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

识别类囊体膜中参与氧化还原调节和光系统 II 组装的新开放阅读框

• 批准号: 0111058
• 负责人: Willem Vermaas
• 金额: $ 33万
• 依托单位: Arizona State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2005-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0111058&HistoricalAwards=false
• 关键词: Identifying; New; Open; Reading; Frames

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.

随着越来越多的基因组序列信息的可获得性，古老的谚语“我们知道的越多，我们知道的我们不知道的越多”听起来是正确的。在原核生物的数千个基因中，有近一半的基因编码了功能未知的蛋白质。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架被认为编码与功能蛋白复合体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，人们已经清楚地看到，即使在关系相对密切的生物体中，许多功能未知的假定蛋白质也不容易识别出对应的蛋白质。然而，对参与调节或稳定生理过程的蛋白质的功能鉴定对于理解作为其基因组信息功能的生物体的分子生理学和新陈代谢具有重要意义。相当全面的粘粒中断文库，每个文库包含来自聚胞体蓝杆菌的30-45kb的基因组DNA片段。已经在随机位置整合了转座子的PCC6803将被用来识别与光合作用有关的新基因。这样的转座子中断文库适用于大约90个不同的宇宙，总共覆盖了聚球藻80%以上的面积。PCC6803基因组。缺乏光系统I的集胞藻菌株将用这些中断文库进行转化，并对转化子进行高光耐受性筛选。光系统I缺失菌株是光敏感的，因为在高光强下，类囊体膜中的质醌池的过度减少显然是致命的。第二种方法将使用假逆转(即恢复特定表型的第二位点突变体，该表型可以被选择为阳性)。这两种方法(在基因组相对较小的区域随机失活基因导致正向选择的表型，以及使用基因组限制图进行假逆转作图)一起提供了一种将特定的开放阅读框架与特定功能联系起来的极好的方法。由于这种方法使用特定表型的出现作为第一选择标准，而鉴定受影响的基因是简单的第二步，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醌池周围电子流动的未知开放读框。因为存在明显的表型，所以对不分离野生型和突变型基因组拷贝或不表现表型(在定向反向遗传学方法中通常是挫折的来源)的突变体的工作基本上被消除了。这个项目将大大有助于鉴定新的开放阅读框，其产物涉及诸如质体对苯二酚库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知基因的功能，这些基因尚未通过其他手段进行鉴定。