RUI: The Gene Stream: From Sequence to Cell Function

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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.