CHARACTERIZATION OF MOTIFS REGULATING EMBRYONIC & SPERMATOGENIC GENE EXPRESSION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们的实验室有兴趣在识别序列基序，调节基因表达的关键事件，如胚胎发生和精子发生。胚胎发生是脊椎动物中高度保守的发育过程，导致具有相似表达模式的直向同源基因受到进化上保守的机制控制的假说。新的转录因子结合位点的计算预测是一项艰巨的任务，这在很大程度上是由于它们的小尺寸。然而，有几种方法可用于发现具有高功能潜力的位点，包括基序发现算法（隐马尔可夫模型），统计抽样方法（吉布斯抽样）和与某种类型的基序发现方法（系统发育足迹）相结合的序列比较方法。 为了验证我们的假设，即具有保守功能的直系同源物由保守的转录机制调节，我们正在寻找人类和模式生物（包括鱼类和啮齿动物）基因上游的基因组区域中的基序。为了评估候选基序的功能，我们正在使用电泳迁移率变动测定来测试它们特异性结合从小鼠、冰鞋和角鲨中纯化的核蛋白的能力。我们已经成功地使用了现有的基因组序列和基因表达信息，从人类，小鼠，大鼠，鲨鱼，小溜冰鞋，斑马鱼和河豚，以确定区域的选择数量的基因上游的序列保守。我们将继续在此基础上构建并验证我们发现的图案。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for 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.