SEA-URCHIN ZYGOTE CHROMATIN EXHIBIT AN UNFOLDED NUCLEOSOMAL ARRAY DURING THE FIRST S-PHASE

To investigate changes in chromatin organization associated with DNA r eplication during the first stages of development of the sea urchin Te trapygus niger, we compared micrococcal nuclease (MNase) digestion pat terns of chromatin from zygotes harvested during the first S phase and from unfertilized eggs. We observed that the majority of DNA fragment s derived from MNase digested zygote nuclei were similar to or smaller than a mononucleosome, while those derived from unfertilized egg nucl ei were larger (1,500 to 410 bp). This result indicates that in zygote s, where active DNA replication is occurring, the major chromatin frac tion is represented as unfolded nucleosomes. In contrast, in unfertili zed eggs chromatin appears to be organized into polynucleosomes. To de termine if the unfolded structure of nucleosomes observed during S pha se is related to the level of poly (ADP-ribosylation) of cleavage stag e (CS) histone variants, zygotes were treated with 20 mM 3-Amino Benza mide (3 ABA) during the interval between 3 and 30 min post-inseminatio n (p.i.). This treatment with 3 ABA decreases the poly (ADP-ribosylati on) of CS histone variants and inhibits the first S phase in zygotes [ Imschenetzky et al. (1991): J Cell Biochem 46:234-241; Imschenetzky et al. (1993): J Cell Biochem 51:198-205]. When the MNase digested patte rns of chromatin from these 3 ABA treated and control zygotes were com pared, we found that the unfolded structure of the nucleosomes remains unaltered by the inhibition of the poly(ADP-ribose) synthetase with 3 ABA. This result indicates that the unfolded nucleosomal structure, p articular to the chromatin of S phase zygotes, is not contemporaneous to DNA replication and is independent of the normal level of poly(ADP- ribosylation) of CS histone variants. (C) 1995 Wiley-Liss, Inc.