Figure 1.

A: Schematic representation of the genomic DNA encoding rat P53. An N-ethyl-N-nitrosourea (ENU)-induced mutation in the sixth exon of the gene results in a premature stop codon, truncating the protein within the DNA binding domain. The sequence trace indicates the T to A transition in a heterozygous rat resulting in a premature stop codon. Asterisk indicates the stopcodon that was introduced by the ENU-induced mutagenesis. B:Tp53^C273X results in the lack of p53 protein and function in primary rat embryonic fibroblasts (REFs). Whereas wild-type REFs show p53 stabilization and translocation to the nucleus on UV radiation, no p53 protein is observed in homozygous mutant REFs. C:Tp53^C273X results in a complete lack of p53 protein. Wild-type and homozygous mutant REFs were treated with 0.5 μmol/L doxorubicin and lysed at the indicated time points. Western Blotting reveal the presence of p53 in wild type REFs, which is stabilized on induction of double strand breaks, whereas no protein can be detected in homozygous mutant cells. Notably, the epitope of the anti-p53 monoclonal antibody 1C12 is located N-terminally from the ENU-induced premature translational stop, demonstrating complete absence of potentially truncated p53 protein.