PHOTOSYNTHESIS, MUTANTS, AND CLONING IN CYANOBACTERIA

本提案的目的是了解 光合膜的结构和功能。 我们 方法将是通过离散的，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和氧气释放装置。 我们 策略将包括七个主要的实验技术：（1） 分离和表征一种放氧复合物， 光系统II和藻胆体;（2）位点特异性标记和 蛋白质的交联以确定它们在膜中的位置;（3） 光合电子传递从H_2O到NADP ~+的重建 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 突变体，菌株将被分离，必须保持异养 生长;（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）正常人和正常人叶绿素蛋白复合物的荧光分析， 发育细胞 本项目是一个结合遗传学和生物化学研究膜 结构和功能，因此对膜生物学非常有益， 将军 应从中获得的具体信息 建议包括：（1）主要膜的蛋白质组成 复合物，如O2释放装置和叶绿素蛋白;（2） 蛋白质在膜中的精确位置;（3） 突变对细胞膜结构和功能的影响 将光合作用基因克隆到野生型和突变体细胞中;（5）鉴定 编码光合作用蛋白的基因;（6） 产生77 K荧光发射的叶绿素组分 （7）缺铁细胞膜组装的机制 在光诱导后的异养细胞中。 的 生物化学和遗传学的结合使A. nidulans一个非常宝贵的 生物能量学和膜结构的研究。

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.