogaster females. Surprisingly, distribution of spontaneous and induced lethals along the X-chromosome of 1(1) ts 403 strain appeared to be non-random: they primarily are located in its distal portion (1-44 cM of genetic map or in I-II sections of the Bridges cytogenetic map). This correlates with non-random distribution of mobile elements in the X-chromosome of D. melanogaster (Leibovich, 1990).     

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.     

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.     

Studies on mutagen-sensitive strains of Drosophila melanogaster I. The enhanced X-ray sensitivity of a mutant strain (ebony) which is UV-sensitive and also deficient in photorepair

Yegorova and colleagues (1978) showed that a mutant strain of Drosophila melanogaster (ebony) was more sensitive to UV-induced killing of embryos and also less proficient in photoreactivating (PR) ability than a wild-type (Canton-S) strain and that the genes governing UV sensitivity and PR ability were different and presumably located on the autosomes. The experiments reported in the present paper were designed to compare the patterns of sensitivity of these 2 strains and their hybrids to X-irradiation. The sensitivity of the larvae to the killing effects of X-irradiation, and of male and female germ-cell stages to the X-ray induction of genetic damage was studied.It was found that the larvae of the ebony strain are more sensitive to X-ray-induced killing than those of the Canton-S strain. The frequencies of radiation-induced dominant lethals and sex-linked recessive lethals are higher in spermatozoa sampled from ebony males than in those of Canton-S males. In spermatozoa sampled from hybrid males, the yields of dominant lethals are no higher than in those sampled from Canton-S males and do not seem to depend on the origin of the X-chromosome. There are no statistically significant differences between the ebony and Canton-S strains in the sensitivity of their spermatozoa to the induction of autosomal translocations.Stage-7 oocytes sampled from ebony females are more sensitive to the X-ray induction of dominant lethality than are those from Canton-S females; oocytes sampled from hybrid females manifest a level of sensitivity that is significantly lower than that in either parental strain. The frequencies of X-chromosome losses induced in in this germ-cell stage are significantly lower in ebony than in Canton-S females at least at the exposure level of 3000 R at which 3 experiments were carried out. There are no measurable differences in the amount of dominant lethality induced in stage-14 oocytes of ebony, Canton-S and hybrid females.When X-irradiated Berlin-K males are mated to ebony or Canton-S females, the yields of dominant lethals are higher when ebony females are used, showing that there is a “maternal effect” for this kind of damage. Such a maternal effect is also found for sex-linked recessive lethals (irradiated Muller-5 males mated to ebony or Canton-S females). However, when irradiated ring-X-chromosome-carrying males are mated to ebony or Canton-S females, the frequencies of paternal sex-chromosome losses (scored as XO males) are lower when ebony females are used.These results have been interpreted on the assumption that the ebony strain is homozygous for recessive, autosomal genes that confer increased radiosensitivity and that the Canton-S strain carries the normal, wild-type alleles for these genes. The higher yields of dominant and recessive lethals in mature spermatozoa and of dominant lethals in stage-7 oocytes are a consequence of an enhanced sensitivity to the mutagenic (in particular, to the chromosome-breaking) effects of X-irradiation and/or of defective repair of radiation-induced genetic damage. The lower yield of XO males from irradiated stage-7 oocytes of ebony females is probably a consequence of a defect in the repair of chromosome-breakage effects, resulting in the conversion of potential X losses in females into dominant lethals. The “maternal effects” for dominant lethals, sex-linked recessive lethals and for the loss of ring-X chromosomes are assumed to have a common causal basis, namely, a defective repair of chromosome-breakage events in the females of the ebony strain.     

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.     

Genetic Analysis of Recessive Lethal Mutations Induced by the Influenza Virus in the X Chromosome of Drosophila melanogaster

Mutagenic potential of the influenza virus was evaluated. Based on its capacity of inducing recessive lethal mutations in the X chromosome of Drosophila melanogaster, the influenza virus can be classified as a moderate-activity mutagen. Its mutagenicity does not depend on ability to reproduce in the cell system. This virus was shown to disrupt formation of the wing, particularly wing vein M_{1 + 2}. Cytogenetic examination of polytene X chromosomes bearing recessive lethal mutations in Drosophilasalivary glands did not reveal chromosome rearrangements. These lethals are assumed to be small deletions or point mutations. The determination of the lethal activity stage of these mutations showed that they disrupt the expression of genes functioning at various developmental stages of Drosophila.Two of them were conditionally lethal (temperature-sensitive). Two of 15 mutations analyzed were mapped to region 2B9-10–3C10-11.     

