The primary goal of this study is to elucidate the molecular mechanisms regulating gene expression during spermatogenesis in mouse testis. The basic assumption underlying the experimental strategies proposed, is that the precision and regularity of the program of germ cell proliferation and differentiation is regulated primarily at the level of gene transcription. To accomplish this goal cDNA libraries will be constructed with RNA isolated from fractionated gem cells representative of all the morphologically distinct stages of spermatogenesis. Subtractive hybridization will be used to enrich for stage-specific transcripts in molecular probes utilized to screen these libraries. Stage and cell-type specificity of the cDNAs isolated will be determined by developmental and cell-type specific Northern blots and also by in situ hybridization assays. Sequences from the 5' end of these cDNAs will be used to construct a nested set of gene-specific primers. These primers will be utilized in ligation mediated single-sided PCR strategies to isolate promotor/enhancer elements of the respective genes and also to analyze patterns of stage and cell-type specific transcription factor binding using in vivo footprinting assays. Future studies utilizing these promotor/enhancer elements are expected to establish functional roles for transacting factors and other regulatory molecules known to be expressed during different stages of spermatogenesis. Students will participate in all recombinant DNA methodologies including construction of cDNA libraries, plaque hybridizations, Northern and Southern hybridizations, and DNA sequencing.
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