[Mutation processes in the natural populations of Drosophila and Hirundo rustica from Ukrainian radiation contaminated territories].

Natural populations of Drosophila melanogaster and Hirundo rustica that reside at the territories with different levels of radioactive pollution were investigated. The levels of visible mutations, sex-linked mutations and gonad reduction of Drosophila and the rate of interphase markers of chromosomal instability in erythrocytes of birds were selected as parameters for population monitoring. The results point out to possible reverse dependence among the level of chromosomal instability of birds, the rate of lethal mutations in sex chromosome of Drosophila and the density of radioactive pollution.

The distribution of randomly recovered X-ray-induced sex-linked genetic effects in Drosophila melanogaster.

Cytogenetic analysis of more than 1500 randomly recovered lethal X chromosomes derived from 2000 and 3000 r X-ray exposures of post-meiotic male germ cells has made possible a plot of the distribution in different regions of the X chromosome of: (1) gene mutations associated with cytologically normal chromosomes, (2) mutations associated with chromosomal rearrangement breakpoints, (3) deficiencies, and (4) rearrangement breakpoints whether or not they are associated with mutations. The distribution of point mutations, vital loci and rearrangement breakpoints in different regions of the X chromosome is not proportional to either the number of bands or the relative DNA content. Further, the density of vital loci (those capable of mutating to a lethal allele) is quite different in some regions as compared to others. For example, vital loci in the 3AB region, which has been thoroughly studied by Judd and others, are at least as numerous as bands; whereas, the 3CD region, equally long, has only two vital loci. Other regions densely populated with vital loci include 1B, 1F-2A, 10A, 11A, and 19EF; sparsely populated regions include 6EF and 10B-10E. It seems reasonable to conclude that the recovered X-ray-induced mutants available for analysis do not represent a random sample of those initially induced in the exposed male germ cells.

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.

In support of the telomere concept.

The frequency of recovered X-ray-induced (4000R) rearrangements that, in all probability mimic terminal deletions of the X chromosome was only one of, roughly, 10-5 X chromosomes screened for tip deficiencies. Although the single exception looks terminally deleted, it is probably capped by a very short or nonpolytene telomeric segment. It is apparent from these data that the probability of "healing" or stabilization of a terminally deleted X in the zygotic nucleus or developing embryo of Drosophila melanogaster is vanishingly small. The telomeric caps in two obviously interstitial deficiencies that were recovered represent, roughly, 1/500 of the length of a mitotic chromosome. These findings give some indication of the extreme difficulty of detecting short telomeric segments capping either deleted polytene chromosomes or deleted metaphase chromosomes of, for example, humans.

Analysis of the chromosome aberrations induced by x-rays in somatic cells of Drosophila melanogaster.

A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.-The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chromatid aberrations. They also display a greater frequency of spontaneous aberrations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G(2) and G(1). These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferentially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translocations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.

Some radiation effects on segregation in Drosophila.

Translocation induced in the immature oocyte, in meiotic prophase, affects division I orientation and segregation, the usual result being that the two halves of translocations are directed to opposite poles. Since interchange is usually (if not exclusively) between chromatids, this is to be expected from the creation of illegitimate conjunctions. Good agreement is obtained between patterns of segregations deduced from recovered half-translocation bearing exceptions and the kinds of disomic gametes expected as alternative recoveries from the same division I configurations. Inferences drawn from the study of compound-X females have been found to apply as well in the case of females of normal karyotype. Numerical errors occur predominantly, possibly exclusively, in division I. The rate of induced nondisjunction of specific chromosome pairs varies in relation to the structure of the entire complement, as required if radiation-induced nondisjunction is interchange dependent, but which would be unexpected if the mechanism involved effects on individual spindle fibers, chromosomes, or chromosomal bivalents.

Nature and consequences of induced chromosome damage in mammals.

