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. Author manuscript; available in PMC: 2007 Sep 25.
Published in final edited form as: Mamm Genome. 2002 Jan;13(1):5–19. doi: 10.1007/s0033501-2109-8

Genetic, physical, and comparative map of the subtelomeric region of mouse Chromosome 4

Xia Li 1, Alexander A Bachmanov 1, Shanru Li 1, Zhenyu Chen 1, Michael G Tordoff 1, Gary K Beauchamp 1,2, Pieter J de Jong 3,4, Chenyan Wu 4, Lianchun Chen 4, David B West 4, David A Ross 4, Jeffery D Ohmen 4, Danielle R Reed
PMCID: PMC1994206  NIHMSID: NIHMS26596  PMID: 11773963

Abstract

The subtelomeric region of mouse chromosome (Chr) 4 harbors loci with effects on behavior, development, and disease susceptibility. Regions near the telomeres are more difficult to map and characterize than other areas because of the unique features of subtelomeric DNA. As a result of these problems, the available mapping information for this part of mouse Chr 4 was insufficient to pursue candidate gene evaluation. Therefore, we sought to characterize the area in greater detail by creating a comprehensive genetic, physical, and comparative map. We constructed a genetic map that contained 30 markers and covered 13.3 cM; then we created a 1.2-Mb sequence-ready BAC contig, representing a 5.1-cM area, and sequenced a 246-kb mouse BAC from this contig. The resulting sequence, as well as approximately 40 kb of previously deposited genomic sequence, yielded a total of 284 kb of sequence, which contained over 20 putative genes. These putative genes were confirmed by matching ESTs or cDNA in the public databases to the genomic sequence and/or by direct sequencing of cDNA. Comparative genome sequence analysis demonstrated conserved synteny between the mouse and the human genomes (1p36.3). DNA from two strains of mice (C57BL/6ByJ and 129P3/J) was sequenced to detect single nucleotide polymorphisms (SNPs). The frequency of SNPs in this region was more than threefold higher than the genome-wide average for comparable mouse strains (129/Sv and C57BL/6J). The resulting SNP map, in conjunction with the sequence annotation and with physical and genetic maps, provides a detailed description of this gene-rich region. These data will facilitate genetic and comparative mapping studies and identification of a large number of novel candidate genes for the trait loci mapped to this region.

The subtelomeric region of mouse Chr 4 harbors loci that cause alterations in development, behavior, and disease susceptibility (Table 1) but sparse physical and genetic mapping resources. Identification of candidate genes for these traits has been hampered because genetic and physical maps are difficult to construct accurately near the telomere (Riethman 1997). As a result, the quality of available mapping data is poor in this region. Examples of this include inconsistencies in marker and gene order, both in individual studies and composite maps, low marker density, poor YAC-coverage, lack of a sequence-ready BAC contig, and no available draft or finished genomic sequence.

Table 1.

Loci with developmental, behavioral, or disease susceptibility phenotypes mapped to the subtelomeric region of mouse Chr 4.

Name Symbol cM Reference
Friend virus susceptibility 1 Fv1 76.5 (Stoye et al. 1995)
Gastritis type A susceptibility locus 2 Gasa2 77.5 (Silveira et al. 1999)
Epistatic circling gene of C57L/J ecl 78.4 (Taylor 1976)
Fluctuating asymmetry QTL 1 Faq1 79.1 (Leamy et al. 1997)
Beta-carboline-induced seizures 1 Bis1 80.0 (Martin et al. 1995)
Insulin-dependent diabetes susceptibility 9 Idd9 82.0 (Rodrigues et al. 1994)
Testis-determining autosomal 1 tda1 82.0 (Eicher et al. 1996)
Saccharin preference Sac 83.0 (Fuller 1974)
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Gene names and symbols are from the Mouse Genome Database (MGD), maintained at The Jackson Laboratory. Distances from the centromere (cM) are from the integrated MGD map.

This region contains the saccharin preference locus (Sac), which affects the intake of sweeteners (Bachmanov et al. 1997). A Sac candidate gene in the area was identified (Tas1r1; Hoon et al., 1999), and then excluded (Li et al. 2001). Sac has effects on the peripheral sensitivity to sweeteners (Bachmanov et al. 1997; Inoue et al. 2001), and is allelic between the C57BL/6ByJ (B6; high sweetener preferring) and 129P3/J (129) and DBA/2 (low sweetener preferring) strains (Fuller 1974; Lush 1989; Belknap et al. 1992; Lush et al. 1995; Blizard et al. 1999). The Sac locus selectively affects sweet and alcohol intake; other taste stimuli, such as sour, salty, and bitter, are unaffected by Sac (Bachmanov et al. 1996a; 1996b).

To increase the resources available for the identification of Sac and other neighboring loci, a detailed characterization of this region was undertaken. First, a dense genetic map was constructed, with markers previously mapped to the region, and updated with new markers generated during the project. The B6 and 129 strains were chosen to create the genetic map because they are allelic for the Sac locus and are distantly related. The newly generated genetic map resulted in the construction of a physical map with overlapping BAC clones. A BAC clone covering the Sac critical region was sequenced and analyzed for the presence of genes. In addition, comparative mapping was conducted because previous work suggested that significant conserved synteny existed between the telomeric region of mouse Chr 4 and the telomeric region of the p-arm of human Chr 1.

Materials and methods

DNA markers and sequences

Markers on distal Chr 4 used for mouse genotyping and physical mapping were obtained from several sources. The locations of ESTs R75150, M134G01, M136B1, K01599, AA408705, M134C6, D4Xrf497, D4Xrf215, Al225779, and K00231 were found in the radiation hybrid databases of the Whitehead Institute for Biomedical Research/MIT Center for Genome Research (http://www-genome.wi.mit.edu/), the Medical Research Council (http://genex.hgu.mrc.ac.uk/), and the Mouse Genome Database (http://www.jax.org) maintained by The Jackson Laboratory. Markers based on BAC end sequences were obtained through The Institute for Genomic Research web site (http://www.tigr.org) or by direct sequencing, when necessary. Some STSs, e.g., R74924, D18402, and U37351 and genes, e.g., BG228522, Ski, Pkcz, Gnb1, Cdc212, DvI1, Tnfrsf4 and Agrn were available from public databases (National Center for Biotechnology Information [NCBI], Massachusetts Institute of Technology [MIT]). Additional markers, including all D4Mon markers, were designed using sequence from the RPCI-23 BAC 118E21.

Primers were purchased from Research Genetics (Huntsville, Ala.) or synthesized at Integrated DNA Technologies, Inc. (Coralville, Iowa). Most primer pairs and their PCR products were tested for polymorphisms between the B6 and 129 strains. Most polymorphic markers were used for genotyping the F2 mapping panel and to construct the genetic map. Markers located between D4Mit256 and K00231 were used to make the BAC contig. Details about markers and their associated primer sequences are given in Table 2.

Table 2.

Sequence-tagged sites (STS) in the subtelomeric region of mouse Chr 4.

