Seminal vesicle secretory proteins (SVS) IV and V expression in the rat are under the control of androgens. The gene for SVS IV and the genes for SVS V have been isolated from a rat gene library. The DNA sequence has been determined for the transcription unti and flanking sequences of the SVS IV gene. The first objective is to derive the DNA sequence of the flanking regions and transcription unti for the SVS V genes.
The aim i s to compare 5'-flanking regions for potential regulatory sites in SVS IV and SVS V genes which may have some role in androgen action. Two major Sl nuclease sensitive sites in supercoiled plasmid pSVS 3.3 (containing flanking and transcription unit for SVS IV) have been identified. The 5'-Sl site is at or near an inverted repeat at -113 to -117 bp from the transcription initiation site. Along with sequencing the SVS V genes, Sl nuclease sensitive sites will also be mapped in supercoiled plasmids containing the SVS V genes as they are isolated, mapped, and sequenced. DNAse I hypersensitive and Sl nuclease sensitive sites in the SVS IV and V genes will be mapped by digestion of nuclei from normal and castrate seminal vesicle tissue. These data will be compared to sites identified by the Sl nuclease digestion of various supercoiled plasmids containing SVS IV and SVS V gene fragments. A major objective will be to link the SVS IV and SVS V genes to several eukaryotic cloning vectors with dominant selection markers (e.g., pSV-neo) and introduce the SVS genes into recipient cell lines with well characterized androgen receptors or into primary cultures of seminal vesicle cells. Cell lines will be established from the transfection experiments which, when androgen is added to the media, the transcription rate of the introduced SVS genes will significantly increase. With these cell lines and the production of various in vitro deletion mutants and transfection of these mutant DNA forms of the SVS genes into cell lines, the regions in the 5'-flanking, transcription unit and/or 3'-flanking that are necessary for androgen-dependent expression may be established. These data will broaden our knowledge base about steroid hormone action and the control of an apparently coordinately regulated gene set.
