International Mammalian Genome Society


The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

D1 Events of Sequence in the Mouse Genome Project

R. H. Reeves and J. Lund. Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205

The imminent availability of large amounts of mouse and mouse/human comparative sequence brings into focus a number of issues from the pragmatic to the speculative about the generation, analysis and applications of this information. Rapid progress on the production end of sequencing has accelerated efforts in its analysis. Sequence annotation tools and philosophies are developing concurrently with human genomic sequence. The mouse community can provide a "first line" for analysis of mouse sequence, and should play an important role in capitalizing on the opportunities afforded by having comparative genomic sequence from two mammals.

Why do comparative sequencing? What can be gained? Comparative sequence is effective for gene finding, and provides a useful tool for identification of promoter/ regulatory regions. It would not be surprising to find conservation of sequences that encode higher order chromosome structure and function elements, such as signals for replication, tertiary structure, matrix attachment, and locus control regions. Comparative sequence will greatly facilitate understanding of gene and chromosome evolution. Conservation of sequence is one of the most powerful indicators of what is fundamentally important about sequence and may provide insights into the purpose of the majority of unique sequence DNA whose function is unknown.

We have built physical maps and prepared minimal tiling paths of sequence-ready clones representing >8.5 Mb of the mouse genome in regions conserved with HSA22 and HSA21. These clones are in sequencing queues currently; more than 2.0 Mb of sequence has been completed and deposited in public databases. Physical maps were built on the scaffold of a high resolution recombinational map and were substantially supplemented in later stages by YAC STS content maps from the Whitehead Center for Genome Research (WCGR). PAC and BAC STS content maps were built by screening filter arrays of the RPCI-21 and -23 libraries with YACs, BACs, and pools of single copy probes. Extensive end-sequencing of selected clones extended sequence, and the collected set of end sequences provided a useful gene discovery tool when used in BLAST searches. PAC/BAC sets were fingerprinted to identify minimal tiling paths.

Comparative sequence analysis was facilitated by development of the CGAT (Comparative Genome Analysis Tools) software package, http://physiology.med.jhu.edu/roger/seq/Tools.html. Detailed analysis of 634 kb of sequence from the VCFS-homologous region of MMU16 was undertaken. This region has been intensively analyzed for gene content in humans by mapping, exon trapping, direct selection, and sequence analysis in human. Comparative sequence analysis found two new genes in this region and proved far more effective than algorithmic approaches for gene finding. In combination with GRAIL2 and MZEF, 60% of exons including part of every gene in this test segment were identified with no false positives, suggesting a highly accurate approach for first-pass automated sequence annotation.

Minimal tiling paths of mouse clones spanning more than 8.5 Mb have been prepared by the authors and colleagues Tim Wiltshire, Mathew Pletcher, Susan E. Cole, Deborah Cabin, Veronica Aquino, Melissa Villanueva, Elizabeth Rue, Bruce Birren, Jessica Lehoczky, Ken Dewar, Marcia Budarf and Bev Emanuel.

These clones are presently in sequencing queues at: The University of Oklahoma Genome Center (Bruce Roe); The Whitehead Center for Genome Research (Eric Lander, Bruce Birren); The Human Genome Center, Tokyo (Yoshi Sakaki, Masahira Hattori); MPIMG in Berlin (Marie-Laure Yaspo, Juliane Ramser); The Joint Genome Institute (Lisa Stubbs, Elbert Branscomb; and The Human Genome Center, Keio (N. Shimizu).

 


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