Cloning and expression of a novel gene highly expressed in GCH non-DN cells

• Main Authors: Kuei-Yu Chen, 陳桂玉
• Other Authors: Yu-May Lee
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/70425880646961230186

碩士 === 國立臺灣大學 === 生化科學研究所 === 89 === Hereditary progressive dystonia (HPD)/DOPA responsive dystonia (DRD) is caused by mutation of the GTP cyclohydrolase I (GCH) gene which is responsible for the first and rate limiting step of tetrahydrobioterin (BH4) synthesis. BH4 plays an important and essential role in aromatic amino acid monooxygeneases and nitric oxide synthase. HPD/DRD patients showed very low (about 2-20% of normal values) GCH activities compared with normal individuals. Dominant negative mechanism hypothesis for HPD/DRD has been proposed and was examined in the cell model. To understand the molecular regulation different between DN and non-DN cells and to look more details about dominant negative mechanism, we performed PCR-selected cDNA subtraction method to selectively amplify differentially expressed genes. More than thirty clones differentially expressed clones were found in DN and non-DN cells. Clone R13, expressed mainly in non-DN cells as confirmed by northern blot analysis, was a novel gene with unknown function. Deduced amino acids encoded by R13 gene were obtained from the GenBank database. There were three forms of deduced R13 proteins resulted from alternative splicing and different promoter usage. The three proteins were designated as R13-A, R13-B, and R13-C. R13-A may localize in the cytosol or membrane bound using immunocytochemistry. R13-B may have post-translational processing, the predicted signal peptide might be cleaved. The stop codon resided in R13-C was not detected. The function of R13 on the GCH protein was discussed.