| Transcription from the Long terminal repeat (LTR) of Rous sarcoma virus (RSV) in rat 3Y1 fibroblasts was increased 5 fold within an hour of addition of serum to serum-deprived cells. This stimulation did not require synthesis of new proteins, and was mostly dependent on two CCAAT boxes in the LTR, though other upstream sequences played a secondary role. Addition of serum caused the rapid increase of a nuclear protein that bound the two CCAAT boxes in the LTR and this too could be seen in the presence of cycloheximide. Competition with oligonucleotides representing various CCAAT sequences suggested that the serum induced CCAAT factor was related to the CP1/CP2 rather than the NF1 or C/EBP types of CCAAT binding factors.;The serum dependence of transcription from the RSV LTR was lost in v-src transformed 3Y1, and cross-feeding experiments showed that the mechanism did not involve the production of extra-cellular growth factors. Temperature-sensitive (ts) v-src transformed 3Y1 was used to demonstrate that the tyrosine kinase activity of v-src could (a) substitute for the serum-requirement of the RSV LTR, (b) increase the level of transcripts initiated in the LTR even in the presence of serum, (c) exert its serum-sparing effect on the RSV LTR in the absence of new protein synthesis, and (d) increase the amount of CCAAT binding factor in the nucleus. Orthovanadate, an inhibitor of tyrosine-phosphatases, which non-specifically elevated the level of phosphotyrosine in the cells, could mimic the effects of serum and v-src on transcription from the RSV LTR.;The RSV LTR directed accurate initiation of transcripts in Saccharomyces cerevisiae, about 60 bases downstream from the TATA box that is used in animal cells. The authentic TATA box was absolutely necessary for the transcription, and the same CCAAT boxes that were responsive to serum in animal cells, were acting as "upstream activating sequences". The activity of the RSV LTR in yeast was not stimulated by lactate, and was not decreased in HAP2$sp-$ or HAP3$sp-$ yeast, suggesting that the "UAS" activity of the CCAAT boxes was mediated by yeast CCAAT binding factors other than HAP2/HAP3. While a high level of v-src was toxic to the yeast, expression of very low levels of the oncogene stimulated the CAT activity from the RSV LTR about two fold. |
