Sponsored by the U.S. Department of Energy Human Genome Program
Human Genome News Archive Edition
Human Genome News, November 1992; 4(4)
The third meeting of the NIH-DOE Joint Working Group on the Mouse was held March 5-6 in Washington, D.C., to focus on physical mapping tools and strategies. Attendees included working group members, invited speakers, and federal government representatives. Following are some meeting highlights.
Nat Sternberg (Du Pont-Merck Pharmaceuticals) reviewed progress in producing mammalian genomic libraries in the P1 cloning system. The average insert in the mouse library is 70 kb, with about 80 to 90% of mouse gene sequences represented. Polymerase chain reaction (PCR) techniques can be used to isolate any one clone from the library in a week or two. The library, which is now available commercially, is arranged in 300 pools of 400 to 500 clones each. Groups of 10 to 50 pools are screened to identify those to be tested further. In the next 6 months Sternberg anticipates expanding the P1 mouse library threefold to sixfold.
Sternberg pointed out the advantages of P1 clones over cosmid clones and yeast artificial chromosomes (YACs) for mapping. He has included in the newest vector (1) rare restriction sites to size the insert and (2) flanking T7 and Sp6 promoters to generate labeled RNA probes (riboprobes) from the insert ends. With cycle sequencing techniques, he can use 200 to 300 bp of sequence from the insert ends to construct a sequence tagged site (STS). Sternberg also described efforts to develop transposon technology for the orderly dissection of cloned DNA and its use in transforming mammalian cells.
To make the Imperial Cancer Research Fund (ICRF) mouse and human YAC libraries more widely available, Hans Lehrach (ICRF) has for 2 years been testing the feasibility of transferring filter screening technology to laboratories in the United States and Europe. He plans to include in his arrays the Rad 52 YAC and P1 libraries developed by Steve Brown at St. Mary's Hospital in London. If the transfer is successful, this system can combine information originating in the limited number of mapping laboratories with the large amounts of data generated in laboratories working on mouse genetics or embryology.
Lehrach raised the possibility of mapping the genomes of man, mouse, and perhaps other mammals as a single project. He suggested extending the reference library concept to create a biological information net based on common YAC, P1, cosmid, cDNA, and exon trap clone libraries from many different experiments and laboratories. Such a system would allow the efficient accumulation of many types of biological information, reduce unintended overlaps, and simplify the establishment of pertinent relational databases.
Eric Green [Washington University (WU) School of Medicine] discussed his group's early progress in establishing a complete physical map of human chromosome 7. The strategy includes isolating DNA in YACs, assembling YAC contigs based on STS identification in isolated clones, and constructing STS-content maps that reflect spatial relationships deduced from assembled YAC contigs. A detailed summary was published in PCR Methods and Applications 1, 77-90 (1991).
The focus of this project has been to develop high-throughput STS-specific PCR assays from random DNA sequences derived from human chromosome 7. These efforts have produced more than 400 chromosome 7-specific STSs, all of which are sufficiently robust for screening a comprehensive YAC library. Computer simulations by Phil Green (WU) suggested that current STSs probably represent one-third to one-half of those required to construct an STS-content map of human chromosome 7 with 100- to 200-kb resolution.
More than 600 chromosome 7-specific YACs have been isolated from the WU total human DNA library. Characterization of these clones, which together should contain about 15 to 25% of the DNA from chromosome 7, is under way. The new library should have larger cloned inserts and fewer chimeric YACs and reduce the screening required to identify YACs containing particular STSs. Eric Green also discussed the relative merits of PCR vs hybridization-based approaches for identifying physical landmarks in sets of isolated YAC clones.
Pieter de Jong (Lawrence Livermore National Laboratory) discussed important human DNA cloning methods that might be applied to the mouse. He favored using small cloned fragments from chromosome-enriched libraries as anchors in combination with chromosome-enriched YAC or bacterial artificial chromosome arrays to prepare a contig map of the mouse genome. Using cosmid libraries made from flow-sorted chromosomes as an anchor source would require extensive preparation, de Jong felt, and a more realistic approach would be to construct small-fragment PCR libraries for (1) individual mouse chromosomes, using a single-event flow-sorting approach, or (2) individual chromosome regions by microdissection.
The group discussed the need to make available to the mouse community mapping resources such as YAC and cosmid libraries and cDNA arrays. To meet this need, the NCHGR RFA HG-92-002 provides that applicants distribute resources.
Many attendees felt that Human Genome Project goals would be achieved more quickly if mouse genomic analysis were tied closely to functional analysis. Experimental approaches currently allow evaluation on a gene-by-gene basis of the effect of a given gene on the entire phenotype. Given the rapid progress being made in generating genetic and physical maps and accumulating sequence information, new, efficient, cost-effective methods should be developed for functional gene mapping, embryonic stem cell resources, and gene targeting. The mouse offers an appropriate model for these technologies. The working group agreed to devote most of the next meeting to a fuller discussion of these issues and suggested that other NIH components be encouraged to participate.
The P1 mouse library manuscript has been published [Mammalian Genome 3, 550-58 (1992).]
Encyclopedia of the Mouse Genome II (1992), a companion reference tool to the journal Mammalian Genome, is now available. 294 pp. Free with journal subscription; $49 separately. (Springer-Verlag New York, Inc.; Attn: Dean Smith; 175 Fifth Ave., New York, NY 10010 (212/460-1500 or 800/777-4643).
A complete meeting report is available from the Joint Mouse Working Group. Contact:
Reported by Bettie Graham
Roswell Park Memorial Institute
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Human Genome Program, U.S. Department of Energy, Human Genome News (v4n4).
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.