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By Sean Henahan, Access Excellence

BALTIMORE, MD A team of researchers have managed to complete a genetic map of the bacterium Haemophilus influenzae. This accomplishment represents the first complete genetic map of a free-living organism and should speed efforts to sequence the human genome.

The researchers used a new approach called whole genome shotgun sequencing to sequence the 1,749 genes of the bacterium in less than a year. The feat would not have been possible in such a relatively short period of time without impressive new computer software and hardware.

The H. influenzae project was based on an approach to genomic analysis using sequencing and assembly of unselected pieces of DNA from the whole chromosome. The result was the complete nucleotide sequence- 1,830,137 base pairs. This approach eliminates the need for initial mapping efforts involving overlapping 40 kb DNA segments and should be applicable to mapping other microbial species for which gene maps do not exist, the researchers note.

Other ongoing genome projects, including the human genome project are based on the sequencing of clones derived from mapped restriction fragments, or lambda or cosmid clones. This approach has been slower, lacking sufficient computations muscle to support the assembly of the tens of thousands of random sequences found in a genome into a single assembly.

The current research relied on newly developed software programs that allowed rapid and accurate sequencing of large segments of DNA. The so-called 'shot-gun sequencing strategy' begins with the preparation of a single random DNA fragment library by first mechanically breaking the organism apart. The resulting fragments were then sequenced and assembled to produce the complete genome with the help of a software program called TIGR ASSEMBLER. .

In addition to a creating a physical map of the genome, the research team has catalogued many of genes' locations and many of their functions. Having access to a complete genome will help researchers determine how living organisms are organized genetically and how they have evolved. It should also help them determine the pathogenic elements of the genome.

The map of H. influenza was a nationwide collaboration by a team of researchers from Johns Hopkins University (including Nobel Prize winner Hamilton Smith), the State University of New York in Buffalo, the National Institute of Standards and Technology and the Institute for Genomic Research.

The success of this approach is also a vindication for study director Craig Venter of the Institute for Genomic Research. Others researchers had previously expressed skepticism about the utility of a database of gene fragments for complete genome mapping, and the National Institutes of Health had declined to fund the H. influenzae genome project. The team has now been invited to collaborate on 30 different microbial genome mapping projects.

Haemophilus influenzae is a Gram-negative bacterium that depends on a human host. The organisms causes recurring childhood ear infections and is also linked to meningitis in children. A vaccine has recently become available for this bacterium.

While this is the first successful mapping a complete genome sequence of a living organism, researchers have previously determined the complete genome sequence for several viruses, including cytomegalovirus, vaccinia and vareola (smallpox). Indeed, the smallpox virus was the first to be sequenced using the automated technology.

The current research provides considerable encouragement to scientists involved in other genome mapping projects. In addition to the Human Genome Project, scientists are also now mapping the genomes for E.coli, B. subtilis, C.elegans and Drosophila melanogaster.

"The success of whole genome shotgun sequencing offers the potential to accelerate research in a number of areas. Comparative genomics could be advanced by the availability of an increased number of complete genomes from a variety of prokaryotes and eukaryotes," report the researchers.

The new methods used to map H. influenza could lead to identification of the complete genomes of other pathogenic organisms. This in turn could lead to the development of new vaccines. The new strategy should also speed up the sequencing of the human genome.

For more information on this research see: Science Vol. 269, 7/28/95, Smith et al, pp. 495-511; and associated commentary, pp.468-470.

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