Amount, location, priming capacity, circularity and other properties of cytoplasmic DNA in sea urchin eggs.

Abstract

1. Values of 8.26 ± 0.30 pg. (9.5 χ haploid amount) for Lytechinus pictus and 3.30 ± 0.25 pg. (4.3 χ haploid amount) for Strongylocentrotus purpuratus have been obtained for the DNA content per egg of these sea urchins. The methods involved repeated CsCl-buoyant density centrifugations, digestion of interfering polysaccharide, and monitoring of the procedures with added radioactively labeled DNA. The final determinations were made on material characterized by several criteria as highly purified.2. Mitochondrial (M) and yolk (Y) fractions of differentially centrifuged homogenates of L. pictus eggs contain the bulk of the cytoplasmic DNA. It is uncertain to what extent the smaller variable amount (11.5 to 26.5%) found in the supernatant may be derived from breakdown of M- and Y-particles.3. For distribution between M- and Y-fractions the best present value is considered to be about 3:1. Since yolk spherules are approximately one-third as numerous as mitochondria, the amount of DNA is estimated to be the same per particle, namely, 2.3 χ 10-17 grams.4. Evidence is presented that the cytoplasmic DNA of eggs of L. pictus and S. purpuratus can serve as primer in a DNA-dependent RNA-synthesizing system with approximately the same activity as nuclear DNA.5. The cytoplasmic DNA of L. pictus eggs shows a buoyant density of 1.703 gm./cm.3 ac compared with 1.693 for the nuclear. A third nucleic acid band, equal in amount to the nuclear, has been found at a density of 1.719. The amount of nuclear DNA corresponds to the sperm (haploid) value.6. Melting temperatures in standard saline-citrate are 84.0° C. for sperm and gastrula DNA and 86.8° C. for whole-egg DNA, indicating a guanine-cytosine content calculated from these values as 35% and 42%, respectively. These are similar to the values (34% and 44%) calculated from the buoyant densities.7. Electron microscopic observations of DNA prepared from mitochondria of L. pictus show almost exclusively circles that measure about 4.45 microns in circumference. It is estimated that there are one or two such circular filaments of double-stranded DNA per mitochondrion or yolk particle.8. Centrifugation of egg DNA of L. pictus in preformed CsCl gradients has revealed two main components with sedimentation coefficients of ca. 23S and 28S indicative of the two forms of circular DNA.