Using a sensitive homologous pairing/DNA strand transfer assay, we detected formation of joint molecules in the presence of nuclear extract from cultured mosquito C7-10 cells in a reaction containing single stranded circular ml3 DNA and a linear, double stranded DNA 5'-end-labeled on the strand complementary to a portion of the single-stranded substrate. Joint molecules were detected by the reduced electrophoretic mobility of labeled probe on agarose gels, which indicated that the 5'-end labeled strand of the linear duplex had formed a hybrid with the single-stranded substrate. Characterization of the activity detected initially in crude nuclear extracts provided a basis for a 5-fold enrichment of activity after a two-step KCl elution from heparin-Sepharose. Further purification by preparative electrophoresis yielded a band at approximately 35 kDa, which, when transferred to Immobilon P membrane, specifically bound the labeled, complementary strand probe. Optimal activity 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 activity from mosquito cells is the first such activity to be described from an insect other than Drosophila melanogaster.
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- Gene transfer
- RecA protein
- Single strand transferase