Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Automated Multiplex Microbial Genome Sequencing

Joshua L. Cherry[1], Debi Nelson[1], Peter Cartwright[1], Mark Stump[1], Diane Dunn[1] and Robert B. Weiss [1,2].
[1]Department of Human Genetics, University of Utah, Salt Lake City, UT 84112. [2]Corresponding author.

We are initiating large-scale genomic DNA sequencing of microorganisms of industrial and biological interest. Our first project will be the complete sequence of the genome of Pyrococcus furiosus (DSM 3638), a hyperthermophilic member of the "Archaea." This organism was isolated from hot marine sediments with temperatures near 100degreesC. The Archaea constitute one of the three great phylogenetic branches of living things. They are the least well studied of these three groups. The complete sequencing of an archaea genome will yield a tremendous quantity of information about this largely unexplored territory. The data will have much use in phylogenetic analyses and sequence comparison involving widely diverged taxa. Many archaeons, especially methanogens and thermophiles, have potential economic importance. Interest in thermophilic organisms has been growing in recent years, and thermostabile enzymes have a wide variety of industrial uses. The sequencing of the entire genome of a thermophile would amount to the cloning of a large number of such enzymes. It has been observed by X-ray crystallographers that proteins from thermophiles generally form usable crystals more easily than their mesophilic homologues. Many structure determinations have depended upon the cloning of a thermophilic homologue. Our sequencing efforts will enable us to provide such clones easily and rapidly. Comparison of thermophilic protein sequences to homologous mesophilic sequences will provide much information concerning thermal stability, and protein stability in general.

Novel methods and instrumentation for enzyme-linked fluorescent multiplex sequencing will be utilized [1]. The contiguous sequence of individual 20 kb inserts will be determined by a transposon-based directed strategy. A P. furiosus library of 20 kb insert size is being built in 10 multiplex vectors. These vectors provide multiplex tags for both the end sequencing and transposon mapping phases of the process. These fragments will be fed into the transposon system in an ordered fashion via an end sequencing strategy which obviates the need for physical mapping. The first step in the process will be end sequencing of the P. furiosus genomic library This end sequence will consist of ~2000 single-pass sequence ladders from the ends of the 20 kb inserts. This sequence will itself yield much information and will result in the discovery of many genes for hyperthermophilic proteins. Sequence matching between newly completed insert sequences and the database of end sequences will allow selection of new inserts to be sequenced, leading ultimately to completion of the genome.

This work is funded by DOE grant DE-FG03-94ER-61950 (R.B. Weiss, P.I.)

[1] Cherry, J.L., Young, H., Di Sera, L.J., Ferguson, F.M., Kimball, A.W., Dunn, D.M., Gesteland, R.F., and Weiss, R.B. (1994). Enzyme-linked fluorescent detection for automated multiplex DNA sequencing. Genomics 20, 68-74