Polyomavirus DNA synthesis in vitro: Characterization of wild-type and ts DNA- mutant Chinese hamster ovary cell extracts.
Item
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Title
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Polyomavirus DNA synthesis in vitro: Characterization of wild-type and ts DNA- mutant Chinese hamster ovary cell extracts.
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Identifier
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AAI9207092
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identifier
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9207092
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Creator
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Lawlor, Kenneth Gerard.
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Contributor
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Adviser: Harvey L. Ozer
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Date
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1991
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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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Chemistry, Biochemistry | Biology, Cell | Biology, Molecular
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Abstract
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Several ts DNA{dollar}\sp-{dollar} mutant CHO cell lines affected in cellular and polyomavirus DNA synthesis have been isolated in this laboratory. I have adapted a system for polyomavirus DNA synthesis in vitro and developed an in vitro complementation assay, using chromatographic fractions prepared from wild-type (wt) CHO cell extract, in an effort to identify cellular factors which stimulate polyoma in vitro DNA replication activity of defective CHO ts DNA{dollar}\sp-{dollar} mutant cell extracts.;The polyomavirus in vitro DNA replication activity is completely dependent upon the addition of polyomavirus large T antigen and a polyoma origin of replication. Analysis of in vitro DNA replication products synthesized by wild-type (wt) CHO cell extract, after deproteinization, included novel form I DNA monomeric products.;Detailed analysis of one CHO ts DNA{dollar}\sp-{dollar} mutant, JB3-O, has been performed. Cell extracts derived from JB3-O are defective for polyoma in vitro DNA replication, compared to wt CHO cell extract, at all temperatures assayed in vitro, suggesting that JB3-O extract exhibits a temperature-independent defect in vitro. JB3-O extract is not only deficient in in vitro DNA replication activity but also synthesizes an aberrant replicative form. These replicative forms are molecules which are greater than 75% replicated. Poor cell growth is not the cause of the defective polyoma in vitro DNA replication activity.;We have attempted to complement the defective polyoma in vitro DNA replication activity of JB3-O cell extract by addition of chromatographic fractions prepared from wt CHO cell extract. One phosphocellulose fraction showed a low level or stimulation leading to an increase of incorporation into the aberrant replicative form typically synthesized by JB3-O cell extract. Addition of topoisomerase II led to stimulation and synthesis of more monomeric product but net synthesis was still significantly lower than that of wt CHO cell extract. These data suggest that JB3-O extracts are defective in more than one function required for efficient polyoma in vitro DNA replication.
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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.