Locus Accessiona Forward primer 5′ → 3′ Reverse primer 5′ → 3′ Product size (bp)b Polymorphic B6/129c Detection methodd
D4Mit33 MGI92868 GGAGGTGTCAGGAGCCCT CTCCTGACTGAGGTACCAAACC 126 Yes SSLP
D4Mit190 MGI92820 CAGTGAAGCAAAAAGATTCGG TTCCAGTCTTGGGAATGTATCC 144 Yes SSLP
D4Mit42 MGI92875 CATGTTTGCCACCCTGAAAC CCTCACTTAGGCAGGTGACTC 100 Yes SSLP
Tas1r1 AF301161 GGAGTTGCAGCCTCTACAGC CAGGAGAGGAAAATGGCAAA 100 Yes SSCP
D4Mit254 MGI100505 AATACAATTATCATTTGCATTGTGG TCGTGGTGGACCTTATCTCC 135 Yes SSLP
Trp73 AF138873 TGAGATCTGGTGCCCTCTCT GCCTGATCTAGGCTGGAAAA 229 Yes Both
D4Mit209 MGI92840 CCTTGCTGGTTCTGGCTAAG CTATCTCCTGAGTTGCTCTGAGC 122 Yes SSLP
D4Mit256 MGI100503 CTGGAGAGTTAGAATGGGGTACC ACAGAGGCGCTTCCTAAC 132 Yes SSLP
R75150 R75150 ACAGGACAAATGCTGGGTTG GTGGTAAAGAACGCTTGGCT 217 Un NA
BG228522 BE137821 GCCGATCCTGGTGATGTACT ACAATGGCTCAAAACCGTTC 200 Un NA
M136B1 AU042805 TCCTTTATGTCCAACAGCCA CATGGTCTGTGATGTGACCA 164 No SSCP
K01599 AA062172 GGAAAAGGGAGTCGCCATA GAGCCGCCTAACTCTCACAC 166 No SSCP
D4Smh6b X58261 CTGTAGGCTGCTTTTATCTTTTG TGCCCCTTCAGCACATGCCA 102 Yes SSLP
AA408705 TC87041 GCTTCAGAAAATCGAGGCAC GCATGGGCTATGATAGGTGG 232 No SSCP
Ski U14173 ATCTCTTGTTTCGTGGTGGG AGGATGCCCATGACTTTGAG 173 No SSCP
399I12-T7 AZ052540 TGGTGGTGTAATACTATTCCTTTG TCTTTAATTTTTGGCTTTTTGATACA 102 No SSCP
Pkcz NM_008860 ACACATTAAGCTGACGGACT CAAACATAAGGACACCCAGT 164 Yes SSCP
R74924 R74924 AGTGCCACCAACCTGGTAAG AAGTGCCTGCAGGGATGC 165 No SSCP
M134C6 AI098070 CAAAACCACATGGTTACCGA GCCCTATTGCCAAATGACTT 264 No SSCP
438C18-SP6 AZ077160 CGGGACCTAAAACTGGACAA TGGGGACAGTTACCAGGAAG 254 Yes SSCP
472O18-T7 AZ114562 CTTTCCATTCTCCACCCTCA AGGTCCTAGGGAGAGGTCCA 260 No SSCP
399I12-SP6 AZ052537 CAGCTGTGTGCATGTTGACC CATCATGAAGACTCAGGGCA 106 No SSCP
360M12-SP6 AZ027787 AATGATGAAGTGTCAGCCTCAG CAACAGAACTCAAAGCCTGG 100 Yes SSCP
151E4-SP6 AZ263331 GACCTTCGGAAGAGCAGTTG AGTGTGTGTCGCCATATCCA 223 No SSCP
147A15-T7 AZ273113 GTGGTTGCTGGGATTTGAAC CAAGCAACCAAACAACCAAA 101 No SSCP
307E5-SP6 AQ982924 GCTAGTTGGGGAACAAACCA ACTGCAAATGTCCAACTCCA 149 No SSCP
Gnb1 NM_008142 GGGCATCTGGCAAAGATTTA AGATAACCTGTGTGCCCGC 281 No SSCP
37D20-T7 AZ088686 GAAAACAATCGGGGAGAAGTC TGAAATTATCACACGCCAGG 109 No SSCP
415A22-SP6 AZ057087 GTCCACACCTGGCTTTTGTT CAGCACTCAGTGAGGTTCCA 199 Yes SSCP
415G24-SP6 AZ059760 ATGTAATGGAAGGGCTGCTG CAGCACTCAGTGAGGTTCCA 113 No SSCP
34M15-SP6 AZ238889 CGCTACTTCGCTTTTATCCG ATGATGACGTACGACGACGA 150 No SSCP
Cdc2L2 NM_00766 GGCTTCAGCCTCAAGTTCTG AAAACAACCAAGTTGCCCTG 101 No SSCP
436P10-T7 NA CACAGGCCAAGTTGTTGTTG CAGGGGACCTTCTGAATGAT 115 Un NA
D18402 D18402 GGAGTTCTCCTACCCTGGCT GAGGCTCTGAGCAGTGTCAA 167 No SSCP
360M12-T7 AZ027785 CGGTCAGGAGTAGTGTGGGT CAGCAGCTGATATTGAGGCA 123 No SSCP
138D7-T7 AZ281648 ACCTCTAGGGTTTACGGGGA CCTCAGGTAGTGCAAGCTCC 199 No SSCP
151E4-T7 AZ263335 GTCCCAAAAGCTAGCACAGG TCATGAGCCAACCATGTGATT 240 No SSCP
457N22-T7 AZ089282 CCGGAGGACCATAAATCTGA CCTCAAAAACAAGCCTGAGC 129 No SSCP
37D20-SP6 AZ088681 GCTAGCCTTGAAGCCAACAC TGAACAGCATGCTTACCCAG 122 No SSCP
147A15-SP6 AZ273111 TCCGGAGGACCATAAATCTG CACAGTCCCAGTCATTCCCT 249 Yes SSCP
D4Xrf497 AA117049 GCGAGACGAGTGGGTAGTTC ACACTGAAACCTCGCTTGCT 129 No SSCP
280G12-T7 AZ986251 GGGCTGGGAATTGAACCTAT TGAATCCCTTACAGCCTTGC 420 Yes SSCP
49O2-T7 AZ253249 TCCCTAGAGGCCTGTCTGTC TCGTCTCGGAGCCTCTTCTA 169 Yes SSCP
34M15-T7 AZ238896 GGGTTTATGTGGCAAGCACT ACTCCATTTGCCTTTTGTGG 118 No SSCP
118E21-SP6 AZ274152 GGGGCAGGTGGGTAATAAGT CAAAAGCCCAACTCCTTGAG 271 Yes SSCP
U37351 U37351 GTGGCTTGGTGCTATTGACA GGGGCTATTAAGGCCATTTT 160 No SSCP
338N4-T7 AQ998700 AGTTACACAGCTGGGACGA GCAAGAGCCTAGCAATCCAC 245 Yes SSCP
284D21-SP6 AQ932576 ACAGAAATCCCTCATGCGA TCAGTGTGGACCAGAAAGTCC 105 No SSCP
387F5-SP6 AQ998225 CAGTTACACAGCTGGGACGA GCAAGAGCCTAGCAATCCAC 245 Yes SSCP
D4Mon7 G67778 CCCACTATGGTCCCAGAGAA GCGCTGACATCCTCCTATGT 187 No SSLP
M134G01 AI31686 CACATTAGCCATTGTCCTGG GGCAGAAAGGAATCAGAAGC 161 Yes SSCP
D4Mon4 G67775 ACATGCCTGCCTATCTTTGC GGAACCTGTTTTCCATGGTG 197 No SSLP
Dvl NM_010091 AAGTTCATGGGCCTCACCACCTGTG TACTAGCTACCCTTCACATACC 238 No SSLP
D4Mon6 G67777 TGAGTGTCCTCTGCCTGATG CCATGGGAGACCAGAAGGTA 206 Un SSLP
Tas1r3 AF311386 GCAACAGAGTCACAACTGCC ACACACAGTACCAACCCCGT 293 Yes SSCP
D4Mon3 G67774 GCTTACGATGGTCGTGAGGT GCAAGCAACCTGAACATGAA 188 No SSLP
D18346 D18346 ATGTCCAGGGTAGAGAGCCC TGTCCGCAGTGTGGAAACTA 165 Yes SSCP
D4Xrf215 F07942 TTCCAAGCTCACACATCAGC GTGCTGCTCTGCATTGAGTG 124 No SSCP
350D2-T7 AZ032147 TGTAGGGAATGTTTCTGCACC ACATGGAACAGGATTCTGGC 295 Yes SSCP
D4Mon41 G67754 TGCATCACTATTAAGCCTCAACC AAGAATTTGCAAAGACTGTGAGA 260 No Both
D4Mon43 G67755 GGTGGCTCAAACCATCCATA GAGGGCAATGAGCAAAATGT 203 Yes SSCP
D4Mon44 G67756 GGTCCTGTCTCTGGTTCAGG TAACACCCACATCAGGCAAC 201 Yes Both
D4Mon48 G67757 CAGCAGGCAAGATGACCTC GTCCCTCACCAGCCATGTTA 205 Yes Both
D4Mon49 G67758 AGCCTGGGCTAAGTTGTGTG TATGGGCCAATGTTGTTCCT 204 Yes Both
D4Mon50 G67759 ATGGTGGCTCACAACCATCT AT TTGTCCTCTGATTGCAGC 193 Yes SSCP
D4Mon52 G67760 ATGCTCAGCCTGCTTTGTTT GCTGATAGCCCTGGGTTCTA 198 No Both
D4Mon53 G67761 TGTACGCACAAATTGACTTGC GAATCCACATTGCAAAGCCTA 222 Yes SSCP
D4Mon54 G67762 CACAGGCAAATGAAGGGAAG CCAGACTTCTCCAGCTCTCC 187 Yes Both
D4Mon31 G67747 CAGATTCTCCAGCTGTCAGG CTGTGTTTCCGCACCAAGT 229 No SSCP
D4Mon56 G67763 CCTGTGGTTGACTAGGCAGAA GCCTGATAGCCTGGAATACA 406 Yes SSCP
D4Mon5 G67776 ACCTTGTTCCTGGTGTGAGC TAGCTGGGACGTGGTATGGT 200 No SSLP
D4Mon58 G67764 TGCACTGACCGTGATAGAGG CGGTGTAGCTCTGGCTGTCT 200 No SSCP
D4Mon59 G67765 CATCTCACCAACTCGCACTT TTTCTGGGAACAAAGAGGCTA 418 No Both
D4Mon60 G67766 GAACCCAAGTGTTGGGGTAA TGGAAGCCCATCTGTCTCTT 222 Yes Both
D4Mon33 G67748 CAGCAGAGGTGATGGGTTCT TTGTCACACAGTGGTTAAATGC 203 No SSCP
D4Mon62 G67767 GGTGTGCACCACCATATTCA GGGAATTATCAGCCAAAAAGC 201 Yes Both
D4Mon34 G67749 TAGAACCGTGGCTGAGGACT CCGTAAGATATGAAAGAACTTGGA 201 No SSCP
D4Mon63 G67768 GCCCAACTGAAAGCTCAACT GGAAGGGGGATAACAATTGAA 263 Yes Both
D4Mon64 G67769 TGCTAATTTCAAGCACAGTGAGA AGCTTGACACCTTGACAGCA 369 Yes SSCP
D4Mon36 G67750 CCTTCCTCGTCTGAGCTGTT TTGGGACGTGACCTGAGAAT 232 Yes SSCP
D4Mon66 G67770 TTCAATTGAGTTTCTCTCCTCTGA TGCAGGACCAAGAAGTAGGC 200 Yes SSCP
D4Mon37 G67751 TATGTGTCTGGCCGTTGTTC GATGTGGGTGCAGGTGAAG 206 No SSCP
D4Mon39 G67752 GCTGAGCAGCCTCTAGCAA ACCATGGCTTTTCCCAGTAA 241 No SSCP
139J18-T7 AZ260477 ACCACTTGGGGGCTACTTCT GCTGATCCCCCTGTGATTTA 232 Yes SSCP
D4Mon40 G67753 CTGTGCCTTTGGTGATCAGA TGTGGCACTCTACGGCATAA 261 No Both
D4Mon1 G67772 GCAGTGAGCTGCAGAGTTTG AGGCCTACCCAAGGACATCT 201 Yes SSCP
417B22-SP6 AZ061836 AAACAGGCATGAAACTCAGGA GGGTATCATTGTCACCTCCA 116 No SSCP
227G4-T7 N/A GGAAGCAAATGCTCCACTAAA TATCCCTAGCCCCTTGTGTG 243 No SSCP
D4Mon28 G67746 GACCTTTGGAAGAGCAGTCG TGGCAGCTCACAATGTCTTT 296 No SSLP
D4Mon27 G67745 TGCCTTTTTCTCACATTGTCTC TTAGAAGCAGAGGCAGAGGC 250 No SSLP
D4Mon26 G67744 TCAGACATCTCTGGCCTCCT TTCACTAAGTTGCCCAGGCT 160 No SSLP
284D21-T7 AQ977902 GGTTTGGGAGTGTTAGGCAA ACTCAGTTGGCCTCTCCTCA 138 No SSCP
280G12-SP6 AZ001739 GCCCCATAAAATCCACTCCT GCTCCGGAAGGCTAGAAGAT 233 Yes SSCP
387F5-T7 AQ998226 GCCCCATAAAATCCACTCCT TTGCCTAACACTCCCAAACC 214 Yes SSCP
49O2-SP6 AZ253245 GATAGTCCCTTAGCCAGCCC GCCATAGCTCCTCACTGCTC 218 No SSCP
338N4-SP6 AQ998697 CAGTTTAGCACCCCACCCTA TCTGCACCTCTGTTCACCTG 115 No SSCP
130A12-SP6 AZ266745 CAGTTAGCACCCCACCCTAA TCTGCACCTCTGTTCACCTG 114 Yes SSCP
D4Mon25 G67743 GTTTCACATGTTGTGGTGGC GGGACCTTTGGGATAGCATT 131 No SSLP
Tnfrsf4 NM 011659 AGGCAGAAAGCAGACAAGGA CGACAGCACTTGTGACCACT 138 Yes SSCP
D4Mon24 G67742 CAGCAGCAAATGACCTTTCA GAGGCAGGCAGATTTCTGAG 147 No SSLP
D4Mon22 G67741 GTTGGGGCTGCTCATAGAAA GCTGTGGCTCTCTTGGAGTT 422 Yes Both
D4Mon21 G67740 CAGTTCTTCCCGAAAACCAC TTTCTGGGAACTGAGATGGC 174 No Both
A1225779 AI225779 GCTGTTAGTGAGGTCAGGGC CGTAGGTGGCACAGTTGAGA 104 Yes SSCP
D4Mon20 G67739 TTAGCGTTAGGGTGAGGGTG GGAGACTACGGACTTGTGGC 150 No Both
D4Mon19 G67738 GGAAGGTAGGGCCTGGTAAT GCTCCAAGATCTGTGCGATT 277 No Both
D4Mon18 G67737 GCTCAGCCCCTGAATCAATA GGGATCTGCCTGTCTTACCA 111 No Both
D4Mon17 G67736 GCTCAAGGAAGGACACACCT TGCTCTTAACATTTTGAGCCAT 201 No SSLP
D4Mon16 G67735 ATCAGACAGCCCACAACCTC TATGTGCCACCACACCTGTC 206 No SSLP
D4Mon68 G67771 ACATGTCCACTGTGGCAAAA TGTCATGAGTTTGAGGCCAG 246 No SSLP
D4Mon2 G67773 TGATCTTTCCAAACGCATAAGA AGACACCCTAGGTCCTGCTG 151 No SSLP
D4Mon15 G67734 TGTTCCTGAGTTCACAACGC ATTCCCAGCAACTACATGGC 269 No SSLP
D4Mon9 G67779 GGACAGGTAGCTCACCCAAC CCAAGAGTCAGCCTTGGAGT 200 No SSLP
D4Mon14 G67733 TACTTCCCCTTTCCCGAACT TCCTTGGTGCTTACCCTCAC 232 Yes SSLP
D4Mon13 G67732 CTAGGGGACTCTGCCAAGTG CAAGACACCCAGTCCCAACT 195 No SSLP
D4Mon12 G67781 GCAAGTTTCAGGAGCTAGGG CCCCAGAACCAGAGACCATA 166 Yes SSLP
D4Mon11 G67780 AGCCAGGGCTACACAGAGAA ACCCTGCTACAACGCAGACT 153 No SSLP
118E21-T7 AZ274183 CCCAGAACTCCATCCTCAAA CCCAACCTGTGGTCAGCTAT 185 No SSCP
382A8-SP6 AZ018016 AGCAGGCACAGGTCTCTTGT AAGAACAGGACAGTGGTGGG 202 No SSCP
139J18-T7 AZ260480 TCAGTTACCAAGGGTTTCGG ATAGGTTGTCACAGGCCAGG 122 Yes SSCP
Agrn M92654 TGTGACTTCCTCTTCCCCAC TGAGCCACTCCAGATGTCAG 156 No SSCP
28.MMHAP7FLB4.seq M92657 CACTAGAGCTGCCACCTTCC CCCTCAGCACCACTTTTTGT 162 No SSCP
K00231 AA410003 GTGCTCTGCAGACAAACCAA GAGCCATTTTGACCCTTAAA 154 Yes SSCP
V2r2 AF053986 CAATTGAGGAATGGCTACCAA TGGCTTCATGTCCATTGTGT 170 Yes SSCP
238O5-T7 NA TATAAGCAGCCCCTCATTGG CAGGCCAGACACTGCTTACA 244 Yes SSCP
D4Ertd296e C80036 AGGCATATTGTATAATAAATTTGTAGT CCGGATGACTCTACTTGAC 201 Yes SSCP
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a