There are marked qualitative and quantitative differences in the patterns of chromosomal damage observed after irradiation of spermatogonia, spermatozoa and oocytes of mice. These differences often result from reduced or zero transmission of particular classes of abberration arising in pre-meiotic germ cells. Probably this is the reason why the level of X-chromosomal and autosomal monosomy is not increased after spermatogonial irradiation. Similarly, the reduced transmission of certain d-se deficiencies may help to explain their low F(1) frequency after pre-meiotic as compared with later irradiation. Spermatozoal irradiation has revealed no Robertsonian translocations, but has produced some types of reciprocal translocations which apparently are not transmitted to the F(1) after spermatogonial treatment because they prevent maturation of the male pre-meiotic germ cell. Thus they cause sterility in males, but not in females. They include X-autosome and Y-autosome translocations, those giving a metacentric or sub-metacentric chromosome (with reciprocal product present) and those in which one break-point is in or near the centromeric heterochromatin while the other is more distally placed. This last group (which grades into male sub-fertile conditions) gives a preponderance of chain configurations (often with one separate univalent) in heterozygotes of both sexes at meiosis and a high incidence of somatic marker chromosomes. Nondisjunction associated with the univalent generates tertiary trisomics, which are usually male-sterile also and may show phenotypic abnormalities. Sterile males with complete separation of X and Y chromosomes have also been reported after mutagenic treatment of meiotic and post-meiotic germ cells. Such separation seems to prevent a primary spermatocyte from forming a secondary one. The usual derivation (in mouse and man) of tertiary trisomics from mothers rather than from fathers may be due to a similar block, together with a general tendency for male heterozygotes for the parental balanced translocation to be sterile or sub-fertile. Mature oocytes tend to resemble spermatoza in the types of aberration produced by irradiation, which include the male-sterile translocation, but more data are needed. Many of the aberrations described contribute to the human cytogenetic load and can be studied in the mouse from this point of view.

Microtus oeconomus (Rodentia), a useful mammal for studying the induction of sex-chromosome nondisjunction and diploid gametes in male germ cells.

Preliminary data indicate that chemicals can also increase the frequency of sex-chromosome nondisjunction. Positive results--which certainly need further confirmation--have been obtained for MMS, p-fluorophenylalanine, vincristine, procarbazine, carbendazim, and bleomycin. Nocodazole, benomyl, colcemic, 6-mercaptopurine, and halothane were all negative at the concentrations tested. For the induction of diploid spermatids positive results were only obtained for MMS and parafluorophenylalanine. In view of the results obtained, the Microtus system is considered a very useful tool for analyzing factors contributing to the high frequency of aneuploidy and triploidy among abortuses and of aneuploidy in liveborn infants of men. A method is described for the detection of sex-chromosome nondisjunction and diploid spermatids in male germ cells of the field vole Microtus oeconomus. The method is based on the unique distribution pattern of heterochromatin in Microtus cells, which makes it possible to identify X and Y chromosomes in early spermatids with a simple C-banding procedure. Slide preparation is easy. Scoring of early spermatids for extra sex-chromosomes is simple and 2000-4000 cells per hour can be examined. With the Microtus system it has now been demonstrated that radiation of spermatocyte stages with doses of 50, 100 and 200 R results in a higher frequency of sex chromosome nondisjunction and of diploid gametes. Both types of aberrant gametes can be produced during the first and second meiotic division.

A translocation X;Y system for detecting meiotic nondisjunction and chromosome breakage in males of Drosophila melanogaster.

A nondisjunction and chromosome breakage screening system devised by Craymer and modified in our laboratory, involves an X;Y translocation with the short arm of the Y (Ys), marked with the wild type allele of yellow, attached to the distal end of an X (break point 11D) carrying the recessive marker y; and the long arm of the Y chromosome (YL), marked with the dominant locus Bar of Stone (BS), attached to the proximal end of the X. A female tester strain carrying normal chromosomes homozygous for the yellow allele is employed in the mating scheme. Following normal disjunction in the male, all zygotes, which in this case receive aneuploid paternal sex-chromosomes and a normal euploid maternal complement, will die as a result of genetic imbalance. Thus all survivors from this corss can be classified as exceptions arising from: (1) nondisjunction in the female; (2) gross deletion of the paternal X;Y chromosome; (3) complete loss of the paternal X;Y chromosome; or (4) primary meiotic nondisjunction in the male. Results indicate the sensitivity of this scheme for the detection of events induced by x-rays and various chemicals. Positive results have been obtained with the known mutagens EMS and x-radiation.

