Analysis of the chromatin structure of SV40 wt776 and a mutant blocked in the initiation of virus assembly
Abstract
In order to study the structure/function relationship of eukaryotic chromatin, several approaches were used to examine and to compare the chromatin structure of SV40 wt776 and tsC219, a temperature sensitive SV40 mutant. At 40$\sp\circ$C, tsC219 is defective in the initiation of virus assembly due to a mutation in the major capsid protein, VP1. Restriction enzyme digestion analyses showed that the tsC219 regulatory region is very accessible ($>$90%) to BgII and SphI. This accessibility is reduced in wt-infected cells or tsC-infected cells at the permissive temperature (33$\sp\circ$C). These results indicate that the virion assembly process plays an active role in modulating the structure of SV40 chromatin in vivo. A topoisomerase I assay was used to examine the flexibility of SV40 DNA in the wt and ts219 40$\sp\circ$C chromatin. The results showed that histones constrain the DNA from twisting whereas the linker DNA was found to be free to twist in response to changes in temperature. The degree of this flexibility was dependent upon the linker length. The topological analyses further revealed that the number of nucleosomes on the wt and tsC219 chromatin appear to be similar and that the number of nucleosomes on the chromatin varies by 3-4 nucleosomes on either side of the mean of 26 nucleosomes. Micrococcal nuclease combined with M13 cloning methodology and DNA sequencing was used to determine the location of the nucleosomes assembled in vivo on the wt and tsC219 DNA. Several nucleosomes were identified at and near the replication origin, the enhancer sequences, the early and late transcription initiation sites, the termination region and regions containing bent DNA. The overall arrangement of the nucleosomes along the SV40 DNA appears very complex; overlapping nucleosomal clones were found throughout the genome for both the tsC219 and wt chromatin. The results indicate that the nucleosomes do not occupy unique positions. However, the distribution of nucleosomes does not appear to be random.
Degree
Ph.D.
Advisors
Bina, Purdue University.
Subject Area
Biochemistry
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