We and others have shown that there may be as many as 150,000 different mRNAs expressed in the rodent brain, most of which are brain-specific, arise after birth and are present at very low concentrations when whole brain is examined. Based on RNA complexity and analysis of clonal neural cell lines and brain sections, most of these mRNAs result from the composite nature of the brain, and any given cell requires only a subset of the entire brain repertoire. Surprisingly, half these mRNAs are not polyadenylated at their 3' termini, unlike most eukaryotic RNAs. We have begun to clone DNA sequences encoding some of these rare transcripts and would like to expand this array to include more that are 1) brain-specific; 2) developmentally regulated; 3) poly A-; and 4) cell-type specific; i.e. expressed in certain neural cell types or in specific regions of the brain such as the olfactory epithelium. By hybridization to the appropriate cellular RNAs, we can determine if such clones are post-transcriptionally regulated or modulated by environmental factors. Interesting clones that encode polysomal RNAs will be sequenced and the predicted peptides synthesized to generate antiserum for use in immunohistochemistry in brain sections as well as for biochemical identification of the putative proteins. In addition, direct in situ hybridization will be attempted. Southern blot analysis and isolation of longer genomic clones (from a Lambda library) should allow us to assess whether such clones rearrange during development, are members of brain-specific multi-gene families or exhibit unusual properties, such as being intronless, as has been suggested for the poly A- mRNAs. Using the same cloning approaches, we would like to study a simpler neuronal system, the olfactory epithelium. Basal cells in the epithelium continually divide to replace old olfactory neurons, and division can be synchronously induced by cutting the axons of the receptor neurons which project back to the olfactory bulb in the brain. We propose to prepare libraries (both cDNA and rare transcript-genomic libraries) to RNA extracted from rat olfactory epithelium to select clones that are specific to the receptor cells and those that are specific for the dividing stem cell.
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