This research will focus on investigating the pattern of distribution of histone proteins in nucleosomes during cell growth, replication and transcription, in the broad context of trying to understand how eukaryotic cells maintain the differentiated state. We will ask whether certain histones or other proteins remain associated with DNA that was synthesized concurrently in the same generation, and whether there are special conformations of nucleosomes at each stage in development. To do this we must first succeed in separating chromatins synthesized in different cell generations. The separation should be under conditions which are gentle and preferably do not alter chromatin or require fixing of proteins to the DNA. As discussed below, we have developed methods for fractionation of chromatin that allow us to obtain a nucleosome subfraction that is enriched in cleavage stage histone H2A and depleted in (beta, gamma, and delta) H2A histone. We have also identified a nucleosome subfraction that appears to be enriched in newly replicated DNA. Experiments will be carried out to follow the distribution of cleavage stage (CS) and alpha histone subtypes to daughter chromatin during sea urchin embryogenesis. Cohen et al. (1975) and Newrock et al. (1978) have shown that CS subtypes of histones H1, H2A, and H2B are synthesized from fertilization to about the 100-cell embryo; synthesis of alpha subtypes extends from the third cleavage division through blastula, after which the beta, gamma, and delta subtypes are synthesized. The CS and alpha subtypes still persist in prism-stage chromatin. Their distribution will be studied to determine whether they are randomly distributed or stay with the DNA synthesized at the time of their synthesis.
| Sowers, L C; Shaw, B R; Veigl, M L et al. (1987) DNA base modification: ionized base pairs and mutagenesis. Mutat Res 177:201-18 |
