The repair-deficient mei-9 alpha Drosophila female potentiates chromosome loss induced in the parenteral genome by diethylnitrosamine

Following matings of DEN-treated Xc2/BSYy+ males with repair-deficient mei-9 alpha females and ordinary females, significant increases in complete and partial sex chromosome loss as well as dramatic shifts in sex ratio were found with mei-9 alpha but not ordinary females. Accordingly, the mei-9 alpha female enhances the detection of chromosome lesions leading to chromosome loss induced in the male genome by DEN. To date, the 4 compounds tested in this way (DMN, DEN, MMS and procarbazine) exhibit strong potentiation of chromosome loss with mei-9 alpha females suggesting the possibility that a protocol involving treatment (or not) or Xc2/BSYy+ males mated with mei-9 alpha females may hold promise as an alternative to traditional tests for chromosome loss using repair-proficient females. Comparison with published translocation data on the 4 compounds indicated above suggests an overall greater sensitivity of the described mei-9 alpha chromosome-loss test compared with the traditional translocation test in the detection of chemically induced chromosome lesions.     

Induced chromosome loss following treatment of postmeiotic cells of the Drosophila melanogaster male with MMS and DMN and matings with repair-proficient females and the repair-deficient females mei-9a and st mus302

Drosophila melanogaster ring-X males carrying a double marked Y chromosome, BsYy+, were treated with MMS or DMN and mated with repair-proficient females or the repair-deficient females mei-9a and st. mus302. Frequencies of induced complete loss (principally the ring-X) and partial losses of the Y chromosome (loss of Bs or Y+) decreased in the sequence st must302 greater than mei-9a greater than repair-proficient females agreeing with the sequence obtained previously with procarbazine and DEN. With MMS and DMN, some 30-40% or more or partial Y chromosome losses are mosaics from mei-9a and only 0.4% from st mus302 females and a delay in mei-9a females. Similar findings with procarbazine and DEN are indicated. That the higher sensitivity of st mus302 relative to mei-9a results from impairments in both postreplication and excision repair in the former remains to be determined.     

O-alkylation in DNA does not correlate with the formation of chromosome breakage events in D. melanogaster

Postmeiotic cell stages of repair-proficient ring-X (RX) males were treated with methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), diethylnitrosamine (DEN) or ethylnitrosourea (ENU) and then mated to either repair-defective (mei-9L1) or to repair-competent females (mei-9+). Absence of the mei-9+ function resulted in a hypermutability effect to all alkylating agents (AAs) when they were assayed for their ability to induce chromosomal aberrations (chromosome loss; CL), irrespective of marked differences in distribution of DNA adducts brought about by these AAs. This picture is different from that described previously for the induction of point mutations (Vogel et al., 1985a). There, evidence was presented indicating that reduction in DNA excision repair does not affect point mutation induction (recessive lethals) by those AAs most efficient in ring-oxygen alkylation such as ENU, DEN, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and isopropyl methanesulfonate (iPMS): the order of hypermutability of AAs with mei-9L relative to mei-9+ was MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females were plotted against those determined for mei-9+ females, straight lines of following slopes were obtained: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4, and iPMS = ENU = DEN = ENNG = 1. Those findings, together with the recent observation that AAs do not split into two groups when assayed for their ability to cause CL, point to the involvement of different DNA alkylation products in ENU- and DEN-induced chromosome loss vs. that of point mutations. It is concluded that with ENU and DEN chromosomal loss results from N-alkylation products whereas point mutations (SLRL) are the consequence of interactions with oxygen-sites in DNA. Thus, as a consequence of a very dominating role of O-ethylguanine (and possibly O4-alkylation of thymine), N-alkylation in DNA does not contribute measurably to mutation induction in the case of ENU-type mutagens while O-alkylation, very clearly, does not show a positive correlation with the formation of chromosome breakage events in Drosophila. Conversely, it appeared that with MMS-type mutagens (MMS; dimethyl sulfate, DMS; trimethyl phosphate, TMP), alkylation products such as 7-methylguanine and 3-methyladenine, if unrepaired or misrepaired, are potentially mutagenic lesions causing both mutations and chromosomal aberrations.     

