PHOTOSYNTHESIS, MUTANTS, AND CLONING IN CYANOBACTERIA

该提案的目的是了解之间的关系 光合膜的膜结构和功能。 我们的 方法将通过离散但相关的技术使用 单细胞、可转化蓝藻 Anacystis nidulans R2。 的 重点将放在光系统 II 和释氧装置上。 我们的 策略将包括七项主要的实验技术：（1） 析氧复合物的分离和表征，其中包含 光系统 II 和藻胆体； (2) 特定地点的标签和 蛋白质交联以确定其在膜中的位置； (3) 重建从 H2O 到 NADP 的光合电子传递 使用分离的复合物的脂质体； (4)光合突变体的分离 和抗除草剂突变体。 除了对温度敏感之外 突变体、菌株将被分离出来，必须通过异养来维持 成长； (5)与克隆基因的互补分析； (6)克隆 光合作用基因将用于生化和遗传实验； (7) 正常和正常情况下叶绿素蛋白复合物的荧光分析 发育中的细胞。 该项目是膜的遗传和生化综合研究 结构和功能，因此对膜生物学有很大好处 一般。 需要从这里获取的具体信息 提案内容包括：（1）主要膜的蛋白质组成 复合物，例如放氧装置和叶绿素蛋白； (2) 蛋白质在膜中的精确位置； (3) 的影响 膜结构和功能的突变； (4)添加效果 将光合作用基因克隆到野生型和突变细胞； (5)身份 编码光合蛋白的基因； (6) 身份 产生 77 K 荧光发射的叶绿素成分 峰； (7)缺铁细胞中膜组装的机制 添加铁后和异养细胞中光诱导后。 的 生物化学和遗传学的结合使构巢构巢菌成为非常有价值的 用于研究生物能量学和膜结构的有机体。

The purpose of this proposal is to understand the relationship between membrane architecture and function in photosynthetic membranes. Our approach will be via discrete, yet correlative, techniques using the unicellular, transformable cyanobacterium Anacystis nidulans R2. The emphasis will be on Photosystem II and the oxygen-evolving apparatus. Our strategy will consist of seven major experimental techniques: (1) the isolation and characterization of an oxygen-evolving complex that contains Photosystem II and phycobilisomes; (2) site-specific labeling and cross-linking of proteins to determine their position in the membrane; (3) reconstitution of photosynthetic electron transport from H2O to NADP+ in liposomes using isolated complexes; (4) isolation of photosynthetic mutants and herbicide-resistant mutants. In addition to temperature-sensitive mutants, strains will be isolated that must be maintained by heterotrophic growth; (5) complementation analysis with cloned genes; (6) cloned photosynthesis genes will be used for biochemical and genetic experiments; and (7) fluorescence analysis of chlorophyllprotein complexes in normal and developing cells. This project is a combined genetic and biochemical study of membrane structure and function and is thus of great benefit to membrane biology in general. The specific information that should be obtained from this proposal includes: (1) the protein composition of the major membrane complexes, such as the O2-evolving apparatus and chlorophyll-proteins; (2) the precise location of proteins in the membrane; (3) the effects of mutation on membrane structure and function; (4) the effect of adding cloned photosynthesis genes to wild-type and mutant cells; (5) the identity of genes coding for photosynthetic proteins; (6) the identity of the chlorophyll components that give rise to the 77 K fluorescence emission peaks; and (7) the mechanism of membrane assembly in iron-deficient cells after iron addition and in heterotrophic cells after light induction. The combination of biochemistry and genetics makes A. nidulans a very valuable organism for the study of bioenergetics and membrane structure.