In all plants, one of the isoprotein variants of histone H3, a packaging protein for genes in chromatin, appears conserved. It is not, as most histones, synthesized only when cell DNA is doubled. Such a continuously synthesized histone H3 form is known in mammals and birds as a Replacement Variant because it replaces DNA packaging whenever chromatin packaging units, nucleosomes, have been lost. This can occur when polymerases transcribe or replicate genes. Nucleosome re-assembly uses available histones and these are the Replacement histones in cells not involved in DNA replication. These histones can be different in structure and this allows one to follow them by gel electrophoresis and high performance liquid chromatography. This project studies, in alfalfa and Arabidopsis, the expression in the cell cycle of the first core histone variant in plants to test the predictions of a function as Replacement Variant. RNA is measured by variant specific RT-PCR and protein by histone labeling and purification. Is its high level of acetylation, a modification of histones in transcriptionally active chromatin, the result of preferential assembly on active genes? Does histone turnover vary with chromatin conformation? Possible modulation of expression by development and environment (osmolarity, heat, phytohormones) will be evaluated. Histones are a major class of DNA-associated proteins which are involved in the "packaging" of DNA into chromatin, particularly into the nucleosome structure which represents the "bead" of the "bead on a string" description of chromatin organization. This project will elucidate the role of a putative Replacement Variant histone which is synthesized throughout the cell cycle and serves to package pre-existing DNA, unlike other histone variants which are synthesized during the period of DNA synthesis and serve to package the newly synthesized DNA prior to mitosis.
