Prolactin-deficient GH3B3 cells are defective in the utilization of the endogenous prolactin promoter yet are fully competent to initiate transcription from a transfected prolactin promoter.
Transcription of the prolactin (PRL) gene has been analyzed in wild-type D6, PRL-deficient B3, and revertant r16 GH3 cells. Levels of processed nuclear transcripts from the PRL gene were substantially reduced in the deficient line compared to wild-type cells and returned to greater than wild-type levels in the revertant line. Rare PRL transcripts in the deficient line contained the same 5' end found on transcripts in wild-type and revertant cells as judged by primer extension and S1 nuclease protection assays, implying that the cells are deficient in utilization of the normal wild-type promoter. Deficient cells also contained wild-type levels of the PRL- and growth hormone-specific transcription factor pit-1/GHF-1, and no difference was found in the ability of extracts from wild-type and deficient cells to retard various restriction fragments from both the proximal and the distal PRL promoter regions. The deficient and wild-type cells were equally competent in initiating transcription from a transfected rat PRL promoter containing both the distal and proximal promoter elements. These observations imply that PRL-deficient cells are not defective in a trans-activating factor functioning on these PRL promoter fragments (trans model). Rather, inefficient use of the PRL promoter in the variant cells may reflect an increased methylation state of the PRL gene itself (cis model).
Arnold
T E
TE
Department of Genetics, University of Georgia, Athens 30602.
Farrance
I K
IK
Morris
J
J
Ivarie
R
R
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D015152:Blotting, Northern; D002460:Cell Line; D002874:Chromosome Mapping; D004247:DNA / Q000187:drug effects; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D005786:Gene Expression Regulation; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D012333:RNA, Messenger; D011388:Prolactin; D004247:DNA; D005020:Ethyl Methanesulfonate; D001374:Azacitidine