Ethylation pf DNA and protamine by ethyl methanesulfonate in the germ cells of male mice and the relevancy of these molecular targets to the induction of dominant lethals

The molecular dosimetry of ethyl methanesulfonate (EMS) in the germ cells of male mice has been investigated. The mice were injected i.p. with 200 mg/kg of [³H]EMS and the ethylations per sperm head, per deoxynucleotide, and per unit of protamine were then determined over a 2-week period. The ethylations per sperm head closely paralleled the dominant-lethal frequency curve for EMS, reaching a maximum of 5 to 6.5 million ethylations per vas sperm head at 8 to 10 days after treatment. Ethylation of sperm DNA was greatest at 4 h after treatment, with 5.7 ethylations/10⁵ deoxynucleotides, and gradually decreased to 2.2 ethylations/10⁵ deoxynucleotides at 15 days after treatment. The ethylation of sperm DNA did not increase in the germ-cell stages most sensitive to EMS, ans was not correlated with the dominant-lethal frequency curve for EMS. However, ethylation of sperm protamine did increase in the germ-cell stages most sensitive to EMS, and showed an excellent correlation with the incidence of dominant lethals produced by EMS in the germ cells.A model is presented to explain, at a molecular level, how dominant lethals may be induced in mouse germ cells by EMS. Ethylation by cysteine sulfhydryl groups contained in mouse-sperm protamine could block normal disulfidebond formation, preventing proper chromatin condensation in the sperm nucleus. Stresses in the chromatin structure could then eventually lead to chromosome breakage, with resultant dominant lethality.     

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.     

Unscheduled DNA synthesis in spermatogenic cells of mice treated in vivo with the indirect alkylating agents cyclophosphamide and mitomen

Cyclophosphamide (CPA) and mitomen (DMO) are chemical mutagens that require metabolic activation to produce their biological effect. We have used an in vivo UDS assay in various meiotic and postmeiotic germ-cell stages of male mice to study DNA repair after treatment with these chemicals. EMS, a compound requiring no metabolic activation, was also used for comparative purposes.CPA and DMO induced UDS in meiotic through early-to-midspermatid stages, but no UDS was detected in late spermatids and mature sperm. While EMS produced a maximum UDS response in the germ cells immediately after treatment, CPA and DMO did not produce a maximum response until ～0.5 to 1 h after injection. This delay is attributed to the time required for CPA and DMO to be enzymatically vonverted active alkylating metabolites.Unlike the results found with EMS, mutation frequencies (dominant lethals, translocations, specific-locus mutations) following CPA treatment are not noticeably reduced in germ-cell stages in which UDS occurred. In the case of DMO, mutations are induced only in mature spermatozoa, and these germ-cell stages represent only a fraction of those in which no UDS is detected. The results with CPA and DMO thus still leave unclear the relationship between DNA repair and the differential spermatogenic response of mice to genetic damage.     

The use of alkaline elution procedures to measure DNA damage in spermiogenic stages of mice exposed to methyl methanesulfonate

The ability of methyl methanesulfonate (MMS) to induce DNA breakage in spermiogenic stages of the mouse was studied using an alkaline elution technique. At daily intervals over a 3-week period following i.p. injection of 50 mg MMS/kg, mature spermatozoa were recovered from treated (3H-labeled) and control (14C-labeled) animals, lysed together on polycarbonate filters, and eluted with a high pH (12.2) buffer. Elution of germ-cell DNA from MMS-treated animals was found to increase in stages in which genetic damage from MMS is greatest. In general, the pattern of DNA elution from treated, spermiogenic stages paralleled the pattern of sensitivity to dominant lethals, specific-locus mutations and heritable translocations found by other investigators. It also paralleled the pattern of sperm-head methylation and protamine methylation measured in an earlier study (Sega and Owens, 1983). At 9 days post treatment (sperm sampled were in mid-to late-spermatid stages at the time of MMS exposure) the elution of sperm DNA did not change significantly over a pH range of 11.6-12.8, suggesting that, at the time of assay, DNA breaks were already present in the sperm. Because of the parallelism found between increased sperm DNA elution and increased genetic damage after mutagen treatment, alkaline elution may prove useful in monitoring potential genetic damage in human sperm.     

Molecular dosimetry of the mutagen ethyl methanesulfonate in Drosophila melanogaster spermatozoa: linear relation of DNA alkylation per sperm cell (dose) to sex-linked recessive lethals.

