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In this issue...
DOE '99 Oakland Highlights
In the News
Ethical, Legal, and Social Issues
Web, Other Resources, Publications
Meeting Calendars & Acronyms
Analysis of microbial genomes can provide clues to genome organization and evolution, contribute to a healthy citizenry, and offer potential solutions to long-standing challenges in renewable energy production, chemical and materials production, and environmental cleanup. To take advantage of these opportunities for fulfilling key missions, in 1994 DOE initiated its Microbial Genome Program (MGP), a spinoff of the Human Genome Program. At the Oakland Contractor-Grantee meeting, MGP researchers reported exciting progress.
David Schwartz (now at University of Wisconsin, Madison), Owen White (The Institute for Genomic Research), and Kenneth Minton [now retired from Uniformed Services University of the Health Sciences (USUHS)] described mapping, sequencing, analyzing, and genetically engineering the 3-Mb genome of D. radiodurans. This microbe can survive radiation exposure thousands of times greater than doses that are lethal to humans. Although its chromosomes shatter into hundreds of fragments when hit with millions of rads of gamma radiation, the organism can stitch itself back together in about a day.
Scientists hope that analyzing D. radiodurans' genome will give some clues to its remarkable DNA-repair mechanisms and that the microbe will be useful in cleaning up toxic mixed-waste sites around the globe. Schwartz's optical mapping of the D. radiodurans genome was critical to the discovery that it has four chromosomal elements rather than just one [see Science 285(5433), 1558-62; HGN 10(1-2), 12].
Schwartz described using a single-molecule approach to produce ordered restriction maps from individual DNA molecules. The technique, proven useful for producing high-resolution detailed maps of clones and entire genomes, is expected to facilitate large-scale sequencing projects. Optical mapping also generates an in situ picture of the entire genome's architecture, revealing the number of chromosomes and the existence of extrachromosomal elements. The team plans to use optical mapping to complete a human reference map that will include 10x to 15x coverage and link with other physical maps by aligning restriction-mapped BAC contigs.
White discussed results of an early survey of the D. radiodurans genome to determine how this organism withstands extraordinarily high levels of radiation and oxidative stress. But so far, analysis of the DNA-repair genes has turned up nothing unique that would account for the capability to knit double-stranded breaks back together.
"They're pretty much the garden variety of DNA-repair genes," White noted. A survival strategy unique to this organism is that the genome is partitioned into regions of genes that have specific functions.
Minton discussed efforts to annotate the D. radiodurans sequence, focusing on properties that render this organism resistant to radiation. This work has been taken over by Michael Daly (USUHS). Features noted to date include a novel enzyme that combines potential repair domains from three independent repair proteins. Minton reported on Daly's work to engineer this genome to enhance the potential for organopollutant degradation in radioactive mixed-waste environments; for example, Daly introduced genes to degrade toluene to less-dangerous substances and showed that the genes retain effectiveness even at high-radiation doses.
Minton also described Daly's engineering of heavy-metal resistance by transferring a mercury-resistance gene from Escherichia coli and the team's plans to transfer a uranium-reduction gene from the microbe Shewanella putrefaciens to D. radiodurans. In Shewanella, uranium acts as a final electron acceptor, being reduced from uranium+6 to uranium+4, which settles like a stone or mineral and does not enter the groundwater.
Carol Giometti (Argonne National Laboratory) described the Archaeal Proteomics Project, whose goal is to identify proteins and regulatory pathways relevant to bioremediation and energy technology. She explained that proteomics includes information on relative protein abundance, posttranslational modifications, changes in stimuli-response kinetics, and subcellular location. Important proteomics tools include two-dimensional gel electrophoresis (2-DGE) and mass spectrometry.
Initial work is focused on the Pyrococcus furiosus and Methanococcus jannaschii proteomes (both genomes have been sequenced completely). Both are hyperthermophilic archaea with growth temperatures near 100oC and enzymatic capabilities that have promise for bioremediation, energy conversion, and chemical-processing systems. Investigators are using 2-DGE to purify and quantitate proteins expressed in archaea that are grown under a variety of conditions designed to modulate specific metabolic pathways. Giometti discussed preliminary results, which provide a foundation for studying the M. jannaschii and P. furiosus proteomes.
Projects have begun to leverage microbial sequence information to sequence other strains more rapidly. Gary Andersen (Lawrence Livermore National Laboratory, LLNL) spoke about using suppressive subtractive hybridization to identify genomic differences among enteropathogenic strains of Yersinia enterocolitica and Y. pseudotuberculosis. The technique uses PCR amplification to enrich for unique segments of restricted DNA and simultaneously limits nontarget amplification by suppression PCR. Of the two pathogens, Y. enterocolitica is more often associated with human infection.
These explorations are likely to reveal unique DNA regions that define the genetic basis for the underlying differences in their phenotypic variation. Streamlining, automating, and increasing this technique's throughput should enable large-scale genomic comparison among closely related strains and generation of strain-specific oligonucleotide probes for molecular epidemiology studies. Ultimately, this technology could enable the much more rapid determination of closely related microbial sequences based on completed reference strain sequences.
The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v10n3-4).
The Human Genome Project (HGP) was an international 13-year effort, 1990 to 2003. Primary goals were to discover the complete set of human genes and make them accessible for further biological study, and determine the complete sequence of DNA bases in the human genome. See Timeline for more HGP history.