Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
Human Genome News, September 1993; 5(3)
The Sixth Genome Mapping and Sequencing Meeting was held May 12-16 at Cold Spring Harbor, New York. Organized by Rick Myers (Stanford University), Bob Waterston (Washington University, St. Louis), and David Porteous [Medical Research Council (MRC), Edinburgh], the conference was attended by about 400 participants. Some 300 posters and platform presentations provided a rich and lively basis for discussion of recent advances and outstanding problems in the field.
Human Disease Genes
The first session, on human disease genes, opened with James Gusella (Massachusetts General Hospital) presenting the recent success of the Huntington's Collaborative Research Group in cloning and identifying the predisposing mutation in this important but hitherto elusive gene. The mutation is identified as a triplet expansion in a gene whose abnormal DNA sequence sheds little light on its function.
Although most abstracts concerned human genome mapping, the session devoted to model organisms was one of the highlights. Gerald Rubin (University of California, Berkeley) reminded attendees of the role played by Drosophila melanogaster geneticists in laying the foundations for the Human Genome Project. The ability to correlate Drosophila's
very high resolution cytogenetic map with a vigorously developed genetic mutational map illustrates the power of this model system.
Last year, attendees heard how chromosome 3 of Saccharomyces cerevisiae had been sequenced by the European Yeast Genome Collaborative Project; now, chromosomes 2 and 9 are close to completion.
Greg Elgar (Cambridge University, U.K.) introduced the project initiated by Sydney Brenner (Scripps Research Institute) to map and sequence the genome of the puffer fish or Fugu. This fish is distinguished by having the most compact vertebrate genome (~400 Mb), with a coding complexity very similar to that of mammalian DNA. Its sequence similarity to mammalian gene homologues is high, and its exon structure is highly conserved. With considerably smaller introns, this organism yields much more coding information from genomic sequencing than others do. As yet, little genetic data has been published for Fugu.
The Caenhorabditis elegans project, led jointly by Waterson and John Sulston [Laboratory of Molecular Biology (LMB), Cambridge, U.K.], continues to set the pace with 2 Mb of finished sequence, half of which is contiguous, at an average gene density of 1 per 4.5 kb. Sequencing of an additional 3 Mb of the estimated 100-Mb genome is expected this year. Finishing the sequence of this 16,000-gene organism, with its elegant genetics and completed cell-fate map to track each cell's movement during development, is a major near-term goal.
Comparative studies of mouse and human molecular composition, genetics, development, and pathophysiology is of powerful and mutual benefit to both communities. Steve Brown (St. Mary's Hospital Medical School, U.K.) of the European Collaborative Interspecific Backcross project illustrated this point by explaining how the 500-kb-resolution mouse genetic map can complement and significantly improve the human meiotic map constructed by typing microsatellite markers in the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees [Jean Weissenbach (Institut Pasteur); James Weber (Marshfield Medical Research Foundation)].
While shotgun sequencing-the generally favored large-scale sequencing method--is becoming more efficient, other technologies are also being explored. Possible improvements include the use of inexpensive walking primers assembled from hexamers or pentamers [Levi Ulanovsky (Weizmann Institute of Science, Rehovot, Israel)] or selected from small libraries of nonamers [Jerry Slightom (Upjohn Company)]. Use of genomic coupled amplification and sequencing eliminates the need for cloning and prior amplification by polymerase chain reaction and may be valuable for studying sequence variation [Gualberto Ruano (BIOS Laboratories)]. However, modifications of conventional protocols will probably not provide routine 1-kb reads in the near future. Andre Rosenthal (LMB) suggested that investigators need to move away from gel casting and consider such alternatives as the use of fluorescently labeled nucleotides to sequence by addition, removal, and extension of primed template DNA.
