Analysis of p53 mutants for transcriptional activity.
The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.
Raycroft
L
L
Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Schmidt
J R
JR
Yoas
K
K
Hao
M M
MM
Lozano
G
G
D000818:Animals; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D016158:Genes, p53; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53