Date of Award

9-18-1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Barry Milavetz

Abstract

A large fraction of simian virus 40 (SV40) chromosomes found in lytically infected cells have been shown to contain a large nucleosome-free region (NFR) over the promoter. Despite the importance of this NFR in transcription, little is known about what actually controls its formation. Recently, it has been shown that specific, short DNA sequences may act as organizing centers for nucleosome phasing in other eukaryotic systems. The primary goal of this study was to determine if specific SV40 early promoter sequences existed that were capable of phasing nucleosomes and thus directing the formation of a NFR.In order to identify specific SV40 enhancer and other early promoter sequences capable of nucleosome phasing, an SV40-based reporter system was used which consisted of a polylinker of unique restriction endonuclease sites cloned into nucleotide (nt) 2666 of an SV40 recombinant lacking the T intron and one copy of the enhancer. Sequences of interest were introduced at one or both ends of the reporter and the ability of the sequences to phase nucleosomes and form a NFR was measured by quantitating the proportion of SV40 chromosomes cleaved at each of the restriction sites within the reporter.In a deletional analysis of the SV40 enhancer, the AP-1 site was identified as being able to confer the highest levels of nuclease sensitivity either alone or in combination with an intact early domain. To ensure that other important early promoter elements capable of nucleosome phasing hadn't been overlooked, an insertional analysis was done in which various early promoter elements were analyzed for their abilities to phase nucleosomes. Two copies of the Sp1 site were found to confer levels of nuclease sensitivity comparable to those levels seen with the AP-1 site, whereas, one copy of the Sp1 site and the TATA box conferred low levels of sensitivity. All other elements analyzed showed intermediate levels of nuclease sensitivity.

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