ABSTRACT
DNA was prepared from sperm of Clypeaster japonicus and Echino-cardium cordatum: acetone dried spermatozoa were suspended in a solution of 0.1 M EDTA, 4 mM Tris, pH 8.2, and 1% SDS, and treated overnight with Pronase P at 37°C. The digest was shaken several times with a chloroform-octanol mixture and DNA was precipitated by 2 volumes of ethanol. Prepared DNA was sheared in a Virtis 60 homogenizer to short fragments. Fragmented DNA was denatured at 100°C for 5 min and then reassociated in 0.12 M or 0.4 M phosphate buffer, pH 6.8, containing 2 mM EDTA and 0.1% SDS at 60°C or 64°C, respectively. Renatured double-stranded DNA fragments were separated from non-renatured single-stranded fragments by absorption on an hydroxyapatite column. After elution, their concentrations were optically measured. By the analyses of Cot curves obtained, the fold back component of Clypeaster DNA (330 nucleotide long) was found to be 0.08 and those of Echinocardium DNA (370 nucleotide long) was 0.02. Fractions of the fast and slow repetitive, and single copy compongnts were 0.20 (K = 0.66 M−1 .S−1 ), 0.20 (0.043) and 0.46 (0.0013) in Clypeaster DNA, and 0.25 (55), 0.25 (0.22) and 0.39 (0.0017) in Echinocardium DNA, respectively. The single copy components (Cot 102 - 104) were separated, labeled with 3H-thymidine triphosphate by a nick translation reaction and reassociated or hybridized with Clypeaster (Clypeasteroid), Echinocardium (Spatangoida) and Hemi centrotus pulcherrimus (Echino-idea) DNA fragments. The labeled DNA was reassociated more than 80% with the DNA of the same species, but practically no hybridization was observed with DNA of different sea urchin species belonging to different orders.