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants at both loci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An anlysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show verly little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104^D1 and mei-41^D5 are located in th eregion 14B13±?14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101^D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103^D1 is between bands 12A1,2 and 12A6,7, and mus(1)-109^A1 is in section 8F3-9A2.     

The Utilization during Mitotic Cell Division of Loci Controlling Meiotic Recombination and Disjunction in DROSOPHILA MELANOGASTER

To inquire whether the loci identified by recombination-defective and disjunction-defective meiotic mutants in Drosophila are also utilized during mitotic cell division, the effects of 18 meiotic mutants (representing 13 loci) on mitotic chromosome stability have been examined genetically. To do this, meiotic-mutant-bearing flies heterozygous for recessive somatic cell markers were examined for the frequencies and types of spontaneous clones expressing the cell markers. In such flies, marked clones can arise via mitotic recombination, mutation, chromosome breakage, nondisjunction or chromosome loss, and clones from these different origins can be distinguished. In addition, meiotic mutants at nine loci have been examined for their effects on sensitivity to killing by UV and X rays.—Mutants at six of the seven recombination-defective loci examined (mei-9, mei-41, c(3)G, mei-W68, mei-S282, mei-352, mei-218) cause mitotic chromosome instability in both sexes, whereas mutants at one locus (mei-218) do not affect mitotic chromosome stability. Thus many of the loci utilized during meiotic recombination also function in the chromosomal economy of mitotic cells.—The chromosome instability produced by mei-41 alleles is the consequence of chromosome breakage, that of mei-9 alleles is primarily due to chromosome breakage and, to a lesser extent, to an elevated frequency of mitotic recombination, whereas no predominant mechanism responsible for the instability caused by c(3)G alleles is discernible. Since these three loci are defective in their responses to mutagen damage, their effects on chromosome stability in nonmutagenized cells are interpreted as resulting from an inability to repair spontaneous lesions. Both mei-W68 and mei-S282 increase mitotic recombination (and in mei-W68, to a lesser extent, chromosome loss) in the abdomen but not the wing. In the abdomen, the primary effect on chromosome stability occurs during the larval period when the abdominal histoblasts are in a nondividing (G2) state.—Mitotic recombination is at or above control levels in the presence of each of the recombination-defective meiotic mutants examined, suggesting that meiotic and mitotic recombination are under separate genetic control in Drosophila.—Of the six mutants examined that are defective in processes required for regular meiotic chromosome segregation, four (l(1)TW-6^cs, ca^nd, mei-S332, ord) affect mitotic chromosome behavior. At semi-restrictive temperatures, the cold sensitive lethal l(1)TW-6^cs causes very frequent somatic spots, a substantial proportion of which are attributable to nondisjunction or loss. Thus, this locus specifies a function essential for chromosome segregation at mitosis as well as at the first meiotic division in females. The patterns of mitotic effects caused by ca^nd, mei-S332, and ord suggest that they may be leaky alleles at essential loci that specify functions common to meiosis and mitosis. Mutants at the two remaining loci (nod, pal) do not affect mitotic chromosome stability.     

Retinol (vitamin A) inhibition of dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) induced sister-chromatid exchanges in V79 cells and mutations in Salmonella/microsome assays

When the Chinese hamster cell line V79 and the tester strain of Salmonella typhimurium TA100 were treated with the precarcinogens dimethylnitrosamine (DMN) or diethylnitrosamine (DEN) in the presence of S9 mix, a dose-dependent increase of sister-chromatid exchanges (SCE) in V79 cells and His+ revertants in TA100 resulted. DMN was a far more efficient SCE inducer than DEN, while DEN was a more efficient inducer of His+ revertants than DMN. Retinol (Rol) effectively inhibited DMN and DEN induced SCE in V79 cells and His+ revertants in TA100. Concurrent treatment of V79 cells with Rol at various doses and one dose of DMN or DEN in the presence of S9 mix caused a significant reduction of SCE as compared to SCE induced by DMN or DEN without Rol. Rol inhibition of DMN-induced SCE was dose-dependent. Rol was less efficient in inhibiting DEN-induced SCE, and no consistent dose-dependent inhibition was observed. At all doses, Rol significantly inhibited DMN and DEN induced mutation frequencies in TA100. At the highest dose of Rol (40 micrograms/plate), the inhibition of DMN and DEN induced His+ revertants reached about 90% and 60%, respectively. The possibility that Rol exerts its antimutagenic activities by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of precarcinogens such as DMN and DEN is discussed.     

