Molecular cloning studies on DNA polymerase alpha

• Main Author: Caird, Mandy Ruth
• Published: University of Aberdeen 1989
• Subjects: 572.8; Genetics
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292388

The DNA polymerase α assay systems and partial purification techniques already in use in the laboratory were improved by isolating better-activated DNA and freezing cells prior to sonication. The suitability of monoclonal antibodies against <i>Xenopus laevis</i> and human KB cell (Tanaka <i>et al</i>., 1982) DNA polymerase α was established as a prelude to screening expression libraries. All were found to inhibit polymerase activity by approximately 50%. Two out of three anti-<i>Xenopus laevis</i> DNA polymerase α antibodies and three out of four anti-human DNA polymerase α antibodies bound antigen in HeLa cell cytoplasmic extracts. Human hepatoma, human foetal liver, human fibroblast and mouse bone marrow lambda gt11 expression libraries were screened with the above antibodies using the method of Young and Davis (1983) and various horseradish peroxidase and alkaline phosphatase conjugated second antibody systems in an attempt to isolate a human or mouse DNA polymerase α gene. A 1.9kb cDNA clone from a calf thymus expression library (Foster <i>et al</i>., 1986) was characterised. The molecular weight of 165-175kDa and polymerase activity of the β-galactosidase-calf fusion protein was confirmed but the fusion protein could not be detected with any of the antibodies mentioned above. The 1.9kb cDNA clone was used to show the regulation of DNA polymerase α activity in growth-regulated bovine kidney cell to be at the transcriptional level. The DNA sequence of the calf thymus cDNA fragment was determined. The DNA sequence of 2042 bases and all predicted open reading frames were compared with sequences contained in the GenBank, EMBL and NBRF (nucleic acid and protein) databanks of the University of Wisconsin Genetics Computer Group molecular biology package. No homology to any DNA polymerase α nucleic acid or protein sequence was found, suggesting that the sequence of Foster <i>et al</i>. (1986) might not code for a DNA polymerase α. In searches of international sequence databanks the 1.9kb DNA fragment shows the greatest similarity to human, bovine and rodent sequences. However, the similarity scores are very low suggesting that this sequence is unique and will require the determination of a longer sequence before location of an identity.