A GENETIC SYSTEM TO DETECT MITOTIC RECOMBINATION BETWEEN REPEATED CHROMOSOMAL SEQUENCES IN DROSOPHILA SCHNEIDER LINE-2 CELLS

In order to study mitotic homologous recombination in somatic Drosophi la melanogaster cells in vitro and to learn more on the question how r ecombination is influenced by mutagens, a genetic system was developed where spontaneous and drug-induced recombination could be monitored. Two recombination reporter substrates were stably introduced in multip le copies into the genome of established D. melanogaster Schneider lin e 2 cells: one plasmid (pSB310) contained the 5' and 3' deleted neomyc in phosphoribosyltransferase alleles neoL and neoR as direct repeats; the other (pSB485) contained similar deletions (lacZL and lacZR) of th e beta-galactosidase gene (lacZ). Restoration of a functional neo gene upon mitotic recombination between homologous sequences allowed direc t selection for the event, whereas recombination in single cells harbo uring the integrated lacZ-based reporter plasmid was detected by histo chemical staining or flow cytometric analysis (FAGS). The neo-based co nstruct in the clonal transgenic cell line 44CD4 showed a spontaneous recombination frequency of 2.9 X 10(-4), whereas the 485AD1 cell line harbouring the lacZ-based construct exhibited a frequency of 2.8 X 10( -4). The alkylating agents EMS and MMS and the clastogen mitomycin C w ere able to induce recombination in the 485AD1 cell line in a dose-dep endent manner. The results obtained from these studies suggest that th e transgenic cell lines are potentially useful tools for identifying a gents which stimulate direct repeat recombination in somatic Drosophil a cells. (C) 1997 Elsevier Science B.V.