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

随着基因组序列信息的可用性不断增加，“我们知道的越多，我们知道我们不知道的就越多”这句老话听起来是真的。在原核生物的数千个基因中，接近一半的基因编码着功能未知的蛋白质。虽然其中一些可能编码结构蛋白，但许多未知的开放阅读框有望编码与功能蛋白复合物的组装或稳定有关的重要调节蛋白。令人惊讶的是，在过去的几年里，人们已经清楚地认识到，即使在相对密切相关的生物体中，许多这些假定的功能未知的蛋白质也没有容易识别的对应物。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。相当全面的cosmid中断文库将用于鉴定参与光合作用的新基因，每个文库包含30-45 kb的基因组DNA片段，这些片段来自于褐杆菌Synechocystis sp. PCC 6803，其中转座子被随机整合到其中。这些转座子中断文库可用于约90种不同的cosmids，总共覆盖了Synechocystis sp. PCC 6803基因组的80%以上。缺少光系统I（类囊体膜中质体醌池中电子的主要吸收源）的联胞菌菌株将通过这些中断文库进行转化，并筛选具有高光耐受性的突变体。无光系统i型菌株之所以具有光敏性，是因为在高光强下类囊体膜中质体醌池的过度还原显然是致命的。第二种方法将使用假回复性（第二位点突变体恢复特定的表型，可以选择为阳性）。这两种方法（在相对较小的基因组区域中随机失活基因导致正选择表型，以及使用基因组限制图谱进行伪反向定位）共同提供了一种将特定开放阅读框与特定功能联系起来的绝佳方法。由于该方法使用特定表型的外观作为首选选择标准，并且受影响基因的鉴定是简单的第二步，因此该项目提供了一种强大的手段来识别影响类囊体膜中质体醌池周围电子流的未知开放阅读框。由于存在明确的表型，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的挫折来源）基本上被消除了。该项目将有助于识别新的开放阅读框，其产物参与诸如塑料醌池的氧化还原调节或光系统II的组装和稳定性等过程。预计将发现一些与光合作用有关的未确定基因的功能，这些基因尚未通过其他方法确定。

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.