Interpretation of cytogenetic damage induced in the germ line of male mice exposed for over 1 year to 239Pu alpha particles, fission neutrons, or 60Co gamma rays.

The relative biological effectiveness (RBE) of 239Pu alpha particles, fission neutrons (0.85 MeV), and 60Co gamma rays has been evaluated for the induction of reciprocal chromosome translocations in spermatogonia and of chromosome/chromatid fragments and chromatid rearrangements in the primary spermatocyte of adult male B6CF1 mice. Age concurrency was maintained for both internal and external radiations which were delivered at about 1 rad/week for 239Pu (single intravenous dose of 10 microCi/kg), 0.67, 1.67, and 2.67 rad/week for neutrons, and 6.95, 17.4, and 32 rad/week for gamma rays for at least 60 weeks. In terms of frequency of translocations, the response to the alpha emitter was nonlinear (concave downward) with little dose-response predictability; to cumulative neutron exposures the response was linear, without evidence of a dose-rate effect; and to gamma radiation the responses were linear, and a significant dose-rate effect was seen. RBE estimates are variable. For translocations, the n/gamma ratio is between 10 and 24, depending upon weekly dose level, and the ratio is 1 or less for the alpha particle relative to the neutron. For fragments, the n/gamma ratio is 18 to 22, depending upon age factors, and alpha/n is 1.5. For chromatid rearrangements, n/gamma is 7 and alpha/n is essentially indeterminate, but much below one. The overall response to the alpha emitter is interpreted to be a complex function of (a) microdosimetric heterogeneity, (b) a nearly invariant deposition pattern in the gonad, (c) the high sensitivity of differentiating spermatogonia to cell killing, and (d) the capacity of stem cells in relatively radiation-free areas to progressively assume the major spermatogenic role.

Is there a proportionality between the spontaneous and the X-ray-induction rates of mutations? Experiments with mutations at 13 X-chromosome loci in Drosophila melanogaster.

The X-ray induction of recessive visible specific locus mutations at 14 X-chromsome loci was studied in Drosophila melanogaster using the "Maxy" technique. The X-ray exposure was 3000 R to 5-day-old males and the sampling of germ cells was restricted to mature spermatozoa. Presumptive mutant females recovered in the F1 generation were tested for transmission, allelism, fertility and viability in males. A total of 128 mutations (115 completes and 13 mosaics including those that were male viable as well as male-lethal) recovered among 38 898 female progeny were found to be transmitted. On the basis of the above frequency, the average mutation rate can be estimated as 7.8 X 10(-8)/locus/R; for mutations that were viable and fertile in males, the rate is 3.0 X 10(-5)/locus/R (49 mutations among 38 898 progeny). The frequency of mutations at the different loci encompassed a wide range: while no mutations were recovered at the raspberry and carnation loci, at others, the numbers ranged from 1 at echinus to 31 at garnet; in addition, the proportion of mutations that was male-viable was also different, depending on the locus. Schalet's extensive data on spontaneous mutations at 13 (of the 14 loci employed in the present study) loci permit an estimate of the spontaneous rate which is 6.1 X 10(-6)/locus (a total of39 mutations among 490 000 progeny); for mutations that were viable and fertile in males, the rate is 3.0 X 10(-6)/locus (19 mutations among 490 000 progeny). The mutability of the different loci varied over a 9-fold range. When the different loci are ranked depending on their relative mutability (for spontaneous and induced mutations) it is found that in general, loci that mutate spontaneously relatively more frequently are also those at which more mutations have been recovered in the radiation experiments and likewise, those that are less mutable spontaneously are also those that mutate less after irradiation. Since the data are limited, it is concluded that the above finding is not inconsistent with the assumption of proportionality between spontaneous and induction rates of mutations. On the basis of the above results, a doubling dose of 100 R can be calculated for the X-ray induction of specific-locus mutations in Drosophila spermatozoa.

