Characterization of the 5' flanking region and potential control elements of the ovine follitropin receptor gene.

In the preceding two reports, we presented evidence for the structure and functional characteristics of two different, yet related variants of the sheep testicular follicle-stimulating hormone receptor (oFSH receptor) cDNA. To shed further light on the structural basis of the formation of such receptor forms with different motifs and the eventual understanding of gene regulation, we initiated studies to clone the gene. An 8 kb EcoR I fragment containing the exon-1 and 5' flanking sequence was cloned and characterized from among the 14 clones that were isolated from the genomic library. Although not all other clones were fully characterized, we believe that the entire gene of 85-100 kb has been secured as we adopted a successive screening strategy to accommodate currently known alternatively spliced variants of the receptor in this species. This has led us to propose a revised model that includes an 11th exon for the oFSH receptor gene. The 11th exon that lies beyond the currently postulated 10th exon contributes important DNA sequence that results in two different structural/functional motifs. One creates a dominant negative receptor and the other leads to the formation of a growth factor type I receptor for the hormone. In the 2.1 kb 5'-upstream region, there are a number of potentially interesting regulatory elements that resemble sites for estrogen response element (ERE-like), CRE, and orphan receptor (SF-1/ NGF I-A) transcription factors among others. Other interesting features include the presence of potential germ cell specific and methylation sites. By performing primer extension with testicular RNA, we could identify a single major transcription start site at -163 relative to +1ATG. The availability of the structure of FSH-receptor gene in this domestically important seasonal breeder could spur investigations into the control of receptor gene expression.