RUI: The Gene Stream: From Sequence to Cell Function

这个项目将在圣奥拉夫学院(一个主要是本科生的机构)进行，是一个了不起的跨学科合作，涉及生物信息学、蛋白质组学以及细胞和分子生物学，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接合生物学。核-皮质相互作用启动减数分裂并调节一系列事件，包括核交换、程序性核退化以及决定体细胞核和生发核的命运。在参与一个目标广泛的基因发现计划时，PI和他的合作者和学生将探索3种特定基因产物的作用：T-Mec1，它在减数分裂中发挥作用；Fenestrin，它在介导原核交换和受精方面发挥作用；以及T-Vasa，它在决定核命运方面发挥作用。皮埃里克·S·科尔开发了一种分离完整的核交换连接并用荧光标记的凝集素分析其丰度的方法。他的实验室还擅长通过操纵目标基因表达来创建GFP标记的结构和基因敲除。科尔实验室在传统和共聚焦荧光显微镜技术方面受到了特别好的培训。联合PI罗伯特·卢瑟福德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以寻找基因同源物。他的“CCT”计划已经确定了与酵母和果蝇模式基因同源的减数分裂检查点基因。联合PI道格拉斯·博斯曼将最先进的蛋白质组学带入合作。博斯曼的实验室可以从聚丙烯酰胺凝胶中提取蛋白质，将它们碎裂，并通过质谱分析确定精确的分子量。这些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学分析。该项目的科学目标：研究人员将继续搜索最近发表的四膜虫基因组，寻找与减数分裂检查点基因a/o同源的同源基因，以及其产物参与建立胚胎极性和后生动物胚胎中的极质决定的基因。他们已经为该项目确定了目标基因，包括Mec1的四膜虫同源物(一种介导酵母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确定和极颗粒形成相关的基因)。他们还分离和分析了构成核交换连接的蛋白质。这些蛋白质将被用来产生免疫印迹和免疫荧光定位的抗体，并进行质谱学，以便为未来的分子分析识别相应的基因。该项目的教学目标：本科生将从他们教员导师的研究实验室或圣奥拉夫学院生物、化学和计算机科学系的学术课程发起的独立项目中进入拟议的“基因流”。学生们将把喜欢的基因序列或蛋白质从一个课堂实验室带到另一个课堂实验室，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研究体验。智力优势：四膜虫的配偶发育告诉我们许多关于核-皮质相互作用如何管理减数分裂、受精和决定核命运的知识。科尔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生殖系核命运的机制最初出现在单细胞原生动物中。广泛的影响：“基因流”项目将对所有背景的学生进行邀请和培养，不分性别或种族。科尔博士和他的合作者在吸纳女性和少数族裔本科生，为研究生提供参加州级、全国性和偶尔的国际会议的机会方面有着长期的记录。该团队还被纳入了Research Links 2010年的倡议，将四膜虫作为一个模式研究系统与全国各地的本科机构分享。

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.