X-ray induced sex-chromosome loss, when ring-X chromosome males are irradiated and mated to females carrying mei-9, mei-41 or mei-218.

Radiation damage induced in the sperm nucleus is repaired after this nucleus has entered the oocyte. The yield of induced genetic damage is known to be dependent on maternal genotype and can also be modified by treatment of the females with metabolic inhibitors. The experiments reported here were designed to find out whether a more specific analysis of the interaction between male gamete and oocyte cytoplasm can be carried out using mutants that are known to affect repair processes. Males carrying ring-X chromosomes were exposed to X-ray doses up to 1000 R and mated to females homozygous for a repair-deficient mutant. The mutants used were mei-9a, mei-9L1, mei-41A10, and mei-41D5. In addition a yellow (y) X chromosome was used as a control and an attempt was made to obtain data using mei-21815, a mutant at a locus not thought to affect repair. With mei-9 mutants there is an enhancement of the spontaneous and induced rates of paternal sex-chromosome loss. The mei-41 mutants did not affect the rates of paternal sex-chromosome loss. Mei-218 females were difficult to work with because they gave very few progeny. From these data it can be argued that repair-deficient mutants will indeed be useful for an analysis of the fixation of radiation-induced genetic damage.

A genetic study of the effects of the repair-deficient mei-9a mutation in Drosophila on spontaneous and X-ray-induced paternal sex chromosome loss.

The repair-deficient mutant, mei-9a in Drosophila melanogaster was investigated regarding its effect on spontaneous and X-ray-induced chromosome loss in male postmeiotic cells. From matings of males carrying a mei-9a or an ordinary ring-X and a doubly marked Y chromosome (BSYy+) with mei-9a or ordinary females, the spontaneous frequencies of complete loss, partial loss, and inferred ring-X loss (based on shifts in sex ratio female:male) were significantly higher with mei-9a than with non-mei-9a. When males were given 3000 rad X-irradiation, frequencies of induced partial loss, inferred ring-X loss and the reduction in the number of progeny per female were significantly greater with mei-9a than with non-mei-9a. The results provide evidence that the mei-9a is a potentiator of both spontaneous and X-ray-induced chromosome lesions in sperm of the Drosophila male. Evidence is presented which implicates the presence of mei-9a in the P1 female and not the male as (at least) largely responsible for the characteristic mei-9a effects.

The timing of the restitution of chromosome breaks induced by X-rays in the mature sperm of Drosophila melanogaster.

Drosophila melanogaster males with marked X and Y chromosomes were irradiated, and mature sperm sampled by mating the males to females carrying attached-X chromosomes. Induced loss and partial loss of the paternal sex chromosomes was studied. F1 females were scored according to their phenotype, and transmitted fragments were analyzed genetically. Half of the exceptional F1 females could be scored as "partial losses". Of the apparent total loss exceptions, which were tested, half were carrying detectable fragments. 21% of the transmissible fragments is an under-estimate because only 6 of the 10 chromosome tips were marked in such a way that duplications could be detected. In addition, the markers used were located near, but not at, the chromosome ends. These data are interpreted as indicating that a high proportion of the chromosome loss and partial loss, induced by irradiation of mature sperm, is a consequence of chromatid rearrangements arising from chromosome breaks which stay open until replication. It is suggested that, during the transition from sperm head to mature pronucleus, repair of breaks and chromosome replication are two processes that occur in overlapping time intervals. It is therefore possible for chromosome breaks induced in mature sperm to give rise to chromosome and chromatid rearrangements.

Neutrons and X-rays, comparative studies with Drosophila melanogaster. 2. Sex-chromosome loss and partial loss, evidence for the induction of chromatid aberrations in spermatozoa.

