This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator. Our laboratory is interested in identifying sequence motifs that regulate gene expression during critical events such as such as embryogenesis and spermatogenesis. Embryogenesis is a highly conserved developmental process in vertebrates, leading to the hypothesis that orthologous genes with similar expression patterns are controlled by evolutionarily conserved mechanisms. Computational predictions of novel transcription factor binding sites is a daunting task due in large part to their small size. However, several approaches are useful for uncovering sites with high functional potential including motif-finding algorithms (Hidden Markov Models), statistical sampling methods (Gibbs sampling) and sequence comparison methods coupled to some type of motif finding methodology (Phylogenetic footprinting). To test our hypothesis that orthologs with conserved function are regulated by conserved transcriptional mechanisms, we are searching for motifs in the genomic regions upstream of genes from humans and model organisms, including fish and rodents. In order to evaluate the function of candidate motifs, we are using the electrophoretic mobility shift assay to test them for the ability to specifically bind nuclear proteins purified from mice, skates and dogfish sharks. We have successfully used the available genomic sequence and gene-expression information from human, mouse, rat, dogfish shark, little skate, zebrafish and Fugu to identify regions of sequence conservation upstream of a select number of genes. We continue to build upon this set and validate the motifs we discover.
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