Multiple binding sites for nuclear proteins of the anterior pituitary are located in the 5'-flanking region of the porcine follicle-stimulating hormone (FSH) beta-subunit gene.
Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH), are synthesized specifically in the gonadotropes of the anterior pituitary. The aim of this study was to investigate nuclear factors that bind specifically to the porcine FSH beta-subunit gene. We examined nuclear protein binding to 2.75 kilobase pairs (kbp) of DNA adjacent to the porcine FSH beta-subunit gene: about 2.32 kbp of upstream DNA and 0.43 kbp of downstream DNA. The upstream region contains only TATA box, CACCC element, and some imperfect sequences of cAMP-responsive element, activator protein-1 binding site, and activator protein-2 binding site. Gel mobility shift assay using nuclear proteins extracted from the porcine anterior pituitary revealed that the proteins bound to a limited region of DNA, 107 bp long (designated as Fd2), located about -800 bp upstream from the transcription initiation site. Competitive binding assays demonstrated that the protein binding was sequence specific; the addition of excess amounts of several putative regulatory sequences and plasmid (non-homologous) DNA fragments did not reduce the binding. Furthermore, all five subfragments of Fd2 were also bound by the pituitary nuclear proteins, showing that the entire region of Fd2 is involved in this interaction. Southwestern blotting demonstrated that at least seven protein species of 110, 98, 78, 63, 52, 42, and 35 kDa recognize Fd2. Nuclear proteins from several other porcine tissues were also able to bind to the Fd2 fragment but the gel shift patterns were different and the bindings were weak, although only the cerebellum showed a pattern of binding that was similar to that of the anterior pituitary. These data suggest that multiple proteins of the anterior pituitary recognize a specific region of the porcine FSH beta-subunit gene.
Kato
Y
Y
Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
Tomizawa
K
K
Kato
T
T
D020121:5' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D015139:Blotting, Southern; D005640:Follicle Stimulating Hormone / Q000235:genetics / Q000378:metabolism*; D037201:Follicle Stimulating Hormone, beta Subunit; D009687:Nuclear Proteins / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012045:Regulatory Sequences, Nucleic Acid; D013552:Swine
D020121:5' Untranslated Regions; D037201:Follicle Stimulating Hormone, beta Subunit; D009687:Nuclear Proteins; D005640:Follicle Stimulating Hormone