Chromatin Structure Of Sv40/pbr322 Recombinant Plasmids And Viral Insertion Mutants

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology


Simian virus 40 (SV40) chromatin isolated from infected cells contains a nuclease-sensitive region which overlaps the early and late gene promoters and the origin of replication. The role of the viral late proteins in the occurrence of the nuclease-sensitive region was investigated by studying the nucleoprotein structure formed by SV40/pBR322 recombinant plasmid DNA molecules in nuclei of transfected COS-1 cells. Recombinant plasmids can be extracted from the nuclei of COS-1 cells as nucleoprotein complexes and have sedimentation properties similar to those of SV40 nucleoprotein complexes. Two DNase I-sensitive regions were detected in the SV40 portion of the plasmid at the same sites that were DNase I-sensitive in SV40 chromatin in infected cells. COS-1 cell RNA does not hybridize with the late region of the viral genome, suggesting that late proteins are not required for the occurrence of the nuclease-sensitive feature. When plasmid-transfected COS-1 cells were superinfected with SV40, no change in the distribution of DNase I-sensitive sites in plasmid chromatin was seen, suggesting that late proteins do not modify the nuclease-sensitive chromatin structure.The nuclease-sensitive chromatin structure is organized by multiple genetic elements contained within a small segment of the viral genome. In an effort to determine where these elements are, insertion mutations were placed within this region of the genome between the SV40 origin of replication and sequences known to be essential for viral early and late gene expression. DNA replication was evaluated by transfecting recombinant plasmids containing the SV40 origin of replication and insertion mutations into COS-1 cells. Replication was inhibited to an increasing extent with increasing size of the inserted DNA segment. The effect on replication efficiency was cis-acting. The endogenous endonuclease and DNase I cleavage sites in mutant chromatin were displaced in the late direction from the SV40 origin and accessibility to BgII was reduced. These results suggest that efficient function of the viral origin of replication is correlated with its location within the nuclease-sensitive chromatin structure.


Biology, Genetics

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