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.     

Investigations on radiosensitive and radioresistant populations of Drosophila melanogaster. IV. The genetics of the relative radioresistance in stage-7 oocytes of the irradiated population RO I

The genetic system that controls the relative radioresistance in an irradiated laboratory population of Drosophila melanogaster (RÖ I) was studied. Comparisons were made between an unirradiated control population (+60, +K), the population RÖ I (after 227–333 generations of irradiation at 2100 R per generation), the sub-population RÖ I₀ (derived from RÖ I after 260 generations of irradiation and kept without irradiation for up to 74 generations), the F₁ hybrids +60/RÖ I, various homo- and heterozygous carriers of the 3 major chromosomes of RÖ I and +60, respectively, in combination with suitable balancers, and several chromosome substitution stocks of +K and RÖ I. The criteria used to assess the magnitude of radiosensitivity were dominant lethals, X-chromosome loss, and sex-linked recessive lethals induced in stage-7 oocytes at various exposure levels of X-irradiation.The data show that the radioresistance in RÖ I is controlled by a stable and homozygous genetic system. The system is semidominant. With respect to the induction of dominant lethals and sex-linked recessive lethals, the relative resistance is mainly contributed by chromosomes I and II. The effects of the two chromosomes are additive, each contributing about half the relative resistance. Resistance to the X-ray induction of X-chromosome loss is solely contributed by chromosome II.The findings suggest that at least 2 different and independent mechanisms are involved in determining the resistance of the RÖ I population.     

Genes Controlling Development: Morphoses, Phenocopies, Dimorphs, and Other Visible Expressions of Mutant Genes

We studied facultative dominant lethal mutations obtained earlier inDrosophila melanogaster. In some genotypes, these mutations were expressed as lethals, but in other genotypes they lacked this expression. The mutations were maintained in the following cultures: (1) females Muller-5 heterozygous for the mutation; (2) males crossed to attached-Xfemales; and (3) females and males homozygous for the mutation. During culturing, many mutations were found to give rise to phenotypically abnormal progeny. Generally, these abnormalities were morphoses involving various body parts; they were mostly asymmetric and nonheritable. Maternal and paternal effects in the formation of morphoses were observed. In four cases, dimorphic mutations were recorded: a female homozygous for the mutation had mutant phenotype whereas its male counterpart was phenotypically normal. The mutations were recessive with regard to the norm. New phenotypes behaving as mutations with incomplete penetrance arose during culturing. In cultures of mutant homozygotes phenocopies would appear en masse; they would persist for one or two generations and disappear. One wave of phenocopies succeeded another. Visible phenotypes appeared, which further behaved as ordinary recessive mutations. We concluded that these visible manifestations are characteristic for regulatory mutations controlling ontogeny. Their appearance is explained by the activation of new regulatory scenarios caused by blocking standard regulatory pathways.     

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.     

Test of recessive lethals in the mouse

The presence of spontaneous lethal mutations in inbred strains is discussed with special reference to their variation and influence on estimates to induced mutations.A model is presented that will facilitate classification of lethal-free and lethal heterozygotes.The model is used in classification of sons to lethal heterozygous males carrying a spontaneous mutation.The observed results are in good agreement with the model.From experience it is concluded that the most efficient way to use the facilities in lethal tests is to examine 10 or more full brothers to the P parents. By doing so pre-existing spontaneous lethals can be excludde by eliminating families in which any of the P parents were lethal heterozygous. The observed total rate of recessive lethals gives slight over-estimation of the induced rate of mutations, as spontaneous mutations in the gametes forming the F₁ cannot be excluded.     

Genetic instability at the cut locus of Drosophila melanogaster induced by the MR-h12 chromosome

Summary Unstable mutations were generated at the cut locus by the MR-h12 factor which induces male recombination. The unstable allele ct ^MR2, containing the MR-transposon in the cut locus is a very powerful mutator producing a number of different viable and lethal mutations both in the cut locus and outside it.I describe several types of mutations: stable reversion to wild type, which were sometimes associated with the appearance of unstable mutations in other loci; of stable deficiencies at the cut locus (lethals); new unstable mutations at different loci with the ct ^MR2 allele conserved; new unstable cut alleles with a phenotype other than that of ct ^MR2. The possible mechanisms of these mutational events are discussed. The genetic system constructed in the present work affords an opportunity for molecular studies of the cut locus and the MR-transposon, as a sequence from the cut locus has recently been cloned (Tchurikov et al. 1981).     

