| Co-Investigator(Kenkyū-buntansha) |
SAKAI Masao Fujita Health Univ., School of Med., Dept. of Anatomy, Assistant, 医学部解剖学, 助手 (90196048)
YAMADA Keiki Fujita Health Univ., School of Med., Dept. of Anatomy, Assistant Prof., 医学部解剖学, 教授 (60084604)
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| Research Abstract |
(1) Our coworkers in T. Nagatsu's laboratory have systematically isolated cDNA and genomic clones encoding catecholamine-synthesizing enzymes. In human tyrosine hydroxylase (hTH), four types of mRNA were produced by alternative RNA splicing from a single gene. The cDNA probe (probe 1 : 0.25kb SacI-EcoRI), which hybridized only with hTHmRNA, or the cDNA probe (probe 2 : 1.3kb EcoRI-SacI), which hybridized with both human and mouse mRNAs was labeled with [a-35S] dCTPaS by the multiprime DNA labeling method. Using probe 2, TH expression was observed in the ventral tegmental area (VTA) and substantia nigra (SN), but no expression was found using probe 1 (manuscript in preparation).
(2) We employed an 11 kb DNA fragment of the TH gene consisting of 2.5 kb of 5'-upstream region containing promoter information, the entire exon-intron structure, and 0.5 kb of the 3'-franking region for the production of transgenic (Tg) mice. Using 35S-labeled protein-coding region of hTHcDNA as a probe (probe 2
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), extremely high-level expression of the mRNA was observed in the SN and VTA from Tg mice. Using 35S-labeled hTH-specific cDNA fragment (probe 1), specific expression of hTHmRNA was observed in Tg brain, whereas no hybridization signal was observed in non-Tg brain sections (Ref. 6).
(3) We have described tissue-specific expression of the transgene in catecholaminergic (CAergic) neurons and adrenal glands. In addition to these hTH gene expression in CAergic regions, we also found atypical hTH gene expression in non-catecholaminergic (nCAergic) regions of Tg mouse brain by immunocytochemistry at light and electron microscopic levels and by in situ hybridization. These cells were AADC-, DBH-, PNMT-, DA-, and 5HT-negative. We have previously reported the transient expression of TH in the anterior olfactory nucleus (AON) during late embryonic and postnatal stages (Ref. 3). Moreover, we reported an existence of only PNMT-positive neurons in the AON from postnatal 10 days to adult stage. At electron microscopic level, synaptic formation was observed (Ref. 1). Transient appearance of TH or PNMT immunoreactive neurons was found (Ref. 5).
(4) We succeeded to produce antibodies raised to the different oligopeptide segments of human DBH (Ref. 4). Less
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