ISOLATION OF DNAS THAT SELECTIVELY BIND A BACULOVIRUS PRODUCED MUTANTP53 (ALA-143) PROTEIN BUT NOT AN RRL OR WGL PRODUCED MUTANT P53 PROTEIN

A PCR-based technique was used to generate a large pool of random sequ ence double-stranded DNAs. Four DNA sequences that selectively bound i n vitro to a mutant p53 143A protein, synthesized in baculovirus infec ted cells, were characterized. The four DNA sequences all approximated the known consensus sequence for wild-type p53. Wild-type p53 also bo und the four DNA sequences. Two other mutant p53 proteins (His 175 or Trp 248) did not bind. The ability of mutant p53 143A protein to bind to DNA is totally dependent on the irt vitro system used to synthesize the p53 protein. Rabbit reticulocyte lysate (RRL) or wheat germ lysat e (WGL) produced mutant p53 143A is unable to bind to DNA but does bin d to a known protein partner, hdm2, thus these activities can be uncou pled.