TRANSCRIPTION FACTOR IN CONTROL OF CALBINDIN EXPRESSION IN CEREBELLUM

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。Calbindin是一种在神经元中表达的钙缓冲蛋白，在小脑的浦肯野细胞中表达水平最高。先前的工作提供了体外证据，证明在calbindin上游调控区的40个碱基对Purkinje细胞的基因表达是必要的和充分的。这些研究最初是在组织切片培养中进行的，后来在转基因小鼠模型中重复进行。初步结果表明，这些结果在体内也是正确的。根据这些结果，推测应该有一种转录因子特异性地识别该序列，从而控制小脑中Calbindin的表达水平。一个目标是分离这种假定的转录因子。采用传统的生化方法，采集野生型小鼠的小脑，并分离核提取液。用凝胶漂移实验检测含有DNA结合活性的组分，这些提取物在一系列柱子上进行分级。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载体结合并倒入柱中的寡核苷酸序列的串联重复组成。双柱法的设计目的是只分离那些以序列特异的方式与结合序列结合的蛋白质。事实上，这种方法已经确定了激活的转录因子。

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.