Inactivation of the p53 tumor suppressor gene is the most common genetic abnormality of human cancers. The wild type protein is highly regulated by phosphorylation affecting its tertiary structure, DNA binding and function in transcriptional control. Mutant p53 gene products fold into abnormal conformations which fail to perform wild type protein functions for negative growth regulation. While the rise and fall of total p53 protein is important to several p53 functions such as G1 arrest after DNA damage, it does not reflect the dynamic changes in phosphorylation which appear to also accompany increased p53 levels during cellular stress responses and cell cycle regulation. We have previously shown that 2D PAGE can separate p53 isoforms which should be valuable in comparing posttranslational differences between normal and transformed cells. In human cells we have found that all isoforms are phosphorylated except for a single isoform which is apparently the unmodified polypeptide. Since p53 is expressed at a low level,isolation of sufficient quantities for biochemical studies was furthered by baculovirus expression of human p53 gene. Sequence specific binding to consensus sequence oligonucleotides is being measured by mobility shift assay as a reflection of p53 function. Development of these systems will enable us to test the hypothesis that specific p53 isoforms are bioactive. Use of mutant p53 proteins would serve to distinguish specific and non-specific p53 interactions compared to the wild type protein. Comparison of DNA binding with recomninant p53 and native p53 using mobility shift assay should determine if specific isoforms are capable of sequence specific binding. Since DNA binding probably involves p53 oligomerization, native and denatluring conditions will be examined. Another experimental method being developed to assess DNA binding potential of various p53 isoforms is northwestern blotting.