Accession numbers beginning with MGI are from the Mouse Genome Database and all other Accession numbers are from GenBank.

b

Product size is obtained from B6 genomic DNA.

c

Un = Unknown, not attempted.

d

N/A = not available.

Methods of detection: SSLP = simple sequence length polymorphisms; SSCP = single-strand conformation polymorphisms. Several markers were within known genes, e.g., R74924 (Pkcz); D18402 (Mmp23); U37351 (Pcee) and 28.MMHAP7FLB4.seq (Agrn). The STSs are ordered from the centromeric end to the telomeric end.

Genetic mapping and linkage analysis

Mouse genomic DNA was purified from tails by NaOH/Tris (Truett et al. 2000), or phenol/chloroform extraction. Markers that differed in length of the PCR product (simple sequence length polymorphism; SSLP) were tested by using a standard protocol (Dietrich et al. 1992), with minor modifications (Bachmanov et al. 1997; Li et al. 2001). Other markers were tested by using a single-strand conformation polymorphism (SSCP) protocol (Orita et al. 1989), or by sequencing the PCR products of B6 and 129 DNA, to detect single nucleotide or small insertion/deletion polymorphisms. Linkage analysis and construction of the genetic map were conducted by using MAPMAKER/EXP 1.1 (Lander et al. 1987).

Construction of a BAC contig

The RPCI-23 female (C57BL/6J) mouse BAC library (Osoegawa et al. 2000) was used for library screening. All probes were radioactively labeled by the random hexa-nucleotide method (Feinberg and Vogelstein 1983). Hybridization and washing of membranes followed standard protocols (Church and Gilbert 1984). The library was screened twice, first with a probe generated from YAC 178B3, and second, with pooled probes of markers on the distal end of Chr 4.

