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
Y. Kato et al., 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, MOL C ENDOC, 158(1-2), 1999, pp. 69-78
Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone
(LH), are synthesized specifically in the gonadotropes of the anterior pitu
itary. The aim of this study was to investigate nuclear factors that bind s
pecifically to the porcine FSH beta-subunit gene. We examined nuclear prote
in 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 downstrea
m 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 th
at the proteins bound to a limited region of DNA, 107 bp long (designated a
s Fd2), located about -800 bp upstream from the transcription initiation si
te. Competitive binding assays demonstrated that the protein binding was se
quence specific; the addition of excess amounts of several putative regulat
ory 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 invol
ved in this interaction. Southwestern blotting demonstrated that at least s
even 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 bindi
ngs were weak, although only the cerebellum showed a pattern of binding tha
t was similar to that of the anterior pituitary. These data suggest that mu
ltiple proteins of the anterior pituitary recognize a specific region of th
e porcine FSH beta-subunit gene. (C) 1999 Elsevier Science Ireland Ltd. All
rights reserved.