The dosage-response curve for EMS was determined with dose measured as ethylations of DNA per sperm cell, and response measured as the relative frequency of sex-linked recessive lethals induced in sperm cells of Drosophila melanogaster. Dose can be converted to ethylations per nucleotide of DNA by dividing ethylations of DNA per sperm cell by 3 X 10(8) nucleotides per sperm cell. Adult males were exposed to equal amounts of either [3H]EMS for determining dose or nonlabeled EMS for determining mutational response. By feeding EMS for 24 h in a concentration of 25 mM, a high dose of 1.4 X 10(-2) ethylations per nucleotide was observed. With 1.4% of the nucleotides ethylated, 57% of the X-chromosomes were hemizygously viable; therefore, ethylation per se is not very efficient in inducing mutations. The relative frequency of mutations increased linearly with the dose from a dose of 2.1 X 10(-4) to 1.4 X 10(-2) ethylations per nucleotide. No threshold was apparent, and the statistical limits of the exponent, 1.0 +/- 0.1, excluded an exponent as high as 1.2. This linear relation suggests no change in mechanism of mutagenesis occurs from low to high dose in Drosophila. A nonlinear relation was found between exposure and dose; when exposure was increased by a factor of 250 (from 0.1 to 25 mM EMS in the feeding medium) dose was increased by a factor of only 68. By extrapolating down from our lowest dose of 2.1 X 10(-4) ethylations per nucleotide with an observed frequency of 0.55% +/- 0.08% sex-linked recessive lethals, we estimate the doubling dose for sex-linked recessive lethals to be 4 X 10(-5) ethylations per nucleotide.     

Dosimetry studies on the ethylation of mouse sperm DNA after in vivo exposure to (3H)ethyl metanesulfonate

Methods for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of mouse spermatozoa in the vasa deferentia and epididymides have been developed. These include procedures for the removal of contaminating protamine, which, like DNA, possesses nucleophilic sites that can be ethylated by EMS. At least 99% of all sperm protamine (at a 95% confidence level), as well as any other cellular contaminants, is removed during purification of the DNA. The purified DNA recovered from spermatozoa gives no indication of a preferential recovery of either (G+C)-rich or (A+T)-rich regions of the mouse genome: the [¹⁴C]dT/[³H]dC ratios for whole sperm and sperm DNA were the same for each animal tested.The spermatozoa of males used in the dosimetry studies were labeled with [¹⁴C]thymidine, and then the animals were given various [³H]EMS doses intraperitoneally. A constant exposure time of 4 h was used. The ratios of ³H and ¹⁴C activities in whole sperm and purified sperm DNA were used to measure the percentage of the total sperm ethylation occurring in the DNA. The maximum percentage found was about 18% in the dose range of 100–400 mg/kg. Values for the ethylations per nucleotide (E/N) ranged from ～ 10^?7 at 3.3 mg/kg up to ～ 10^?4 at 400 mg/kg, and the data indicated that E/N increased with the 1.5 power of the dose. E/N was also measured in testicular DNA, and the values obtained were close to those found for spermatozoan DNA.The results of such chemical dosimetry studies will be far-reaching in the interpretation of molecular events responsible for genetic alterations. As an example, dominant lethal studies by others, using EMS in the dose range considered in the present paper, have shown little or no effect until two or more days after injection of the mutagen into male mice. Since many sperm DNA ethylations are found after a 4-h exposure to EMS it appears that most of these DNA ethylations are not genetically important, at least in the production of dominant lethals, and that perhaps genetic damage occurs only at rarely ethylated DNA sites.     

Somatic cell mutagenicity in Drosophila melanogaster in comparison with genetic damage in early germ-cell stages

With the intention of assessing the general performance, sensitivity and the underlying mechanisms of somatic cell mutagenicity assays in Drosophila, a study was undertaken to compare the effectiveness of 5 procarcinogens and 4 direct-acting agents in the white/white-coral eye mosaic assay (SMART) with their activity in early (premeiotic) male and female germ-cell stages, after exposure of Drosophila larvae. The outcome indicated a lack of agreement in the results from recessive lethal assays (SLRL) in comparison with the somatic mutation and recombination test (SMART). The procarcinogens 2-naphthylamine (NA), 3-methylcholanthrene (MC), 9,10-dimethylanthracene (DA) and 7,12-dimethylbenz[a]anthracene (DMBA), and the direct-acting mutagens bleomycin (BM), methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), were quite efficient in producing somatic recombination and mutations in white/white-coral larvae, as opposed to only weak effects in early germ-cell stages. 2-Acetylaminofluorene (2AAF) showed marginal effects in both germ cells and somatic tissue after exposure of female larvae, but was inactive in testis. The discrepancy in mutational response between somatic cells and premeiotic germ cells is most impressive for MMS and BM. There is sufficient evidence for attributing a good sized proportion of the encountered variation to efficient error-free DNA repair of premutational damage and to segregational elimination during meiosis of deleterious mutations: (1) The efficient point mutagen ENU was the but one agent producing high levels of viable genetic alterations in early germ cells and in somatic cells. A similar behaviour was previously described for diethylnitrosamine, which ethylates DNA in the same fashion as ENU. (2) In early germ-cell stages of mei-9L1 male larvae, MMS induced multiple mutations (putative clusters) at a low dose differing by a factor 20-40 from those needed to produce an equivalent response in repair-competent strains. This is consistent with the concept of an active excision repair in premeiotic cells. (3) In the case of EMS, next to DNA repair, germinal selection seems to restrict the realization of EMS-induced genetic damage in premeiotic cells. (4) Bleomycin-induced chromosome aberrations caused high mortality rates in males (hemizygous for an X-chromosome) but not in females. MMS and BM, agents known to show preference for chromosome aberration induction, produced 3-6-fold higher rates of somatic mutational events (SME) in female genotypes as compared with the other sex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recessive lethals induced by ethyl methanesulfonate and diethyl sulfate in Drosophila melanogaster post-meiotic male cells pre-treated with butylated hydroxytoluene

