The tilapia prolactin I gene: evolutionary conservation of the regulatory elements directing pituitary-specific expression.
To study the elements involved in the pituitary specific transcriptional regulation of the tilapia prolactin I gene (tiPRL I), we have cloned and entirely sequenced a 3.4-kb genomic fragment immediately upstream from the first exon. In footprinting experiments, three tilapia sequences are protected from DNase I digestion by rat pituitary extracts (base pair coordinates -643 to -593, -160 to -111, and -73 to -46). Computer analysis of the nucleotide sequence reveals significant homology to mammalian binding sites for Pit-1, a transcription factor that is known to mediate pituitary-specific expression of the PRL genes in mammals. The tiPRL I 5'-flanking sequences can direct transient expression of a linked luciferase reporter gene in transfected rat pituitary cell lines and tilapia pituitary primary cell cultures. Transient expression experiments with 5'-deletion mutants reveal three regulatory regions. Two have a stimulatory effect on transcription and one an inhibitory effect. Electrophoretic mobility-shift assays (EMSA) demonstrate that the rat Pit-1 factor specifically binds to tilapia DNA sequences. Several such tilapia Pit-1 binding sites mediate activation of a linked heterologous promoter in transfected rat and tilapia pituitary cells. As evidenced by EMSA, a Pit-1-like protein is present in tilapia pituitary extracts. All these data point to a high conservation of the molecular mechanisms involved in pituitary-specific expression of the PRL genes in vertebrates.
Poncelet
A C
AC
Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liége, Sart-Tilman, Belgium.
Levavi-Sivan
B
B
Muller
M
M
Yaron
Z
Z
Martial
J A
JA
Belayew
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002457:Cell Extracts; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D005796:Genes / Q000235:genetics; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000166:cytology / Q000502:physiology*; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D017210:Tilapia / Q000235:genetics*; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D002457:Cell Extracts; D004268:DNA-Binding Proteins; C494186:Pou1f1 protein, rat; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D011388:Prolactin; D004247:DNA