It has been generally accepted that yeast Saccharomyces cerevisiae do not contain H1 histones, the histones which are involved in the condensation of the nucleosome fibre in higher eucaryotes. We discovered, by carrying out functional assays for HI histones, that yeast chromatin consists of chromatosomes, the nucleosomes containing H1 histones and 168 base pair (bp) DNA. Furthermore, the H1-like activity can be extracted by 0.6 M NaC1, and chromatosomes can be assembled from H1-depleted chromatin in the presence of the 0.6 M extract. In this application, we propose to investigate the structure and function of the H1 histones in yeast with the use of genetics and biochemical approaches. We will purify the yeast H1 histones from the 0.6 M NaC1 extract and clone and sequence the H1 gene(s). The deduced amino acid sequence will be compared with those of mammalian H1 histones, and the similarity and differences in the structure of H1 will be determined. The H1 gene in the yeast chromosome will be inactivated by replacement of the H1 gene with marker genes, and the biological function of H1 histones in yeast will be investigated by studies on the effects of depletion of H1 histones on the cell cycle, gene transcription, and chromatin structure. The role of H1 in the condensation of yeast nucleosome fibre will be investigated by observation of ionic strength-dependent folding of nucleosome fibre under an electron microscope, and the regions of the H1 important in the condensation of nucleosome fibre will be investigated by partial deletions of the H1 gene. The phosphorylation of Hi during the cell cycle will be investigated. It will be determined if Hi is indeed the substrate for the cell cycle control protein kinase (CDC28) in vivo as others have suggested.
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