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

昼夜节律是重要的生物信号，几乎存在于从细菌到人类的所有主要生命群体中。然而，目前还不清楚生命的第二个主要原核域--古生代是否有成员也拥有生物钟。从进化论的角度来看，对古生物的研究具有很大的相关性，因为它们的起源以及它们与真核生物和细菌的关系迄今仍未解决。有趣的是，DNA序列信息揭示了昼夜节律基因同源物的存在，即众所周知的kaiC，存在于许多不同的古生菌基因组中。KaiC是蓝藻生物钟的主要驱动力，它调控有节奏的基因表达和控制细胞分裂的时间。到目前为止，还没有实验证据来解释这些古生菌kaiC同源物的功能作用。这个项目的重点是检查在模式系统基因组中发现的四个kaiC同源物的遗传调控，以及进行有针对性的基因敲除以确定它们的真正功能。有证据表明，这四种基因受昼夜周期的控制。这项研究的具体目标是定义和表征这些昼夜节律基因在古生物中扮演的角色(如果有的话)。讨论了以下问题：四个类似昼夜节律的基因对昼夜光感应有多重要？这些基因对于正常的功能是相互依赖的吗？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。这些实验将给出一个关于在古生代中发现的普遍存在的kaiC同源物的性质的更完整的图景。这些结果将为更好地理解古生代是否确实有某种类型的生物钟--类似蓝藻的生物钟--提供必要的第一步。或者，如果在这些实验之后消除昼夜节律假说，结果仍将对于更好地理解嗜盐古菌kaiC同源物所起的作用以及通过关联在古生菌中发现的kaiC同源物所起的作用仍将是重要的。更广泛的影响所有拟议的研究都将在莱德大学进行，这是一所小型的文科学校，没有理科研究生课程。因此，为了及时完成拟议的实验，本科生参与这项工作是必不可少的。PI致力于对本科生进行板凳研究、公共演讲和科学写作方面的培训。在过去九年中，在比德尔实验室接受指导的学生中，超过70%(n=40)是女性，30%是代表不足的少数民族(非洲裔、加勒比裔、拉丁裔或阿拉伯裔美国人)。这40名学生中有近一半被研究生(包括博士和硕士)或专业(医学、护理、牙科、兽医)录取。在博德实验室接受指导的学生获得了国家微生物学研究奖，在同行评议的期刊出版物上被列为作者，并在每年的国家科学会议上展示他们的数据。这些学生接受了涵盖遗传学、分子生物学、生物信息学和微生物学等多个学科的基本技术培训。实践、体验式学习是在BIDLE实验室培养的，导致训练有素的下一代生命科学家毕业。

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.