To determine the BAC clones corresponding to each probe, positive clones identified by the initial screenings were picked into 384-well microtiter plates and regridded onto 8 × 12 cm Hybond-N+ membranes (Amersham Pharmacia Biotech, Piscataway, NJ) by using a Biogrid robot (Biorobotics, Haslingfield, Cambridge, UK). Each filter was then hybridized against individual probes, under the same conditions as the primary screening. The secondary screening results were confirmed by PCR STS-content mapping, using overnight cell suspension as templates. BAC end STS markers were used to confirm the BAC physical map and identify polymorphisms.

To determine the size of individual BAC clones, we extracted the DNA using a modified alkaline lysis procedure (Sambrook et al. 1989) and analyzed after digestion with NotI (New England Biolabs, Beverley, Mass.) by pulse-field gel electrophoresis (PFG), performed on a Chef Mapper (Bio-Rad, Hercules, Calif.). Samples were run for 16 h on 1% agarose gels using 0.5× TBE buffer, at 14°C; the settings were 200 V (6V/cm) and a linear pulse time ramp from 0.3 to 33 s with a pulse angle of 120°. The gels were subsequently stained with ethidium bromide for photography. Approximate insert fragment sizes were determined by comparison with the low-range PFG size markers (New England Biolabs). Finally, the BAC contig was assembled by using SEGMAP, version 3.48 (University of Washington, Seattle, Wash.).

BAC ends sequencing and STS development

BAC DNA for sequencing was prepared from 200-ml overnight cultures, according to the modified protocol for BACs by using P100 midi-prep columns (Qiagen, Inc., Valencia, Calif.). Unique sequences from BAC ends were identified by analysis with Repeatmasker (Smit and Green 2000), and used to design PCR primers for genotyping F2 animals and to screen BAC clones to refine STS content mappling.

BAC DNA sequencing and analysis

BAC 118E21 was isolated by using an alkaline lysis protocol, followed by CsCl density gradient centrifugation. Following ethanol precipitation, the CsCl-purified DNA was resuspended at 15 ng/μl in TE. Two hundred microliter of this solution was sonicated for 2 s, with a Misonix XL2015 ultrasonicator (Farmingdale, New York). The sheared DNA was end-filled with T4 DNA polymerase, cloned into pUC18, and cut with SmaI. Twenty-five hundred transformants were grown, plasmid DNA was isolated, sequenced forward and reverse with Big Dye Terminators (PE Biosystems, Foster City, Calif.), and data were collected on an ABI3700 DNA sequencer. Sequence data were processed and assembled with the Phred, Phrap, and Consed package of programs on an SGI 02 workstation.

After BAC 118E21 was sequenced, the STS content of this BAC and overlapping BACs was confirmed by aligning the STS and BAC end sequences with the 118E21 sequence (Sequencher, Gene Codes Corporation, Ann Arbor, Mich.). Another genomic clone, AF185591, overlapped with 118E21, and their sequences were assembled by Sequencher. Repeat sequences were identified with RepeatMasker. Genscan (Burge and Karlin 1998) was used to predict gene content and intron/exon organization. The predicted proteins were submitted to a tBLASTn search through the nr and the mouse EST database at NCBI.

For identifying genes through their similarity to ESTs or gene sequences, portions of the 284-kb genomic sequence were submitted to iterative BLAST searches to find matching genes and ESTs, and the cDNAs were aligned with genomic sequence to identify intron/exon organization.

Analysis of gene expression and detection of SNPs

Primers were designed to span intron/exon junctions suggested by Genscan (Tables 4 and 5) and were used to amplify genomic and tongue cDNA. Genomic DNA was either extracted as described above or purchased from The Jackson Laboratory. Tongue cDNA was prepared as follows: total RNA was extracted by using TRIZol Reagent (Life Technologies Inc., Rockville, Md.) from enzymatically separated mouse lingual epithelium (Ruiz et al. 1995; Spielman and Brand 1995), which included fungiform, foliate, and circumvallate taste papillae. The RNA was reverse-transcribed (Superscript reverse transcriptase, Life Technologies). The cDNA samples were amplified by using AmpliTaq DNA Polymerase with GeneAmp (Perkin Elmer Corporation, Branchburg, N.J.). Single bands of expected sizes were excised from the gel, purified, and sequenced to determine the intron/exon junctions. The resulting sequence was used to confirm that the product was from the expected sequence, and to find polymorphism between strains. Sequences were aligned and polymorphisms were identified with Sequencher.

Table 4.

EST or cDNA matches and exon/intron junctions of genes in the distal region of Chr 4.

