Wild-type murine p53 represses transcription from the murine c-myc promoter in a human glial cell line.

Here we analyzed the effect of the suppressor proto-oncogene p53 on transcription from the P2 promoter of the murine c-myc gene. c-myc promoter constructs were coupled to the chloramphenicol acetyl-transferase (CAT) gene and were transiently transfected into a human glial cell along with plasmids overexpressing wild-type or mutant p53. It was found that significant repression of c-myc transcription took place following cotransfection with wild-type but not mutant p53. However wild-type p53 did not suppress transcription from the SV40 early promoter or from the MHC promoter. Promoter-CAT constructs containing only the ME1a2 or E2F elements, from the P2 promoter, were repressed by p53, indicating that p53 may exert its effect at these two sites within the P2 promoter. Finally, when the SV40 T antigen and wild-type p53 were expressed together in glial cells the repressive effect of p53 was abolished.

Three distinct elements within the murine c-myc promoter are required for transcription.

We have undertaken a detailed analysis of the cis-acting elements that are required for optimal transcription initiation from P2, the major promoter of the murine c-myc gene. We find that three elements contribute to promoter strength, termed ME1a2, E2F and ME1a1 at positions -85, -64 and -46 respectively (relative to P2). Individually the elements are weak, but combined they contribute to full promoter activity, all acting in a positive fashion. The E2F element is the site at which the SV40 large T antigen transactivates the c-myc promoter. However, transactivation requires the presence of the ME1a2 or ME1a1 elements in addition to E2F. By a number of criteria it appears that the ME1a2 and ME1a1 elements bind the same or a very closely related protein (monomer Mr 94,000). It was found that Hela cells contain a novel factor that is capable of binding to the ME1a1 and ME1a2 elements but only in the presence of the protein-dissociating agents deoxycholate or formamide. Finally, the E2F factor binds DNA both as a monomer and as a multiprotein complex; the latter is cell type specific. Both types of bound E2F factor form less stable protein-DNA complexes than the ME1a2/ME1a1 factor.

Tyrosine phosphorylation of a yeast 40 kDa protein occurs in response to mating pheromone.

Tyrosine phosphorylation of proteins in the yeast Saccharomyces cerevisiae has been examined following exposure to the mating pheromone alpha-factor. When a cells are treated with alpha-factor a protein of approximately 40 kDa molecular weight is tyrosine phosphorylated. This tyrosine phosphorylation response requires an intact signal transduction pathway, is not restricted to a short interval of the cell division cycle, and requires protein synthesis for its maximal accumulation. Mating competent fus3 deletion strains fail to elaborate the phosphotyrosine response. The possibility that FUS3 encodes the 40 kDa protein is discussed.

Analysis of the c-myc P2 promoter.

The cis-acting elements governing transcription from the murine c-myc P2 promoter have not been well defined. To gain a better understanding of the nature of the protein-DNA interactions that take place on the P2 promoter, protein binding assays were performed. The ME1a2 and E2F factors appear to be the predominant proteins bound to a region spanning positions -140 to -24 relative to the P2 transcription start site. By a number of criteria, these factors appear to be distinct. When c-myc promoter sequences were coupled to the chloramphenicol acetyltransferase gene (CAT) and transiently transfected into tissue culture cells it was found that optimal transcription from P2 was heavily dependent on the ME1a2 element.