Molecular cloning of mei-41, a gene that influences both somatic and germline chromosome metabolism of Drosophila melanogaster

The mei-41 gene of Drosophila melanogaster plays an essential role in meiosis, in the maintenance of somatic chromosome stability, in postreplication repair and in DNA double-strand break repair. This gene has been cytogenetically localized to polytene chromosome bands 14C4-6 using available chromosomal aberrations. About 60 kb of DNA sequence has been isolated following a bidirectional chromosomal walk that extends over the cytogenetic interval 14C1-6. The breakpoints of chromosomal aberrations identified within that walk establish that the entire mei-41 gene has been cloned. Two independently derived mei-41 mutants have been shown to carry P insertions within a single 2.2 kb fragment of the walk. Since revertants of those mutants have lost the P element sequences, an essential region of the mei-41 gene is present in that fragment. A 10.5 kb genomic fragment that spans the P insertion sites has been found to restore methyl methanesulfonate resistance and female fertility of the mei-41 ^D3 mutants. The results demonstrate that all the sequences required for the proper expression of the mei-41 gene are present on this genomic fragment. This study provides the foundation for molecular analysis of a function that is essential for chromosome stability in both the germline and somatic cells.     

The mei-9a Mutant of DROSOPHILA MELANOGASTER Increases Mutagen Sensitivity and Decreases Excision Repair

The mei-9^a mutant of Drosophila melanogaster , which reduces meiotic recombination in females (Baker and Carpenter 1972), is deficient in the excision of UV-induced pyrimidine dimers in both sexes. Assays were performed in primary cultures and established cell lines derived from embryos. An endonuclease preparation from M. luteus , which is specific for pyrimidine dimers, was employed to monitor UV-induced dimers in cellular DNA. The rate of disappearance of endonuclease-sensitive sites from DNA of control cells is 10–20 times faster than that from mei-9^a cells. The mutant mei-218, which is also deficient in meiotic recombination, removes nuclease-sensitive sites at control rates. The mei-9^a cells exhibit control levels of photorepair, postreplication repair and repair of single strand breaks. In mei-9 cells DNA synthesis and possibly postreplication repair are weakly sensitive to caffeine. Larvae which are hemizygous for either of the two mutants that define the mei-9 locus are hypersensitive to killing by the mutagens methyl methanesulfonate, nitrogen mustard and 2-acetylaminofluorene. Larvae hemizygous for the mei-218 mutant are insensitive to each of these reagents. These data demonstrate that the mei-9 locus is active in DNA repair of somatic cells. Thus functions involved in meiotic recombination are also active in DNA repair in this higher eukaryote. The results are consistent with the earlier suggestions (Baker and Carpenter 1972; Carpenter and Sandler 1974) that the mei-9 locus functions in the exchange events of meiosis. The mei-218 mutation behaves differently in genetic tests and our data suggest its function may be restricted to meiosis. These studies demonstrate that currently recognized modes of DNA repair can be efficiently detected in primary cell cultures derived from Drosophila embryos.     

Frequent Imprecise Excision among Reversions of a P Element-Caused Lethal Mutation in Drosophila

RpII215^D50 (= D50) is a lethal mutation caused by the insertion of a 1.3-kb P element 5' to sequences encoding the largest (215 kilodaltons) subunit of Drosophila RNA polymerase II. In dysgenic males D50 reverted to nonlethality at frequencies ranging from 2.6 to 6.5%. These reversions resulted from loss of P element sequences. Genetic tests of function and restriction enzyme analysis of revertant DNAs revealed that 35% or more of the reversion events were imprecise excisions. Two meiotic mutations that perturb excision repair and postreplication repair (mei-9^a and mei-41^D5, respectively) had no influence on reversion frequency but may have increased the proportion of imprecise excisions. We suggest that these excisions are by-products of, rather than intermediates in, the transposition process.     

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6: SEQUENTIAL OXIDATION TO ALDEHYDES AND CARBOXYLIC ACIDS AND ANALYSIS OF REACTION STEPS*

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH₃CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (^Dk_app ∼ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The ^Dk_app for DEN was ∼3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO₂H and CH₃CO₂H, respectively. In neither case was a lag observed, which was unexpected considering the k_cat and K_m parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde).     

