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K. Kshitish Acharya1 , P. Prabhakara Reddi1 , John C. Herr1
CRGCV, Department of Cell Biology, University of Virginia, Charlottesville, VA 22908 1
The testis-specific gene SP-10 is expressed during spermiogenesis. Transgenic mice demonstrated that the cis-acting elements critical for round spermatid-specific transcription lie in the -266 to -91 bp region of the SP-10 promoter. In the present study, electrophoretic mobility shift assay (EMSA) and southwestern analysis (SWA) were employed to specify smaller regions of the SP-10 promoter which could potentially interact with transcription factors. Four contiguous (A - D) and 3 overlapping segments (AB, BC and CD) of the -266 to -91 bp region, each 45 bp in length, were analyzed. EMSAs revealed that each oligomer formed more than one sequence-specific complex with testis nuclear proteins. However, most of these complexes were common to at least one of the somatic tissues studied, viz. brain, heart, lung, liver, spleen and kidney. Fragments A and D, which consist of GATA-1 and Ets-1 binding sites, respectively, formed one testis-specific complex each. Age-dependent expression of testicular proteins binding to the SP-10 promoter was examined next. A and BC formed specific complexes with testicular proteins from 24 but not 5, 8 or 17 day old mice. Thus, these binding proteins first appear in the pre-pubertal testis coincident with the abundance of round spermatids which express SP-10. SWA demonstrated presence of a 40 - 45 kD protein in the testis, but not kidney nuclear extracts, that binds to all the SP-10 oligomers (except CD) and has the highest affinity to C. This 40 - 45 kD protein did not bind to 3 other unrelated oligomers. In addition, oligomer C bound to 70 and 84 kD, and D bound to 51 and 95 kD testicular proteins. The study thus identified specific SP-10 promoter fragments and indicated the presence of cognate binding proteins of possible testis-specificity. Supported by NIH HD 29099, HD 36239 and Fogarty International Center.
This abstract is being presented on Monday, August 2 at 2:30 PM at Todd 276.