REGULTION IN STRONGYLOIDES STERCORALIS

寄生线虫使全世界数百万人患病或衰弱。在绝大多数线虫中，第三幼虫阶段（L3）构成脊椎动物宿主的感染阶段。无论这些L3在感染过程中是如何获得的，它们都可能被视为过渡性的，处于发育停滞状态，只有在暴露于最终宿主中存在的线索时才会被重新激活。这些寄生虫调控L3发育的机制尚不清楚，由于缺乏涉及寄生线虫的分子遗传系统，这一领域的研究受到阻碍。相比之下，L3的发育生物学，包括连续发育和阻滞发育之间的切换，已经在自由生活的秀丽隐杆线虫中得到了积极的研究，从而获得了丰富的相关分子遗传信息。这项拟议研究的总体目标是确定与秀丽隐杆线虫相似的机制是否也能调节粪圆线虫的发育。之所以选择S. stercoralis作为模型，是因为在许多其他功能和形态上的相似性中，这种蠕虫具有另一种自由生活周期，使人联想到C.秀丽隐杆线虫的连续发育周期。本研究的具体目的是：首先，确定在粪线虫中存在与控制线虫L3发育的daf通路胰岛素样分支上的四个关键基因同源的基因。为了实现这一目标，我们将强调采用PCR方法，包括基于已发表的秀丽隐杆线虫基因序列的引物。研究的目标基因是秀丽隐杆线虫daf-2、age-1、daf-18和daf-16的同源基因。其次，我们将研究推测的粪球菌同源物的功能。假定的诱导基因的功能同源性将通过用特定的双链RNA消融转录本来确定。dauer诱导剂和推测的dauer抑制基因的同源性将在适当的秀丽隐杆线虫突变体中进行互补研究。最后，我们将努力开发粪球菌种系转化的方法，以评估可能的调控基因的功能。采用显微注射将秀丽隐杆线虫DNA转化为性腺合胞的方法。

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 , in a state of developmental arrest, which are reactivated only then exposed to cues present in the definitive host. The mechanism 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 alternative free-living cycle reminiscent of the continuous development cycle of C., elegans. The specific aims of this proposal are, first, to ascertain the existence in S. stercoralis of homologs to four key genes on the insulin-like branch of the daf pathway which controls development in C. elegans L3. Work toward this aim w3ill stress a PCR approach involving primers based on published C. elegans gene sequences. Genes targeted for study are homologs of C. elegans daf-2, age-1, daf-18 and daf-16. Second, we will investigate the function of the putative S. stercoralis daf homologs. Functional homology of putative dauer inducing genes will be ascertained by ablating transcripts with specific double stranded RNA. Homology of dauer inducers and putative dauer suppressing genes will be investigated by complementation studies in appropriate C. elegans mutants. 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.