This proposal addresses specific mechanistic questions about general biological priniciples utilizing a powerful genetic system. We wish to continue our studies on the """"""""Structure of the Subunits of Eukaryotic Chromosomes"""""""" by focusing attention on non- nucleosomal elements that define the functional boundaries of genes and chromosomal loops. For all of these studies we have selected Saccharomyces cerevisiae as the system of choice. Our experimental approach combines biochemical, cell biological, immunological, genetic, and reverse genetic techniques to address the four following Specific Aims: (1) To determine the relationship between the cis-acting DNA sequences necessary for creating a nuclease hypersensitive region in chromatin at the 5'-end of a gene and for mediating transcriptional initiation. (2) To determine the relationship between the cis-acting DNA sequences necessary for establishing an abrupt transition in chromatin structure at the 3'-end of a gene and for mediating transcriptional termination. (3) To delineate the evolutionarily conserved elements participating in the """"""""chromosomal loop attachment"""""""" of a mouse MAR sequence (""""""""matrix association region"""""""") that has been introduced into yeast. o precise DNA sequences o role of topoisomerase II o other DNA binding proteins o intranuclear location of binding-sites (4) To determine the functions of homologous MAR elements in yeast.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022201-17
Application #
3271012
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1978-05-01
Project End
1993-04-30
Budget Start
1991-05-01
Budget End
1992-04-30
Support Year
17
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Type
Schools of Medicine
DUNS #
City
Dallas
State
TX
Country
United States
Zip Code
75390
Sathyanarayana, U G; Freeman, L A; Lee, M S et al. (1999) RNA polymerase-specific nucleosome disruption by transcription in vivo. J Biol Chem 274:16431-6
Qiu, P; Kupfer, K C; Garrard, W T (1997) A method for genome comparisons and hybridization studies using known megabase-scale DNA sequences as a reference. Genomics 43:307-15
Liang, C P; Garrard, W T (1997) Template topology and transcription: chromatin templates relaxed by localized linearization are transcriptionally active in yeast. Mol Cell Biol 17:2825-34
Fishel, B R; Sperry, A O; Garrard, W T (1993) Yeast calmodulin and a conserved nuclear protein participate in the in vivo binding of a matrix association region. Proc Natl Acad Sci U S A 90:5623-7
Blasquez, V C; Hale, M A; Trevorrow, K W et al. (1992) Immunoglobulin kappa gene enhancers synergistically activate gene expression but independently determine chromatin structure. J Biol Chem 267:23888-93
Lee, M S; Garrard, W T (1992) Uncoupling gene activity from chromatin structure: promoter mutations can inactivate transcription of the yeast HSP82 gene without eliminating nucleosome-free regions. Proc Natl Acad Sci U S A 89:9166-70
Freeman, L A; Garrard, W T (1992) DNA supercoiling in chromatin structure and gene expression. Crit Rev Eukaryot Gene Expr 2:165-209
Whitehurst, C; Henney, H R; Max, E E et al. (1992) Nucleotide sequence of the intron of the germline human kappa immunoglobulin gene connecting the J and C regions reveals a matrix association region (MAR) next to the enhancer. Nucleic Acids Res 20:4929-30
Lee, M S; Garrard, W T (1991) Positive DNA supercoiling generates a chromatin conformation characteristic of highly active genes. Proc Natl Acad Sci U S A 88:9675-9
Lee, M S; Garrard, W T (1991) Transcription-induced nucleosome 'splitting': an underlying structure for DNase I sensitive chromatin. EMBO J 10:607-15

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