RUI: Characterization of Four Cryptic Circadian Gene Homologs In The Haloarchaeon Haloferax Volcanii

RUI：Haloarchaeon Haloferax Volcanii 中四个隐秘昼夜节律基因同源物的表征

• 批准号: 1051782
• 负责人: Kelly Bidle
• 金额: $ 39.47万
• 依托单位: Rider University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1051782&HistoricalAwards=false
• 关键词: RUI; Characterization; Four; Cryptic; Circadian

Circadian rhythms are important biological signals that have been found in almost all major groups of life from bacteria to man. Yet, it remains unclear if any members of the second major prokaryotic domain of life, the Archaea, also possess a biological clock. From an evolutionary perspective, the study of the Archaea is of great relevance, as their origin and relationship to the eukaryotes and bacteria thus far remains unresolved. Interestingly, DNA sequence information has revealed the presence of circadian gene homologs, known as kaiC, throughout a number of diverse archaeal genomes. KaiC is a major driver of the cyanobacterial circadian clock that acts to regulate rhythmic gene expression and to control the timing of cell division. To date, experimental evidence has not been provided to explain a functional role for any of these archaeal kaiC homologs. This project focuses on examining both the genetic regulation of four kaiC homologs found in the genome of the model system, the obligate halophilic, or salt-loving, archaeon Haloferax volcanii, as well as performing targeted gene knockouts to ascertain their true function. Evidence suggests that these four genes are regulated by diurnal cycles of light and darkness. Specific objectives for this research have been designed to define and characterize what role, if any, these circadian rhythm genes are playing in the Archaea. The following questions are addressed: How important is each of the four H. volcanii circadian-like genes in contributing to diurnal light sensing?; Are these genes inter-dependent for proper function? A multi-faceted experimental approach will involve classical genetics and microbiology, molecular biology, and protein biochemistry. These experiments will give a more complete picture regarding the nature of the ubiquitous kaiC homologs found among the Archaea. These results will provide the necessary first steps for better understanding if Archaea do indeed have some type of circadian clock, cyanobacterial-like, or otherwise. Alternatively, if the circadian clock hypothesis is dispelled following these experiments, the results will nevertheless be important in improved understanding of the role played by the haloarchaeal kaiC homologs, and, by association, the kaiC homologs found among the Archaea. Broader impactsAll of the proposed research will take place at Rider University, a small, liberal arts school that does not have graduate programs in the sciences. Thus, undergraduate involvement in this work is imperative for the timely completion of the proposed experiments. The PI is committed to the training of undergraduates in bench research, public speaking, and scientific writing. Greater than 70% of students mentored in the Bidle lab over the last nine years (n=40) have been female and 30% were underrepresented minorities (African-, Caribbean-, Latin-, or Arab-Americans). Nearly half of these 40 students have been accepted into graduate (both Ph.D. and Master's level) or professional (Medical, Nursing, Dental, Veterinary) programs. Students mentored in the Bidle lab have been the recipients of national research awards in microbiology, have been listed as authors on peer-reviewed journal publications and present their data at national scientific meetings each year. These students are trained in fundamental techniques covering a host of disciplines including genetics, molecular biology, bioinformatics, and microbiology. Hands-on, experiential learning is fostered in the Bidle lab, leading to the graduation of well-trained, next-generation life scientists.

昼夜节律是重要的生物信号，在从细菌到人类的几乎所有主要生命群体中都有发现。然而，尚不清楚第二大原核生物领域——古生菌是否也拥有生物钟。从进化的角度来看，古生菌的研究是非常重要的，因为它们的起源和与真核生物和细菌的关系至今仍未得到解决。有趣的是，DNA序列信息揭示了在许多不同的古细菌基因组中存在昼夜节律基因同源物，称为kaiC。KaiC是蓝藻生物钟的主要驱动因素，其作用是调节有节奏的基因表达和控制细胞分裂的时间。迄今为止，还没有实验证据来解释这些古细菌kaiC同源物的功能作用。这个项目的重点是研究在模型系统（专性嗜盐或嗜盐古菌）的基因组中发现的四种kaiC同源物的遗传调控，以及进行靶向基因敲除以确定它们的真正功能。有证据表明，这四种基因受昼夜循环的光和暗的调节。这项研究的具体目标是定义和描述这些昼夜节律基因在古细菌中发挥的作用（如果有的话）。解决了以下问题：四种H. volcanii昼夜节律样基因对昼夜光感知的贡献有多重要？这些基因是否相互依赖以维持正常功能？一个多方面的实验方法将涉及经典遗传学和微生物学，分子生物学和蛋白质生物化学。这些实验将对古生菌中普遍存在的kaiC同源物的性质提供更全面的了解。这些结果将为更好地了解古细菌是否确实有某种类型的生物钟、类似蓝藻的生物钟或其他生物钟提供必要的第一步。或者，如果生物钟假说在这些实验之后被消除，那么这些结果对于提高对盐古菌kaiC同系物所起作用的理解，以及通过关联，在古菌中发现的kaiC同系物，将是重要的。更广泛的影响所有拟议中的研究都将在莱德大学（Rider University）进行，这是一所规模较小的文理学院，没有理科研究生课程。因此，为了及时完成所提出的实验，本科生参与这项工作是必不可少的。PI致力于培养本科生的实验研究、公开演讲和科学写作能力。在过去九年中，在Bidle实验室接受指导的学生中，超过70% （n=40）是女性，30%是代表性不足的少数民族（非洲裔、加勒比裔、拉丁裔或阿拉伯裔美国人）。这40名学生中近一半已被研究生（博士和硕士）或专业（医学，护理，牙科，兽医）课程录取。在比德尔实验室指导的学生已经获得了国家微生物学研究奖，被列为同行评审期刊出版物的作者，并在每年的国家科学会议上展示他们的数据。这些学生接受的基本技术培训涵盖了许多学科，包括遗传学、分子生物学、生物信息学和微生物学。比德尔实验室提倡动手、体验式学习，培养训练有素的下一代生命科学家。