PHYSIOLOGY OF BACTERIO-OPSIN GENE EXPRESSION IN HALOBACTERIUM

盐杆菌中细菌视蛋白基因表达的生理学

• 批准号: 3734550
• 负责人: RICHARD F SHAND
• 金额: --
• 依托单位: NORTHERN ARIZONA UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3734550
• 关键词: Archaea; Escherichia coli; bacterial genetics; bacteriorhodopsins; environment; gene complementation; gene expression; gene mutation; genetic manipulation; genetic regulation; genetic transcription; light intensity; messenger RNA; northern blottings; nucleic acid probes; nucleic acid sequence; oxygen microelectrode; oxygen tension; physiology; regulatory gene; transfection /expression vector; transposon /insertion element

Bacteriorhodopsin is an integral membrane photoreceptor found in Halobacterium halobium, an extremely halophilic member of the domain Archea. It possesses near structural identify to rhodopsin, a photoreceptor integral to the vision process in animals. The long term goal of this research is to elucidate the regulatory circuitry involved in bacterio-opsin (bop) gene expression. H. halobium occupies unique environmental and evolutionary niches, and elucidation of new regulatory circuitry in this organism may provide new models for genes in eukaryotes and eubacteria in which the regulatory mechanism(s) are unknown. One aim is to use the techniques, methodologies and experimental design of this project to train Native American Minority Biomedical Students in a variety of relevant and current biomedical skills. Students will have the opportunity to learn how to (i) grow and manipulate both H. halobium and E. coli; (ii) quantitate growth spectrophotometrically and by viable counts; (iii) isolate plasmids and prepare 32P riboprobes; (iv) isolate RNA and probe Northern blots to quantitate bop gene cluster mRNA levels; (v) isolate and quantitate bacteriorhodopsin protein spectrophotometrically, etc. In addition to learning various techniques, students will learn how to design their own experiments and analyze the data generated from these experiments. The results of the research carried out by the student will be presented by the student at regional and national meetings, and if possible, be published in a peer-reviewed journal. A second aim is to quantitate mRNA levels in a series of experiments that will: (i) determine the order in which bop, brp and bat transcripts appear when induced both by light and by low oxygen tension, (ii) test the hypothesis that continued exposure to high levels of dissolved oxygen will suppress induction of the bop gene cluster, and (iii) determine if the bop gene cluster is induced by other environmental conditions such as amino acid starvation. A third aim is to determine the hierarchical regulatory relationships among the bop, brp and bat genes. This will be done by placing mutants that contain mutations in one of the genes under inducing conditions and quantitating transcript levels from the other two genes. A fourth aim is to construct deletion mutations in the chromosomal genes of the bop gene cluster. Deletion mutations will provide unambiguous genetic backgrounds in which to evaluate physiological experiments and will be suitable for genetic complementation studies.

细菌视紫红质是一种完整的膜光感受器， 盐生盐杆菌（Halobacterium halobium），一种极端嗜盐的成员， Archea 它与视紫红质，a 动物视觉过程中不可或缺的光感受器。 长期 这项研究的目的是阐明涉及的调节回路 在细菌视蛋白（BOP）基因表达中。 H.盐生菌占据着独特的 环境和进化的利基，并阐明新的监管 这种有机体中的电路可能为真核生物中的基因提供新的模型 和调节机制未知的真细菌。 一个目的是使用技术，方法和实验设计 该项目旨在培养美国土著少数民族生物医学学生， 各种相关和当前的生物医学技能。 学生将有 有机会学习如何（i）生长和操纵H。盐生植物 和E.（ii）定量生长的微生物和活的 计数;（iii）分离质粒并制备32 P核糖核酸探针;（iv）分离 RNA和探针北方印迹定量bop基因簇mRNA水平; (v)分离和定量细菌视紫红质蛋白 除了学习各种技术外， 学生将学习如何设计自己的实验和分析， 这些实验产生的数据。 研究结果 由学生进行的将由学生在区域 和国家会议，并在可能的情况下，在同行审查的 日报 第二个目标是在一系列实验中定量mRNA水平， 将：㈠确定国际收支平衡表、预算审查方案和最佳可行技术记录的顺序 出现时，诱导光和低氧张力，（ii）测试 持续暴露于高水平溶解氧的假设 将抑制BOP基因簇的诱导，和（iii）确定是否 BOP基因簇由其它环境条件诱导， 如氨基酸饥饿。 第三个目标是确定层级监管关系 在bop brp和bat基因中。 这将通过将突变体 其在诱导条件下包含其中一个基因的突变， 定量其它两个基因的转录水平。 第四个目标是在染色体基因中构建缺失突变 bop基因簇。 缺失突变将提供明确的 遗传背景，以评估生理实验， 将适合于遗传互补研究。