The response of Drosophila melanogaster male germ cells to the induction of mutation by ethyl methanesulfonate (EMS) and diethyl sulfate (DES) and the influence of pre-treatments with butylated hydroxytoluene (BHT) were studied. Careful sampling of cell stages revealed that fully mature motile sperm were less sensitive to the induction of sex-linked recessive lethals by EMS than late spermatids, and that the remaining cell stages presented a fairly homogeneous response to the mutagen. The frequency of lethals induced by DES could be grouped into two plateaus: the first one, with a higher mutation rate, comprised motile and immotile sperm and late spermatids, the second one, medium and early spermatids. No sparing action of BHT was detected in any of the developing germ cells treated with EMS or DES, whereas an increase in sex-linked recessive lethal frequency was observed in some experiments in early spermatids. The enhancement of damage is attributed to impairment of repair achieved through the ability of BHT to modify enzymic activity.     

Sex-specific dynamics of global chromatin changes in fetal mouse germ cells

Mammalian germ cells undergo global reprogramming of DNA methylation during their development. Global DNA demethylation occurs around the time when the primordial germ cells colonize the embryonic gonads and this coincides with dynamic changes in chromatin composition. Global de novo DNA methylation takes place with remarkably different dynamics between the two sexes, prospermatogonia attaining methylation during fetal stages and oocytes attaining methylation postnatally. Our hypothesis was that dynamic changes in chromatin composition may precede or accompany the wave of global DNA de novo methylation as well. We used immunocytochemistry to measure global DNA methylation and chromatin components in male and female mouse fetal germ cells compared to control somatic cells of the gonad. We found that global DNA methylation levels sharply increased in male germ cells at 17.5 days post coitum, but remained low in female germ cells at all fetal stages. Global changes in chromatin composition: i, preceded global DNA methylation in fetal germ cells; ii, sex specifically occurred in male but not in female germ cells; iii, affected active and repressive histone marks and iv, included histone tail and histone globular domain modifications. Our data suggest that dynamic changes of chromatin composition may provide a framework for the pattern of male-specific de novo DNA methylation in prospermatogonia.     

An autoradiographic study of unscheduled DNA synthesis in the germ cells of male mice treated with X-rays and methyl methanesulfonate

Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.     

Prophage induction in Haemophilus influenzae and its relationship to mutation by chemical and physical agents

It is known that UV, X-rays, MMC and MMS are not mutagenic for H. influenzae, whereas HZ, EMS and MNNG are potent mutagens for this bacterium. All of these agents, however, are known to be both mutagenic and able to induce prophage in E. coli. We report here that all the agents except HZ induce prophage in H. influenzae, and EMS even induces in the recombination-defective recl mutant, which is non-inducible by UV, MMC, MNNG and MMS. MMS did not cause single-strand breaks or gaps in DNA synthesized after treatment of H. influenzae, but EMS and MNNG produced them. EMS caused more breaks in DNA synthesized before treatment than in that synthesized after treatment. On the other hand we did observe such breaks or gaps induced in E. coli in DNA synthesized posttreatment by EMS as well as by MMS and MNNG, at comparable survival levels.     

Adaptive response to alkylating agents in the Drosophila sex-linked recessive lethal assay

The adaptive response to alkylating agents was studied in Drosophila assays under various treatment procedures. Pre-treatment of males as well as treatment of females with low doses of EMS (0.05-0.1 mM) did not affect sex-linked recessive lethal (SLRL) rates induced by high doses of this mutagen (10 mM, various feeding duration) in mature sperm cells. Pre-treatment of males with a low dose of MMS (0.1 mM) enhanced mutagenesis induced by the high dose of EMS (10 mM) at different stages of spermatogenesis, the observed effects exceeding the additive action of both mutagens. On the contrary, larval pre-treatment with the adaptive dose of EMS (0.05 mM) resulted in resistance of their germ cells to higher doses of EMS (1 mM). Specifically, offspring production increased while dominant lethality in F(1) as well SLRL frequency in F(2) was significantly reduced as compared with the effects of larval exposure to the challenge dose. Under the conditions tested, the adaptive response of germ cells to alkylating agents was demonstrated in larvae, but not in adult flies.     

