Analysis of the changes in DNA polymerase Alpha as a result of age and transformation in cultured human fibroblast cell lines : a dissertation /
ABSTRACT: Eukaryotic DNA replication is a complex and tightly regulated process. DNA polymerase [alpha] (pol [alpha]) is essential for the initiation of DNA synthesis. We were interested in how pol [alpha] differs in normal and transformed cells as well as in cells from young and old donors. We e...
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| Format: | Thesis Book |
| Language: | English |
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College Station, TX :
Texas A&M University,
1993.
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| Summary: | ABSTRACT: Eukaryotic DNA replication is a complex and tightly regulated process. DNA polymerase [alpha] (pol [alpha]) is essential for the initiation of DNA synthesis. We were interested in how pol [alpha] differs in normal and transformed cells as well as in cells from young and old donors. We examined pol [alpha] isolated from fetal human female fibroblasts transformed with Simian virus 40 (SV40). Pol [alpha] from this transformed cell line, 2RA, showed more than 3-fold higher specific activity than pol [alpha] from its normal cell correlate (WI38). We also examined pol [alpha] isolated from GM3529, a fibroblast line established from cells of a 66 year old man, and the pSV3.neo-transformed cell correlate (2-1). DNA pol [alpha] isolated from transformed cell line, 2-1, revealed a more than 9-fold higher specific activity than that of pol [alpha] from the untransformed cell line, GM3529. These data suggest that pol [alpha] isolated from pSV3.neo-transformed cells exhibits altered physical and catalytic characteristics compared with its untransformed cell counterpart, and that those alterations may be associated with increased replication of the genome in plasmid-transformed cells. A DNA polymerase [alpha] accessory protein was isolated from a transformed murine cell line, L1210, with techniques originally developed by Goulian et al. (1990). We showed that [alpha]AP binds to the SV40 origin of synthesis in the absence of pol [alpha]/primase. DNA pol [alpha]/primase isolated from GM3529 showed increased binding affinity for SV40 DNA in the presence of [alpha]AP, but pol [alpha]/primase from L1210 cells showed little increase in DNA binding affinity in the presence of [alpha]AP. We showed that pol [alpha] from GM3529 exhibited significantly increased specific activity in the presence of exogenous [alpha]AP. [alpha]AP was also examined for binding affinity to multiple DNA templates. [alpha]AP bound to a 113 bp nonspecific DNA sequence from pUC18 and to a 32 bp oligonucleotide of the bovine papilloma virus origin of replication. These results indicate that [alpha]AP is not a sequence-specific double stranded binding protein. |
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| Item Description: | "Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 1993". "Major Subject: Genetics". Approved as to style and content by: David L. Busbee, Robert C. Burghardt, Evelyn Tiffany-Castiglioni, Van G. Wilson, Gerald R. Bratton, James R. Wild. Vita. |
| Physical Description: | xiii, 111 leaves : illustrations ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 102-110). |