Analysis of new muscle specific genes/proteins and their function in sarcomere assembly in C. elegans using SAGE and Affymetrix Microarray Chip technology including Bioinformatics

我的研究计划的目的是鉴定秀丽隐杆线虫中参与肌节组装的新基因/蛋白。凭借我申请的奖学金，我将在温哥华不列颠哥伦比亚大学Don Moerman教授的实验室进行一个项目。他的实验室专注于肌肉发育，特别是秀丽隐杆线虫肌肉肌节组装的问题。该系统是研究含整合素黏附结构组装的良好体内模型。最近，温哥华Michael Smith基因组科学中心利用秀丽隐杆线虫肌肉特异性RNA，通过基因表达序列分析（SAGE）鉴定出了大量肌肉特异性基因。我将使用这个数据库来帮助找到附着复合体中的新基因/蛋白质，并确定它们在肌节组装过程中的功能。拟开展的具体研究目标如下：1)以亚细胞定位为重点，确定线虫不同候选基因的表达模式。2)利用RNAi或诱变筛选技术生成目标基因的秀丽隐杆线虫突变动物。3)关注肌节发育的突变动物表型分析。4)感兴趣的靶蛋白生化分析。由于有许多具有未知功能的人类同源基因，这一发现将非常有用，因为线虫中的肌丝晶格组装与哺乳动物细胞培养中的局灶粘连组装非常相似。

The aim of my research proposal is to identify novel genes/proteins involved in sarcomere assembly in C. elegans. With the fellowship I am applying for, I will work on a project in the laboratory of Prof. Don Moerman at the University of British Columbia, Vancouver. His lab focuses on muscle development and specifically on the problem of muscle sarcomere assembly in C. elegans. This system is an excellent in vivo model to study the assembly of integrin-containing adhesion structures. Recently, a large number of muscle specific genes have been identified by serial analysis of gene expression (SAGE) using C. elegans muscle specific RNA at the Michael Smith Genome Sciences Center, Vancouver. I will use this database to help find new genes/proteins placed in adhesion complexes and to determine their function during sarcomere assembly. The specific goals of the proposed research are as follows: 1) Determination of the expression pattern of different candidate genes in C. elegans focusing on subcellular localization. 2) Generating C. elegans mutant animals for the genes of interest using RNAi or a mutagenesis screen. 3) Phenotyp analysis of mutant animals focusing on sarcomere development. 4) Biochemical analysis of interesting target proteins. Since there are many genes having human homologues with unknown function the findings will be very useful as myofilament lattice assembly in the nematode is remarkably similar to the assembly of focal adhesions in mammalian cell culture.