The long-term objectives of this project are to elucidate the organization, function and evolution of repeated genes in the ciliated protozoan, Tetrahymena thermophila. The total number of histone proteins and of different histone genes in Tetrahymena is sufficiently small that a complete characterization of this mutigene family is feasible. The H1 type histones of macro- and micronuclei are different and there are two macronuclear-specific core histone variants. Using gene-specific cloned probes, we shall test the hypothesis that the nuclear-specific localization of histones is due to expression of these genes only at the stage of the cell cycle during which the appropriate nucleus is replicated. There is also differential regulation of multiple mRNAs for the same histone (H3 and H4) in growing and starved cells. In attempts to elucidate the mechanism(s) underlying the growth-dependent regulation of histone mRNAs and the putative cell-cycle specific regulation of genes for macro- and micronuclear histones, we shall attempt to develop an in vitro transcription system in which the expression of normal and modified histone genes can be studied. The macronuclear-specific histone variant, hv1, is evolutionarily conserved and appears to be preferentially associated with active genes. We propose to test this hypothesis by using an antibody to hv1 as an affinity probe for the DNA sequences to which hv1 is bound in Tetrahymena macronuclei and by characterizing the effects of the antibody on a nuclear run-on transcription system. We will also perform in situ immunocytochemistry to localize hv1 in nuclei at the EM level. We have cloned a portion of the yeast gene coding for hv1 using and antibody against Tetrahymena hv1 and a yeast (genomic) expression library in Lambdagt11. This antigenically active portion of the yeast gene will be subcloned, sequenced and used as a probe to isolate the complete gene from yeast, Tetrahymena and Drosophila. These genes will be sequenced and their organizations in the respective genomes will be studied. In yeast, we will determine if the hv1 gene is an essential gene. If it is, in vitro mutagenesis will be used to dissect the important parts of the molecule.
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