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|Title:||Purification and Characterization of Simian Virus 40 Large T Antigen|
|Author(s):||Giacherio, Donald Allen|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||Simian Virus 40 is a small DNA virus which can replicate normally following productive infection of permissive monkey cells, transform various cell lines of different species in tissure culture, and induce tumor formation following injection into newborn hamsters. Large T antigen is one of two viral-coded non-structural proteins, and is produced early in productive infection and continuously in transformed cells. Several lines of evidence indicate that large T antigen of SV40 is involved in the initiation of viral DNA replication during productive infection, the stimulation of host cell DNA synthesis following SV40 infection, the regulation of viral transcription, and the initiation and maintenance of transformation. In order to better understand its role in the above processes, large T antigen was purified from a line of SV40 transformed human fibroblasts (SV80 cells) and its biochemical activities studied.
T antigen was purified to near homogeneity from the nuclei of SV80 cells by a procedure that involved high salt extraction of nuclei, a 100,000 x g ultracentrifugation to clarify the extract, ammonium sulfate fractionation, then successive column chromatography steps on Ultrogel AcA34, DEAE-Sephadex, and heparin-Sepharose. T antigen was monitored through the purification by the complement fixation reaction. This procedure yielded an approximately 200-fold purification from the clarified nuclear extract, with two protein species of molecular weights 92,000 and 84,000 detectable. Both of these species represent large T antigen, with the smaller being a protease degradation product of the larger.
Purified large T antigen was tested for various biochemical activities. No nicking activity of SV40 DNA could be detected, and a protein kinase activity was separable from the complement fixing activity of large T antigen. A weak ATPase activity was found which copurifies with T antigen and is inhibitable by hamster anti-SV40 tumor serum. This ATPase activity was shown to be stimulated approximately seven-fold by the addition of poly dT. Longer chain length poly dT molecules stimulate better than short chain length oligo dT molecules, with 30-40 nucleotides and longer being the size for optimal stimulation. The Km for ATP was found to vary somewhat with enzyme concentration, which may be indicative of an aggregation of T antigen.
Because of large T antigens role in the initiation of viral DNA synthesis and its demonstrated DNA binding properties, large T antigen was analyzed for an ability to alter the helix structure of SV40 DNA. This was done by incubating purified large T antigen with relaxed circular SV40 DNA in the presence of topoisomerase I, and analyzing for incorporation of superhelical turns into the DNA.
The incubation of highly purified large T antigen with relaxed, circular SV40 DNA in the presence of topoisomerase I resulted in the introduction of negative superhelical turns in the DNA. ATP was not required for this reaction. A similar introduction of superhelical turns could also be obtained when a recombinant plasmid DNA (Y 182) which contains sequences of both SV40 DNA and pBR322 DNA was used. However, no effect was observed when pBR322 DNA was incubated with T antigen in the presence of topoisomerase I. These results are consistent with the hypothesis that large T antigen can recognize and unwind specific sequences on SV40 DNA.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
|Date Available in IDEALS:||2014-12-14|