RUI: Control Mechanisms for G1 Cyclin Subcellular Localization in Budding Yeast

当考虑调节基本细胞过程（如细胞分裂）的途径时，调节蛋白质进出细胞核的不同机制至关重要。该项目的重点是对蛋白质进出细胞核的调节运动重要的基因的鉴定和表征。这项工作将广泛地影响我们对核定位信号（NLS）和核输出信号（内斯）依赖的蛋白质运动的细胞基因的理解，同时澄清在芽殖酵母G1型细胞周期蛋白的特殊情况下调节蛋白质运动的机制。将使用基于绿色荧光蛋白（GFP）的报告基因和全基因组自动高通量显微镜鉴定对NLS和内斯活性重要的基因。不同的NLS携带GFP报告基因的比较将用于鉴定该过程通常所需的那些基因以及特异性NLS序列的那些基因。这些基因调节蛋白质运动的生理相关性将解决G1细胞周期蛋白。定位，结合，和遗传分析将进行阐明G1细胞周期蛋白Cln 3的调节运动中的细胞因子的作用，并测量这种regulatedprotein运动对细胞周期进程的影响。智力优势：这项工作将导致显着的进展，在芽殖酵母G1期细胞周期蛋白的调控定位的机制的理解。这项研究还将确定对信号序列依赖的核质运动重要的基因，这将对更广泛的科学界有价值。 这项工作代表了PI的重点转移，并将以自动化高通量显微镜的形式将新技术引入研究实验室。为了促进这项研究，合作已经开发，以获得专业知识和资源，不提供在罗兹College.Broader影响：这项工作将显着影响PI的能力，给本科生研究人员的重大和从事的经验。罗兹学院和多伦多大学的合作，在洛克菲勒大学和弗吉尼亚大学的支持下，为不同背景的本科研究人员提供了一个独特而宝贵的机会，不仅可以在使用先进技术的同时共同工作，而且可以对核质蛋白质转运的基本生物学理解产生重大影响。来自美国科学界代表性普遍不足的群体的个人将积极参与这项工作，特别是通过与田纳西州孟菲斯的LeMoyne Owen学院（一所私立的历史上黑人的小型文理学院）的安排。这项工作特别适合本科研究人员。它利用清晰，直接和完善的方法来解决基本和基本的生物学问题，同时也向学生介绍功能基因组学竞技场的尖端技术。 该项目将在学术和夏季资助本科研究人员，增加罗兹学院生物系的研究文化。 学生和PI将通过在区域，国家和国际会议上的演讲，以及在高度重视的同行评审期刊上发表数据，将本研究的结果广泛传播给科学界。

Distinct mechanisms for regulated movement of proteins into and out of the nucleus are critical when considering pathways that regulate basic cellular processes, such as cell division. This project focuses on the identification and characterization of genes important for the regulated movement of proteins into and out of the nucleus. The work will broadly impact our understanding of the cellular genes critical for nuclear localization signal (NLS) and nuclear export signal (NES) dependent protein movement, while clarifying the mechanism of regulated protein movement in the specialized case of budding yeast G1 type cyclins. Genes important for NLS and NES activity will be identified using Green Fluorescent Protein (GFP) based reporters and automated genome wide high throughput microscopy. Comparisons of distinct NLS bearing GFP reporters will be used to identify those genes generally required for this process as well as those genes specialized for specific NLS sequences. The physiological relevance of these genes to regulated protein movement will be addressed for the G1 cyclins. Localization, binding, and genetic assays will be carried out to elucidate the role of cellular factors in regulated movement of the G1 cyclin Cln3 and to measure the impact that this regulatedprotein movement has on cell cycle progression. Intellectual Merit: This work will lead to significant progress in the understanding of the mechanisms of regulated localization of G1 cyclins in budding yeast. This research will also identify genes important for signal sequence dependent nucleo-cytoplasmic movement that will be of value to the broader scientific community. The work represents a shift in focus for the PI and will introduce new technologies in the form of automated high throughput microscopy into the research laboratory. To facilitate this research, collaborations have been developed to gain access to expertise and resources not available at Rhodes College.Broader Impacts: This work will significantly impact the ability of the PI to give significant and engaged experiences to undergraduate researchers. The collaboration of Rhodes College and the University of Toronto, with the support of The Rockefeller University and the University of Virginia, provides a unique and invaluable opportunity for undergraduate researchers of various backgrounds to not only work together while using advanced technologies, but to make a significant impact on the basic biological understanding of nucleo-cytoplasmic protein transport. Individuals from groups generally underrepresented in US science will be actively recruited for this work, specifically through arrangements with LeMoyne Owen College in Memphis, TN (a private historically black small liberal arts college). This proposed work is especially well suited for undergraduate researchers. It utilizes clear, straight forward and well established methods to address basic and essential biological questions while also introducing the students to cutting edge technology in the arena of functional genomics. This project will fund undergraduate researchers during academic and summer months, increasing the culture of research in the Department of Biology at Rhodes College. Students and the PI will disseminate results from this study broadly to the scientific community through presentations at regional, national, and international meetings, as well as publication of data in highly regarded peer-reviewed journals.