| Co-Investigator(Kenkyū-buntansha) |
HAGIHARA Masako Department of Biochemistry, Nagoya University School of Medicine, Research Worke, 医学部, 教務員
ICHINOSE Hiroshi Departmnt of Biochemistry, Nagoya University School of Medicine, Assistant Profe, 医学部, 助手 (90192492)
KANEDA Norio Department of Biochemistry, Nagoya University School of Medicine, Associated Pro, 医学部, 講師 (00144139)
NAOI Makoto Department of Bioscience, Nagoya Institute of Technology, Professor, 教授 (50022786)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1990: ¥3,400,000 (Direct Cost: ¥3,400,000)
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| Research Abstract |
We cloned fullーlength cDNAs and genomic DNAs of human catecholamine synthesizing enzymes, i. e., tyrosine hydroxylase (TH), aromatic L-maino acid decarboxylase (AADC), dopamine betaーhydroxylase (DBH), and phenylethanolamine Nmethyltransferase (PNMT), and determined the nucleotide sequences and the deduced amino acid sequences. Multiple mRNAs of human TH, human DBH, and human PNMT were discovered by cDNA cloning. Four types of human TH mRNAs are produced by alternative splicing mechanisms from a single gene. The multiple forms of human TH may give additional regulation to the human enzyme in relation to neuropsychiatric diseases such as Parkinson's disease. Two types of TH mRNA corresponding to type 1 and type 2 were detected in adrenal gland and brain of two species of primate. In contrast, only a single from of TH mRNA was detected in Sunkusmurinus and rat. We have succeeded in expressing human TH gene in transgenic mice. The 5'-flanking of the genes of human TH, DBH and PNMT contain
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possible transcription regulatory elements such as cyclic AMP responsive element (CRE) (TH, DBH, PNMT), glucocorticoid responsive element (GRE) (DBH, PNMT), and Sp 1 (TH, PNMT) binding site. We cloned a fullーlength hAADC cDNA encoding a protein of 480 amino acids with calculated molecular mass of 53.9 kDa. COS cells transfected with the hAADC cDNA produced a major immunoreactive band and the expressed enzyme decarboxylated both L-DOPA and L-5hydroxytryptophan. We isolated two different mRNA types (trpes A abd B) for hDBH and its genomic DNA, and showed that these mRNAs are generated through alternative polyadenylation from a single gene.
These results indicate that the studies on the molecular structures and the gene expression of human catecholamineーsynthesizing enzymes are significant in the basic studies of neuropsychiatric diseases as Parkinson's disease. In this project we have succeeded in cloning fullーlength cDNAs and genomic DNAs of all human catecholamineーsynthesizing enzymes ; human TH, AADC, DBH, and PNMT in order to elucidate gene expression. Less
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