Characterization of acDNA highly enriched in CNS

Abstract

MUD 5 is one of several cDNAs isolated in a differential screen to identify messages induced during apoptosis in differentiated PC12 cells following removal of NGF. Although subsequent studies indicated that MUD 5 mRNA was not consistently modulated during apoptosis, its characterization was continued because it was expressed primarily or exclusively in the nervous system. Northern blots indicate a single 1.4 kb transcript in all brain regions with expression beginning around postnatal day 3 and increasing gradually until it reaches adult levels at postnatal day 25. In situ hybridization studies of adult rat brain localize MUD 5 mRNA to neurons in cortical and limbic structures as well as cerebellum and brain stem. The most heavily labeled neurons include Purkinje cells, and neurons in the hippocampus, dentate gyrus, deep cerebellar nuclei, and several brainstem nuclei. A full length MUD 5 was isolated from a PC12 cDNA library. The cDNA is 1299 bases, contains a strong Kozak sequence, and predicts an acidic protein of 308 amino acids. The transcript has a high GC content, 62% overall, with some areas as high as 71%. The protein has a predicted molecular weight of 35 kDa; however, following in vitro translation and gel electrophoresis it runs as a 40 kDa protein. The protein has no membrane spanning regions and is predicted to be a globular polypeptide. MUD 5 is highly homologous to OCP1, one of two major proteins in the organ of Corti. Mud 5 also contains a region of 140 amino acids that is similar to proteins in the ICE/ced-3 family of cell death proteases. This homology includes the active site cysteine and flanking amino acids (QACRG/L). The N terminal region of MUD 5 contains a glutamic acid rich PEST sequence which is known to decrease protein stability and an F box that is speculated to be involved in ubiquitin mediated proteolysis. MUD 5 mRNA is decreased following exposure of PC12 cells to metabolic stress induced by a proline analogue but is not modulated by heat shock or reactive oxygen species