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Molecular cloning of the human Substantia innominata : characterization of a brain large mRNA

Brain tissue samples were collected from individuals with histologically and biochemically confirmed Alzheimer's Disease (AD), as well as from a group of individuals without any signs of neurological disease (NNC). Ribonucleic acid (RNA) was extracted from these tissues, characterized by several chemical methods, and the yields were compared between AD and NNC groups. High molecular weight RNA could be effectively extracted from frozen postmortem human brain. In comparison to the NNC group, tissue RNA levels were reduced in the AD hippocampus, but not in the temporal cortex or substantia innominata (SI). No difference in the physical integrity of the RNA was apparent between AD and NNC groups. A high complexity complementary deoxyribonucleic acid (cDNA) library was prepared using RNA extracted from the NNC SI. Differential hybridization screening using a variety of cDNA probes was employed to identify mRNAs expressed di f f e r e n t i a l l y between AD and NNC tissue, and between SI and other human tissues. Many selected mRNAs were examined for specificity of expression in brain tissue and brain regions. The cDNA clone pSI3a-24 identified an mRNA, which, on Northern blot hybridization, was expressed in brain tissue but not in the other human tissues examined. The identified mRNA was unusually large, with a chain length estimated at 15,500 bases. Quantification of the brain tissue levels of this mRNA was carried out using a ribonuclease protection assay. Tissue levels were higher in the SI (40 pg/jug RNA) than in the temporal cortex (28.6 pg/Mg), and were lowest in the cerebellum (11.2 pg/M9)• Levels of the mRNA in temporal cortex samples were increased 29% in the AD group, relative to NNC. No significant difference in the SI tissue levels was observed between AD and NNC groups. Hybridization analysis of human genomic DNA indicated that the mRNA was encoded by a single copy gene. Sequence analysis of the f u l l 3 kilobases of cloned cDNA was completed. Computer database searches failed to identify any known nucleic acid sequence with homology to the cDNA. Examination of the cDNA sequence for potential polypeptide coding regions suggested that the corresponding mRNA has a 3 untranslated region of at least 3 kilobases.