Formation of native-like mammalian sperm cell chromatin with folded bull protamine

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.     

Comparative mutagenicity of alkylsulfate and alkanesulfonate derivatives in Chinese hamster ovary cells.

Mutation induction and cell killing produced by selected alkylsulfates and alkanesulfonates have been quantitated using the Chinese hamster ovary/hypoxanthine--guanine phosphoribosyl transferase (CHO/HGPRT) system. Dose--response relationships of cytotoxicity and mutagenicity are presented for two alkylsulfates [dimethylsulfate (DMS), diethylsulfate (DES)] and three alkyl alkanesulfonates [methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and isopropyl methanesulfonate (iPMS)]. Under the experimental conditions employed, cytotoxicity decreased with the size of the alkyl group. DMS was more toxic than DES, and MMS was more toxic than EMS and iPMS. All agents produced linear dose--response of mutation induction: DMS was more mutagenic than DES, and MMS was more mutagenic than EMS and iPMS based on mutants induced per unit mutagen concentration. However, the following relative mutagenic potency was observed when comparisons were made at 10% survival: DES greater than DMS; EMS greater than MMS greater than iPMS.     

Quantitative evaluation of radiation-induced changes in sperm morphology and chromatin distribution

Sperm head cytometry provides a useful assay for the detection of radiation-induced damage in mouse germ cells. Exposure of the gonads to radiation is known to lead to an increase of diploid and higher polyploid sperm and of sperm with head shape abnormalities. In the pilot studies reported here quantitative analysis of the total DNA content, the morphology, and the chromatin distribution of mouse sperm was performed. The goal was to evaluate the discriminative power of features derived by high resolution image cytometry in distinguishing sperm of control and irradiated mice. Our results suggest that besides the induction of the above mentioned variations in DNA content and shape of sperm head, changes of the nonhomogeneous chromatin distribution within the sperm may also be used to quantify the radiation effect on sperm cells. Whereas the chromatin distribution features show larger variations for sperm 21 days after exposure (dpr), the shape parameters seem to be more important to discriminate sperm 35 dpr. This may be explained by differentiation processes, which take place in different stages during mouse spermatogenesis.     

The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10⁸ daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl-N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10⁸ daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10⁸ daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O⁶-methylguanine (O⁶-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN₁ character of MNUA would cause a greater formation of phosphotriesters than would MMS.     

Induction of specific-locus and dominant lethal mutations in male mice in the low dose range by methyl methanesulfonate (MMS)

Methyl methanesulfonate (MMS) induces specific-locus and dominant lethal mutations in spermatozoa and spermatids of mice. A dose of 15 mg/kg b.w. of MMS induces 9% dominant lethal mutations in the most sensitive germ-cell stages, corresponding to the mating intervals 5-8 and 9-12 days post treatment. A dose of 150 mg/kg b.w. of MMS in the same mating intervals induces 100% dominant lethal mutations. The sensitivity pattern for the induction of dominant lethal and specific-locus mutations is the same. In the mating interval 5-8 days a dose of 20 mg/kg b.w. of MMS induced 3.8 x 10(-5) mutations per locus per gamete. The yield of specific-locus and dominant lethal mutations in the low dose range increases proportionally with the dose. A dose given in 2, 4 or 5 fractions yields the same frequency of mutations as a single injection of the total dose. The additivity of small doses proves that the pre-mutational lesions are not or only partially repaired in these stages and that MMS is not or only partially detoxified. In addition, the frequency of dominant lethal and specific-locus mutations depends on the germ-cell stage.     

Increment kinetics of recessive lethal mutations and dominant lethals in offspring of Drosophila melanogaster on storage of methyl-methanesulfonate-treated sperm in females

The frequencies of sex-linked recessive lethal mutations in F1 males after feeding adult male Drosophila melanogaster with 0.25 and 0.5 mM methyl methanesulfonate (MMS) orally for 24 h increased approximately linearly with storage of the treated spermatozoa in females, whereas the number of hits of dominant lethals in the sperm after feeding 0.3 and 0.5 mM MMS increased approximately with the square of the storage time. Chromosome losses and mosaics in F1 males also increased with the dose of MMS to males, but their yields were too low to be analyzed quantitatively, only indicating a slight increase of chromosome loses and a slight decrease of mosaics with the time of storage of sperm. Maternal non-disjunctions (or chromosome losses), detected in F1 males, decreased with the dose of MMS to spermatozoa and their yield decreased with the time of storage of sperm of both MMS-treated and the control groups. A unitary model is proposed to explain the effect of storage on the dominant lethals and recessive lethal mutations.     

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.