Gene/EST Exon/CD Exon Position Begin-End Exon length (bp) Detected by Genscan Intron length (bp) Acceptor site (intron/EXON) Donor site (EXON/intron)
BG176077
 E1 22670–23413 744 No N/A N/A N/A
AA546397
 E1 23756–24249 494 No N/A N/A N/A
AI428898
 E1 26304–26696 393 No N/A N/A N/A
Pcee
 E1a 26817–26832 16 No N/A N/A N/A
 E1b (CD1) 26833–27114 282 No N/A N/A CTGCCGCAGgtaagggcc
 E2 (CD2) 27460–27534 75 Yes 345 tgtccctagGTGGCCATG TCCATGGAGgtaggcttc
 E3 (CD3) 27750–27859 110 Yes 215 acatttcagCATGTGTCC AGAGAAAAAgtgagtcct
 E4 (CD4) 29533–29653 121 Yes 1673 tccctgcagGAAGCCTGT CCTCACAAGgtgggtttg
 E5 (CD5) 32160–32224 65 Yes 2506 atcttccagATAATCGTT GACTGCATGgtgagttga
 E6 (CD6) 34565–34664 100 Yes 2340 tggtttcagGAACTACAT ACACTGGAGgtaagtatg
 E7 (CD7) 35017–35121 105 No 352 tgttcccagATCCCTTTA CGGAAAAAGgttcttttg
 E8 (CD8) 35206–35347 142 Yes 84 ggttctcagGTTGATTTG CCAAGCTGGgtgagtgtc
 E9 (CD9) 36039–36159 121 No 691 gattcagtaGAATCCCCA TGTAAATGGgtaaggtgc
 E10 (CD10) 36243–36336 94 No 83 ctgtttgcaGCTTGCTGA AAAGAGAAGgtctgtgtc
 E11a (CD11) 37631–37985 355 No 1294 tctctacagGAAAAGTGA 33UTR after stop codon
 E11b 37986–38638 653 No N/A 33UTR 33UTR
NM 025338
 E1 45939–46028 90 No N/A N/A GTCTGTTTGgtgcgtgcg
 E2a 46276–46308 33 No 247 gtgttctagCTTTGAGCG N/A
 E2b (CD1) 46309–46363 55 No N/A N/A CCTGGGCAGgtaagacgc
 E3 (CD2) 46645–47090 446 Yes 281 tttccccagGTTTCAGTC AAGAAGCAGgtgaatatg
 E4 (DC3) 47184–47350 167 No 93 cccttgcagATCAAATTT N/A
AK004732
 E1a 54090–54134 45 No N/A N/A N/A
 E1b (CD1) 54135–54283 149 Yes N/A N/A TTCTTCAGAgtgagtgtg
 E2 (CD2) 55071–55091 21 Yes 787 ttcttccagGCTCTGCAG TGTCCTCAGgtcagtatc
 E3 (CD3) 55199–55501 303 Yes 107 cccgacaagGGCCTTCAG TCATTCGCGgtacactgt
 E4 (CD4) 55738–55839 102 Yes 236 gccaagcagCTGTGGACA CAGAGGATCgtgagtgtg
 E5 (CD5) 55955–56425 471 Yes 115 catccgcagCCCTATTAA CTAGCCCAGgtgtgtgtg
 E6 (CD6) 56846–57004 159 Yes 420 tacatctagATCCCACCC ACAGCGGAGgtgaggctg
 E7 (CD7) 57082–57121 40 Yes 77 tcttcacagGATGCAAGA GTCAAAGGGgtgagtttg
 E8 (CD8) 57194–57270 77 Yes 72 tccctttagGAAGGATGT TCCAGCTAGgtaggggtg
 E9 (CD9) 57348–57428 81 Yes 77 cccaaccagATTACAAAA TGGATAAAGgtgagcact
 E10a (CD10) 57594–57619 26 No 165 ggcttccagAGTTCAGGA 33UTR after stop codon
 E10b 57620–58382 763 No N/A 33UTR 33UTR
Dvl
 Promoter 59138–59177 40 No N/A N/A N/A
 E1 (CD1) 62060–62229 170 No N/A N/A GGACTTCGGgtcagtggg
 E2 (CD2) 67743–67812 70 Yes 5513 tgaccacagGGTGGTGAA GTTTCCTGGgtgagtcta
 E3 (CD3) 67962–68083 122 Yes 149 ccttcacagCTGGTCCTG CTCCTTCCAgtaaggccc
 E4 (CD4) 68308–68411 104 Yes 224 tctatgcagTCCAAATG GCGATGAGGgtactatgg
 E5 (CD5) 68655–68793 139 Yes 243 tccccacagCTGCCCGG TACGAGCCGgtgggtgac
 E6 (CD6) 68891–68984 94 Yes 97 ctgcctcagGCTGAGCAG ACAGACCGGgtaggcagc
 E7 (CD7) 69056–69125 70 Yes 71 cctccgcagGCATCCTCC CTCTCAACAtgggtgagg
 E8 (CD8) 69231–69350 120 Yes 105 ctcctgcagAGAGGCACC TTGCTGCAGgtgggagcc
 E9 (CD9) 69429–69505 77 Yes 78 accccgtagGTGAACGAT CCAGACAGGgtgaggtgg
 E10 (CD10) 69597–69664 68 Yes 91 actctgtagGCCCATCAG TCCCAAGGGgtgagttac
 E11 (CD11) 69804–69944 141 Yes 139 cccacacagCAGCACTGA GCGCCCCACgtaagtggc
 E12 (CD12) 70457–70588 132 Yes 512 gctctgcagAGCTTGAGG CTGTCATTGgtgagtccc
 E13 (CD13) 70687–70854 168 Yes 98 tgtgcccagGGGCGGATG TGTGCAGTAgtgagtagg
 E14 (CD14) 70939–71145 207 Yes 84 tcctgccagACCTCGCAT AGAGTGAAGgtgagaggt
 E15 (CD15) 72318–72691 374 Yes 1172 tccccctagGGAGCAAGA 33UTR after stop codon
Tas1r3
 CD1 77659–77469 191 Yes UN 53UTR GTGCAACAG/gtatggagg
 CD2 77360–77060 301 Yes 108 tggcctcag/GTTCTCACC ATGCCACAG/gtgagccca
 CD3 76979–76176 804 Yes 80 tcaccacag/GTCAGCATA CCCTGGCAG/gtaagggta
 CD4 76064–75867 198 Yes 111 actccacag/CTCCTGGAG GGCAACCAG/gtaaggaca
 CD5 75768–75648 121 Yes 98 catgtacag/GTGCCAGTC AGCATCCAG/gtgaaccgt
 CD6 75472–74511 962 No 175 tcactacag/ATGACTTCA 33UTR after stop codon
NM_024472
 E1a 81628–81560 69 No N/A N/A N/A
 E1b (CD1) 81628–81438 122 Yes N/A N/A ACTTGTCAGgtgcgtcta
 CD2 81210–79608 1603 No 227 ctttcgcagGTTTCTAAA N/A
AK010425
 E1a 83931–84017 87 No N/A N/A N/A
 E1b (CD1) 84018–84045 28 No N/A N/A CTCCCTTGGgtgagtccg
 E2 (CD2) 86487–86584 98 Yes 2421 tttgagtagGGGCTGGCC CAATGATGACgtgagtact
 E3 (CD3) 87210–87283 74 Yes 625 tctcatcagAGGCGCTTT GATCATCAGgtaggttgt
 E4 (CD4) 89498–89726 229 Yes 2214 ttcccccagCCACTTCCA ACAGTTCAGgtcaggatc
 E5 (CD5) 99458–99556 99 No 9731 cctctgcagGTGGATGAT GTCTACACGgtcagtgga
 E6 (CD6) 99647–99681 35 No 90 tttggccagGGTGACTAT GCATTTGGGgtaagtagg
 E7 (CD7) 99925–100063 139 Yes 243 ctgttctagGGCTGCGTG GGAGGAAAGgtattgact
 E8 (CD8) 100452–100516 65 Yes 388 cgctcccagGTGCTGATT GACCTTCTGgtaggtgca
 E9 (CD9) 101158–101347 190 Yes 641 gacacacagGGAGCGCAT GGTCCCATGgtaaggccc
 E10 (CD10) 101476–101559 84 Yes 128 accctgcagGTTGTGTTT AAGAACATGgtaagggtt
 E11 (CD11) 101792–101881 90 Yes 232 tgcttgcagGTCATCATG CGCCAGATGgtgagtgcc
 E12 (CD12) 101965–102127 163 Yes 83 atgccacagCTGGAGGTA AGGAATTCCgtaggcagc
 E13 (CD13) 102215–102322 108 Yes 87 tttcctcagGGGTCAGCT TGGTGCAGGgtatgaagg
 E14 (CD14) 102408–102469 62 Yes 85 tcctcacagGACTGTTGC AAAGACAGTgtaagtgta
 E15 (CD15) 102544–102686 143 No 74 gccccacagAATTTCCGG TCTCAAGAGgtgcgccct
 E16 (CD16) 102756–102885 130 Yes 69 tctccttagCACCCTCAA ACTTACCAGgtaaggagc
 E17a (CD17) 102980–103045 66 No 94 tccttacagGATGAAGAG 33UTR after stop codon
 E17b 103046–103228 183 No N/A 33UTR 33UTR
AA435261
 E1 105481–105412 70 No N/A N/A GGCCATTCGgtgagcccg
 E2 105262–105113 150 Yes 149 ttgccacagTGTACTGAA CAGATCTGCgtgagctaa
 E3 105029–104802 228 No 83 gctctgcagGTACCTGGA N/A
KIAA1716-like
 E1 (CD1) 110459–110528 70 No N/A N/A CTGGACAAGgtaagagac
 E2 (CD2) 110927–111046 120 Yes 398 cacttgtagCTGGTCAAA GTCATCTCGgtgaggcac
 E3 (CD3) 111244–111297 54 Yes 197 tttcttaagGAATGTCTG TATCACACGgtgagcccc
 E4 (CD4) 113735–113793 59 Yes 2437 ctatcccagATCCTGTTT GTCAAAGAgtaagtgac
 E5 (CD5) 114281–114464 184 Yes 487 ggcacccagGGATGTGCG GTGCTGCAGgtcagctgc
 E6 (CD6) 114823–114867 45 Yes 358 ctctggcagATCAATGTC TTAGATTCTgtgagtgct
 E7 (CD7) 114953–115048 96 Yes 85 cactcgcagATGCTGTCC GCAGCTGAGgtgagtcta
 E8 (CD8) 115151–115225 75 Yes 102 tccccacagTTGGACCAG CAGCAACGGgtgaggccc
 E9 (CD9) 116539–116651 113 Yes 1313 cttcctcagGACTTTTCC ATGGAACCGgtaagggcc
 E10 (CD10) 116740–116791 52 Yes 88 ctcactcagACGGTGGTT AAACTCAAGgtattctgg
 E11 (CD11) 116996–117096 101 Yes 204 tgcccacagGATGCACTC ACCTACCAAgtgaggagg
 E12 (CD12) 117169–117280 112 Yes 72 tccctacagGAGCTGTAT TACAGTGAGgtgcactgc
 E13 (CD13) 117522–117730 209 Yes 241 atccaccagAGGCTGGAC CATCCACAGgtagtcagt
 E14 (CD14) 117824–117893 70 Yes 93 gtctaggagCTTGGGCGT CTGCTAAAGgtgtgtgag
 E15 (CD15) 118003–118097 95 Yes 109 gccccgcagCTGATGTGT CAGTTCCAGgtgaggttg
 E16 (CD16) 118175–118377 203 Yes 77 ctggtccagGCAGGACAA TGTCCTCAGgtgggaggg
 E17 (CD17) 118615–118722 108 Yes 237 cccttgcagCTGCTACTA GTCCACGCAgtgagtgct
 E18 (CD18) 119877–120097 221 Yes 1154 ctcccacagAGGGTGCCG GTACTAGGGgtgagcaca
 E19 (CD19) 120274–120383 110 No 176 gctgtccagGGTTCCTTG CCGTACCGGgtgagttct
 E20 (CD20) 120476–120582 107 Yes 92 cccagccagGTCTGTCTG TGTCACATTgtgagtctg
 E21a (CD21) 120677–120825 149 No 94 caaccccagGCTTCGCCT 33UTR after stop codon
 E21b 120826–120847 22 No N/A 33UTR 33UTR
AA967911
 E1 116539–116651 113 Yes N/A N/A ATGGAACCGgtaagggcc
 E2 116740–116791 52 Yes 88 ctcactcagACGGTGGTT AAACTCAAGgtattctgg
 E3 116996–117096 101 Yes 204 tgcccacagGATGCACTC ACCTACCAAgtgaggagg
 E4 117169–117280 112 Yes 72 tccctacagGAGCTGTAT N/A
BG243209
 E1 116996–117096 101 Yes N/A N/A ACCTACCAAgtgaggagg