The informatics session addressed challenges and recent successes in handling and interpreting the vast wealth of new DNA sequence information and the complex data being generated by meiotic mapping and contig-assembly projects. Ingenuity and enthusiasm within the informatics community has made possible the accommodation of chimeric clones and assembly of coherent maps [Steven Lincoln, Massachusetts Institute of Technology (MIT); Bruno Lacroix, CEPH]. Remarkably accurate gene identification is now achievable from raw sequence data [Gary Stormo, University of Colorado; David Searls, University of Pennsylvania; Phil Green, Washington University]. Existing databases may already contain representatives of most ancient conserved regions of protein sequence (Green).
Sessions on cDNA and other mapping methods illustrated the need and potential for further technical innovations, including (1) microdissection and amplification of single cuts from metaphase chromosomes for use as painting probes [Jeff Trent (University of Michigan, Ann Arbor)]; (2) targeting of human dispersed repeats by homologous recombination for chromosome tagging and manipulation in somatic cells [Viv Watson (MRC, Edinburgh)]; (3) direct cDNA selection by genomic DNA carried in yeast artificial chromosomes (YACs) or cosmids [Mike Lovett (University of Texas Southwestern Medical Center, Dallas)]; and (4) construction of a truly normalized gene library by selective cloning of CpG islands using methyl-CpG binding protein [Sally Cross (University of Edinburgh)]. The presence of chimerism in CEPH mega-YAC libraries has been criticized, but users repeatedly defended the enormous value of the libraries for building maps and bridging gaps. Eric Lander (MIT) conveyed preliminary but very encouraging data suggesting a minimal level of chimerism in mega-YACs cloned in a new recombination-defective yeast strain. Nevertheless, cloning large (up to 350 kb) DNA fragments in bacteria is a promising alternative because of the simple insert purification offered by circular recombinant molecules [Hori Shizuya (California Institute of Technology)].
The closing session on large maps focused on efforts by human genome center investigators, who reported that large-insert clone physical maps of several chromosomes are either complete or nearing completion. Recurrent themes were the need for (1) multiply redundant cosmid and YAC libraries and (2) considerable duplication of effort-particularly because of significant levels of chimerism. Glen Evans (Salk Institute) described genomic sequencing strategies based on obtaining very dense cosmid coverage of chromosome 11 and exploitation of this coverage. His plans include using only T3 and T7 primers and end sequencing ~500 bp of every ordered cosmid for rapid construction of a frame or outline for the whole chromosome. He believes the resulting map of sequence tagged sites will provide a solid platform for biological investigations.
Participants expressed a growing sense that established methods and reagents are capable of yielding high-resolution clone maps of the human genome, chromosome by chromosome. However, some serious problems remain in making a global map, as illustrated by the computational difficulties in assembling coherent contig maps from the fingerprint analysis of the CEPH/Genethon mega-YAC library [Ilya Chumanov (CEPH)]. In collaboration with CEPH/Genethon, Lander plans to acquire and type microsatellite repeats on an industrial scale, but most of the meiotic events represented by the pedigrees are identifiable. Establishing relative order and distance with meiotic intervals requires a different approach. Integration with the physical map will depend heavily on overcoming the chimerism problem, either computationally or by constructing additional YAC libraries in recombination-deficient hosts.
David Cox (Stanford University) presented his latest mapping results using whole-genome radiation hybrids. The radiation map has the dual advantages of significantly increased resolution (now ~500 kb) and portability (~100 independent hybrids harboring a total of ~7000 independent and essentially random breaks) while still showing complete congruence with the meiotic map. His work and that of many other participants emphasized the importance of continued efforts in conceptual and experimental innovation and integration.
[David Porteous, MRC, Edinburgh; Bob Waterston, Washington University; and Rick Myers, Stanford University]
The electronic form of the newsletter may be cited in the following style:
Human Genome Program, U.S. Department of Energy, Human Genome News (v5n3).
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.
Published from 1989 until 2002, this newsletter facilitated HGP communication, helped prevent duplication of research effort, and informed persons interested in genome research.