Cloning and expression of a novel neural gene

• Main Author: Marshall, Daina Elisabeth
• Published: University of Edinburgh 1997
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.657342

Five novel cDNA clones were isolated and identified from a differentiated glial subtractive library. They were shown to be specific to the nervous system by Northern blotting analysis. In situ hybridisation (ISH) studies were carried out on cultured glial cells to determine if any of the clones were expressed by specific glial cell types. Oligodendrocytes were identified by the monoclonal antibody O4 and astrocytes were identified with an anti-GFAP monoclonal antibody. Clone OL0755, one of the novel brain specific clones identified, was chosen for further investigation. Two cDNA clones, believed to be full length, were obtained by hybridisation screening of a rat cDNA library. Clone OL0755-A is 2.7 kb and encodes a protein of approximately 60 kD. Clone OL0755-B is 2 kb in size and encodes a protein of approximately 50 kD. Sequence analysis showed these clones to be two alternatively spliced forms of a common gene. An antibody (Ab755) raised against a 23 amino acid N-terminus peptide common to both clones (EASFVQTTMALGLPSKKASSRNV) identifies two proteins with the predicted sizes, 50 kD and 60 kD. Both the mRNAs and the proteins are developmentally regulated. Both mRNAs increase in abundance in the postnatal rat brain peaking at postnatal day 21 (P21). Expression of the smaller, more abundant protein also increases during development peaking at P21 while the larger protein is less abundant and downregulates slightly with age. Neither protein is phosphorylated or N-glycosylated. ISH studies on p21 rat brain sagittal sections with clone OL0755 (common to both OL0755-A and OL0755-B) showed a very strong neuronal pattern of expression but also a weaker expression in glial cells which was confirmed by ISH on rat optic nerves and which was consistent with the ISH studies on cultured glial cells. The appearance of these proteins in both glial cells and neurons during the active phase of myelination suggests a possible role in this process either as a structural component or as part of a signalling mechanism between the cell types.