EVIDENCE FOR A DNA HOMOLOGOUS PAIRING ACTIVITY IN NUCLEAR EXTRACTS FROM MOSQUITO CELLS

Using a sensitive homologous pairing/DNA strand transfer assay, we det ected formation of joint molecules in the presence of nuclear extract from cultured mosquito C7-10 cells in a reaction containing single str anded circular m13 DNA and a linear, double stranded DNA 5'-end-labele d on the strand complementary to a portion of the single-stranded subs trate, Joint molecules were detected by the reduced electrophoretic mo bility of labeled probe on agarose gels, which indicated that the 5'-e nd labeled strand of the linear duplex had formed a hybrid with the si ngle-stranded substrate, Characterization of the activity detected ini tially in crude nuclear extracts provided a basis for a 5-fold enrichm ent of activity after a two-step KCl elution from heparin-Sepharose. F urther purification by preparative electrophoresis yielded a band at a pproximately 35 kDa, which, when transferred to Immobilon P membrane, specifically bound the labeled, complementary strand probe, Optimal ac tivity of the electroeluted enzyme required both magnesium and ATP and was sensitive to the ratio of single-stranded and double-stranded DNA substrate and to the amount of protein, This homologous pairing activ ity from mosquito cells is the first such activity to be described fro m an insect other than Drosophila melanogaster. Copyright (C) 1996 Els evier Science Ltd.