 E2 117169–117280 112 Yes 72 tccctacagGAGCTGTAT TACAGTGAGgtgcactgc
 E3 117522–117730 209 Yes 241 atccaccagAGGCTGGAC CATCCACAGgtagtcagt
 E4 117824–117893 70 Yes 93 gtctaggagCTTGGGCGT CTGCTAAAGgtgtgtgag
 E5 118003–118097 95 Yes 109 gccccgcagCTGATGTGT N/A
BF181401
 E1 117824–117893 70 Yes N/A N/A CTGCTAAAGgtgtgtgag
 E2 118003–118097 95 Yes 109 gccccgcagCTGATGTGT CAGTTCCAGgtgaggttg
 E3 118175–118377 203 Yes 77 ctggtccagGCAGGACAA TGTCCTCAGgtgggaggg
 E4 118615–118722 108 Yes 237 cccttgcagCTGCTACTA GTCCACGCAgtgagtgct
 E5 119692–119782 91 No 969 gtcctacagGTCTAAGCA N/A
BF120370
 E1 120274–120383 110 No N/A N/A CCGTACCGGgtgagttct
 E2 120476–120582 107 Yes 92 cccagccagGTCTGTCTG TGTCACATTgtgagtctg
 E3 120677–121189 513 No 94 caaccccagGCTTCGCCT 33UTR after stop codon
AA547414
 E1 126572–127112 541 No N/A N/A N/A
BG085299
 E1 127208–127796 589 No N/A N/A N/A
AI550648
 E1 127744–128382 639 No N/A N/A N/A
BG070843
 E1 129539–128723 817 No N/A N/A N/A
AA763221
 E1 129440–130032 593 No N/A N/A N/A
AA218417
 E1 131044–130398 647 No N/A N/A N/A
BG066449
 E1 136781–135944 838 No N/A N/A N/A
AA199518
 E1 136706–137369 664 No N/A N/A N/A
BF140854
 E1 139653–140275 623 No N/A N/A N/A
AA823914
 E1 141472–142259 788 No N/A N/A N/A
BE956856
 E1 158678–159251 574 No N/A N/A N/A
AI530700
 E1 160031–159473 559 No N/A N/A N/A
Ubc6p
 E1 (CD1) 163829–163959 131 Yes N/A N/A TCTTGAATGgtaagacta
 E2 (CD2) 166294–166334 41 Yes 2334 ctccgttagGCATTATGT CTTATGAAGgttagtaga
 E3 (CD3) 170056–170158 103 Yes 3721 ttggtgtagGTGGCTATT CAACACAAGgtaagcttc
 E4 (CD4) 170244–170382 139 Yes 85 ttctttcagGCTGTGTCT GACTTCACGgtgaggtgt
 E5 (CD5) 171190–171270 81 Yes 807 tcttcctagAAAAAACAG GTTGTGGAGgtaagaagg
 E6a (CD6) 171903–172187 285 Yes 632 ttctctcagGAAATTAAA 33UTR after stop codon
 E6b 172188–172454 267 No N/A 33UTR 33UTR
NM_026125
 E1a 177140–177400 261 No N/A N/A N/A
 E1b (CD1) 177401–177559 159 Yes N/A N/A TCCGTCAAGgtaggcaga
 E2 (CD2) 179365–179481 117 Yes 1805 acttttcagCCCCCGGAA AAGAAGTCGgtaaggccc
 E3 (CD3) 179590–179710 121 Yes 108 ctcttttagCGAGGCCTC TACTGAAAGgtaagattg
 E4 (CD4) 179802–179953 152 Yes 91 tctccacagAGGCCACAG TTCCAAGCTgtgagtggg
 E5 (CD5) 180416–180524 109 Yes 462 tttccacagCCTACTACT TGCACGTGGgtgagccag
 E6 (CD6) 180688–180778 91 Yes 163 tacccgtagACCACAGTG TCGTCATACgtgagtccc
 E7 (CD7) 180904–180982 79 Yes 125 tatgcccagGTCCCTGGA CATCTACAGgtaagcaac
 E8a (CD8) 181226–181334 109 No 243 tgtctttagTCTGGACAG 33UTR after stop codon
 E8b 181335–181451 117 No N/A 33UTR 33UTR
AW743506
 E1 206936–206461 476 No N/A N/A N/A
Cab45a
 E1 207908–208231 324 No N/A N/A GAAGCCGGGgtgagtgag
 E2 (CD1) 211086–211415 330 No 2854 accttgcagGAGTCCCAT CTTTTCCAAgtaagtgtc
 E3 (CD2) 214029–214165 137 Yes 2613 tgctactagGGTAGACGT ACGGTGACGgtacagtgg
 E4 (CD3) 215301–215414 114 Yes 1135 tctttttagGCCATGTTT ATGAGGAGAgtgagtgct
 E5 (CD4) 223418–223576 159 Yes 8003 ctcttgcagCACAGGAAG GGGACTTGGgtaagacct
 E6 (CD5) 223775–223950 176 Yes 198 ttatctgcagATCAGGATG GAGCTAGAGgtgagcaag
 E7a (CD6) 224405–224581 177 Yes 454 gtctcacagAACTACATG 33UTR after stop codon
 E7b 224582–225468 887 No 33UTR 33UTR
Tnfrsf4
 E1a 228399–228577 179 No N/A N/A N/A
 E1b (CD1) 228578–228710 133 Yes N/A N/A GCCAGCCAGgtgagtagc
 E2 (CD2) 228932–229054 123 Yes 221 ccactacagGCCATGGTA GCAACCATCgtgagtcc
 E3 (CD3) 229606–229713 108 Yes 551 cttccctagGAAGTGGAA TTGGAGTTGgtgagcctc
 E4 (CD4) 230090–230156 67 No 376 ttgctgcagACTGTGTTC CTGGACCAAgtgagtact
 E5 (CD5) 230582–230778 197 Yes 425 gtcccacagTTGTACCTT CTCCTGAGGgtaagggac
 E6 (CD6) 230858–230977 120 No 79 tcctcatagGCCCTGCAT AACCTTGTTgtgagtatc
 E7a (CD7) 231066–231136 71 Yes 88 ttctcttagGGGGAAACA 33UTR after stop codon
 E7b 231137–231293 157 No N/A 33UTR 33UTR
AW544835
 E1 235140–235698 559 No N/A N/A N/A
Tnfrsf18
 E1a 241046–241090 45 No N/A N/A N/A
 E1b (CD1) 241091–241241 151 Yes N/A N/A ATGCTCCAGgtaaggcaa
 E2 (CD2) 242020–242142 123 Yes 778 cttgtccagGCAAGGAGG AGTCTCAAGgtatgttcc
 E3 (CD3) 242666–242753 88 Yes 523 tatccacagGGGATATTG TTGGACCAAgtaagtcct
 E4 (CD4) 242925–243127 203 Yes 171 gtgtcccagCTGTTCTCA GTCCTCGAGgtcagttgt
 E5a (CD5) 243199–243320 122 Yes 71 ttactacagAGACCCAGC 33UTR after stop codon
 E5b 243321–243593 273 No N/A 33UTR 33UTR
AI225779
 E1 248278–247905 374 No N/A N/A N/A
BF319334
 E1 249905–249544 362 No N/A N/A N/A
AI132502
 E1 259999–259859 141 No N/A N/A ACCAATCAGgtaagaacc
 E2 259420–259280 141 Yes 438 gtgttgtagTTCATGCAG ACCTTCACGgtcagtcga
 E3 252725–252550 176 No 6554 attttacagGGCTGGTGA N/A
AI595135
 E1 264205–264135 71 No N/A N/A TTCCTACTGgtaaggagg
 E2 263876–263796 81 Yes 256 ggtcttcagGCTGGACCC AAGATGCAGgtggggctt
 E3 263715–263583 133 Yes 80 tatccacagACGACCTGG AGCATCAATgtgagtaga
 E4 263438–263320 119 Yes 144 accctgcagAATCAGCAA TTCGGGAAGgtaggagac
 E5 263243–263020 224 No 76 tctgcccagGCCAGCAGT CCAAACCAAgtgagccca
 E6 261643–261605 39 No 1376 tctcccaagGCTGGACCC N/A
AK014686
 E1 275577–275229 349 No N/A N/A GCCTCAGAGgtggggtgca
 E2 266501–266434 68 No 8727 cttcttcagGTTTAAACG TCTGATCAGgtaaacatg
 E3 264937–264525 413 No 1496 tctttgcagCAGACACAG AGGTGCCAGgtgaatgca
 E4 263715–263583 133 Yes 809 tatccacagACGACCTGG AGCATCAATgtgagtaga
 E5 263438–263320 119 Yes 144 accctgcagAATCAGCAA TTCGGGAAGgtaggagac
 E6 263243–263117 127 Yes 76 tctcgccagGCCAGCAGT CCAAACCAAgtgagccca
 E7 261643–261558 86 Yes 1473 tctcccaagGCTGGACCC AAGATGCAGgtgagaagc
 E8 259420–258537 884 No 2137 gtgttgtagTTCATGCAG N/A
AI425800
 E1 275441–275352 90 No N/A N/A TTAGAGCAGgtgggttca
 E2 266501–266434 68 No 8850 cttcttcagGTTTAAACG TCTGATCAGgtaaacatg
 E3 264937–264618 320 No 1496 tctttgcagCAGACACAG GTCCAAGATgtccaagat
 E4 260993–260952 42 No 3624 taccgcaagATGCCCTTC GGTGCAGAGgtgtgggcg
 E5 260030–259995 36 No 921 accccctagATATGTCCA N/A
AW987045
 E1 270132–269542 591 No N/A N/A N/A
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Positions are given from the start of the 284-kb sequence, with the first base pair = +1. E = Exon, CD = Coding region, UN = Unknown, N/A = Not Applicable. The region of 1–40143 bp is from a genomic sequence identified as Cyclin ania 6b (Accession #AF185591), but because only a small part of mRNA for Cyclin ania 6b is known (Accession #AF246633), and because AF246633 is almost fully overlapped by Pcee, we denote this sequence as Pcee. For most of the KIAA1716-like gene, no mouse EST or cDNA was available, but the human sequence was a close match to the mouse genomic DNA, so the intron-exon structure of the human KIAA1716 was used to show the intron-exon structure in its mouse ortholog. This was true for all but the first exon, which was not available in human, but there was a mouse EST, which was used to predict the boundary. When there was a Unigene cluster associated with a given EST, the longest EST within the cluster was used to align genomic and cNDA sequence (BG176077 = Mm.23825, AI428898 = Mm.23492, AK004732 = Mm.28978, BF 140854 = Mm.172945, AW544835 = Mm.102764, AI595135 = Mm.159514). Several ESTs, shown above, group with Unigene clusters and therefore are likely to represent the same gene. These instances are as follows: AA547414, BG085299 and AI550648 are part of Unigene cluster Mm.56803; BG070843, AA823914 and AA763221 are part of Mm.182484; BG066449 and AA199518 are part of Mm.133906; BE956856 and AI530700 are part of Mm.322248, and AI132502, AK014686 and AI425800 are part of Mm.42006. Gene and cNDA names follow the rules outlined in Figs. 2 and 3.

