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学信号，在从细菌到人的几乎所有主要的生活群体中都发现。然而，尚不清楚第二个主要的生命原核生物领域的成员是否也具有生物钟。从进化的角度来看，对古细菌的研究具有很大的意义，因为迄今为止，它们与真核生物和细菌的关系和关系尚未得到解决。 有趣的是，DNA序列信息揭示了许多多种古细胞基因组的昼夜节律同源物（称为KAIC）的存在。 KAIC是蓝细菌昼夜节律时钟的主要驱动力，该时钟起着调节节奏基因表达并控制细胞分裂的时间。 迄今为止，尚未提供实验证据来解释这些古细菌KAIC同源物中的任何一个功能作用。 该项目的重点是研究模型系统基因组中发现的四个KAIC同源物的遗传调节，专性的卤素或热爱盐的haloferax colloferax dolcanii，以及执行有针对性的基因敲除以确定其真实功能。证据表明，这四个基因受光和黑暗的昼夜周期的调节。这项研究的特定目标旨在定义和表征这些昼夜节律基因在古细菌中发挥的作用（如果有）。解决了以下问题：四个h. l. l. dolcanii昼夜节律基因在有助于昼夜光传感方面的重要性？这些基因是否相互依赖于适当的功能？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。这些实验将提供有关古细菌中无处不在的KAIC同源物的性质的更完整的图片。 这些结果将提供必要的第一步，以更好地理解古细菌的确确实具有某种类型的昼夜节律，类似蓝细菌的时钟。 另外，如果在这些实验之后消除了昼夜节律假设，那么结果对于改善Haloarchaeal KAIC同源物的作用以及通过关联而言，结果在古细菌中发现的KAIC同源物将至关重要。拟议研究的更广泛的影响力将在骑手大学（Rider University）进行，这是一所小型文科学校，在科学中没有研究生课程。 因此，本科参与这项工作必须及时完成拟议的实验。 PI致力于在替补席研究，公开演讲和科学写作中对大学生的培训。在过去的九年中，超过70％的学生在Bidle Lab中受到指导（n = 40岁），而女性则为30％，而代表人数不足（非洲，加勒比海，拉丁裔或阿拉伯 - 美国人）。在这40名学生中，将近一半已被录取为毕业生（博士学位和硕士学位）或专业（医疗，护理，牙科，兽医）课程。 在Bidle Lab中受到指导的学生已经获得了微生物学国家研究奖，并已被列为同行评审期刊出版物的作者，并每年在国家科学会议上介绍他们的数据。这些学生接受了涵盖许多学科的基本技术培训，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 Lab中培养的，导致训练有素，下一代生命科学家的毕业。