It has been reported by another laboratory that the proto oncogene protein, myc, regulates transcription of the rat prothymosin alpha gene by binding to an enhancer element, CACGTG, located in the first intron. Previous data obtained in this laboratory using reporter constructs expressed in Mouse L cells did not concur. The controversy has been reconsidered using the endogenous prothymosin alpha gene as well as a series of transfected wild type and mutant genes as """"""""reporters."""""""" Expression of the transfected human wild type prothymosin alpha gene in COS-1 cells was extremely robust and not significantly affected by disruption of putative enhancer elements or removal of the first intron. When the expression of the wild type prothymosin alpha gene in the presence of a cotransfected normal myc gene was compared with that in the presence of a dominant negative mutant myc gene, the dynamic range of the response was 1.4-fold. Cotransfection of a dominant negative Max gene failed to reduce transcription of the endogenous prothymosin alpha gene or the wild type transfected gene. The data do not support the idea that myc activates transcription of the intact prothymosin alpha gene, or reporter constructs that mimic its structure. They suggest that the prothymosin alpha promoter and downstream elements are buffered so as to respond poorly, if at all, to transient fluctuations in transcription factors which regulate other genes. Prothymosin alpha is a phosphorylated protein located in the nucleus of all mammalian cells. Although the protein is an absolute requirement for cell growth and division, there is no evidence that it is regulated in a cell cycle dependent manner; its half-life is virtually identical to the generation time of the cell. We have investigated the half-life of its phosphate moiety in HeLa cells, which were pulse-labeled with radioactive orthophosphoric acid, using three chase techniques: a phosphate chase; or digitonin treatment followed by incubation in nonradioactive ATP; or electroporation in the present of ATP. The results indicate that the half-life of the phosphate is very short relative to the half-life of prothymosin alpha, itself.
