Collaborative Research: Functional Analysis of the Synechococcus PCC 7942 Genome

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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.