Date of Award

11-1-1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

We proposed that SV40 T-antigen binding SiteI and adjacent sequences are involved in the regulation of the proportion of SV40 chromosomes containing a nucleosome-free promoter (NFP) region. Wild-type and SiteI/T antigen defective SV40 chromosomes were analyzed by promoter-specific restriction endonuclease digestion and electron microscopy. SiteI/T antigen mutant chromosomes showed a significant increase in the proportion of chromosomes containing a nuclease hypersensitive promoter region. This increase corresponded to a similar increase in the proportion of chromosomes with a LARGE (greater than 4 times the average internucleosomal distance) nucleosome-free region in the SiteI/T antigen mutants as visualized by electron microscopy. The proportion of chromosomes containing a SMALL NFP (3 to 4 times the average internucleosomal distance) remained the same for SiteI deletion mutants as wild-type. These results suggest that there are at least two independently regulated forms of NFP regions in SV40 chromosomes. T antigen binding at SiteI decreased the proportion of chromosomes with a LARGE nucleosome-free region, while the SMALL size nucleosome-free region was independent of T antigen binding at SiteI. Analysis of a series of insertion and deletion mutants suggested that the elements involved in regulation of the Early boundary of the NFP region were sequences located near SiteI that share homology with known enhanson elements (OCT, SRE, TC, and GT) in SV40 and other eukaryotic enhancers. Deletion of SiteI greatly increased the proportion of chromosomes with a LARGE nucleosome-free region, while deletion of SiteI plus one copy of the Early OCT element resulted in an intermediate increase compared to wild-type. Deletion of both Early OCT elements, or the TC sequences, generated nonviable viruses. Insertion of 7 bases between SiteI and the Early OCT did not change the proportion of chromosomes with a LARGE or SMALL NFP, but an internal restriction site appeared to be blocked. The above results suggested that the putative Early enhanson elements are important regulatory elements. One of the mechanisms by which enhansons activate transcription may be through placement or exclusion of nucleosomes within regulatory regions.

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