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.
Center for Molecular Biology and Genetics, Mie University, Tsu, Mie, 514-8507, Japan.
T
T
Ohkubo
M
M
Tanaka
K
K
Nakashima
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