The primary objectives of this project are to elucidate how replication-- dependent and replication-independent histone synthesis are interrelated and regulated with regard to the stoichiometry of the various histone subtypes in the nucleosome, and how replication-dependent histone synthesis is regulated with regard to the rate of DNA synthesis. We have characterized two H2A cDNA clones which correspond to the two most highly transcribed human H2A genes found to date. With the four H2A cDNAs we have isolated and three more from GenBank, we analyzed several cell lines for the expression of these seven genes. The replication-linked genes were found to be expressed in parallel in four cell lines. The rate of H2A isoprotein synthesis was also analyzed and found to be close to the amounts of each known mRNA. A manuscript describing these two cDNA's is in preparation. We have evidence suggesting that soluble histone in mammalian cells is bound in a negatively charged complex that sediments at 8-9s. Because histones have been so conserved during evolution, it has not been possible to prepare high affinity antibodies. To determine whether or not it would be possible to incorporate an altered histone protein, perhaps carrying an epitope to a known monoclonal antibody, into chromatin, we altered the gene for histone H2A.la to encode a H2A histone with an extended C-terminal sequence designed so that it would be separable on 2D acrylamide gels. The protein was found to incorporated into chromatin, but in small amounts. A manuscript describing this work is in preparation.
