RUI: The Gene Stream: From Sequence to Cell Function

该项目将在圣奥拉夫学院（以本科生为主）进行，是一个涉及生物信息学、蛋白质组学、细胞和分子生物学的跨学科合作项目，同时将实验室研究与课堂实验室活动联系起来。该项目的科学核心是淡水纤毛虫（嗜热四膜虫）的结合生物学。核皮质相互作用启动减数分裂并介导一系列事件，包括核交换、程序性核退化以及体细胞和生发核命运的决定。在从事广泛的基因发现项目时，PI及其合作者和学生将探索3种特定基因产物的作用：T-MEC1，在减数分裂中起作用；卵泡甾体素，在细胞核交换和受精中起调节作用；以及T-VASA，它在决定核命运中起着作用。PI Eric S. Cole开发了一种分离完整核交换连接的方法，并用荧光标记凝集素测定其丰度。他的实验室也有能力通过操纵目标基因表达来制造GFPtagged结构和基因敲除。科尔实验室在常规和共聚焦荧光显微镜技术方面训练有素。罗伯特·卢瑟福（Robert Rutherford）为该项目带来了生物信息学资源，使团队和他们的学生能够搜索四膜虫基因组，寻找基因同源物。他的“CCT”项目已经确定了减数分裂检查点基因，这些基因与酵母和果蝇的模式基因同源。道格拉斯·伯斯曼（Douglas Beussman）为合作带来了最先进的蛋白质组学。Beussman的实验室可以从聚丙烯酰胺凝胶中提取蛋白质，将其分解，并通过质谱测定精确的分子量。该数据可用于扫描最近发表的四膜虫基因组，提取相应的基因序列进行分子遗传分析。该项目的科学目标：研究人员将继续在最近发表的四膜虫基因组中寻找减数分裂检查点基因的同源物a/o同源物，以及在后生动物胚胎中参与建立胚胎极性和极质测定的基因。他们已经确定了该项目的靶基因，包括MEC1（在酵母中介导减数分裂检查点的基因）和VASA（其产物与后生动物胚胎的种系决定和极性颗粒形成有关的基因）的四膜虫同源基因。他们还分离和分析形成核交换连接的蛋白质。这些蛋白质将用于产生抗体，用于western blots和免疫荧光定位，并进行质谱分析，以鉴定相应的基因，用于未来的分子分析。项目教学目标：本科生将从其导师的研究实验室或从圣奥拉夫学院生物、化学和计算机科学系的学术课程中开展的独立项目中进入拟议的“基因流”。学生将携带自己喜欢的基因序列或蛋白质从一个教室实验室到另一个教室实验室，有效地在班级之间建立“研究桥梁”，这将为每个学生提供项目连续性和跨学科的研究经验。智力优势：四膜虫的配偶发育告诉我们核皮质相互作用如何控制减数分裂、受精和决定核命运。Cole博士和他的合作者特别感兴趣的是，在后生动物胚胎中区分体细胞和种系核命运的机制最初是在单细胞原生动物中出现的。更广泛的影响：“基因流”项目将邀请和培养所有背景的学生，不分性别或种族。长期以来，科尔博士和他的合作者一直在招募女性和少数族裔本科生，并为研究生提供参加州、国家和偶尔的国际会议的机会。该团队还被纳入了“2010年研究链接”计划，该计划旨在与全国各地的本科院校共享四膜虫模型研究系统。

This project, which will be performed at St. Olaf College (a predominantly undergraduate institution), is a remarkable cross-disciplinary collaboration that involves bio-informatics, proteomics, and both cellular and molecular biology, while linking laboratory research to classroom laboratory activities. The scientific core of the project is the biology of conjugation in the freshwater ciliate, Tetrahymena thermophila. Nuclear-cortical interactions initiate meiosis and mediate a cascade of events including nuclear exchange, programmed nuclear degeneration, and the determination of somatic and germinal nuclear fates. While engaged in a broadly targeted program of gene discovery, the PI and his collaborators and students will explore the roles of 3 specific gene products: T-MEC1, which plays a role in meiosis; fenestrin, which has a role in mediating pronuclear exchange and fertilization; and T-VASA, which has a role in the determination of nuclear fates. PI Eric S. Cole has developed a method of isolating intact nuclear exchange junctions and assaying their abundance with fluorescently tagged lectins. His lab is also competent at creating GFPtagged constructs and genetic knock-outs by manipulating targeted gene expression. The Cole lab is particularly well trained in conventional and confocal fluorescence microscopy skills.Co-PI Robert Rutherford brings bioinformatic resources to the project, allowing the team and their students to search the Tetrahymena genome for genetic homologs. His "CCT" program has identified meiosis checkpoint genes homologous to those in yeast and pattern-gene orthologs from Drosophila, among others.Co-PI Douglas Beussman brings state-of-the-art proteomics to the collaboration. Beussman's laboratory can take proteins from polyacrylamide gels, fragment them, and determine precise molecular weights via mass spectrometry. This data can be used to scan the recently published Tetrahymena genome pulling out corresponding gene sequences for molecular genetic analyses.Scientific Objectives of the project: The investigators will continue searching the recently published Tetrahymena genome for homologs a/o orthologs to meiotic checkpoint genes, and genes whose products participate in establishing embryonic polarity and pole-plasm determination within metazoan embryos. They have already identified target genes for the project, including Tetrahymena homologs for MEC1 (a gene mediating a meiotic checkpoint in yeast) and VASA (a gene whose product is associated with germ-line determination and polar granule formation in metazoan embryos). They are also isolating and analyzing proteins that form the nuclear exchange junction. These proteins will be used to generate antibodies for western blots and immunofluorescence localization, and to perform Mass Spectrometry in order to identify the corresponding genes for future molecular analysis.Teaching Objectives of the project: Undergraduate students will enter the proposed "Gene Stream" from their faculty mentor's research laboratories or from independent projects launched from academic courses in the biology, chemistry and computer science departments at St. Olaf College. Students will carry a favorite gene sequence or protein from one classroom laboratory to another, effectively creating "research bridges" between classes which will provide project continuity and an inter-disciplinary research experience for each student.Intellectual Merit: Conjugal development in Tetrahymena has much to teach us about how nuclear-cortical interactions govern meiosis, fertilization and determination of nuclear fates. Of particular interest to Dr. Cole and his collaborators is the possibility that the mechanism for distinguishing somatic and germline nuclear fates within metazoan embryos arose initially within unicellular protozoans.Broader Impact: The "Gene Stream" program will be inviting and nurturing to students of all backgrounds regardless of gender or ethnicity. Dr. Cole and his collaborators have a long track record of including women and minority undergraduates and providing research students with opportunities for attending state, national and occasionally international meetings. The team has also been included in the Research Links 2010 initiative to share Tetrahymena as a model research system with undergraduate institutions across the nation.