Identifying New Open Reading Frames Involved in Redox Regulation and Photosystem II Assembly in Thylakoid Membranes

随着越来越多的基因组序列信息的可用性，古老的格言“我们知道的越多，我们就越知道我们不知道”听起来是真的。在原核生物的数千个基因中，近一半的基因编码功能未知的蛋白质。 尽管其中一些可能编码结构蛋白，但许多未识别的开放阅读框预计编码参与功能蛋白复合物组装或稳定的重要调控蛋白。令人惊讶的是，在过去的几年里，已经很清楚，许多这些功能未知的推定蛋白质没有容易识别的对应物，即使在相对密切相关的生物体。 然而，参与调节或稳定生理过程的蛋白质的功能鉴定对于理解生物体的分子生理学和代谢作为其基因组信息的功能具有重要意义。 将使用相当全面的粘粒中断文库来鉴定参与光合作用的新基因，所述文库各自含有来自集胞藻属细菌PCC 6803的基因组DNA的30-45 kb片段，其中转座子已在随机位点整合到所述文库中。这样的转座子中断文库可用于约90种不同的微囊藻，共覆盖集胞藻属PCC 6803基因组的80%以上。 将用这些中断文库转化缺乏光系统I（类囊体膜中质体醌库中电子的主要汇）的集胞藻属菌株，并筛选转化体的高光耐受性。光系统I-少的菌株是光敏感的，因为在高光强下发生的类囊体膜中质体醌库的过度还原显然是致命的。 第二种方法将使用假回复突变体（第二位点突变体，恢复可被阳性选择的特定表型）。这两种方法（在基因组的相对较小区域中基因的随机失活导致正选择表型，以及使用基因组限制性图谱的假回复突变体作图）一起提供了将特定开放阅读框与特定功能联系起来的极好方式。由于这种方法使用特定表型的外观作为第一选择标准，并作为受影响的基因的识别是一个简单的第二步，该项目提供了一个强大的手段来识别未知的开放阅读框架，影响电子流周围的质体醌类囊体膜。由于存在明确的表型，因此基本上消除了对不分离野生型和突变基因组拷贝或不显示表型的突变体的研究（通常是靶向反向遗传学方法中的挫折来源）。这个项目将大大有助于识别新的开放阅读框架，其产品参与的过程，如氧化还原调节的质体醌池或组装和稳定的光系统II。 预计将发现许多与光合作用有关的未鉴定基因的功能，这些基因尚未通过其他方法鉴定。

With the availability of ever increasing amounts of genomic sequence information, the old adage "the more we know, the more we know that we don't know" rings true. Of the thousands of genes in prokaryotes, close to half code for proteins of as yet unknown function. Whereas some of these may code for structural proteins, many unidentified open reading frames are expected to encode important regulatory proteins involved with assembly or stabilization of functional protein complexes. Surprisingly, over the past years it has become clear that many of these putative proteins of unknown function do not have easily recognizable counterparts even in relatively closely related organisms. Yet the functional identification of proteins involved in regulation or stabilization of physiological processes is of major importance in understanding the molecular physiology and metabolism of an organism as a function of its genomic information. Fairly comprehensive cosmid interruption libraries that each contain 30-45 kb fragments of genomic DNA from the yanobacterium Synechocystis sp. PCC 6803 into which transposons have been integrated at random sites will be used to identify new genes involved in photosynthesis. Such transposon interruption libraries are available for about 90 different cosmids, together covering more than 80% of the Synechocystis sp. PCC 6803 genome. Synechocystis strains lacking photosystem I, the major sink for electrons in the plastoquinone pool in the thylakoid membrane, will be transformed with these interruption libraries and transformants will be screened for high-light tolerance. Photosystem I-less strains are light-sensitive because over-reduction of the plastoquinone pool in the thylakoid membrane, which occurs at high light intensity, apparently is lethal. A second approach will use pseudorevertants (second-site mutants restoring a particular phenotype that can be selected for positively). The two approaches (random inactivation of genes in a relatively small region of the genome resulting in positively selectable phenotypes, and pseudorevertant mapping using genomic restriction maps) together provide an excellent way to link specific open reading frames to specific functions. As this approach uses the appearance of specific phenotypes as the first selection criterion and as identification of the affected gene is a simple second step, this project provides a powerful means to identify unknown open reading frames affecting electron flow around the plastoquinone pool in thylakoid membranes. Because a clear phenotype exists, work on mutants that do not segregate wild-type and mutant genome copies or that do not show a phenotype (often a source of frustration in targeted reverse-genetics approaches) is essentially eliminated. This project will contribute significantly to the identification of new open reading frames whose products are involved in processes such as redox regulation of the plastoquinone pool or assembly and stability of photosystem II. It is anticipated that the function of a number of unidentified genes relating to photosynthesis will be found that have not been identified by other means.