The Genom of Homo sapiens.pdf

176 GUYON ET AL.million reads (mean read length, 576 bases), representing~1.5x coverage of the 3-Gb haploid canine genome(Vinogradov 1998).For 187 genes known to span HSA1p, the associatedpeptide sequence was searched against the complete collectionof dog reads using tblastn. For each peptide, allhomologous dog reads that were identified by the blastsearches were assembled at high stringency (99% nucleotideidentity) using TIGR Assembler (http://www.tigr.org/softlab/assembler/). Each assembly, or unassembledread, was then searched back against the Ensembl(release 1.1) collection of confirmed cDNAs and peptides(using blastn and blastx, respectively). If the highest scoringhits (for both the DNA- and protein-sequencesearches) were to the gene that was used originally forsearching, the assembly was considered a fragment of aputative ortholog. The coordinates of each human geneon HSA1p were obtained from NCBI build 31 of the humangenome (http://genome.ucsc.edu/).Radiation Hybrid MappingGenes were mapped on the 118 cell lines of theRHDF5000-2 panel described previously (Vignaux et al.1999). In brief, PCR primers were selected for mappingusing a standard selection program, i.e., Primer3(http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Whenever possible, both primerswere selected in the two introns flanking the annotatedexon sequence. Alternatively, to better ensure amplificationof the correct gene, in some cases one primer was selectedfrom a flanking intron and the other from a correspondingexon. Primers were preferentially selected to be25 bp in length and to work under a single optimal set ofPCR conditions (salt, Tm, Mg ++ , etc.) generating PCRproducts of 200–250 bp.Typing of markers was done using existing infrastructuredescribed previously (Priat et al. 1998; Mellersh et al.2000; Breen et al. 2001; Guyon et al. 2003). In brief, allreactions are done using a 96-well or 384-well format ina volume of 10–15 µl. An initial screen using 50 ng ofdog DNA, 50 ng of hamster DNA, and a 1:3 mix ofdog/hamster DNA (50 ng) is used to select primers suitableto be placed across the entire panel. PCRs were donewith 50 ng of RH DNA, and products were resolved on1.8% or 2% agarose gels, electrophoresed for 30 minutesas described previously (Priat et al. 1998; Mellersh et al.2000; Breen et al. 2001). Bands were viewed under UVlight after ethidium bromide staining, and an image wasrecorded.All markers were typed in duplicate and were consideredconsistent when the two vectors for each marker hada discrepancy value 9.0. Contiguous groups ofthe same chromosome origin were computed together.RH groups containing at least one HSA1p orthologousmarker were then ordered using the TSP approach asspecified by the CONCORDE computer package(http://www.math.princeton.edu/tsp/concorde.html)(Agarwala et al. 2000). TSP/CONCORDE computes fiveindependent RH maps, and the resulting maps were subsequentlyevaluated to produce a consensus map using amethod developed by us (Hitte et al. 2003). Intermarkerdistances were determined with the rh_tsp_map1.0 versionof TSP/CONCORDE, which produces map positionsin arbitrary TSP units.ACKNOWLEDGMENTSWe acknowledge the American Kennel Club CanineHealth Foundation, U.S. Army Grant DAAD19-01-1-0658 (E.A.O. and F.G.), and National Institutes of HealthR01CA-92167 (E.A.O, E.K., and F.G.). In addition,E.A.O is supported by K05 CA-90754 and is the recipientof a Burroughs Wellcome Award in Functional Genomics.R.G. is partly supported by an AKC and CNRSfellowship, and P.Q. by a Conseil Regional de Bretagnefellowship.REFERENCESAgarwala R., Applegate D.L., Maglott D., Schuler G.D., andSchaffer A.A. 2000. A fast and scalable radiation hybrid mapconstruction and integration strategy. Genome Res. 10: 350.Andersson L., Archibald A., Ashburner M., Audun S., BarendseW., Bitgood J., Bottema C., Broad T., Brown S., Burt D.,Charlier C., Copeland N., Davis S., Davisson M., Edwards J.,Eggen A., Elgar G., Eppig J.T., Franklin I., Grewe P., Gill T.,III, Graves J.A., Hawken R., Hetzel J., and Womack J., et al.1996. Comparative genome organization of vertebrates. TheFirst International Workshop on Comparative Genome Organization.Mamm. Genome 7: 717.Breen M., Thomas R., Binns M.M., Carter N.P., and LangfordC.F. 1999a. Reciprocal chromosome painting reveals detailedregions of conserved synteny between the karyotypes of thedomestic dog (Canis familiaris) and human. Genomics 61:145.Breen M., Langford C.F., Carter N.P., Holmes N.G., DickensH.F., Thomas R., Suter N., Ryder E.J., Pope M., and BinnsM.M. 1999b. FISH mapping and identification of caninechromosomes. J. Hered. 90: 27.Breen M., Jouquand S., Renier C., Mellersh C.S., Hitte C.,Holmes N.G., Cheron A., Suter N., Vignaux F., Bristow A.E.,Priat C., McCann E., André C., Boundy S., Gitsham P.,Thomas R., Bridge W.L., Spriggs H.F., Ryder E.J., CursonA., Sampson J., Ostrander E.A., Binns M.M, and Galibert F.2001. Chromosome-specific single-locus FISH probes allowanchorage of an 1800-marker integrated radiation-hybrid/