Table 5.

Polymorphisms between the 129 and B6 strains for genes on mouse distal Chr 4

Gene or EST Source P# Forward Primer Reverse Primer Sizea (bp) Pos (bp) Nt B6 Nt 129 AA B6 AA 129
Pcee CDNA 1 GCCTCACCGATCGCTATT GGGCCTTCGTACACTTAACG 132 N/A Iden Iden Iden Iden
Pcee CDNA 2 TACAGCTGCCTCCCAGGAAC ACCTTCAGGCTCCTTTCCAT 205 66 A A or G Q Q or R
Pcee CDNA 2 TACAGCTGCCTCCCAGGAAC ACCTTCAGGCTCCTTTCCAT 205 69 T T or C V V or A
Pcee CDNA 2 TACAGCTGCCTCCCAGGAAC ACCTTCAGGCTCCTTTCCAT 205 77 A A or G N N or D
Pcee Gen 3 ATCGGCGACAGGCTCTACT CTCCATGGAATGTTTCACGA 577 103 A A or C N/A N/A
Pcee Gen 3 ATCGGCGACAGGCTCTACT CTCCATGGAATGTTTCACGA 577 179 A or G A N/A N/A
Pcee Gen 3 ATCGGCGACAGGCTCTACT CTCCATGGAATGTTTCACGA 577 428 A or DEL A or DEL N/A N/A
NM_025338 CDNA 1 GGGGTGATTGGCAGCTATG GGTAGATCTTCGGGGTCTGC 508 95 C T S L
NM_025338 CDNA 1 GGGGTGATTGGCAGCTATG GGTAGATCTTCGGGGTCTGC 508 344 A A or G H H or R
NM_025338 CDNA 1 GGGGTGATTGGCAGCTATG GGTAGATCTTCGGGGTCTGC 508 392 T or A T D or V V
AK004732 CDNA 1 GCCGAGTCTTCCACCTACAG CAGCAAGATGAAGAGCAGCA 215 127 T T or C I I or T
AK004732 CDNA 1 GCCGAGTCTTCCACCTACAG CAGCAAGATGAAGAGCAGCA 215 182 T C Y Y
Dvl1 CDNA 1 GATGTGGTGGACTGGCTGTA GAAGCCAGGGTCCTGGTAAG 330 73 A A or G S S or G
Dvl1 CDNA 1 GATGTGGTGGACTGGCTGTA GAAGCCAGGGTCCTGGTAAG 330 306 G A P P
Tas1r3 Gen 1 CACCCATTGTTAGTGCTGGA ACGGGGTTGGTACTGTGTGT 553 169 A G S S
Tas1r3 Gen 1 CACCCATTGTTAGTGCTGGA ACGGGGTTGGTACTGTGTGT 553 197 A A or G T T or A
Tas1r3 Gen 1 CACCCATTGTTAGTGCTGGA ACGGGGTTGGTACTGTGTGT 553 213 T C I T
Tas1r3 Gen 1 CACCCATTGTTAGTGCTGGA ACGGGGTTGGTACTGTGTGT 553 216 C T P L
Tas1r3 Gen 2 CGGCTGGGCTATGACCTAT TGCATTGGCCAGACTAGAAA 476 265 T C N/A N/A
Tas1r3 Gen 2 CGGCTGGGCTATGACCTAT TGCATTGGCCAGACTAGAAA 476 306 T C S S
Tas1r3 Gen 3 GGCAGTTGTGACTCTGTTGC CACTCCTCCTGCTGCTTTG 1018 589 A C N/A N/A
NM_024472 CDNA 1 GGATGACTCAGAGAAAGATTTCAA GGTCTAGCAGGGAATGCTCA 641 142 C T A A
NM_024472 CDNA 1 GGATGACTCAGAGAAAGATTTCAA GGTCTAGCAGGGAATGCTCA 641 227 G A A T
NM_024472 CDNA 1 GGATGACTCAGAGAAAGATTTCAA GGTCTAGCAGGGAATGCTCA 641 373 C T D D
NM_024472 CDNA 1 GGATGACTCAGAGAAAGATTTCAA GGTCTAGCAGGGAATGCTCA 641 379 G A L L
AK010425 CDNA 1 AGGTGGATGATGAGCTGGAA CTCTCTGCAGCGTTTGGAAT 228 N/A Iden Iden Iden Iden
AK010425 Gen 1 AGGTGGATGATGAGCTGGAA CTCTCTGCAGCGTTTGGAAT 560 174 T G N/A N/A
AK010425 Gen 1 AGGTGGATGATGAGCTGGAA CTCTCTGCAGCGTTTGGAAT 560 177 TTT DEL N/A N/A
AK010425 Gen 1 AGGTGGATGATGAGCTGGAA CTCTCTGCAGCGTTTGGAAT 560 180 A DEL N/A N/A
AK010425 Gen 1 AGGTGGATGATGAGCTGGAA CTCTCTGCAGCGTTTGGAAT 560 181 TTTT DEL N/A N/A
AK010425 Gen 1 AGGTGGATGATGAGCTGGAA CTCTCTGCAGCGTTTGGAAT 560 490 A or G G V V
AK010425 Gen 2 AACTTCGCTTCACCTGTCGT TCCACAGTCACAGAGCCATC 208 N/A Iden Iden Iden Iden
AK010425 CDNA 2 AACTTCGCTTCACCTGTCGT TCCACAGTCACAGAGCCATC 137 N/A Iden Iden Iden Iden
AA435261 Gen 1 AAATCATGTGTCCCGAGGAG CTGCTCCTGGCCTAATCAAC 206 N/A Iden Iden Iden Iden
KIAA1716-like Gen 1 CATGTGAGGACATTGAACGG GAGTCAGCCTGCAGCATACA 148 N/A Iden Iden Iden Iden
NM_026125 CDNA 1 TACTACTCAGGGCGCCTTCC CGTATGACGATGACACAAGGA 198 N/A Iden Iden Iden Iden
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Gen = genomic DNA. Pos = the nucleotide number, where the first nucleotide of the Forward primer is considered nucleotide 1. Iden = B6 and 129 DNA do not differ. N/A = not applicable; because nucleotide substitution is within an intron, there is no amino acid substitution. The 129 strain has many within strain polymorphism, e.g. A to A or G, means that the nucleotide is A for the B6 strain, and it is polymorphic (A or G) within the 129 strain.

a

Size in cDNA is the size suggested by aligning an EST with genomic DNA; where no EST was available, the Genscan prediction was used.

#

P = primer pair.

Comparative analysis of mouse and human sequences

To compare the mouse and human sequences over the entire contig, we used BLAST searches to identify conserved genes between mouse and human. Homologous human genes were identified and used as queries against the human htgs and gss databases. BAC or PAC clones positive for these human sequences were scanned by electronic PCR (http://www.ncbi.nlm.nih.gov/genome/sts/epcr.cgi) to determine the map location. Those BACs or PACs that mapped to human Chr 1p36 were identified and then were assembled by overlapping BLAST alignment to determine a minimum tiling path (Fig. 2).

Fig. 2.

Fig. 2

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Mouse and human comparative 1.2-Mb map. The minimum tiling paths of the human BACs (upper) and mouse BACs (lower) are shown. Human BACs are identified by Accession number, and mouse BACs are identified by clone name. Mouse and human genes are denoted by approved gene symbols, if available. If no approved symbol was available in either species, Accession numbers were given. If no Accession number was found, but there was a sequence identity (see Fig. 3) the “like” suffix was added to the human gene name (KIAA1716-like).

