A gene encoding chinook salmon (Oncorhynchus tschawytscha) gonadotropin alpha subunit: gene structure and promoter analysis in primary pituitary cells.

The cDNA clones coding for the chinook salmon gonadotropin alpha (sGTH alpha) subunit were isolated from a salmon pituitary cDNA library. The full-sized cDNA clone, sGTH alpha-C2, was 678 bp in size and encoded a precursor polypeptide of 114 amino acids, including a signal peptide of 22 amino acids. The deduced mature sGTH alpha subunit polypeptide is identical with the major alpha subunit of chum salmon gonadotropin. Genomic clones coding for the sGTH alpha subunit were isolated from a partial BamHI genomic library in lambda EMBL3 using the cDNA as a probe. Seven positive clones were identified from 1 x 10(6) plaques. Restriction mapping of positive clones suggested that there were two copies of the GTH alpha subunit gene in the salmon genome. The 2 kb of 5'-flanking region and the 1.1 kb of the entire sGTH alpha subunit coding region were sequenced from the genomic clone, sGTH alpha-G1. The sGTH alpha subunit gene contains four exons and three introns. However, its introns are smaller than those in the mammalian hormones. Inspection of the sGTH alpha subunit promoter region revealed several potential cis-acting elements, including the gonadotrope-specific element (GSE), Ap-1, AP-2, and others. Functional analysis of the sGTH alpha subunit promoter by the transient transfection of several sGTH alpha/CAT chimeric plasmids into rainbow trout pituitary cells suggests that its pituitary-specific expression is GSE-dependent. The activity of the chimeric plasmids can be stimulated by 8-bromo-cAMP. These experiments indicate that pituitary primary cells can be used to dissect various cis-acting elements important for salmon GTH alpha subunit gene expression.