TRANSCRIPTION FACTOR IN CONTROL OF CALBINDIN EXPRESSION IN CEREBELLUM

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来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 钙结合蛋白是在神经元中表达的钙缓冲蛋白，在小脑的浦肯野细胞中观察到最高水平的表达。 以前的工作提供了体外证据，在上游调控区的钙结合蛋白的40 bp的区域是必要的和足够的基因表达在浦肯野细胞。 最初在组织切片培养物中进行的这些研究在转基因小鼠模型中重复。 初步结果表明，这些结果在体内也是真实的。 基于这些结果，推测应该存在特异性识别该序列的转录因子，因此其作用是控制小脑中钙结合蛋白的表达水平。 一个目标是分离这种假定的转录因子。 采用传统的生物化学方法，其中收获野生型小鼠的小脑并分离核提取物。 使用凝胶迁移试验检测含有DNA结合活性的组分，将这些提取物在一系列柱上分级分离。 目前的方法包括阳离子交换柱，然后是非特异性寡核苷酸柱，然后是特异性寡核苷酸柱。 寡核苷酸柱由与固体支持物结合并倒入柱中的寡核苷酸序列的串联重复组成。 双柱方法被设计为仅分离以序列特异性方式与结合序列结合的那些蛋白质。 事实上，这种方法已经确定了活性转录因子。

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. Calbindin is a calcium-buffering protein expressed in neurons, with the highest levels of expression seen in the Purkinje cells of the cerebellum. Previous work provided in vitro evidence that a 40-bp region in the upstream regulatory region of calbindin was necessary and sufficient for gene expression in Purkinje cells. These studies, originally performed in tissue slice cultures, were repeated in a transgenic mouse model. Initial results indicated that these results were also true in vivo. Based on these results, it was postulated that there should be a transcription factor that specifically recognizes this sequence and therefore acts to control calbindin expression levels in the cerebellum. One goal has been to isolate this putative transcription factor. A traditional biochemical approach was taken where cerebella from wild-type mice were harvested and nuclear extracts isolated. Using a gel shift assay to detect fractions containing DNA-binding activity, these extracts were fractionated over a series of columns. The current approach includes a cation exchange column, followed by a non-specific oligonucleotide column, then a specific oligonucleotide column. The oligonucleotide columns consist of tandem repeats of an oligo sequence bound to a solid support and poured into a column. The two-column approach is designed to isolate only those proteins that bind in a sequence-specific manner to the binding sequence. In fact, this approach has identified the active transcription factor.