Gene expression screening for specific genes associated with the skeletal muscles of patients with distal myopathy with rimmed vacuoles

为了获得关于远端有边缘空泡的肌病发病机制的更多信息，确定这种肌病的分子基础是很重要的。由于DMRV患者骨骼肌的一小块，我们首先使用差异mRNA展示技术分离并鉴定了实验氯喹（CQ）处理大鼠比目鱼肌中特异性表达的基因，被称为人类DMRV动物模型（Liang and Pardee, 1992）。在总共约80条条带中，获得并克隆了11条特定的DNA片段，并将该方法应用于自动DNA测序仪对这些基因的DNA片段进行了测序。在cq处理肌肉和对照肌肉的基因表达比较中，仅在对照肌肉中表达的mRNA的DNA片段与titin、F-1 atp酶、细胞色素C氧化酶（CCO）、肌球蛋白重链和flightless1等基因具有高度的同源性。而仅在cq处理过的肌肉中表达的DNA片段与蛋白磷酸酶A2调节亚基、α -滋养肌球蛋白、脱氢甲酸还原酶、β -原肌球蛋白和nebulin等基因具有高度的同源性。此外，我们获得了两个可能与cq处理肌肉有关的新基因。利用从这些序列中获得的引物进行ART-PCR分析，以控制这些基因在DMRV和对照肌肉中的表达差异。与对照组相比，DMRV肌肉中F-1 atp酶、CCO和flightless-1的表达水平发生了显著变化。果蝇Amogaster基因flightless-1的突变已知会导致间接飞行肌的肌纤维变性而导致飞行能力的丧失。目前，我们正在研究flightless-1在DMRV发病机制中的作用，并试图分离两个与cq处理大鼠肌肉相关的新基因的完整cDNA。

To obtain additional information about the pathogenesis of distal myopathy with rimmed vacuoles, it is important to determine the molecular bases of this myopathy. Because of small piece of the skeletal muscles from patients with DMRV,we first isolated and then identify the genes specifically expressed in the soleus muscles of experimental chloroquine (CQ)-treated rats, known as a animal model of human DMRV,using the defferential mRNA display technique (Liang and Pardee, 1992). Of a total of approximately 80 bands, 11 specific DNA fragments were obtained and cloned, and the DNA fragements of these genes were sequenced by the application of the method to an automatic DNA sequencer. In comarisons of gene expression in CQ-treated muscles and control muscles, the DNA fragments derived from mRNA expressed only in control muscles showed a high level of homology with the genes such as titin, F-1 ATPase, cytochrome C oxidase (CCO), myosin heavy chain, and flightless-1. While, DNA fragments expressed only in CQ-treated muscles showed a high level of homology with the genes such as protein phosphatase A2 regulatory subunit, alpha-trophomyosin, dehydroforate reductase, beta-tropomyosin, and nebulin. Furthermore, we obtained the two novel genes probably related to the CQ-treated muscles. ART-PCR analysis using primers derived from these sequences were performed to control differences in expression of these genes in DMRV and control muscles. Levels of F-1 ATPase, CCO,and flightless-1 expressions showed significant changes in the DMRV muscles as compared to the controls. The mutation of the Drosophila Amogaster gene flightless-1 is known to lead to loss of flight ability due to myofibrillar degeneration in the indirect flight muscles. Now, we are studying a role of flightless-1 in the pathogenesis of DMRV,and try to isolate the complete cDNA of two novel genes related to rat CQ-treated muscles.