CHARACTERIZATION OF C ELEGANS HOMOLOGUES OF PKD1 AND PKD2

由两个常染色体显性多囊肾病基因位点PKD1和PKD2编码的蛋白在细胞中的作用尚未阐明。线虫的PKD1 （lov-1）和PKD2 （pkd-2）同源物在三类成年雄性感觉神经元中高度表达。LOV-1功能缺陷的雄性在交配行为中表现出两种表型：对雌雄同体没有反应，不能有效地定位外阴。这些突变体具有明显正常的感觉神经元，表明lov-1和pkd-2可能参与感觉本身或神经元生理或组织的某些方面。在本项目中，秀丽隐杆线虫分子遗传学鉴定和定义基因作用途径的能力将应用于PKD功能。隐杆线虫的pkd-2基因将被删除并分析其表型。线虫PKD同系物在细胞水平上的功能将通过对这些突变体以及任何其他表型的神经元结构和功能的详细分析来检查。克隆的lov-1基因能够补充染色体突变的行为表型，这使得体内结构/功能研究变得容易。特别是，位点定向诱变研究和转基因动物中这些蛋白质突变形式的表达将允许对蛋白质结构域的作用进行详细分析。此外，通过测试人类PKD蛋白或蠕虫-人嵌合蛋白替代蠕虫蛋白的能力，将验证线虫蛋白详细研究的相关性。候选PKD相互作用蛋白的作用，鉴定为在人类，小鼠或果蝇PKD研究中发现的秀丽隐杆线虫同源蛋白，或在秀丽隐杆线虫中通过双杂交筛选。将通过删除这些基因的染色体拷贝和随后的这些突变的表型和遗传特征进行测试。将进行基因筛选，以确定与PKD功能有关的新基因和新蛋白质，从而确定具有与lov-1突变相同的男性特异性双重感觉缺陷的突变。

The cellular roles of the proteins encoded by the two autosomal dominant polycystic kidney disease loci PKD1 and PKD2 have not yet been elucidated. The C. elegans homologs of PKD1 (lov-1) and PKD2 (pkd-2) are highly expressed in three classes of adult male sensory neurons. Males defective in LOV-1 function display two phenotypes during their copulatory behavior: a failure to respond to hermaphrodites and a failure to efficiently locate the vulva. These mutants have apparently normal sensory neurons, indicating that lov-1 and presumably pkd-2 are involved in sensation per se or in some aspect of neuronal physiology or organization. In this project, the power of C. elegans molecular genetics to identify and define pathways of gene action will be applied to PKD function. The C. elegans pkd-2 gene will be deleted and its phenotype analyzed. The function at a cellular level of C. elegans PKD homologs will be examined by detailed analysis of neuronal structure and function in these mutants, as well as any other phenotypes. The ability of the cloned lov-1 gene to complement a chromosomal mutation for the behavioral phenotypes allows facile structure/function studies in vivo. In particular, site-directed mutagenesis studies and expression of mutant forms of these proteins in transgenic animals will allow a detailed analysis for the role of protein domains. Also, the relevance of detailed studies of the C. elegans proteins will be verified by testing for the ability of human PKD proteins or worm-human chimeric proteins to substitute for the worm proteins, The roles of candidate PKD interacting proteins, identified as C. elegans homologs of proteins found in studies with the human, mouse or fruitfly PKDs, or in C. elegans by two-hybrid screens, will be tested by deletion of the chromosomal copies of these genes and the subsequent phenotypic and genetic characterization of these mutations. Genetic screens for mutations conferring the same male-specific, dual sensory defects as lov-1 mutants, will be carried out to identify new genes and hence new proteins involved in PKD function.