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

昼夜节律是一种重要的生物信号，几乎在从细菌到人类的所有主要生命群体中都发现了这种生物信号。然而，目前尚不清楚第二大原核生命领域-从进化的角度来看，对古细菌的研究具有重要意义，因为它们的起源以及与真核生物和细菌的关系至今仍未得到解决。 有趣的是，DNA序列信息揭示了昼夜节律基因同源物的存在，称为kaiC，在许多不同的古细菌基因组中。 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并控制细胞分裂的时间。 到目前为止，实验证据尚未提供解释任何这些古细菌kaiC同系物的功能作用。 该项目的重点是研究模型系统基因组中发现的四种kaiC同源物的遗传调控，专性嗜盐或嗜盐古菌Haloferax volcano ii，以及进行靶向基因敲除以确定其真正的功能。有证据表明，这四个基因受昼夜光暗周期的调节。本研究的具体目标是定义和描述这些昼夜节律基因在大脑中的作用。下面的问题是解决：如何重要的是每一个四个H。volcanii昼夜节律样基因对昼夜光感应的贡献？这些基因是相互依赖的正常功能吗？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。这些实验将给出一个更完整的图片关于无处不在的kaiC同系物的性质中发现的dichroea。 这些结果将提供必要的第一步，更好地了解，如果真的有某种类型的生物钟，蓝藻类，或其他。 或者，如果在这些实验之后生物钟假说被消除，那么结果将仍然对提高对盐古菌kaiC同系物所起作用的理解很重要，并且通过关联，在盐古菌中发现的kaiC同系物。更广泛的影响所有拟议中的研究将在莱德大学进行，这是一所小型的文科学校，没有理科研究生课程。 因此，本科生参与这项工作是必要的，及时完成拟议的实验。PI致力于培养本科生的实验室研究，公开演讲和科学写作。在过去的九年里，超过70%的学生在Bidle实验室接受指导（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名学生中有近一半已被研究生录取（均为博士）。和硕士水平）或专业（医疗，护理，牙科，兽医）课程。 在Bidle实验室指导的学生一直是微生物学国家研究奖的获得者，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。这些学生接受基础技术培训，涵盖许多学科，包括遗传学，分子生物学，生物信息学和微生物学。动手，体验式学习是在Bidle实验室培养，导致训练有素的，下一代生命科学家的毕业。

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.