Phylogenetic specificity of prolactin gene expression with conservation of Pit-1 function.
In mammals, the pituitary POU homeodomain protein, Pit-1, binds to proximal and distal 5'-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5' sequences in the teleost species, Oncorhynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5'-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least 90-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about 10-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-1 was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-1 POU domain and constructed a rat Pit-1 expression vector that contained salmon Pit-1 POU domain sequences substituted in frame. The chimeric Pit-1 encoded 14 amino acids unique to salmon. Coexpression of rat Pit-1 with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-1 was similar to wild type rat Pit-1 in activating salmon and rat PRL/CAT.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Clinical Biochemistry, University of Toronto, Ontario, Canada.
H P
HP
Elsholtz
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S
Majumdar-Sonnylal
F
F
Xiong
Z
Z
Gong
C L
CL
Hew
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005801:Genes, Homeobox; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010802:Phylogeny; D010902:Pituitary Gland / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011388:Prolactin / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012474:Salmon; D012689:Sequence Homology, Nucleic Acid; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D012333:RNA, Messenger; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase