An investigation of rat DNA polymerase alpha
The aim of this project was to clone the gene encoding the catalytic subunit of the rat DNA replication enzyme, DNA polymerase alpha. A strategy was adopted in which cDNA clones expressing the catalytic subunit sequences would be identified using anti-DNA polymerase antibodies. DNA polymerase alpha was partially purified from regenerating rat liver and exponentially growing rat Y3 myeloma cells. The catalytic subunit was identified as a 170-180kD polypeptide by activity gel analysis of partially purified Y3 cell fractions. The catalytic subunit was found to be susceptible to degradation but without loss of polymerase activity. Glycerol gradient analysis indicated a two stage degradation of DNA polymerase in vivo. Sera were collected from mice immunised with partially purified DNA polymerase alpha from regenerated rat liver. These sera cross-reacted with Western-blotted Y3 cell fractions; removed polymerase activity from solution in plate binding assays and bound alpha polymerase activity (140-180kD) on an immuno-adsorption column cDNA was synthesised using size selected mRNA from exponentially growing Y3 cells and cloned into the expression vectors pUC8 and ?gtll, both of which utilise the lac Z gene to express cloned DNA sequences. Immunoscreening of the ?gtll library was frustrated by non-specific binding of the serum. This non-specific binding was overcome by pre-adsorbing the serum against a lysate of E. coli JM 83. Screening of the pUC8 library revealled 27 out of 2.25x104 colonies which bound pre-adsorbed anti-DNA polymerase alpha serum.