Analysis of the visible mutations induced by x-rays and ethyl methanesulfonate in the mature spermatozoa of Drosophila melanogaster

The recessive visible mutations spectrum of chromosome II induced by X-rays and ethylmethanesulfonata (EMS) in mature Drosophila melanogaster spermatozoa has been studied. Treatment of both mutagens resulted in mutations in all 5 genes in stock mei-9LI and only 4--in D-32. The comparison of mutation frequencies of the same genes in two stocks under EMS-treatment demonstrated the statistical difference of mutation frequencies j, pr, cn of two stocks, genes b and vg did not differ. Under the influence of X-rays the differencies have been observed only for gene b. In stock D-32 the mutation frequency differes from the control for b and vg (EMS treatment) and j, pr, vg (under the action of X-rays), in mei-9LI--the mutation frequency of all 5 genes (under the X-rays) and 4 of 5 genes (EMS treatment).     

Sex-linked lethal mutations induced in Drosophila melanogaster by 7,12-dimethylbenz[a]anthracene

One of the most potent carcinogens, 7,12-dimethylbenz[a]anthracene (DMBA), was tested for the induction of mutations in 2 strains of Drosophila melanogaster. Larvae were fed mixtures containing DMBA, peanut oil and solubilizing agents in darkness. After emergence the males were mated with Basc or FM7a females to test for sex-linked lethals. For Canton-S males, all DMBA treatments produced highly significant increases in mutation frequencies over controls. DMBA was slightly mutagenic for Oregon-R males.     

Increased removal of 3-alkyladenine reduces the frequencies of hprt mutations induced by methyl- and ethylmethanesulfonate in Chinese hamster fibroblast cells.

We have previously reported the isolation of mammalian cell lines expressing the 3-methyladenine DNA glycosylase I (tag) gene from E. coli. These cells are 2-5 fold more resistant to the toxic effects of methylating agents than normal cells (15). Kinetic measurements of 3-methyladenine removal from the genome in situ show a moderate (3-fold) increase in Tag expressing cells relative to normal as compared to a high (50-fold) increase in exogenous alkylated DNA in vitro by cell extracts. Excision of 7-methylguanine is as expected, unaffected by the tag+ gene expression. The frequency of mutations formed in the hypoxanthine phosphoribosyl transferase (hprt) locus was investigated after methylmethanesulfonate (MMS), ethylmethanesulfonate (EMS), N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) exposure. Tag expression reduced the frequency of MMS and EMS induced mutations to about half the normal rate, whereas the mutation frequency in cells exposed to NMU or NEU is not affected by the tag+ gene expression. These results indicate that after exposure to compounds which produce predominantly N-alkylations in DNA, a substantial proportion of the mutations induced is formed at 3-alkyladenine residues in DNA.     

Genetic activity of chemicals in yeast: DNA alterations and mutations induced by alkylating anti-cancer agents.

The simple eukaryotic organism baker's yeast allows demonstration of primary DNA lesions in parallel with measurement of mutagenicity and lethality after treatment with alkylating chemicals. Several anti-cancer drugs formed cross-linked DNA molecules and were genetically active. The mutagenicity and lethality of these drugs varied substantially and were dependent on the function of some processes of DNA dark-repair.     

Retention of induced mutations in a Drosophila reverse-genetic resource

Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.     

Y-Linked male sterile mutations induced by P element in Drosophila melanogaster.

The Y chromosome in Drosophila melanogaster is composed of highly repetitive sequences and is essential only in the male germ line. We employed P-element insertional mutagenesis to induce male sterile mutations in the Y chromosome. By using a combination of two modifiers of position effect variegation, adding an extra Y chromosome and increasing temperature, we isolated 61 P(ry+) elements in the Y chromosome. Six of these Y-linked insertions (approximately 10%) induced male sterile mutations that are mapped to two genes on the long and one on the short arms of the Y chromosome. These mutations are revertible to the wild type in a cell-autonomous and germ-line-dependent manner, consistent with previously defined Y-linked gene functions. Phenotypes associated with these P-induced mutations are similar to those resulting from deletions of the Y chromosome regions corresponding to the male fertility genes. Three alleles of the kl-3 gene on the Y long arm result in loss of the axonemal outer dynein arms in the spermatid tail, while three ks-2 alleles on the Y short arm induce defects at early postmeiotic stages. The recovery of the ms(Y) mutations induced by single P-element insertions will facilitate our effort to understand the structural and functional properties of the Y chromosome.     

Embryonic and post-embryonic lethals induced by diethyl sulfate in mature sperm of Drosophila melanogaster.

It has recently been reported that, in Drosophila melanogaster, when sperm treated with diethyl sulfate was stored in the females, II–III translocations were detected as from the 6th day after the treatment, though none was recovered without storage. Chromosome breaks being currently considered the main cause of dominant lethality and the embryonic period lasting about one day at 25°C, it was thought of interest to study the ability of DES to induce this type of damage with and without storage. It was found that the treatment increased embryonic lethality (measured as frequency of unhatched eggs) and post-embryonic lethality (measured as frequency of larval and pupal death) over the control values. The frequency of embryonic lethals after storage in the females for 6 days was similar to that shown by the unstored samples. In contrast with this, the yield of post-embryonic lethality was markedly raised by that storage time. It is suggested that: (1) lesions are induced as “pre-breaks”, and storage and cell divisions are instrumental in their opening; (2) potential breaks can undergo DNA replication and cell division as such and become open in different cell cycles, impairing embryonic and post-embryonic development; (3) chromosome breaks induced by DES seem to behave in a way similar to those induced by other mono- and poly-functional alkylating agents; and (4) when the potential ability of chemical compounds to induce chromosome breaks is assessed, post-embryonic lethality can be used as a simple one-generation preliminary test, to establish delayed effects.     

