Molecular cloning of the chicken prolactin gene and activation by Pit-1 and cAMP-induced factor in GH3 cells.
Transcription of the prolactin (PRL) gene has been reported to be activated by a nuclear factor, Pit-1. However, the precise molecular mechanisms of the Pit-1-mediated PRL gene activation are still unclear. We have cloned the chicken PRL (cPRL) gene and its 5'-flanking region to analyze their structure and transcription-initiating mechanism. In luciferase assay, forskolin activated the proximal promoter region between -248 and -76 to transcribe the cPRL gene in GH3 cells, although there is no canonical cyclic AMP-responsive element in the promoter region. In gel mobility shift assay, a DNA fragment between -104 and -76 containing a putative Pit-1 binding site was bound by nuclear factors from the GH3 cells. Furthermore, it was observed that Pit-1 protein specifically bound to the DNA fragment in the supershift assay. These results indicate that both Pit-1 and cAMP-induced factor(s) associated with the cis element on the proximal promoter region to activate cPRL gene expression in GH3 cells.
Ohkubo
T
T
Center for Molecular Biology and Genetics, Mie University, Tsu, Mie, 514-8507, Japan.
Tanaka
M
M
Nakashima
K
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens / Q000235:genetics*; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C494186:Pou1f1 protein, rat; D011993:Recombinant Fusion Proteins; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D005576:Colforsin; D011388:Prolactin; D004247:DNA; D000242:Cyclic AMP; D008156:Luciferases