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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD016259-07
Application #
3313546
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1982-09-01
Project End
1990-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
Brownell, J E; Allis, C D (1995) An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei. Proc Natl Acad Sci U S A 92:6364-8
Ward, J G; Davis, M C; Allis, C D et al. (1995) Effects of nullisomic chromosome deficiencies on conjugation events in Tetrahymena thermophila: insufficiency of the parental macronucleus to direct postzygotic development. Genetics 140:989-1005
Sobel, R E; Cook, R G; Perry, C A et al. (1995) Conservation of deposition-related acetylation sites in newly synthesized histones H3 and H4. Proc Natl Acad Sci U S A 92:1237-41
Sobel, R E; Cook, R G; Allis, C D (1994) Non-random acetylation of histone H4 by a cytoplasmic histone acetyltransferase as determined by novel methodology. J Biol Chem 269:18576-82
Madireddi, M T; Davis, M C; Allis, C D (1994) Identification of a novel polypeptide involved in the formation of DNA-containing vesicles during macronuclear development in Tetrahymena. Dev Biol 165:418-31
Stargell, L A; Bowen, J; Dadd, C A et al. (1993) Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila. Genes Dev 7:2641-51
Perry, C A; Allis, C D; Annunziato, A T (1993) Parental nucleosomes segregated to newly replicated chromatin are underacetylated relative to those assembled de novo. Biochemistry 32:13615-23
Perry, C A; Dadd, C A; Allis, C D et al. (1993) Analysis of nucleosome assembly and histone exchange using antibodies specific for acetylated H4. Biochemistry 32:13605-14
Meistrich, M L; Trostle-Weige, P K; Lin, R et al. (1992) Highly acetylated H4 is associated with histone displacement in rat spermatids. Mol Reprod Dev 31:170-81
Davis, M C; Ward, J G; Herrick, G et al. (1992) Programmed nuclear death: apoptotic-like degradation of specific nuclei in conjugating Tetrahymena. Dev Biol 154:419-32

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