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.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.
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