Insulin-like Signaling in Parasitic Nematode Development

性状：（由申请人提供）：寄生线虫 使全世界数百万人衰弱。在绝大多数这些 线虫的第三个幼虫阶段（L3）构成了感染阶段， 脊椎动物宿主不管这些L3是如何在感染过程中获得的 过程中，他们可以被视为过渡阶段，在发展的状态， 逮捕，这是重新激活，只有当暴露于线索存在于 最终宿主这些寄生虫调节发育的机制 在L3中仍然不清楚，这一领域的研究受到缺乏 一个涉及寄生线虫的分子遗传系统。相比之下， L3的发育生物学，包括连续和 被捕（道尔）发展，一直在积极调查中， 自由生活的线虫秀丽隐杆线虫，导致了丰富的相关 分子遗传信息的总体目标 建议的研究是确定是否类似的机制，在C. 线虫还调节寄生虫粪类圆线虫的发育。S. 选择粪虫作为模型是因为，在众多其他功能和 形态相似，这种蠕虫有交替的自由生活周期 使人想起C.优雅具体 这个建议的目的是，首先，以确定在S。粪类 与DAF途径的胰岛素样分支上的四个关键基因的直系同源物， 它控制着C.优雅L3。实现这一目标的工作将强调 PCR方法涉及基于已发表的C. elegans基因序列 研究的目标基因是C. elegans daf-2、age-1、daf-18和 daf-16。其次，我们将研究假定的S的功能。粪类 daf直向同源物。推测的dauer诱导基因的功能同源性将被 通过诸如用嵌合体诱导靶向突变的方法来确定， RNA/DNA寡核苷酸与特异性双链断裂的特异性转录本的切除 单链RNA Dauer诱导物和推定的Dauer抑制基因的同源性 将通过互补和其他转基因研究进行研究， 适当的C. elegans菌株。最后，我们将奋进开发方法， 用于S.以评估功能 推定的调控基因。目前广泛采用的DNA转化方法是C. 通过显微注射到性腺合胞体中将使秀丽隐杆线虫适应。

DESCRIPTION: (provided by the applicant): Parasitic nematodes sicken or debilitate millions of persons worldwide. In the vast majority of these nematodes the third larval stage (L3) constitutes the infective stage for the vertebrate host. Regardless of how these L3 are acquired during the infection process, they may be viewed as transitional stages, in a state of developmental arrest, which are reactivated only when exposed to cues present in the definitive host. The mechanisms by which these parasites regulate development in the L3 remain unclear, studies in this area having been hampered by the lack of a molecular genetic system involving a parasitic nematode. By contrast, the developmental biology of L3, including the switch between continuous and arrested (dauer) development, has been under active investigation in the free-living nematode Caenorhabditis elegans, resulting in a wealth of relevant molecular genetic information on that organism. The overall goal of the proposed study is to ascertain whether mechanisms similar to those acting in C. elegans also regulate development in the parasite Strongyloides stercoralis. S. stercoralis was chosen as a model because, among numerous other functional and morphological similarities, this worm has an alternate free-living cycle reminiscent of the continuous developmental cycle of C. elegans. The specific aims of this proposal are, first, to ascertain the existence in S. stercoralis of orthologs to four key genes on the insulin-like branch of the daf pathway, which controls development in C. elegans L3. Work toward this aim will stress a PCR approach involving primers based on published C. elegans gene sequences. Genes targeted for study are orthologs of C. elegans daf-2, age-1, daf-18 and daf-16. Second, we will investigate the function of the putative S. stercoralis daf orthologs. Functional homology of putative dauer inducing genes will be ascertained by methods such as inducing targeted mutations with chimeric RNA/DNA oligonucleotides and ablating specific transcripts with specific double stranded RNA. Homology of dauer inducers and putative dauer suppressing genes will be investigated by complementation and other transgenesis studies in appropriate C. elegans strains. Finally, we will endeavor to develop methods for germ line transformation of S. stercoralis in order to assess function of putative regulatory genes. Methods widely used for DNA transformation of C. elegans via microinjection into gonadal syncytia will be adapted.