Genetic study on the effects of the repair-deficient mutant females mei-9a, mei-41D5, mus101D1, mus104D1 and mus302D1 of Drosophila on spontaneous and X-ray-induced chromosome loss in the paternal genome

The repair-deficient mutants mei-9a, mei-41D5, mus101D1, mus104D1 and mus302D1 in Drosophila melanogaster were investigated regarding their effects on spontaneous and X-ray-induced chromosome loss in postmeiotic cells. Each mutant was incorporated singly into XC2, and the ring-X male provided with BSYy+. From matings of males carrying mus101D1, mus302D1 or mei-41D5, mutants identifying a caffeine-sensitive (CAS) postreplication-repair pathway, with corresponding mutant females, and non-mutant males to non-mutant females, overall frequencies of spontaneous partial loss and spontaneous complete loss were significantly increased in each mutant cross except for spontaneous complete loss with mus302 where an increase was noted only in brood 2. Similar findings were noted when males carrying the excision-repair mutant mei-9a were mated with mei-9a females. Males carrying the mutant mus104D1, identifying a caffeine-insensitive (CIS) postreplication-repair pathway, tested with mus104D1 females, produced results that were not significantly different from non-mutant controls. When males were given 3000 rad X-irradiation, frequencies of induced partial loss were significantly higher with mus101D1, mus302D1, mei-41D5 and mei91, and not significantly higher with mus101D1, mus302D1, mei41D5 and mei-9a, and not significantly different from controls with mus104D1. It was suggested that the functional CAS postreplication-repair pathway primarily promotes repair of breaks while an alternative pathway(s) not defined by mus104 promotes misrepair. Therefore, the significant increases in both spontaneous and induced partial loss with the excision-repair-deficient mutant mei-9a suggests the possibility that (a) the excision-repair-pathway may not function in misrepair and (b) the undefined misrepair pathway may be dominant pathway for postreplication repair in Drosophila since mei-9a females presumably have functional postreplication repair and misrepair capacity. The suggestion that the CAS postreplication-repair pathway and the excision-repair pathway function primarily in repair, and an undefined pathway in misrepair is in line with the finding that with mus104D1, no significant increase was found in spontaneous complete loss, but with mus101D1, mus302D1, mei-41D5 and mei-9a significant increases were observed. Results on induced complete loss, with the exception of those with mei-41D5, show a poor correlation with other classes of loss of each of the mutants. Possible explanations for this discrepancy are discussed.     

The Interaction of the rad201Gene with Genes mei-9and mei-41in Germline Cells of DrosophilaFemales

The effect of Drosophilamutation rad201 ^G1together with mutations mei-41 ^D5and mei-9 ^aon the sensitivity of oocytes to induction of dominant lethals (DLs) was studied. To this end, the frequencies of spontaneous and gamma-radiation-induced DLs in consecutive egg batches of females carrying double or single mutations were estimated. Since the effects of the mutations examined are expressed only at the previtellogenetic stages of oogenesis, only newly hatched (0–5-hour-old) females, whose oocytes did not develop farther than stage 7, were irradiated. The results obtained indicated that in intact and irradiated oocytes of double mutants mei-9 ^a rad201 ^G1and mei-41 ^D5 rad201 ^G1, mutation rad201 ^G1epistatically suppresses the mutations of the both meigenes.     

Chronic or acute administration of various dialkylnitrosamines enhances the removal of O6-methylguanine from rat liver DNA in vivo

