A PLASMID SYSTEM TO MONITOR GENE CONVERSION AND RECIPROCAL RECOMBINATION INVITRO

A plasmid system allowing for the detection of recombinagenic activiti es in cell-free extracts is described. Two truncated alleles of the ba cterial neomycin resistance gene (neo), differing from each other at a polymorphic restriction site, were constructed. Recombinations involv ing both alleles mediated by Drosophila embryo nuclear protein extract s or Drosophila larva whole cell protein extracts were selected by the ir ability to confer kanamycin resistance to E. coli. Restriction anal ysis of plasmids recovered from E. coli transformants allowed the moni toring of the two molecular mechanisms which can lead to functional ne o genes, gene conversion and reciprocal recombination. A dose dependen t increase in the recombination frequency with increasing amounts of c ell extract was observed. Recombination was further increased by linea rizing one of the two substrate plasmids. The Drosophila cell extracts catalyzed recombination in vitro since after incubation a recombinati on product could be identified by polymerase chain reaction (PCR) tech nology. The recombination was absolutely dependent on the presence of an active cell extract, since no diagnostic PCR product was detected i n a reaction where extract was omitted. Analysis of a representative n umber of recombinant plasmids by restriction analysis revealed that in the absence of an exogenous recombinational system less than 2% of ka namycin resistant recombinant plasmids occurred by gene conversion upo n transformation into E. coli. In contrast, recombinants exhibiting re striction patterns diagnostic for gene conversion were observed at fre quencies between 5.1% and 9.8% after incubation with Drosophila larva cell extracts. These results strongly argued that gene conversion is a prominent mechanism of recombination in Drosophila mitotic cells.