Collaborative Research: Functional Analysis of the Synechococcus PCC7942 Genome

许多生物体，包括细菌、真菌、植物和动物，都有一个生物钟，它协调基因表达和生理的节奏，以预测昼夜周期的变化，并且在没有环境线索的情况下保持不变。光合作用蓝藻是已知的最简单的显示昼夜节律的生物；在蓝藻中，长聚球藻是已知的最小的基因组(~2.7Mb)。这种单细胞生物体需要光才能生长，并为理解生物钟的分子机制提供了许多优势，包括它的变形性和高效的同源重组系统。长链球菌的时钟功能和光同步所需的基因将在确定其完整基因组序列的背景下进行鉴定。由于长链链球菌的基因组非常小，因此它是分离和表征影响时钟机制、对光的敏感性以及与细胞功能联系的突变的模式生物。我们将构建一个包含45kb长链霉菌基因组DNA插入片段的粘粒文库，然后用遗传互补的方法对其进行排序。这种排序方法将揭示这种有机体的生物合成功能的组织，这些功能是所有复杂基因组的共同功能。该文库中的亚克隆将被插入元件MUDS诱变，以产生与基因组每个区域相对应的突变库。这些MUDS插入将被交叉到长链霉菌基因组上以产生突变，并将使用简单的筛选来识别时钟功能和调节所需的基因插入。这些MUDS插入也将被用作确定基因组序列以及这些突变在基因组序列中的位置的起点。这种新的基于功能的基因组测序和测序方法将比目前的随机测序方法提供更多的信息和更低的成本。长链霉菌基因组的序列，当结合对基因组中突变的影响的理解时，将揭示时钟机制和光合代谢所需的最小功能集。由于长链藻与植物叶绿体的进化关系密切，了解长链藻基因组将为理解更复杂的高等植物基因组学提供更简单的基础。

Many organisms, including bacteria, fungi, plants, and animals, have a circadian "clock" that coordinates rhythms of gene expression and physiology that serve to anticipate changes in the day/night cycle, and which persist under constant conditions in the absence of environmental cues. The photosynthetic Cyanobacteria are the simplest organisms known to display circadian rhythms; among the cyanobacteria, Synechococcus elongatus has the smallest known genome (~2.7 Mb). This unicellular organism requires light for growth, and offers many advantages for understanding the molecular mechanism of the circadian clock, including its transformability and efficient systems of homologous recombination. The genes required for clock function and synchronization by light in S. elongatus will be identified in the context of determining its complete genome sequence.Because S. elongatus has a very small genome, it is a model organism in which to isolate and characterize mutations that affect the clock mechanism, its sensitivity to light, and its connection to cellular functions. A cosmid library consisting of 45 kb inserts of S. elongatus genomic DNA will be made, then ordered by the genetic method of complementation. This ordering method will reveal the organization of this organism's biosynthetic functions, functions common to all complex genomes. Subclones in this library will be mutagenized with the insertion element MudS to generate mutant pools corresponding to each region of the genome. These MudS insertions will be crossed onto the S. elongatus genome to generate mutants, and simple screens will be used to identify insertions in genes required for clock function and regulation. These MudS insertions will also be used as starting points for the determination both of the genome sequence, and of the locations of these mutations within the genome sequence. This new function-based approach to genome mapping and sequencing will be more informative and less costly than current, random sequencing approaches. The sequence of the S. elongatus genome, when combined with an understanding of the effects of mutations in the genome, will reveal the most minimal set of functions required for both the clock mechanism and photosynthetic metabolism. Because of the close evolutionary relationship between S. elongatus and plant chloroplasts, understanding the S. elongatus genome will provide a simpler basis for understanding the more complex genomics of higher plants.