Carol J. Bult, Mark Adams, Rob Fleischmann, Jeannine Gocayne, Granger Sutton, Lixin Zhou, Owen White, Lisa FitzGerald, Judy Blake, Rebecca Clayton, Ewen Kirkness, Neil Geoghagen, Jan Weidman, Joyce Fuhrmann, Brian Dougherty[1], Hanna Tomb[1], Claudia Reich[2], Claire Fraser, Gary Olsen[2], Hamilton Smith[1], Carl Woese[2], and J.C. Venter.
The Institute for Genomic Research, 9712 Medical Center Drive, Rockville MD 20850; [1]Department of Molecular Biology and Genetics, The Johns Hopkins University, Baltimore MD 21205; [2]Department of Microbiology, University of Illinois, Urbana IL 61801.
The application of a whole genome shotgun strategy has been applied successfully to obtain complete genomic sequence from two Bacterial (sensu Woese) organisms, Haemophilas influenzae[1] and Mycoplasma genitalium[2] (URL:http://www.tigr.org ). Comparison of these two genomes to each other and to sequence data from other bacteria have provided many insights into genome organization and evolution.[1,2]
We have applied the whole genome shotgun strategy to sequence the genome of Methanococcus jannaschii. M. jannaschii is a barophilic, thermophilic methanogen and a member of the Archaea domain of life (sensu Woese). It has a genome size of approximately 2.0 Mbp and a nucleotide composition which is approximately 70% AT. Phylogenetically, M. jannaschii appears to be basal to other archaeal methanogens and will provide data critical for understanding the genetic basis and origin of methanogenesis.
Five aspects of the whole genome shotgun approach which are critical for its success are 1) the availability of a random genomic small insert plasmid library, 2) the availability of a representative large insert (lambda) library for the creation of a genome sequence scaffold, 3) high quality sequence data from both ends of the plasmid and lambda clones, 4) a robust sequence fragment assembly engine, and 5) data management and analysis tools which are tightly integrated with data production. For the M. jannaschii genome, over 36,000 sequences (~8.5 fold genome coverage) were obtained from the ends of clones from the plasmid library (average insert size 2.5 kbp). Sequence fragments were assembled using TIGR Assembler as the assembly engine.[1,2,3] Physical gaps were closed using PCR, lambda clones, and probes of genomic Southern blots. Sequences from the ends of lambda clones (average size 20 kbp) were used to build a genome scaffold to confirm the orientation and order of contigs generated by TIGR Assembler. Lambda clones also served as templates for primer walking across large repeat regions, such as the ribosomal operons.
The availability of whole genomes from both Bacteria and Archaea domains provides a solid comparative framework for describing the core genetic complement shared by these lineages, understanding what genes and biochemical pathways are unique to each domain, and testing the evolutionary relationship of Bacteria and Archaea to Eukarya (eukaryotes).
*Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FC0295ER61962.A001.
[1] Fleischmann et al., Science 269, 449-604 (1995).
[2] Fraser et al., Science, in press (1995).
[3] Sutton et al. Genome Science and Technology 1, 9-19 (1995).