Collaborative Research: Functional Analysis of the Synechococcus PCC 7942 Genome

许多生物，包括细菌、真菌、植物和动物，都有一个昼夜“时钟”来协调基因表达和生理的节奏，以预测昼夜周期的变化，并在没有环境线索的恒定条件下持续存在。光合作用的蓝藻是已知具有昼夜节律的最简单的生物；在蓝藻中，长聚球菌的基因组是已知最小的（约2.7 Mb）。这种单细胞生物需要光来生长，并且为理解生物钟的分子机制提供了许多优势，包括其可转换性和有效的同源重组系统。在确定其全基因组序列的背景下，将确定长形稻时钟功能和光同步所需的基因。由于S. elongatus具有非常小的基因组，因此它是一种模式生物，可以在其中分离和表征影响时钟机制，对光的敏感性及其与细胞功能的联系的突变。利用遗传互补的方法，构建了一个包含45 kb的长尾螺基因组DNA插入片段的cosmid文库。这种排序方法将揭示这种生物体的生物合成功能的组织，这些功能是所有复杂基因组共有的。该文库中的亚克隆将被插入元件mud诱变，以产生对应于基因组每个区域的突变池。这些mud插入将被交叉到S. elongatus基因组中以产生突变体，简单的筛选将用于识别时钟功能和调节所需基因的插入。这些mud插入也将被用作确定基因组序列和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定位和测序方法将比目前的随机测序方法提供更多信息，成本更低。长叶藻的基因组序列，结合对基因组突变影响的理解，将揭示时钟机制和光合代谢所需的最小功能集。由于长形霉与植物叶绿体之间的密切进化关系，了解长形霉基因组将为了解更复杂的高等植物基因组提供更简单的基础。

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.