Occurrence of spontaneous and nitrosoethylurea-induced mutations and potential changes in repair-deficient Drosophila strains

X-linked recessive lethal frequencies in mature spermatozoa were studied in repair-deficient Drosophila strains. Frequencies of spontaneous and ethylnitrosourea-induced lethals were enhanced in mus(I)104DI+ and unchanged in mei-9IL+. In addition, the majority of lethals was fixed in stages preceding mature spermatozoa. It was shown that premutational lesions (spontaneous and ethylnitrosourea-induced in both mutants) arise in germ cells, these lesions being realized into mutations in the next generations.     

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.     

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.     

Analysis of chromosome 4 inDrosophila melanogaster. I. Spontaneous and X-ray-induced lethals

An analysis of 17 spontaneous and 37 X-ray-induced lethal mutations on the fourth chromosome ofDrosophila melanogaster has revealed a minimum of 22 loci on this microchromosome capable of mutating to lethality. A few of these loci had been identified earlier by their visible alleles but 16 are new discoveries. Seven of the 22 lethal loci are situated within that proximal section of the right arm of chromosome 4 delimited by theMinute-4 deficiency.Genetic tests indicate that two translocations and five deletions are included among the lethals of X-ray origin. No chromosomal aberrations were found among the spontaneous mutants. Allelism was encountered both within and between lethals from the two groups.Three independent estimates of the total number of lethal loci to be expected on this small autosome are presented. These appraisals are based on (1) the size of theMinute-4 deficiency, (2) the number of bands in salivary chromosome 4, and (3) the frequency of recurrence among the lethals. Considering the uncertainties inherent in each determination, the three estimates (34, 35 and 38) show remarkably good agreement.This investigation was supported in part by U.S. Public Health Service Research Grant GM 11627-01, from the Division of General Medical Sciences.     

Autosomal recessive lethal mutations in two mutator stocks of Drosophila ananassae.

The frequency of recessive lethals in the 2nd chromosome was examined in two mutator stocks of Drosophila ananassae, ca and ca; px. They are characterized respectively by possessing an extrachromosomal clastogenic mutator in males, and by the retrotransposon "tom", which induces Om mutability only in females. The frequencies of recessive lethal mutations in the 2nd chromosome among progenies from males and females of the ca; px stock are 0.35 and 0.34 percent, respectively. Similarity of these frequencies indicates that tom does not induce recessive lethals in females. In contrast to the ca; px stock, the frequency of recessive lethals in males of the ca mutator stock was estimated to be 1.54 percent for the 2nd chromosome. No visible mutants except Minutes were recovered. Some recessive lethals derived from ca stock males were associated with chromosomal rearrangements. Being consistent with its high rate of Minute mutation it was demonstrated that the ca clastogenic mutator also induced recessive lethals.     

Isolation of autosomal mutations in Drosophila melanogaster without setting up lines

Summary Mutants of Drosophila melanogaster are being used increasingly for studying different biological mechanisms. However, most attempts to identify new mutations have been restricted to the X-chromosome. It has been very difficult to identify new loci on the autosomes, as recessive mutations have to be made homozygous by setting up independent cultures for each mutagenized chromosome. We introduce a mutagenesis scheme which does not require setting up independent cultures. It uses meiotic recombination in compound autosomes to make recessive mutations homozygous and allows the screening of tens of thousands of mutagenized chromosomes with relatively little effort. In a pilot experiment, we tested about 33,300 chromosomes for temperature-sensitive paralytic mutations. We obtained 62 independent paralytic mutations and a large number of other mutations. Eight out of 25 of the paralytic mutations are on the autosomes. This method makes autosomes, which constitute about 80% of the Drosophila genome, more accessible for mutational analysis of various biological mechanisms.     

A Cytogenetic Analysis of the Chromosomal Region Surrounding the α-Glycerophosphate Dehydrogenase Locus of DROSOPHILA MELANOGASTER

The chromosomal region surrounding the structural gene for α-glycerophosphate dehydrogenase (αGpdh, 2-20.5) of Drosophila melanogaster has been studied in detail. Forty-three EMS-induced recessive lethal mutations and five previously identified visible mutations have been localized within the 25A-27D region of chromosome 2 by deficiency mapping and in some cases by a recombination analysis. The 43 lethal mutations specify 17 lethal loci. αGpdh has been localized to a single polytene chromosome band, 25F5, and there apparently are no lethals that map to the αGpdh locus.