During early embryogenesis cells become determined to differentiate along specialized pathways. The molecular events underlying these processes are not understood. The long-term objective of this project is to obtain an understanding of the steps involved in nuclear determination and differentiation. To this end, specific attention is being paid to changes in histone composition, histone modifications and chromatin structure in specific nuclei undergoing divergent pathways of differentiation. Conjugation in Tetrahymena will be used as a model to study nuclear determination and differentiation during development. In this system, nuclei derived from daughter products of a single mitotic division differentiate into distinctly different products, macro-and micronuclei depending upon their location within the cell. In many ways these events are directly analogous to cellular determination and differentiation which characterize embryogenesis of higher animals. A variety of biochemical, cytological, immunological and micromanipulation approaches will be used to 1) further define the histone composition, histone modification and chromatin structure in synchronous populations of developing nuclei isolated from conjugating cells as well as nuclei in various stages of the cell cycle; 2) fractionate, purify and characterize enzymes which play a role in the histone remodeling process; 3) purify, characterize and compare the linker histones in micro- and macronuclei and study their effects on chromatin higher order structures; 4) generate and use antibodies to specific histones as probes in projects which include protein purification, protein structure-function relationships, chromatin structure and fractionation, cloning of Tetrahymena histone genes as well as immunofluorescent analyses with conjugating cells; and 5) microinject molecules into and micromanipulate nuclei within living cells to ask questions concerning the targeting of histones to the proper nuclear compartment, the consequences on histone metabolism and function by perturbation with specific antibody molecules and the role that cytoplasm plays in directing nuclear determination and differentiation. Understanding these mechanisms in normal development is essential if we are to understand cases of abnormal development, some congenital diseases and certain pathological situations such as neoplastic transformation.
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