The effect of pretreatment of rats with various symmetrical dialkylnitrosamines on the repair of O⁶-methylguanine produced in liver DNA by a low dose of [¹⁴C]dimethylnitrosamine (DMN) has been examined. DMN, diethylnitrosamine (DEN), dipropylnitrosamine (DPN) or dibutylnitrosamine (DBN) were administered to rats for 14 consecutive weekdays at a daily dose of 5% of the LD₅₀. Animals were given [¹⁴C]DMN 24 h after the last dose and were killed 6 h later. DNA was extracted from the liver and analysed for methylpurine content after mild acid hydrolysis and Sephadex G-10 chromatography. While the amounts of 3-methyladenine and 7-methylguanine were only slightly different from controls, the amounts of O⁶-methylguanine in the DNA of the dialkylnitrosamine pretreated rats were about 30% of those in control rats, indicating a considerable increase in the capacity to repair this base. Liver ribosomal RNA from control and dialkylnitrosamine pretreated rats contained closely similar amounts of O⁶-methylguanine suggesting that the induced enzyme system does not act on this base in ribosomal RNA in vivo. Pretreatment with these dialkylnitrosamines also enhanced the repair of O⁶-methylguanine in liver DNA when they were given as a single dose (50% of the LD₅₀) either 3 or 7 days before the [¹⁴C]DMN. In addition, single low doses of DMN or DEN (5% of the LD₅₀) given either 1 or 6 days before [¹⁴C]DMN increased O⁶-methylguanine repair and the magnitude of the effect after DEN was similar to that produced by the other pretreatment schedules. The possible mechanism(s) of the induction of O⁶-methylguanine repair and its relation to hepatotoxicity, DNA alkylation, carcinogenesis and the adaptive response in Escherichia coli are discussed.     

Genetic damage induced by X-rays or neutrons in spermatozoa of Drosophila melanogaster; differential processing in the oocytes of females carrying the DNA-repair-deficient mutants mei-9a and mus-101d1

Radiation-induced premutational lesions on the chromosomes of irradiated mature spertozoa of Drosphila are processed when the sperm nucleus the egg cytoplasm at fertilization. This processing depends on enzymatic repair systems, which are built up in ocytes under the control of the maternal genotype. The present study is concerned with 2 repair-deficient mutants, mei-9^a and mus-101^D1. Irradiated Basc males were crossed to homozygous mei-9^a or mus-101^D1 females, or to repair-proficient control females. The frequencies of recovered sex-link recessive lethal mutations and of II–III translocations were used to assess the effects of impaired maternal repair. Neutrons, as a densely ionizing radiation, and X-rays as a sparsely ionizing one, were used to induce the premutational lesions.The question being asked was whether different radiation qualities cause specific types of lesion that are processed differentially under conditions of impaired maternal repair. The results indicate that this may be so. In comparison with the control, with repair-proficient females, all major effects caused by impaired maternal repair led to frequency reductions in the recovery of lethals and translocations. These reductions in yield were pronounced in all neutron experiments, whereby mus-101^D1 had a stronger effect than mei-9^a. Two possible explanations are considered. The first is based on the idea that specific lesions are processed in a specific way, resulting in a specific mutational end-product, which may not be recovered when repair is impaired. The second is based on the notion that energy deposition in cells exposed to neutrons is not uniform, which leads to clustered damage. Impaired repair may select againts multiply damaged cells much more powerfully than normal repair. Consequently, the surviving fraction of cells is likely to have received less than the average dose. With X-rays, no or only spurious effects of the repair-defective mutants were detected, except in the following case: recovery of translocations (but not of lethals) was strongly reduced when irradiated males were crossed to mus-101^D1 females. It is assumed that mus-101^D1 is defective in repair of DNA double-strand damage, and that the formation of translocations may depend particularly on this repair function.     

Effects of DNA-repair-deficient mutants on somatic and germ line mutagenesis in the UZ system in Drosophila melanogaster

To analyze the underlying mechanisms of the UZ system in Drosophila melanogaster, i.e., the unstable white locus with an IS element included originally described by Rasmuson and Green (1974), the repair deficient mutants mei-9b and mei-4lD5 and the meiotic mutant mei-2l8 were introduced into X-chromosomes containing the UZ system. These strains were submitted to larval feeding of MMS to analyze differences occurring in mutation rate and survival. The mei-9b and mei-4l strains were markedly sensitive to MMS treatment, while mei-2l8 did not affect survival. Only the mutant mei-4l, which is lacking in post-replication repair, affected the somatic mutation rate of the UZ system, decreasing it by 50%. The spontaneous germ line mutation rate of UZ, on the other hand, was increased by introducing mei-4l or mei-9b, possibly as a result of the high frequency of unrepaired spontaneous chromosome breaks that occur in these mutants.     

