PHYSIOLOGY OF BACTERIO-OPSIN GENE EXPRESSION IN HALOBACTERIUM

细菌视紫红质是一种完整的膜感受器，在 嗜盐嗜盐菌，属极端嗜盐菌属 太古宙人。它具有与视紫红质相近的结构特征，一种 光感受器是动物视觉过程中不可或缺的部分。从长远来看 这项研究的目的是阐明涉及的调节电路 细菌视蛋白(Bop)基因的表达。盐生H.halobium占据了独特的 环境和进化的利基，并阐明新的监管 这种生物体中的电路可能为真核生物的基因提供新的模型 以及调控机制未知的真细菌(S)。 一个目标是使用技术、方法论和实验设计 该项目旨在培训美国原住民少数民族生物医学学生 各种相关的和当前的生物医学技能。学生们将拥有 学习如何(I)培养和操纵盐生扁豆 和大肠杆菌；(Ii)用分光光度法和活菌法定量生长 计数；(Iii)分离质粒并制备32P核探针；(Iv)分离 RNA和Northern blots定量检测BOP基因簇的mRNA水平； (V)分离和定量细菌视紫红质蛋白 分光光度法等。除了学习各种技术， 学生将学习如何设计他们自己的实验并分析 从这些实验中产生的数据。研究结果： 由学生进行的活动将由学生在地区 和国家会议，并在可能的情况下发表在同行评议的 日记。 第二个目标是在一系列实验中量化mRNA水平 将：(1)确定BOP、BRP和BAT成绩单的顺序 在光和低氧分压诱导下出现，(Ii)测试 假设持续暴露在高水平的溶解氧中 将抑制BOP基因簇的诱导，以及(Iii)确定是否 Bop基因簇是由其他环境条件引起的，例如 就像氨基酸饥饿一样。 第三个目标是确定层级监管关系 在bop、brp和BAT基因中。这将通过放置突变体来完成 在诱导条件下包含其中一个基因的突变 量化其他两个基因的转录水平。 第四个目标是构建染色体基因的缺失突变。 属于BOP基因簇。删除突变将提供明确的 评估生理实验的遗传背景和 将适用于遗传互补研究。

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.