Glycan alterations in C.elegans surface mutants

描述(由申请人提供)：后生动物的发展需要产生多种细胞类型和复杂的多细胞结构的形态发生。遗传学研究已经确定了糖基化和蛋白多糖生物合成在发育信号中的作用。线虫秀丽线虫是研究糖共轭化合物在多细胞生物体中作用的良好模式系统，也是寄生线虫的原型模型，因为它知道它的完整基因组序列和发育。在这次R2 I(探索性/发展性)拨款申请中，我们希望探索线虫中糖共轭化合物的表达与发育事件之间的一种新的潜在功能联系。SRF-9、SRF-8和SRF-4(表面)是单基因座突变体，具有多种缺陷，包括运动不协调、外阴突出、产卵异常、交配囊和性腺形态缺陷，但它们是基于它们与N-乙酰氨基葡萄糖结合的凝集素的异位表面结合而被分离出来的。SRF-9、SRF-8和SRF-4的突变与LIN-12基因的突变相互作用，LIN-12基因是LIN-12/Noch受体蛋白家族的成员，介导细胞与细胞的相互作用，以决定细胞在发育过程中的命运。我们将验证这样的假设，即SRF-9、SRF-8和SRF-4基因参与糖结合物的生物合成、翻译后加工或分泌，从而将发育事件与糖结合物的表达联系起来。具体地说，我们的工作将：1)通过角质层多聚糖组分的结构特征，确定受SRF-9、SRF-8和SRF-4突变体影响的糖共轭；2)结合单核苷酸多态(SNP)作图和标记挽救实验，通过分子克隆鉴定SRF-9、SRF-8和SRF-4基因。从这些研究中获得的信息将为研究这些基因在线虫发育事件中的作用提供工具。由于复杂的表型是几种人类遗传疾病的标志，包括一些影响糖基化的疾病，拟议的研究将建立使用线虫SRF突变体作为人类糖基化障碍模型的可行性。

DESCRIPTION (provided by applicant): Development of metazoans requires the production of multiple cell types and the morphogenesis of complex multicellular structures. Genetic studies have established roles for glycosylation and proteoglycan biosynthesis in developmental signaling. The nematode Caenorhabditis elegans is a good model system to study the role of glycoconjugates in multicellular organisms as well as a prototypic model for parasitic nematodes because of the knowledge of its complete genomic sequence and development. In this R2 I (Exploratory / Developmental) grant application, we wish to explore a novel potential functional linkage between expression of glycoconjugates and developmental events in C. elegans. Srf-9, srf-8 and srf-4 (surface) are single loci mutants that have multiple defects, including uncoordinated movement, protruding vulva, abnormal egg laying, and defective copulatory bursae and gonad morphology, yet they were isolated based on their ectopic surface binding to a lectin which binds to N-acetylglucosamine. Mutations in srf-9, srf-8 and srf-4 interact with mutations in the lin-12 gene, which a member of the LIN- 1 2/NOTCH family of receptor proteins that mediate cell-cell interactions to specify cell fate during development. We will test the hypothesis that the srf-9, srf-8 and srf-4 genes are involved in the biosynthesis, post-translational processing or secretion of glycoconjugates, and therefore link developmental events with glycoconjugates expression. Specifically, the proposed work will: 1) Define the glycoconjugates affected in srf-9, srf-8 and srf-4 mutants, through the structural characterization of cuticle glycan fractions, 2) Identify the srf-9, srf-8 and srf-4 genes through molecular cloning using a combination of Single Nucleotide Polymorphism (SNP) mapping, and marker rescue experiments. The information derived from these studies will provide the tools to study the role of these genes in developmental events in the nematode C. elegans. Because complex phenotypes are the hallmark of several human genetic disorders, including some that affect glycosylation, the proposed studies will establish the feasibility of using C. elegans srf mutants as a model for glycosylation disorders in humans.