Comparison of frequencies of complete and incomplete translocations induced in mature sperm of Drosophila]

A frequency of induction of translocations taking place in mature sperm of Drosophila between heterochromatic portions of the 2L compound and 2R acrocentric was estimated to be 9.62 X 10(-3). This value is being compared with the previously estimated frequency of incomplete translocations in this system which is (2.5-2.9) X 10(-3). The ratio of frequencies of complete and incomplete translocations obtained was intermediate between the Panshin data, on the one hand, and those of Muller and Hershkowitz, on the other. The reasons for these discrepancies are under discussion. Based on the value obtained for this ratio, the probability of reunion of chromosome break (q) is calculated to be 0.79.     

Analysis of brain morphological changes induced by ethyl methanesulfonate in Drosophila melanogaster

Under the influence of ethyl methanesulfonate the series of both morphological and structural mutants with different types of brain changes has been obtained in Drosophila melanogaster Oregon R strain. In the future, this collection of mutants will be used in the investigations of genetic control of brain degeneration and possible ways of brain regeneration.     

Unstable visible mutations induced in Drosophila melanogaster by injections of oncogenic virus DNA into the polar plasm of early embryos

Summary When RSV DNA cloned in pBR 322 or DNA of simian adenovirus Sa7 (C8) is injected into the pole plasm of embryos of various Drosophila stocks, the progeny of 1–70% of the surviving flies display visible mutations. The mutagenesis is partially directed: the loci mutating due to retrovirus and adenovirus DNA do not everlap. The majority of resulting mutants are characterised by high instability: reversions and new mutations occur in them, which sometimes spread over the whole population(explosive instability). The injected sequences are revealed by dot-hybridization in the DNA of many mutant strains, but only rarely by Southern blotting procedures. The results show that the microinjection of oncovirus DNA into embryos is an approach for obtaining highly unstable strains even from wildtype stable Drosophila stocks without crosses with MR lines or the introduction of P elements. The sets of unstable mutations induced by oncovirus DNA is different from those in hybrid dysgenesis.     

Transposition and reversion of mutations induced in Drosophila by viral DNA

Injection of solutions of highly polymerized DNA isolated from nuclear polyhedrosis virus of Galleria mellonella into adult males induced with a considerable frequency visible mutations, two of which were studied in detail. They were detected in about 30 000 flies in the progeny of treated males. Much less than Notched-wings much greater than (Ndw, chromosome 3, location 87.9, dominant) independently arose 12 times, much less than thickened-veins much greater than (thi, chromosome 2, location 71.4, recessive) independently arose 7 times. No mutations were detected in the control of the same size. It was found that both Ndw and thi mutations gave frequent transpositions and reversions in mature, immature germ cells and in somatic cells, in latter cases leading to mosaicism. These results demonstrate for the first time that mutations induced by exogenous DNA are capable of transposition and reversion.     

Restriction fragment pattern analysis of HPRT mutations induced in rat-liver epithelial cells by alkylating and arylating agents.

Structural alterations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in genomic DNA of adult rat-liver (ARL) epithelial cells that were mutated by alkylating and arylating mutagens were studied by restriction enzyme fragment pattern (RFP) analysis. ARL cells were mutated with the direct-acting alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the activation-dependent arylating agents 7,12-dimethylbenz[a]anthracene (DMBA) and N-2-acetylaminofluorene (AAF). Alterations in the HPRT gene of at least 10 independent 6-thioguanine-resistant (TGr) clones mutated by each chemical were analyzed using 8 different restriction endonucleases; Hind III, EcoRI, BamHI, XbaI, Hae III, XhoI, MspI and PstI, and a full-length HPRT cDNA as a probe in molecular hybridization. Among the 10 MNNG-induced mutants, the RFPs obtained with most endonucleases displayed no changes, while an altered RFP was found in only one mutant using XbaI. None of the 10 DMBA-induced mutants displayed altered RFPs. Restriction analysis of the 10 AAF-induced mutants showed no abnormality in HPRT gene structure in most restriction digests, while altered RFPs were detected in one mutant using MspI and in two mutants with XbaI digestion. Overall, the studies reveal an absence of major DNA sequence changes in 26 of 30 induced mutants although the mutant phenotype of 4 of the TGr clones can be attributed to gross chromosomal changes or a point mutation at the restriction site. The absence of detectable alterations in the RFPs of the majority of the mutants is strongly suggestive of base substitution as the major molecular alteration underlying the mutant phenotype. The HPRT activity of 14 of 30 mutants was at least 5% of the wild-type level, which is consistent with a structural alteration in the gene product expressed as partial activity of the enzyme. Therefore, the data are interpreted as indicating that in the ARL cells, all 3 mutagens induced primarily localized alterations in base sequences in the HPRT gene together with a few mutations involving large sequence changes.