Isolation and characterization of cDNA clones of mRNAs expressed selectively in the immature cells of developing brain.

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We first established several improved procedures to isolate cDNA clones of mRNAs expressed selectively in the immature cells of developing brain. A modified differential screening procedure, which utilized two vector(pT7T3D and pBluescript) -system, showed clear signals with low background levels. Southern blot analysis of amplified cDNA library DNAs were shown to be able to replace the Northern blot analysis. These procedures were reliable especially for the analysis of cDNA clones whose corresponding mRNAs were relatively abundantly expressed. An improved subtractive cDNA cloning procedure, named Directional tag PCR subtraction, were demonstrated to be efficient to isolate differentially expressed clones of relatively rarely expressed mRNAs. These three procedures require no additional RNA for the screening after construction of the cDNA libraries, enabling the analysis of tiny brain regions of particular interest.By use of the procedures described above, we then isolated rat fetal brain-enriched (FBE) clones whose corresponding mRNAs were expressed preferentially in the prenatal stages of brain development. We successfully identified 22 distinct FBE clones whose mRNAswere expressed at least 5-fold more in the fetal brain than in the adult brain. The nucleotide sequence analysis of the 22FBE clones revealed that 13 of them had no significant matches to the sequences reported in the databases, whereas 9 of them matched previously reported sequences (alpha tubulin M alpha 1, beta tubulin M beta 5, thymosin beta 10, stathmin, beta tubulin M beta 2, alpha-internexin, ferritin Lg subunit, neuronatin, and amphoterin). In situ hybridization analysis showed that mRNAs corresponding tothe FBE clones decreased during braindevelopment with various expression patterns. The mRNA of a newly isolated FBE clone was shown to be expressed selectively in the immature cells in theexternal germinal layr of the cerebellum and the subventricular zone of developing brain.