Ectopic Mitotic Recombination in Drosophila Probed with Bacterial  -Galactosidase Gene-Based Reporter Transgenes

Abstract

Plasmids were constructed to investigate homologous mitotic recombination in Drosophila cells. Heteroalleles containing truncated but overlapping segments of the bacterial beta-galactosidase gene (lacZ) were positioned either on separate plasmids or as direct repeats on the same chromosome. Recombination reconstituted a functional lacZgene leading to expression of LacZ+activity detectable by histochemical staining. High extrachromosomal recombination (ECR) frequencies between unlinked heteroalleles were observed upon transient co-transfection into Drosophila melanogaster Schneider line 2 (S2) cells. Stably transfected cells containing the lacZ heteroalleles linked on a chromosome exhibited intrachromosomal recombination (ICR) frequencies two orders of magnitude lower than ECR frequencies. Recombination was inducible by exposing the cells to ethyl methanesulphonate or mitomycin C. Recombination products were characterized by multiplex PCR analysis and unequal sister chromatid recombination was found as the predominant mechanism reconstituting the lacZ gene. To investigate recombination in vivo imaginal disc cells from transgenic larvae carrying the reporter gene on the X chromosome were isolated and stained for LacZ+ activity. The presence of a few LacZ+ clones indicated that mitotic recombination events occurred at frequencies two orders of magnitude lower than the corresponding event in cultured cells and late during larval development.

Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms

“…These assays are most commonly based on the reconstitution of a functional reporter gene by one of the DSB repair mechanisms after the induction of a break in a non-functional substrate. Many such assays were constructed to investigate NHEJ and HR in various types of cells ( 4 , 12 – 19 ), and recently also MMEJ ( 20 – 22 ). However, with a growing number of new studies proposing the occurrence of mechanistically distinct MMEJ pathways, the need arises for more specific assays that may allow to better distinguish these mechanisms.…”