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Neuron-specific enolase (NSE) is an isoform of the glycolytic enzyme enolase which is expressed specifically in neurons and neuroendocrine cells in the mammalian nervous system. Its onset of expression coincides with neuronal differentiation and it has therefore become established as a marker of mature, postmitotic neurons (Zomzely-Neurath, 1983). The molecular basis of neuron- specific gene expression is still poorly understood (Twyman and Jones, 1995b) and the panneuronal NSE gene thus represents an excellent model for the investigation of mechanisms responsible for neuronal gene regulation. Recently, the proximal 1.8 kbp of 5' flanking sequence from the rat NSE gene was shown to confer neuron-specific and panneuronal expression upon a heterologous gene in transgenic mice (Forss-Petter et al., 1990). This suggested that the sequence probably contained neuron-specific cis-acting elements which could be investigated using a deletion-reporter strategy in cultured cells and transgenic mice. The 1.8 kbp flanking sequence has also been shown to respond to NGF and retinoic acid in parallel with the endogenous gene (Alouani et al., 1993). In this project, the 1.8 kbp 5' flanking sequence was dissected, and various truncated derivatives were compared to the full length construct in cultured cells of neuronal and nonneuronal origin. It was shown that 255 bp of 5' flanking sequence was capable of conferring full cell type-specific regulation upon a heterologous gene, indicating the presence of neuronal c/s-acting elements within 255 bp of the transcriptional start site. Further transfection experiments, concentrating on this short proximal fragment, showed that elements responsible for neuron-specific gene expression were present in this region and in vitro analysis identified at least one specific DNA-protein interaction. Preliminary analysis of NSE gene regulation was also carried out in transgenic mice. These experiments, taken together with previous studies, showed that the level of transgene expression was variable and subject to both position and gene dosage effects. It was concluded that further analysis should be carried out in transgenic lines, preferably utilising flanking boundary elements which would protect the NSE transgenes from the position effects (to which they were highly susceptible). The impact of the transfection and transgenic experiments was discussed with respect to the published literature and ideas for future experiments were suggested. |