Losses and duplications of BSY y+-chromosome markers were induced by irradiation of spermatozoa with either 0.5-MeV neutrons or 100-kV X-rays. These 2 types of radiation are known to induce significantly different ratios of double:single strand breaks in DNA. Exceptional progeny were grouped into 3 categories; no Y marker, one Y marker, and Y marker duplications + mosaics. The last combination consisted of exceptions derived from only chromatid-type rearrangements. All other classes of exceptions may be derived from either chromatid- or chromosome-type rearrangements. Doses of 15 Gy neutrons and 27 Gy X-rays induced identical frequencies of exceptional progeny, giving an RBE of 1.8. The ratios of the 3 classes of exceptions were similar for both types of radiation. This observation can be interpreted as indicating that, under the conditions used here, chromosome and chromatid rearrangements are not derived directly from double and single DNA strand breaks, respectively.

The effect of repeated microwave irradiation on the frequency of sex-linked recessive lethal mutations in Drosophila melanogaster.

The effect of repeated microwave irradiation (2375 MHz, CW) on mutagenic changes in Drosophila melanogaster was investigated. Oregon-R males were exposed to sublethal doses of microwaves (15 W/cm2 for 60 min, 20 W/cm2 for 10 min, and 25 W/cm2 for 5 min) for 5 days. The Muller-5 cross was used to detect sex-linked recessive lethal mutations. 4 lethals were found in treated groups but their frequency was not significantly different from that of the control group. No cumulative effect of repeated exposures on the mortality of the treated males was observed; on the contrary, their mortality decreased with the number of exposures. Irradiation did not affect the sex ratio of the F1. A significant decrease in the number of F1 offspring was noted in the group exposed to the power density of 15 W/cm2. A negative thermal effect of microwaves on male germ cells was probably manifested by this long exposure.

Partial losses of sex chromosomes induced by X-rays in spermatozoa of Drosophila melanogaster.

The frequency of induced partial and complete losses of sex chromosomes from spermatozoa of Drosophila melanogaster was determined by mating irradiated males to females heterozygous for an attached-X Y chromosome and for a bb-deficient chromosome. The relative involvement of the X- and Y-chromosomes in partial losses was determined by (a) irradiating males with an X-chromosome centromere marked by a y+ translocated to the short right arm, and a Y-chromosome marked by w+, and (b) comparing the frequencies of fragments recovered from irradiated males with a normal, marked Y-chromosome and from irradiated males and a deleted bb Y-chromsome. 50-80% of the induced losses of sex-chromosomes in rod-X/Y males were partial losses, whereas only 25% of those induced in ring-X/Y males were partial losses. More than 70% of the recovered fragments were of Y-chromosome origin. Fragments recovered from attached-X-Y sons of irradiated males wer checked for the presence of the most proximal euchromatic X-chromosome genes. Most fragments of the rod-X were broken more proximally than bb. In the ring-X there was an excess of fragments broken in the proximal euchromatin. It was concluded that many fragments of the Y-chromosome was due to breaks induced by one-hit events, in both arms of the chromosome that were folded upon themselves. A similar mechanism had been postulated earlier for fragments of the ring-X chromosome.

Effect of low temperature on radiation-induced genetic damage in Drosophila melanogaster: response of motile sperm and late spermatids.

The response of fully mature motile sperm and late spermatids when challenged with X-radiation at 0 degrees C has been studied in sex-linked recessive lethals, II-III translocations and dominant lethality experiments. At 0 degrees C a significant increase in both mutagenic and clastogenic damage was detected compared to that obtained at 24 degrees C. Furthermore, the results of experiments performed with different postirradiation temperatures demonstrate that the low temperature during irradiation was the sole factor responsible for the observed increase. In the recessive lethal and translocation tests the response of late spermatids was higher than that shown by motile spermatozoa. As a whole, the results, which are rather similar to data reported on the effect of irradiation in oxygen of the same cell stages, suggest that the low temperature acted as a dose-modifying factor.

The use of a mutationally unstable X-chromosome in Drosophila melanogaster for mutagenicity testing.

Somatic eye-colour mutations in an unstable genetic system, caused by a transposable element in the white locus of the X-chromosome in Drosophila melanogaster, is suggested as an assay system for mutagenicity testing. The system is evaluated by comparison with a corresponding system in a stable X-chromosome. Its sensitivity is confirmed with X-ray and EMS treatment, and it is found to be confined to the specific segment of the X-chromosome where the transposable element is localized.