Date of Award

9-18-1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Barry Milavetz

Abstract

Simian Virus 40 (SV40) chromosomes possess chromatin structures very similar to eukaryotic chromatin, including the presence of a nucleosome free region (NFR) over their promoter in a fraction of the chromosomes. SV40 domains of the promoter region include the information necessary for defining the NFR although the specific sequences involved in generating NFR's have not been identified. The goal of this study was to determine the minimal sequence within the late promoter region, nucleotides 255-424, of SV40 capable of phasing nucleosomes as measured by its ability to confer the greatest endonuclease sensitivity on adjacent DNR sequences. The degree of nuclease sensitivity was designed to serve as a measure of DNA accessibility which is critically important for processes such as transcription factor binding. To identify the minimal sequence, a deletional analysis of the late region was performed utilizing a SV40 recombinant reporter system. The reporter system consisted of a series of unique restriction sites introduced into SV40 at nt 2666. The unique restriction sites allowed the insertion of test sequences as well as measurement of endonuclease sensitivity which the sequence was able to confer. Test sequences were made by the polymerase chain reaction (PCR), utilizing SV40 template DNA, or by overlapping complementary oligonucleotides. Initially, the greatest sensitivity was conferred by nt 255-313. Interestingly, this is a region known to contain a number of SV40 transcription factor binding sites. Further analysis demonstrated that constructs capable of conferring increased nuclease sensitivities consistently included nt 255-280. The AP-4 and GTIIC transcription factor binding sequences lie within this region and were analyzed individually. Their abilities to confer nuclease sensitivity upon the reporter nearly matched that of the entire late domain. These results suggest that transcription factors AP-4 and Transcription Enhancing Factor (TEF) which binds the GTIIC sequence are able to confer significant levels of nuclease sensitivity and are likely involved in the formation of the NFR extending over the late promoter domain. Further studies should help shed light on how these sequences relate to mechanisms involved in the formation of the SV40 nucleosome free region.

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