Document Type thesis Author Name Stock, Rachel E. URN etd-0822102-175859 Title DNA Binding Activities in Cerebellar Granule Cell Neurons Recognizing the Promoter for The GABA(A)-alpha6 Receptor Subunit Degree MS Department Biology & Biotechnology Advisors Jill Rulfs, Committee Member Daniel L. Kilpatrick, Advisor Dave Adams, Committee Member Keywords NF-1 granule cell neuron Date of Presentation/Defense 2002-08-16 Availability unrestricted
The objective of this thesis project was to begin identifying which regulatory transcription factors are involved in the up-regulation of the gene promoter for the ƒÑ6 subunit of the gamma-alpha-butyric acid (GABAA-ƒÑ6) receptor in cerebellar granule cell neurons (GCNs). Although a 150 base pair sequence proximal to the GABAA-ƒÑ6 gene promoter had been characterized previously using electrophoretic mobility shift assays (EMSAs), the specific transcription factor(s) needed to express the GABAA-ƒÑ6 gene had not been examined.
This project utilized EMSAs to investigate this 150 base pair sequence further. It was found that when this sequence proximal to the gene promoter was divided into two overlapping halves, both shortened sequences were able to compete for binding with nuclear extracts. The full-length sequence was further divided into six sub-regions, and double-stranded competitors were generated from synthetic oligonucleotides. The only oligonucleotide to compete was the one that corresponded to the region of overlap between the left and right halves. This overlap region contains consensus sites for OCT-1, STAT, and the regulatory transcription factor NF-1. An NF-1 consensus sequence was able to compete DNA-protein complexes. Supershift assays showed that a xenopus NF-1 antibody, previously shown to compete in gel shift assays, caused a mobility shift of the DNA-probe complex. Analysis of extracts from granule cell neurons, cultured from 0 to 6 days in vitro (DIV) indicated NF-1 to be present all time points. Northern analyses were performed using probes for NF-1A, NF-1B, NF-1C and NF-1X. NF-1A transcripts were observed from 0 to 6 DIV, while NF-1B and NF-1X transcripts were present at 2 and 4 DIV. NF-1C RNA was barely detectable at any time point.
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