Mutagenicity of dimethylnitrosamine and diethylnitrosamine for Saccharomyces in an in vitro hydroxylation system

Summary Dimethylnitrosamine (DMN) and diethylnitrosamine (DEN), two potent carcinogens, apparently require conversion to metabolites for their activity. Breakdown products of DMN and DEN obtained in the Udenfriend hydroxylation medium [J. biol. Chem. 208, 731 (1954)] induced cytoplasmic petite mutants and genic canavanine-resistant mutants in Saccharomyces cerevisiae. After 5 hours in hydroxylation medium, DMN treatment resulted in the induction of over 90% petite mutants and DEN treatment in nearly 50%. DMN, after 6 hours in hydroxylation medium, approximately doubled the frequency of canavanineresistant mutants; after DEN treatment, the frequency was 5–6 times higher than the spontaneous frequency. Dimethylamine and diethylamine, which lack the nitroso group of the nitrosamines, did not induce mutants under any of the test conditions.Presented at the 2nd Annual Meeting of the Environmental Mutagen Society, Washington, D. C., March 21–24, 1971.     

Micronucleus test and bone-marrow chromosome analysis: A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations

The sensitivities of 2 cytogenetic tests, chromosome analysis and the micronucleus test, were compared by using mice exposed to the substances methyl methanesulfonate (MMS), mitomycin C (MC) and procarbazine (Natulan®). The lowest dose at which a significant effect could be observed in bone-marrow cells of mice was determined. Both test systems proved equally sensitive for MC and procarbazine. Doses as low as 0.16 mg of MC per kg and 3.12 mg of Natulan® per kg significantly increased both the aberration rates and the micronucleus rates above those of the controls. In contrast, after exposure to MMS, chromosomal aberrations were elevated above control levels at 5 mg/kg, and the micronucleus rate differed significantly from that of the controls after a dose of 10 mg/kg. With the present protocol and sample size one can conclude that the micronucleus test is generally comparable in sensitivity to the chromosome analysis. However, the MMS data indicate that there might be chemicals for which the resolution of the chromosome analysis is higher.

When the mutagens were given in 2 single i.p. injections separated by 24 h, the polychromatic erythrocytes were analyzed for the presence of micronuclei 6 or 24 h after the second injection. The double treatment did not increase the micronucleus rates above the single-treatment results at either sampling interval.     

Mutant alleles of the meiotic locus, mei-9, differ in degree of effects on rod chromosome magnification and ring chromosome transmission in Drosophila

Two mutant alleles of the meiotic locus, mei-9, have been examined for their effect on magnification of a rod Xbb chromosome and transmission of a ring Xbb chromosome under magnifying conditions. Our results indicate that the effects of these two mutations are allele-specific: mei-9a strongly inhibits both rod chromosome magnification and ring chromosome loss under magnifying conditions, while mei-9b has a smaller inhibitory effect on rod chromosome magnification and on the transmission of ring chromosomes under magnifying conditions. These observations can be explained by a difference in leakiness between the two alleles. Our results demonstrate that mutants defective in excision repair and repair replication inhibit ribosomal gene magnification. This suggests that a component of the excision repair pathway is involved in the process of magnification.     

An analysis of frequencies of chromosome configurations in wheat and wheat hybrids

Presynaptic association of homologous chromosomes is a prerequisite to the sequence of events that lead to chiasma formation. This association of homologous chromosomes, as entire units, occurs with probability a, and chiasma formation occurs independently in opposite chromosome arms with probability c. a and c have been estimated from frequencies of different chromosome configurations at metaphase I of euhexaploid wheat and several derived lines. In the euploid, a is essentially unity and c is of the order of 95%. All changes in the aneuploidy studied involved c rather than a, whereas the change induced by colchicine application primarily involved a.—Observed and expected frequencies of configurations were compared in wheat hybrids in which only homoeologues were present. The expected frequencies of configurations were estimated from the data, based on a being unity for entire groups of homoeologues and c being the probability of chiasma formation between random homoeologous arms. Observed and expected frequences of configurations were in general agreement; however, an excess of observed closed bivalents at the expense of multivalents is interpreted to mean that not all homoeologues are effectively associated in all cells.—In euhexaploid wheat, we suggest that homologues associate with almost certainty, whereas homoeologous pairs of chromosomes associate less efficiently. The aneuploidy examined in this study does not appear to affect the association of chromosomes, but rather the number of chiasmata that eventuate and, in the case of deficiency of chromosome 5B, the distribution of chiasmata within homoelogues, perhaps by way of rendering sites for chiasma formation of homoelogues more similar.