A novel method for the parallel monitoring of mitotic recombination and clastogenicity in somatic cells in vivo.
Both homologous mitotic recombination (HMR), causing loss of heterozygosity (LOH) of the wild-type allele, and structural chromosome aberrations (CA) involve the formation of double-strand breaks in DNA. Whether the induction of CAs is always accompanied by HMR, or whether there exist DNA lesions specifically forming only one of the two end-points is unknown. Answering this fundamental question requires a system for the parallel detection of CAs and HMR, because only then is their analysis under strictly identical condition (dose, repair, genetic background) possible. We describe here a novel system for the parallel detection of HMR and loss of a whole chromosome as a measure of CA, utilizing somatic cells of Drosophila. In haploid germ cells of Drosophila, loss of a ring-shaped X-chromosome (rX) constitutes a frequent event providing an efficient method for measuring clastogenicity. For somatic cells, however, it was unclear whether the development of such a system would be feasible. The generally accepted notion has been that in XX female genotypes, loss of an entire X-chromosome acts as a cell lethal when generated at or shortly after blastoderm stage. However, here we show that rX-loss, if induced in pre-ommatidia cells of 3rd instar larvae, generates viable clones visible as small white patches in the red compound eye. To set up optimal conditions for the detection and quantification of rX-loss compared to HMR, several protocols were developed and tested against model carcinogens (methyl methanesulfonate, cisplatin and 7,12-dimethylbenz[a]anthracene). Generally, we find striking differences in the efficiency of these carcinogens for recombination when compared with clastogenicity. The cross-linking agent cisplatin is 4- to 6-fold more clastogenic than recombinagenic. 7,12-Dimethylbenz[a]-anthracene, on the contrary, produced less than a doubling effect for rX-loss but was highly active (20-times the background) for HMR. It appears therefore that both processes can be separated from each other. To the best of our knowledge, this is the first report suggesting, in terms of DNA adducts involved, qualitative differences between homologous recombination and clastogenic effects. Application of our system for studies on DNA repair may therefore provide new insight into the linkage of repair pathways in either of the two mechanisms.
Vogel
E W
EW
Department of Radiation Genetics and Chemical Mutagenesis, MGC, Sylvius Laboratories, Leiden University Medical Centre, The Netherlands. vogel@mail.medfac.leidenuniv.nl
Nivard
M J
MJ
D000818:Animals; D002945:Cisplatin / Q000633:toxicity; D002999:Clone Cells / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004330:Drosophila / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D005123:Eye / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development; D005260:Female; D005821:Genetic Techniques; D006579:Heterozygote; D007814:Larva; D008297:Male; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008938:Mitosis; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000633:toxicity*; D011995:Recombination, Genetic; D013489:Suppression, Genetic; D014960:X Chromosome
D009153:Mutagens; D008741:Methyl Methanesulfonate; D002945:Cisplatin