Monfries, Clinton Alphaeus Lloyd; (1994) Characterization of the human brain n-Chimaerin cDNA. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A human retinal cDNA clone λH631.2 detects human and rat poly(A)+ mRNAs of 2.2kb and 2.3kb, respectively, which are highly enriched in the brain. The DNA sequence encodes a putative protein of 299 amino acid residues, with an estimated Mr of 34,339 and pi 7.35. The N-terminal portion has 38-50% identity with corresponding regions in the C1a and C1b regulatory region of protein kinase C, bearing the motif HXFX10CX2CX13/14CX2CX4HX2CX7C, part of which is also found in many transcription factors. The C-terminal portion containing 171 amino acid residues has 42% identity with the C-terminal region of BCR, the product of the breakpoint cluster region gene involved in Philadelphia (Ph') chromosome translocation associated with chronic myelogenous leukaemia. The link between two distinct protein families led to the designation n-Chimaerin, for this novel brain protein. Portions of the λH631.2 cDNA were cloned into various bacterial expression systems to produce β-galactosidase-(β-Gal-), anthranilate synthase-(frpE-) and glutathione S-transferase- (GST-) fusion proteins of n-Chimaerin, as well as an unfused n-Chimaerin product. TrpE/n-Chimaerin expressed proteins were most effective at producing n-Chimaerin specific antibodies in rabbits. Greater specificity was achieved by further absorption of the antibodies to β-Gal/n-Chimaerin fusion proteins. Affinity purified antibodies detected the presence of n-Chimaerin and related molecules in the brain, various tissue culture cell lines and specifically in cytosol from brain and testes, in agreement with mRNA analyses. Preliminary immunocytochemical analyses of rat brain sections, demonstrated the presence of n-Chimaerin in neurones. Extraneous signals were also detected; these may be to related proteins or proteins with shared epitopes. TrpE/, GST/n-Chimaerin and purified n-Chimaerin C-terminal proteins were used by others to establish functional assays, showing the N-terminal to bind phorbol esters in a zinc-dependent manner and the C-terminal to act as a GTPase-activating protein (GAP) for Rac1, a member of the Ras superfamily. These studies have provided a basis for the functional characterization and localization of n-Chimaerin in the brain, and will facilitate an understanding of its specific role in signal transduction in neurones.

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