Studies on chromatin, topology, and DNA replication of simian virus 40.
Item
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Title
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Studies on chromatin, topology, and DNA replication of simian virus 40.
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Identifier
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AAI9218230
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identifier
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9218230
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Creator
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Chu, Yi.
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Contributor
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Adviser: Ming-Ta Hsu
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Date
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1992
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Microbiology | Biology, Molecular
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Abstract
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Simian virus 40 (SV40) has provided an excellent experimental system to study the molecular biology of mammalian cells. In this dissertation, three findings concerning the SV40 chromatin, DNA topology, and DNA replication are described.;Chapter 2 describes a new nuclease hypersensitivity assay using P1 nuclease to probe active SV40 chromatin. P1 nuclease was found to cut a small subpopulation of intracellular SV40 DNA of the highest superhelical density only once under exhaustive digestion. The cleavage site was mapped to either the replication origin or transcription enhancer. Newly synthesized viral chromatin and replication intermediates were preferentially cleaved by P1 nuclease. In SV40 variants in(Or)1411 and in(Or)1412 containing two functional origins, either origin was sensitive to P1 cleavage. The mutated and nonfunctional origin in the in(Or)1415 was not cleaved by P1 nuclease.;Chapter 3 describes another aspect of the structure of newly synthesized SV40 chromatin. It was found that superhelical density of DNA in this subpopulation is greatly increased by ellipticine, an intercalating agent and a topoisomerase II inhibitor. This increase results from the intercalation of ellipticine followed by fixation of DNA topology by a topoisomerase. The density of intercalation, estimated to be one ellipticine per 10-20 base pairs, suggests that ellipticine can intercalate within the nucleosome core. This result is consistent with the interpretation that newly synthesized chromatin contains a loosened nucleosome structure which upon maturation becomes inaccessible to ellipticine intercalation.;Chapter 4 describes the study on the regulation of SV40 DNA replication. It was observed that the two oris in in(Or)1412 and in(Or)1411 are not used with equal efficiency. The following possibilities for their differential usage were tested: (1) Sequences immediately surrounding the two oris affect their usage; (2) Association with an intact transcription unit activates the ori; and (3) Transcription passing through the second, inserted ori interferes with the initiation from this origin. The first two possibilities were eliminated. Attempts of using human cytomegalovirus (HCMV) major immediate early gene promoter/enhancer (P/E) to test the third possibility unexpectedly revealed a strong distance- and position-dependent suppression effect of the HCMV P/E on SV40 ori. The transcription-interference hypothesis was further tested using a variant of in(Or)1412 with the two oris located at the 3{dollar}\sp\prime{dollar} end of a transcription unit. The results of transfection experiments are consistent with this model.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
