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Keywords :
Animals; Base Sequence; Binding Sites; Cell Extracts; Cells, Cultured; Cloning, Molecular; DNA/metabolism; DNA-Binding Proteins/metabolism; Gene Expression Regulation/*genetics; Genes/genetics; Molecular Sequence Data; Pituitary Gland/cytology/*physiology; Prolactin/*genetics; Promoter Regions, Genetic/genetics; Rats; Regulatory Sequences, Nucleic Acid/*genetics; Sequence Analysis, DNA; Sequence Deletion; Sequence Homology, Nucleic Acid; Species Specificity; Tilapia/*genetics; Transcription Factor Pit-1; Transcription Factors/metabolism; Transcription, Genetic/genetics
Abstract :
[en] 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.
Publisher :
Mary Ann Liebert, Inc., Larchmont, United States - New York
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