RUI: Molecular Properties of the Theromyzon (Annelida: Hirudinae) Cocoon

热茧的分子特性丹尼尔·H·谢恩茧为几个动物群体(例如节肢动物、环节动物、扁虫)的发育提供了必要的微环境。也许最广为人知的蚕茧是由家蚕纺成的，它提供了提取蚕丝的原始材料。鲜为人知的是由阴蒂环节动物(例如蚯蚓、水蚤)分泌的茧，它们本身有几个迷人的特性。例如，水生物种会分泌特殊的蛋白质，这些蛋白质在水下组装成一层膜鞘，两端都被水下“胶水”密封，需要在几个小时内合成数千公里长的“纤维”。这项提议的目标是研究由Thermyzon Rough产生的蚕茧的分子方面，Thermyzon Rough是北美特有的一种水生水蚤，是水禽的食血动物。粗茧茧对变性化学物质和高温(250摄氏度)特别有弹性，但它是一种薄薄、柔软和透明的膜。分离得到粗茧膜的一个主要蛋白质组分，其氨基酸序列包含6个串联重复序列，每个重复序列由一个~62个氨基酸重复单位中的12个有序Cys残基组成。Tcp含有异常高的Cys含量(18%)；其他丰富的氨基酸包括Glu(13%)、Val(10%)和Pro(9%)。每个重复序列的前半部分(Cys残基1-6)与抗他汀类抗凝剂具有很强的序列相似性，而后半部分(Cys残基7-12)与在细胞信号转导中发挥作用的Noch/EGF样蛋白结构域有相似之处。在节肢动物和线虫等不同的动物门中存在着功能未知的TCP样重复序列(即12个有序的半胱氨酸残基)，这表明前寒武纪类似TCP样的祖先蛋白被选为阴蒂环节动物茧的结构角色。在这项建议中，将原位检测TCP基因的表达，以监测导致蚕茧产生的阴蒂内的事件，并将研究TCP启动子的特征，以了解蚕茧合成的调控表达。用经典的电子显微镜研究茧膜的基本结构，并用一种针对磷酸三钙的抗体来确定磷酸三钙单体在茧膜中的组装、贡献和空间排列。总而言之，这些研究目标将在水蛭生物学的背景下探索一种不寻常的生物材料的基本性质，同时在科学上未被充分代表的门(环节动物门)中扩展我们的知识。支持这些研究目标的资源将影响罗格斯大学/卡姆登校区的各种学术活动。生物系的本科生将有机会通过独立的研究计划来解决拟议的研究目标，并将作为每周一次的系级研讨会系列的一部分报告他们的结果。这项研究的内容将被整合到现有的“电子显微镜”和“生物学中的计算机”课程中，这些课程将为学生提供参与系内活跃研究领域的机会。此外，这些资金将促进学生参与州立和地方研讨会(例如，罗格斯大学年度本科生研讨会，新泽西科学院)，并将有助于为PI继续参与研究/教育推广计划提供基础设施。

Molecular Properties of the Theromyzon (Annelida: Hirudinea) CocoonDaniel H. ShainCocoons provide a micro-environment necessary for development in several animal groups (e.g., arthropods, annelids, flatworms). Perhaps the most well known cocoon is spun by the silkworm, Bombyx mori, which provides the crude material for extracting silk. Lesser known are cocoons secreted by clitellate annelids (e.g., earthworms, leeches) which have several fascinating properties of their own. For example, aquatic species secrete specialized proteins that assemble underwater into a membranous sheath sealed at either end by an underwater "glue", requiring the synthesis of thousands of kilometers of "fiber" within a few hours. The goal of this proposal is to examine the molecular aspects of the cocoon produced by Theromyzon rude, an aquatic leech endemic to North America and sanguivorous to waterfowl. The T. rude cocoon is particularly resilient to denaturing chemicals and heat (250C), yet is a thin, flexible and transparent membrane. A major protein component of the T. rude cocoon membrane has been isolated (called Theromyzon cocoon protein, or Tcp) and its amino acid sequence revealed 6 tandem repeats, each comprising 12 ordered Cys residues in a ~62 amino acid repeating unit. Tcp contains an unusually high Cys content (18%); other abundant amino acids include Glu (13%), Val (10%) and Pro (9%). The first half of each repeat (Cys residues 1-6) shares strong sequence similarity with antistasin, a leech anticoagulant with protease inhibitor properties, while the second half (Cys residues 7-12) shares similarity with notch/EGF-like protein domains which play roles in cell signaling. Tcp-like repeats (i.e., 12 ordered Cys residues) with unknown functions are present in disparate animal phyla including Arthropoda and Nematoda, suggesting that a pre-Cambrian Tcp-like ancestral protein was co-opted for a structural role in clitellate annelid cocoons. In this proposal, Tcp gene expression will be examined in situ to monitor events within the clitellum leading up to cocoon production, and the Tcp promoter will be characterized to understand the regulated expression of cocoon synthesis. The underlying architecture of the cocoon membrane will be investigated by classical electron microscopy, and with a Tcp-specific antibody aimed at determining the assembly, contribution and spatial arrangement of Tcp monomer in the cocoon membrane. Collectively, these research aims will explore the fundamental properties of an unusual biomaterial in the context of leech biology, while expanding our knowledge in a scientifically underrepresented phylum (Annelida).The resources to support these research objectives will impact a variety of academic activities on the Rutgers/Camden campus. Undergraduate students within the biology department will gain opportunities to address the proposed research aims through an independent study research program and will report their results as part of a weekly, departmental seminar series. Components of this study will be integrated into existing 'Electron Microscopy' and 'Computers in Biology' courses that will provide students an opportunity to become engaged in an active research area within the department. In addition, these funds will facilitate student participation in state and local symposia (e.g., Rutgers University Annual Undergraduate Symposium, New Jersey Academy of Sciences) and will help to provide the infrastructure for the PI's continued participation in research/education outreach programs.