The two H2A histone species H2A.X and H2A.Z have been conserved as separate H2A species throughout evolution, but how the function of these two proteins differs from that of the other H2A species is unknown. Understanding the function of these two H2A species may lead to new insights into function of chromatin. The following studies are in progress this year to elucidate the different roles of the H2A species. (l) We have developed procedures for expressing the three human H2A histones in bacteria. The purposes of these studies are to prepare enough H2A.X and H2A.Z to use in various structural and functional assays. Preliminary results suggest that H2A.Z has a different affinity for forming the H2A-H2B dimer, the first level of H2A interaction. X-ray crystallographic studies of the octamer with H2A.X and H2A.Z replacing H2A.1 are planned in collaboration with Prof. Moudrianakis. (2) In collaboration with Dr. Randall Morse, we are trying to express the three human H2A species in yeast to see if any of the three can replace the yeast H2A in its chromosomes. Preliminary results suggest that none of the human H2A species can replace yeast H2A; these results are interesting since Tetrahymena H2A is able to replace yeast H2A. (3) In order to determine if mammalian cells can utilize his tone proteins with altered sequences, we prepared a gene for such a protein. The gene was expressed in HeLa cells with the altered H2A accounting for about 10% of the total H2A complement. These studies open the possibility of using histones tagged with a monoclonal antibody epitope or other type of affinity site to isolate specific subpopulations of nucleosomes by affinity methods. (4) We are continuing to study the specific Ca+ -activated phosphorylation of H2A.X in mammalian cells. Using recombinant H2A.X, which is a good substrate for the enzyme, we have shown that the kinase requires the Ser-Gln motif with the Ser four residues away from the C-terminal. The phosphorylation appears to be induced under conditions of stress.