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

Theromyzon（环节动物：水蛭目）茧的分子特性daniel H. shain茧为一些动物类群（如节肢动物、环节动物、扁虫）的发育提供了必要的微环境。也许最著名的蚕茧是由家蚕（Bombyx mori）编织的，它为提取蚕丝提供了原始材料。鲜为人知的是由锁骨环节动物（如蚯蚓、水蛭）分泌的茧，它们自己有几个迷人的特性。例如，水生物种分泌特殊的蛋白质，在水下组装成一个膜鞘，两端被水下“胶水”密封，需要在几个小时内合成数千公里的“纤维”。这一建议的目的是检查由Theromyzon rude产生的茧的分子方面，Theromyzon rude是一种北美特有的水生水蛭，对水禽是嗜血的。粗蚕茧对变性化学物质和高温（250摄氏度）具有特别的弹性，但它是一层薄而柔韧的透明膜。已分离出粗蚕茧膜的一个主要蛋白质成分（Theromyzon cocoon protein, Tcp），其氨基酸序列显示6个串联重复序列，每个重复序列包含约62个氨基酸重复单元中的12个有序的Cys残基。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antistasin（一种具有蛋白酶抑制剂特性的水蛭抗凝血剂）具有很强的序列相似性，而后半部分（Cys残基7-12）与notch/ egf样蛋白结构域（在细胞信号传导中起作用）具有相似性。在包括节肢动物和线虫在内的不同动物门中存在功能未知的cp-样重复序列（即12个有序的Cys残基），这表明寒武纪前的cp-样祖先蛋白在链状环节动物茧中发挥了结构作用。在本研究中，我们将对Tcp基因的表达进行原位检测，以监测阴蒂内导致茧生成的事件，并对Tcp启动子进行表征，以了解茧合成的调控表达。利用经典电子显微镜和Tcp特异性抗体研究茧膜的底层结构，以确定Tcp单体在茧膜中的组装、贡献和空间排列。总的来说，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特性，同时扩大我们在科学上代表性不足的门（环节动物）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学习研究计划来解决拟议的研究目标，并将报告他们的结果，作为每周部门系列研讨会的一部分。这项研究的组成部分将被整合到现有的“电子显微镜”和“生物学中的计算机”课程中，这将为学生提供一个参与系里一个活跃的研究领域的机会。此外，这些资金将促进学生参加州和地方专题讨论会（例如，罗格斯大学年度本科生研讨会，新泽西州科学院），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.