Molecular cloning of a cDNA specific for the murine p53 cellular tumor antigen.

Abstract

A cDNA library was constructed from sucrose-gradient-fractionated mRNA from SVT2, a mouse cell line transformed by simian virus 40. Polysomes containing the p53 messenger were specifically immunoprecipitated with monoclonal antibodies against the protein and used to prepare mRNA. This immunoselected mRNA, enriched 1,000-to 2,000-fold for p53-specific sequences, was used to make a cDNA probe and to screen the cDNA library. When approximately 10,000 colonies were screened by differential hybridization with probes made from immunoselected vs. nonenriched mRNA, a single clone was found that contained p53-specific sequences. The identity of this clone, termed pp53-208, was confirmed by the ability of its DNA to hybridize the mRNA coding for p53 (hybrid selection assay). When hybridized to a blot of EcoRI digested mouse DNA, the pp53-208 insert reacted with a single 3.3-kilobase band, suggesting that it is complementary to a single gene.