GENETIC AND BIOCHEMICAL ANALYSIS OF PHYCOBILISOMES

该提案的目的是了解监管 参与控制基因表达的机制 编码藻胆体的成分，主要的光- 蓝藻和红藻中的收获复合体。 最终 这项研究将扩展我们对光是如何 被感知并转化为生化信号， 特定基因的表达。 技术的发展和 在这项工作的过程中利用可能会扩大我们的 蓝藻的遗传方式的知识， 操纵，并有利于分析这样的过程， 固氮作用、光合作用、逆境适应和 有机化合物的利用和降解。 特定捕光色素藻胆蛋白水平 在某些蓝细菌中对光线有很好的反应。 这种调制，可能是由于 特定藻胆蛋白基因的活性，导致更多 有效吸收光的主要波长， 藻胆体 现在可以获得有关 组织、核苷酸序列和转录 藻胆蛋白和连接多肽的特性 （藻胆体的非色素成分）基因，我们可以 开发体内和体外系统，用于分析 单个藻胆体多肽的功能，以及 这些多肽的基因被调控的方式。 基因转移技术（接合，转化）将是 用于将病变靶向到特定的藻胆蛋白或接头 基因（或与藻胆蛋白和接头成簇的基因 基因）。 这些突变体的特征将有助于确定 这些基因在藻胆体生物合成中的作用。 基因 转让技术也将使我们能够确定 序列元件（顺式作用因子），可能位于5'到 转录起始位点，对于调节 藻胆蛋白基因表达 此外，通过融合这些 编码有毒蛋白质的基因的调节序列 对于蓝藻，我们也许可以选择调节性的 突变体（不能对变化的光线作出反应的生物体） 质量）。 为了补充基因调控的体内检查，我们将 开发体外转录系统（其反映体内 法规），并使用它来定义所需的各个组件 用于胆蛋白基因的调节表达。 这些可以 包括RNA聚合酶独特种类和反式作用 结合调节序列的因子。 体外结合， 体内技术应该能让我们确定 参与藻胆体生物合成的过程。

The aim of this proposal is to understand the regulatory mechanisms involved in controlling the expression of genes encoding the components of the phycobilisome, the major light- harvesting complex in cyanobacteria and red algae. Ultimately this research will extend our understanding of how light is perceived and converted into biochemical signals which alter expression of specific genes. The technology developed and exploited during the course of this work may expand our knowledge of the ways in which cyanobacteria can be genetically manipulated and benefit the analyses of such processes as nitrogen fixation, photosynthesis, acclimation to stress and utilization and degradation of organic compounds. Levels of specific light harvesting, pigmented phycobiliproteins are exquisitely responsive to light quality in some cyanobacteria. This modulation, probably the consequence of changes in the activity of specific phycobiliprotein genes, results in more efficient absorption of prevalent wavelengths of light by the phycobilisome. With information now available on the organization, nucleotide sequences and transcriptional characteristics of phycobiliprotein and linker polypeptide (nonpigmented components of the phycobilisome) genes, we can develop both in vivo and in vitro systems for analyzing the function of the individual phycobilisome polypeptides, and the ways in which the genes for these polypeptides are regulated. Gene transfer techniques (conjugation, transformation) will be used to target lesions into specific phycobiliprotein or linker genes (or genes clustered with the phycobiliprotein and linker genes). Characterization of these mutants will help define the role of these genes in the biosynthesis of the phycobilisome. Gene transfer technology will also enable us to determine which sequence elements (cis acting factors), probably located 5' to the site of transcription initiation, are important to regulated phycobiliprotein gene expression. furthermore, by fusing these regulatory sequences to genes encoding proteins which are toxic to the cyanobacterium, we may be able to select regulatory mutants (organisms which cannot respond to changing light quality). To complement in vivo examination of gene regulation, we will develop an in vitro transcription system (that reflects in vivo regulation) and use it to define the individual components required for regulated expression of the biliprotein genes. These may include both unique species of RNA polymerase and trans acting factors which bind regulatory sequences. Combining in vitro and in vivo technology should enable us to define the molecular processes involved in phycobilisome biosynthesis.