Mammalian .alpha.-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

Publisher Website
Google Scholar
Abstract
A new polyclonal antibody against the .alpha.-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell .alpha.-polymerase. The antibody neutralized .alpha.-polymerase activity and was strong and specific for the .alpha.-polymerase catalytic polypeptide (Mr 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+)RNA in .lambda.gt11. A positive change was identified and plaque purified. This phage, designated .lambda.pol.alpha.1, 2, also was found to be positive with an antibody against Drosophila .alpha.-polymerase. The insert in .lambda.pol.alpha.1.2 (1183 base pairs) contained a poly(A) sequence at the 3'' terminus and a short in-phase open reading frame at the 5'' terminus. A synthetic oligopeptide (eight amino acids) correpsonding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified .alpha.-polymerase by enzyme-linked immunosorbent assay and was capabel of immunoprecipitating .alpha.-polymerase. This indicated the .lambda.pol.alpha.1.2 insert encoded an .alpha.-polymerase epitope and suggested that the cDNA corresponded to an .alpha.-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding .alpha.-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed tht this mRNA is .apprx. 5.4 kilobases.