Fig. 1. RP-A foci form on restriction-enzyme-treated sperm pronuclei. Immunofluorescence with affinity-purified anti-RP-A70 antibodies on intact and restriction-enzyme-digested sperm pronuclei. Demembranated Xenopus sperm heads were pre-incubated for 20 minutes, either with a buffer (no addition) or with 0.05 units of each of the restriction enzymes indicated (EcoRI, DraI, PstI) and then Xenopus egg HSSs were added for 1 hour at 23°C. (B) Immunofluorescence with affinity-purified anti-RP-A70 antibodies on sperm heads preincubated with different amounts of EcoRI (0.01 units, 0.05 units, 1 unit) or with 1 unit of the rare cutting endonuclease Not1 prior to addition of HSS extracts as in (A). (C) Immunofluorescence with purified anti-RP-A70 antibodies on sperm heads pre-incubated with 0.02 units of EcoRI and then mixed with HSS and put at 4°C for 1 hour (4°C), mixed with HSS containing 5 mM AMP-PNP and incubated at 23°C for 1 hour (AMP-PNP), incubated for 1 hour with HSS at 23°C, then 40 µg ml-1 ds or ssDNA was added and incubation prolonged for an additional 30 minutes (+dsDNA, +ssDNA). The control figure (no addition) is also documented. Cy3-conjugated donkey anti-rabbit was used as a secondary antibody. (A-C) represent each a cumulative image of 10-12 confocal sections. Bars, 10 µm.
