Insulin-like Signaling in Parasitic Nematode Development

描述：(申请人提供)：寄生线虫或 使全世界数以百万计的人虚弱。在其中的绝大多数 线虫第三幼虫阶段(L3)构成了线虫的感染期 脊椎动物宿主。不管这些L3是如何在感染期间获得的 过程中，它们可以被视为过渡阶段，处于发展状态 逮捕，只有在接触到 最终的主人。这些寄生虫调节发育的机制 在L3仍然不清楚，这一领域的研究因缺乏而受到阻碍 一种涉及寄生线虫的分子遗传系统。相比之下， L3的发育生物学，包括连续和连续之间的转换 被捕(Dauer)发展，一直在积极调查中 自由生活的线虫秀丽线虫，产生了丰富的相关 关于该生物体的分子遗传信息。该计划的总体目标 拟议的研究是为了确定是否存在与C。 线虫也调节着寄生虫STRUNYLOIDS STER珊瑚的发育。S. 草珊瑚之所以被选为模式，是因为在众多其他功能和 形态相似，这种蠕虫有一个交替的自由生活周期 使人想起线虫的连续发育周期。具体的 这项建议的目的是，第一，确定在司氏链球菌中的存在。 与DAF途径的胰岛素样分支上的四个关键基因的同源基因， 它控制着线虫L3的发育。为实现这一目标而进行的工作将强调 基于已公布的线虫基因序列的引物聚合酶链式反应方法。 研究的目标基因是线虫daf-2、age-1、daf-18和 DAF-16。第二，我们将研究推测的斯特拉氏链球菌的功能。 DAF同源基因。可能的达尔诱导基因的功能同源性将是 通过嵌合体诱导靶向突变等方法确定 RNA/DNA寡核苷酸与特异双链消融特定转录本 搁浅的RNA。达尔诱导子和可能的达尔抑制基因的同源性 将通过互补和其他转基因研究进行研究 合适的线虫菌株。最后，我们将努力开发方法 进行种系转化，以评估其功能。 推测的调控基因。方法广泛应用于DNA转化的研究。 通过向性腺合胞体内显微注射的线虫将被适应。

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.