Identification of Proteins Affecting Assembly, Regulation, and Function of Thylakoid Protein Complexes

本研究将利用蓝藻聚球藻的基因组序列。PCC6803(可通过http://www.kazusa.or.jp/cyano/cyano.html)访问，以从功能上识别参与调节、组装和功能的蛋白质复合体的“新”蛋白质，这些蛋白质复合体催化该蓝藻类囊体膜中的电子转移。这个项目涉及对自发突变体的正选择(1)光系统II活性降低或取消的突变体，或(2)特定基因表达调控被修改的突变体。通过对集胞藻的高光耐受性进行筛选，将进行正选择。缺少光系统I的PCC 6803菌株；这种菌株对强光敏感，但如果光系统II受损或类囊体膜中电子传递过程的速率或特定氧化还原成分的调节发生改变，则可以在高光强下繁殖。聚球藻基因组DNA序列的可用性。PCC 6803提供了一种强大的方法来快速识别导致光系统I缺失菌株的高光耐受性的自发突变的位置。这些阳性选择的突变体的DNA将被限制性内切酶切割；每个消化(用一种酶处理)将在凝胶上进行大小分离，并将测试凝胶切片(每个反映特定大小范围的DNA)是否与自发突变产生的原始(高光敏感)菌株的功能互补。聚球藻的完整限制性内切酶图谱。PCC6803基因组被构建为16种酶，每种酶对这种蓝藻基因组进行300-1000次切割。对于16种限制性内切酶中的每一种，所产生的片段都通过它们的基因组位置进行了鉴定，并按大小进行了排序并汇编成表格。根据可以对每个限制性内切酶进行功能性补充的片段的大小范围，可以从每个表中选择适当的限制片段的大小范围。这些限制片段(每个都通过它们在基因组上的位置识别)被汇集在一起，然后根据每个片段末端的基因组核苷酸编号进行排序。在检查该排序的列表时，可以识别核苷酸编号的簇。通常只能找到单个簇，其中每个酶的每个所选大小区域都有一个代表。通过鉴定在该簇中的所有序列之间共有的区域，可以确定携带感兴趣突变的区域(通常不长于2kbp)。感兴趣的区域将通过聚合酶链式反应进行扩增，突变将被确定，这样就可以简单地定位和鉴定未知基因的突变，即使不进行克隆。一些互补的次级突变可能存在于已知基因中，但另一些将位于开放阅读框中，其功能尚不清楚。这些开放阅读框架将特别令人感兴趣，因为所获得的结果将有助于将角色分配给这些具有未知功能的已知序列。这种方法已经导致从叶绿体中鉴定出一个与ycf39相似的开放阅读框架，并影响突变体光系统II的功能。该项目的下一部分将是对已确定的开放阅读框架的作用进行初步分析。分析的类型将取决于开放阅读框和突变体的表型，但可能涉及以下几个方面：(1)序列同源性搜索，(2)电子传递的功能分析，(3)基因表达研究，以及(4)蛋白质合成和降解测量。这项工作将导致鉴定目前未知的基因，其产物可能参与光系统II的组装，在类囊体膜的质醌池外的电子转移，或在细胞调控过程中。这是一个开放的领域，但现在聚球藻的基因组测序将取得快速进展。PCC 6803已经完成，使有效和全面的分析手段成为可能，就像这里描述的那样。这一领域的研究不仅是大规模测序工作的重要补充，因为它提供了关于开放阅读框架功能的信息，而且有望为类囊体膜中电子传递所需的调节机制和组装过程提供新的见解。

This research project will utilize the genomic sequence of the cyanobacterium Synechocystis sp. PCC 6803 (accessible through http://www.kazusa.or.jp/cyano/cyano.html) to functionally identify "new" proteins involved in the regulation, assembly and function of protein complexes that catalyze electron transfer in thylakoid membranes of this cyanobacterium. This project involves positive selection for spontaneous mutants (1) in which photosystem II activity is decreased or abolished, or (2) in which regulation of expression of particular genes has been modified. Positive selection will occur by screening for high-light tolerance of the Synechocystis sp. PCC 6803 strain that lacks photosystem I; this strain is sensitive to high light but can be propagated at high light intensity if photosystem II is impaired or if rates of electron transport processes in the thylakoid membrane or the regulation by particular redox components have been altered.The availability of the genomic DNA sequence of Synechocystis sp. PCC 6803 provides a powerful approach to a rapid identification of the location of spontaneous mutations that lead to high-light tolerance of the photosystem I-less strain. DNA from these positively selected mutants will be fragmented by restriction enzymes; each digest (treated with one enzyme) will be size-separated on a gel and gel slices (each reflecting a certain size range of DNA) will be tested for functional complementation of the original (high-light sensitive) strain from which the spontaneous mutant was derived. A complete restriction map of the Synechocystis sp. PCC 6803 genome has been constructed for 16 enzymes that cut this cyanobacterial genome 300-1000 times each. For each of the 16 restriction enzymes, the resulting fragments, identified through their genomic position, have been sorted by size and compiled into a table. Based on the size range of the fragments that can functionally complement for each restriction enzyme, an appropriate size range of restriction fragments can be selected from each table. These restriction fragments (each identified through their position on the genome) are pooled, and are then sorted according to the genomic nucleotide number at the end of each fragment. Upon inspection of this sorted list, clusters of nucleotide numbers can be identified. Generally only a single cluster can be found in which there is one representative from each of the selected size regions for each of the enzymes. By identifying the region that is common between all sequences in this cluster, the domain carrying the mutation of interest (generally not longer than 2 kbp) can be determined. The domain of interest will be amplified by PCR, the mutation will be determined, and mutations in unknown genes can thus be simply mapped and identified, even without cloning.Some of the complementing secondary mutations may be in known genes, but others will be in open reading frames for which no function is known yet. These open reading frames will be of particular interest as the results obtained will help to assign a role to these known sequences with unknown function. This method already has led to the identification of an open reading frame that is similar to ycf39 from chloroplasts and that affects photosystem II function in mutants. The next part of the project will be the preliminary analysis of the role of open reading frames that were identified. The types of analysis will depend on the open reading frame and on the phenotype of the mutant, but may involve the following: (1) sequence homology searches, (2) functional analysis of electron transport, (3) gene expression studies, and (4) protein synthesis and degradation measurements.This work will lead to the identification of currently unknown genes whose products may be involved in photosystem II assembly, in electron transfer out of the plastoquinone pool in thylakoid membranes, or in control of regulatory processes in the cell. This is an open field, but rapid progress will be made now the genomic sequencing of Synechocystis sp. PCC 6803 has been completed, enabling effective and comprehensive means of analysis such as the one described here. This area of research not only is an important complement to large-scale sequencing efforts, in that it provides information regarding the function of open reading frames, but also is expected to provide novel insights into the regulation mechanisms and assembly processes required for electron transport in thylakoid membranes.