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

为了获得更多的信息，远端肌病的发病机制与镶边空泡，重要的是要确定这种肌病的分子基础。由于DMRV患者的骨骼肌只有一小块，我们首先分离并鉴定了氯喹（CQ）处理的实验大鼠比目鱼肌中特异性表达的基因，该大鼠被称为人DMRV的动物模型，使用差异表达mRNA显示技术（Liang和Pardee，1992）。在总共约80个条带中，获得并克隆了11个特异性DNA片段，并通过将该方法应用于自动DNA测序仪对这些基因的DNA片段进行测序。在CQ处理的肌肉和对照肌肉中的基因表达的comarisons中，来自仅在对照肌肉中表达的mRNA的DNA片段显示出与诸如肌联蛋白、F-1 ATP酶、细胞色素C氧化酶（CCO）、肌球蛋白重链和flightless-1的基因的高度同源性。而仅在CQ处理的肌肉中表达的DNA片段显示与蛋白磷酸酶A2调节亚基、α-营养肌球蛋白、脱氢甲酸还原酶、β-原肌球蛋白和星云蛋白等基因的高度同源性。此外，我们还获得了两个可能与CQ处理的肌肉相关的新基因。使用来自这些序列的引物进行ART-PCR分析，以控制这些基因在DMRV和对照肌肉中表达的差异。与对照组相比，DMRV肌肉的F-1 ATP酶、CCO和flightless-1表达水平显示出显著变化。已知果蝇Amogaster基因flightless-1的突变导致由于间接飞行肌肉中的肌原纤维变性而丧失飞行能力。现在，我们正在研究flightless-1在DMRV发病机制中的作用，并尝试分离与CQ处理的大鼠肌肉相关的两个新基因的完整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.