Functional analysis of Charcot-Marie-Tooth type 2D related glycyl-tRNA synthetase mutants in yeast

• Main Authors: Meng-Tsen Ke, 柯孟岑
• Other Authors: Chien-Chia Wang
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/46545725179763064291

碩士 === 國立中央大學 === 生命科學研究所 === 98 === Charcot-Marie-Tooth disease, or CMT, is the most common inherited neurological disorder, with a frequency about 1 in 2500 people. Several genes have been identified associated with CMT. Missense mutation in glycyl-tRNA synthetase (GlyRS) leads to peripheral nerve degeneration specified as Charcot-Marie-Tooth disease type 2D (CMT2D). In the past decade, ten mutations that are associated with CMT2D were identified in human GlyRS, an essential enzyme for protein translation. Among conserved sequence between yeast and human, E21G, L69P, I263F, G223R, H402R, andG512R were chosen in this research to study the phenotype changes, enzyme activity, and dimer stability in yeast model. Our data indicated I263F, H402R and G512R were not able to complement both cytoplasmic and mitochondrial function in GRS1 knockout strain. In addition, though these CMT2D-cusing mutants did not affect the dimer association of GlyRS, these mutants did not reveal any dominant negative effects in growth assays, suggesting the mutant proteins were not poison to the wild-type ones. Surprisingly, some of CMT2D-causing mutants had the same effect as yeast transcriptional read-through mutant P552F in dual luciferase assay, implying the putative role of GlyRS secondary function in CMT2D pathogenesis. Easily introduced mutations and genetic simplicity of yeast will be a powerful tool for assaying for the putative function of GlyRS to further dissect the mechanism of CMT2D.