AMOUNT, LOCATION, PRIMING CAPACITY, CIRCULARITY AND OTHER PROPERTIES OF CYTOPLASMIC DNA IN SEA URCHIN EGGS

Abstract

Values of 8.26 [plus or minus]0.30 pg. (9.5 x haploid) tor Lytechinus pictus and 3.30[plus or minus]0.25 pg. (4.3 x haploid) for Strongylocentrotus purpuratus have been obtained for the DNA content/egg of these sea urchins. Methods included repeated CsCl-buoyant density centrifugations, digestion of interfering polysaccharide and monitoring of the procedures with added radioactively labeled DNA. Mitochondrial and yolk fractions of differentially centrifuged homogenates of L. pictus eggs contain the bulk of the cytoplasmic DNA, in a ratio of about 3:1. Since yolk spherules are ca. 1/3 as numerous as mitochondria, the amount of DNA is estimated to be the same/particle; namely, 2.3 x 10-1 grams. Evidence is presented that the cytoplasmic DNA can serve as primer in a DNA-dependent RNA-synthesizlng system with about the same activity as nuclear DNA. The cytoplasmic DNA of L. pictus eggs shows a buoyant density in CsCl of 1.703 gm./cm.3 as compared with 1.693 for the nuclear. A 3rd nucleic acid band, equal in amount to the nuclear, has been found at a density of 1.719. Melting temperatures in standard saline-citrate are 84.0[degree]C for sperm and gastrula DNA and 86.8[degree]C for whole-egg DNA, indicating a guanine-cytosine content of 35% and 42%, respectively. These are similar to the values (34% and 44%) calculated from the buoyant densities. Electron microscopic observations of DNA prepared from mitochondria of L. pictus show almost exclusively circles of about 4.45/i in circumference. It is estimated that there are 1 or 2 such circular filaments of double-stranded DNA/mitochondrion or yolk particle. Centrifugation of egg DNA in preformed CsCl gradients reveals 2 main components with sedimentation coefficients of ca. 23S and 28S indicative of the 2 forms of circular DNA.