RUI: Evolution of Phosphate Starvation Response in Yeast

知识价值。本项目研究了子囊菌系中磷酸盐信号转导（PHO）途径在进化时间中的变化，目的是了解该途径的进化转变如何改变不同生态位的生长。利用酵母念珠菌（Candida glabrata）和裂糖酵母（Schizosaccharomyces pombe），这些研究集中在转录调控、真核生物祖先PHO途径的阐明以及所有真核生物如何感知磷酸盐饥饿。研究将确定转录因子Pho4的结合特异性。pho4调控的C. glabrata启动子不包含典型的CACGTG序列，必须利用与酿酒酵母不同的序列。CgPho4对PHO启动子具有多重特异性的假设将通过体内（启动子片段表达）和体外（迁移转移）方法进行验证。该项目将利用先前的研究来证明草酸磷酸酶蛋白Pmu2的新功能化。Pmu2的结构域将与祖先的Pmu1结构域结合，并测量磷酸酶动力学。允许新功能化的蛋白质序列的变化将被确定，并应适用于理解新功能如何更普遍地进化。对S. pombe的研究将集中于鉴定Pho7作为一个可能的转录因子介导S. pombe的磷酸盐饥饿反应。Pho7结合PHO启动子的假设将通过体内和体外方法进行验证。最后，Chlamydomonas（一种绿藻）和S. pombe的PHO通路将在S. cerevisiae中进行遗传重组，从而确定所有真核生物的共同代谢物是否信号磷酸盐饥饿。磷酸盐饥饿影响重要的细胞生物学反应，改变磷酸盐的吸收以进行生物修复或改变细菌和真菌的致病性将有益于社会。这项工作将促进我们对转录因子特异性、新功能化过程、所有真核生物中磷酸盐饥饿代谢信号的可能保存以及对物种形成途径所需的进化转变的更深入理解的认识。更广泛的影响。这些研究是相对较新的科学家，如本科生和硕士水平的学生。实验被分解成适合本科生的小项目，如蛋白质纯化、酶分析和质粒构建。学生将获得遗传学、菌株构建、分子生物学技术、无菌技术和生物化学方面的宝贵经验。在一个更大的项目框架内的任务所有权对学生的自信和鼓励未来的自力更生是很有价值的。这项工作将支持维拉诺瓦大学的众多本科生和硕士生继续进行科学研究。通过积极的研究经历和强有力的指导，积极招募和鼓励来自弱势背景的学生留在科学领域。

Intellectual merit. This project investigates how the phosphate signal transduction (PHO) pathway changes over evolutionary time in the Ascomycota lineage, with the goal of understanding how evolutionary transitions in this pathway alter growth in different niches. Using the yeasts Candida glabrata and Schizosaccharomyces pombe, these studies focus on the regulation of transcription, elucidation of the ancestral eukaryotic PHO pathway, and how phosphate starvation in all eukaryotes is sensed. Studies with C. glabrata will determine the binding specificity of the transcription factor Pho4. Pho4-regulated promoters in C. glabrata do not contain canonical CACGTG sequences and must utilize different sequences relative to Saccharomyces cerevisiae. The hypothesis that CgPho4 has multiple specificities for PHO promoters will be tested with in vivo (expression from promoter fragments) and in vitro (mobility shift) approaches. This project will capitalize on previous studies demonstrating the neofunctionalization of a C. glabrata acid phosphatase protein, Pmu2. Domains of Pmu2 will be combined with ancestral Pmu1 domains and phosphatase kinetics will be measured. The changes in protein sequence allowing for neofunctionalization will be identified and should be applicable to understanding how new functions evolve more generally. Studies with S. pombe will focus on the identification of Pho7 as a likely transcription factor mediating the phosphate starvation response in S. pombe. The hypothesis that Pho7 binds PHO promoters will be tested utilizing in vivo and in vitro approaches. Finally, the PHO pathway from Chlamydomonas (a green algae) and S. pombe will be genetically reconstituted in S. cerevisiae, allowing for the determination of whether common metabolites in all eukaryotes signal phosphate starvation. Phosphate starvation impacts important cell biological responses and altering the uptake of phosphate for bioremediation or altering the pathogenicity of bacteria and fungi would benefit society. The proposed work will advance our knowledge of transcription factor specificity, the process of neofunctionalization, the possible conservation of metabolic signals of phosphate starvation in all eukaryotes, and a deeper understanding of the evolutionary transitions required in pathways for speciation. Broader impacts. These studies are accessible to relatively new scientists such as undergraduate students and Master's level students. The experiments are parsed into small projects suitable for undergraduates, such as protein purification, enzymatic assays, and plasmid construction. Students will gain valuable experience working with genetics, strain construction, molecular biological techniques, sterile techniques, and biochemistry. Ownership of a task within the framework of a larger project is valuable for students' self-confidence and encourages future self-reliance. This work will support numerous undergraduate and Master's students at Villanova University who will go on to perform scientific research. Students from disadvantaged backgrounds are actively recruited and encouraged to stay in science by positive research experiences and strong mentorship.