Isolation of Nuclei from Sea Urchin Eggs and Embryos

Eggs of the sea urchin Arbacia punctulata were collected by the electroshock method (1), washed in artificial seawater, and sedimented by centrifugation at 500 g for 3 min . Embryos were obtained by fertilizing eggs in artificial seawater with a drop of fresh dilute sperm suspension . Only those cultures in which fertilization was 95-1007 effective were used for the isolation of nuclei. The sedimented cells were resuspended in 9 volumes of a 1 M sucrose solution containing 2% (w/v) of the nonionic detergent Triton X-100 and 2 MM MgCI2. Subsequent steps were monitored by light microscopy. The suspension was homogenized in a Potter-Elvehjem homogenizer with 15 slow upand-down strokes of a loose Teflon pestle . The homogenate contained cytoplasmic debris, occasional unbroken cells, and fragments of fertilization membrane when unhatched embryos were used . The homogenate was next centrifuged at 2000 g for 30 min, the supernatant discarded, and the detergent wash repeated . The sediment, containing nuclei and membranes, was then suspended in 3 volumes of 1 .4 mI sucrose, and layered on a discontinuous gradient composed of 1 ml of 2.5 mI sucrose layered on the bottom, 1 ml of 2 .2 mI sucrose in the middle layer, and 1 ml of 1 .9 M sucrose on the top . The preparation was then centrifuged at 25,000 rpm for 45 min at 4°C in a Spinco SW-39 swinging bucket rotor . The layers containing lysed cytoplasm were carefully removed from the top, with a pasteur pipette .