SERUM RESPONSIVENESS OF THE RAT PCNA PROMOTER

Expression of proliferating cell nuclear antigen (PCNA) gene is growth -regulated. The growth dependence of the rat PCNA gene promoter activi ty was investigated. Cultured cells were transfected with the promoter containing plasmid and recovered for 48 h in serum-free medium to bec ome quiescent, Cells were then cultured in serum-containing medium and harvested at certain intervals after serum-stimulation, and the promo ter-directed chloramphenicol acetyltransferase activities in cell extr acts were measured. The promoter used in this study contained sequence s between -693 and +125 in relation to the transcription initiation si te. The promoter activity was found to be serum-responsive, However, t he serum-responsiveness of the promoter became less obvious when the a mount of the promoter increased; meanwhile, the promoter became active in the control unstimulated (or quiescent) cells. It was suspected th at the dosage effect was due to the titration of the negative regulato ry factor in quiescent cells. The titration experiment with a reporter less construct as competitor for regulatory factors showed that the ex cess of promoter molecules reduced the promoter activity in serum-stim ulated cells, while causing a transiently increase of promoter activit y in quiescent cells. Based on these results, it is postulated that th e serum-responsiveness of the rat PCNA promoter is controlled by both negative and positive regulatory factors. Consistent with this proposi tion, promoter binding proteins of 105 and 114 kDa were identified onl y in serum-stimulated and quiescent cells, respectively, in addition t o several other promoter binding proteins (ranging from 76 to 110 kDa) which were seen in both serum-stimulated and quiescent cells. (C) 199 5 Academic Press, Inc.