From the minimum tiling path of ~1.2 Mb of human DNA, the sequence which corresponded to the 284-kb mouse sequence was extracted. A computer algorithm, Pipmaker (http://nog.cse.psu.edu/pipmaker), was used to analyze the sequenced mouse and human region (284 kb). The intron/exon organization used as input to Pipmaker was produced from alignment of the identified cDNAs and genes with genomic DNA (Table 4). In an initial analysis, all mouse cDNA from Table 4 was included; however, for ease of presentation, only those sequences with human–mouse similarity were retained in the final analysis.

Results and Discussion

Creation of a high-resolution linkage map

A genetic map of the subtelomeric region of mouse Chr 4 was produced by using an F2 generation of mice (N = 628) originating from the C57BL/6ByJ (B6) and 129P3/J (formerly 129/J; abbreviated here as 129) strains (Bachmanov et al. 1997). Thirty markers, which were polymorphic between the 129 and B6 strains, were genotyped, and the constructed genetic map spanned a 13.3-cM region from D4Mit33 (proximal) to D4Ertd296e (distal; Fig. 1a). Marker details are provided in Table 2.

Fig. 1.

Fig. 1

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(A) Linkage map of the distal part of mouse Chr 4. Distances between markers were estimated in cM based on data from the B6 × 129 F2 intercross (n = 628). The genetic interval from D4Mit33 to D4Ertd296e is 13.3 cM. Thick line indicates region that was physically mapped. (B) Physical map and the BAC contig of the subtelomeric region of mouse Chr 4 between D4Mit256 and K00231. BAC sizes (kb) are shown in parentheses. Dots indicate the presence of markers detected by hybridization and PCR, and in some cases, by direct sequencing.

Construction of a BAC contig

The RPCI-23 BAC library (Osoegawa et al. 2000) was screened twice to identify BAC clones covering the Sac locus. A probe prepared from a YAC clone (178B3) was used for the first screening. This YAC was mapped to the subtelomeric region of Chr 4, and it appeared to be nonchimeric. Pooled probes including all markers mapped to the subtelomeric region of Chr 4 were used for the second round of screening. To determine the BAC clones positive for each probe, we arrayed all putative clones identified from both screenings to prepare small high-density filters. Each small filter was then hybridized against individual probes. Only positive clones confirmed by both hybridization and PCR were used to construct a contig. The BAC end sequences were either obtained from databases or generated by direct sequencing. Consequently, STS markers were designed from the unique sequences of BAC ends and used to perform STS content mapping against all positive clones.

The BAC-insert sizes were determined by pulse-field gel electrophoresis. Analysis of the 42 BAC clones was conducted with the SEGMAP computer program (Green et al. 1991) and resulted in the assembly of an estimated ~1.2-Mb sequence-ready map representing a 5.1-cM region between D4Mit256 and K00231 (Fig. 1b). Since BAC clone 118E21 was the largest (246 kb) clone located within the Sac critical region, we chose it to prepare shotgun clones to generate a sequence contig with 5–6 × coverage (Accession #AF389853). Consequently, we generated 50 new STS markers (Table 2), fourteen (28%) of which were polymorphic between 129 and B6 mouse strains. These new markers were developed to fill gaps between previously described polymorphic markers, and were selected to amplify simple repeats where possible (Table 3).

Table 3.

Marker positions within the 284-kb sequenced region of distal Chr 4.

Gene/Marker Beginning End
280G12-T7 11715 11858
49O2-T7 27642 27810
118E21-SP6 29363 29633
U37351 38244 38403
34M15-T7 44601 44218
338N4-T7 44905 45148
387F5-SP6 44904 45148
284D21-SP6 45018 45122
D4Mon7 53569 53755
M134G01 58000 58160
D4Mon4 58522 58718
D4Mon6 73982 74187
D4Mon3 84280 84467
D18346 103341 103506
D4Xrf215 117690 118021
350D2-T7 143131 143431
D4Mon41 144063 144322
D4Mon43 144734 144936
D4Mon44 145429 145629
D4Mon48 151165 151369
D4Mon49 151468 151671
D4Mon50 153730 153925
D4Mon52 155679 155876
D4Mon53 155857 157434
D4Mon54 157579 157765
D4Mon31 157787 158016
D4Mon56 160305 160710
D4Mon5 161128 161327
D4Mon58 164439 164638
D4Mon59 165441 165858
D4Mon60 167377 167598
D4Mon33 167809 168011
D4Mon62 171062 171262
D4Mon34 172821 173022
D4Mon63 173883 174145
D4Mon64 175196 175564
D4Mon36 182757 182988
D4Mon66 183512 183711
D4Mon37 187705 187955
D4Mon39 197739 197979
139J18-SP6 199176 199407
D4Mon40 202605 202865
D4Mon1 206251 206451
417B22-SP6 213552 213669
227G4-T7 214230 214473
D4Mon28 221574 221869
D4Mon27 225540 225789
D4Mon26 227773 227932
284D21-T7 230924 231061
280G12-SP6 231023 231255
387F5-T7 231042 231255
49O2-SP6 231113 231331
338N4-SP6 231321 231436
130A12-SP6 231328 231443
D4Mon25 231647 231777
D4Mon24 240095 240241
D4Mon22 244624 245045
D4Mon21 246689 246862
AI225779 247955 248055
D4Mon20 250004 250153
D4Mon19 252161 252437
D4Mon18 253757 253867
D4Mon17 254169 254369
D4Mon16 254415 254620
D4Mon68 255365 255610
D4Mon2 256018 256168
D4Mon15 267313 267581
D4Mon9 268606 268805
D4Mon14 273253 273484
D4Mon13 273608 273802
D4Mon12 276854 277019
D4Mon11 279935 228277
118E21-T7 282595 282779
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Marker locations are given as bp within the sequenced region (1–284,000 bp; see Fig 3). Accession numbers for the markers are in Table 2.

Annotation of raw sequence

BLAST searches using all STSs in the region were conducted to identify any other large genomic clones that overlapped with the sequenced BAC 118E21, but only one was found (Accession #AF185591). These two clones were assembled and yielded a total sequence of 284 kb. After the sequence of the 284 kb was complete, exact STS positions within the region were identified (Table 3).

Two methods were used to identify genes within the 284-kb sequence: predicting genes by computer algorithm (the ab initio method), and by the examination of sequence similarly with other genes and ESTs. Initially, the 284-kb sequence was analyzed by masking the repetitive elements, and a computer algorithm (Gen-scan) was used to predict genes. These predicted genes and the raw sequences were submitted to iterative BLAST searches to determine their similarity to known genes or ESTs. For a predicted gene or genomic sequence to be considered an exact match to a gene or EST deposited in GenBank, the sequence similarity had to be >92% for at least 50% of the sequence (Venter et al. 2001). This degree of similarity allowed for sequencing errors or stain differences, but minimized the possibility that closely related genes were misidentified as exact matches or that alternatively spliced forms were discarded inadvertently.

Of the genes in this region, seven had locus designations and were considered known genes (Fig. 2 and 3): dishevelled, Drosphila dsh homolog (Dvl, GenBank Accession number NM_010091), Paneth cell enhanced expression gene (Pcee, Accession #NM_018856), sweet taste receptor family 1 member 3 gene (Tas1r3, Accession #AF311386), calcium-binding protein (Cab45a, Accession #U45977), a mammalian homolog of yeast UBC7 (Ubc6p, Accession #U93242), and two members of the tumor necrosis factor receptor superfamily, Tnfrsf4 (Accession #X85214; aliases Txgp1 and ox40) and Tnfrsf18 (Accession #NM_009400). Dishevelled (Dvl) is one of the best-characterized genes in the region. It was originally discovered because it is an ortholog of the Drosophila dishevelled (dsh) gene, a gene that influences the development of the fly body plan. In the mouse, Dvl influences complex social and sensorimotor behavior (Lijam et al. 1997; Lee et al. 1999). Several Dvl homologs are located on other mouse chromosomes. The tumor necrosis factor receptors Tnfrsf4 and Tnfrsf18 are involved in immune function and cancer biology. Ubc6p is a homolog of the ubiquitin-conjugating enzyme, but to the best of our knowledge, functional studies with this gene and associated enzyme have not been conducted. The calcium-binding protein Cab45a is present in the lumen of the Golgi apparatus (Scherer et al. 1996). Tas1r3 belongs to a small family of taste receptors (Hoon et al. 1999), is expressed in taste receptor cells, and has been suggested as a candidate gene for the Sac locus (Bachmanov et al. 2001; Kitagawa et al. 2001; Max et al. 2001; Montmayeur et al. 2001; Nelson et al. 2001; Sainz et al. 2001).

Fig. 3.

Fig. 3

Fig. 3

Fig. 3

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Percentage identity plot-based comparative map of mouse and human genomes. The mouse genomic sequence is upper, and the human genomic draft sequence is lower, assembled from three overlapping human BAC clones (Accession # AC026283, AL139287, and AL162741). Both the mouse and human sequences are oriented from the centromere (left) to telomere (right), and the percentage identity is shown on the vertical axis (50–100%). The portions of the figure corresponding to exons with exact matches to known genes are yellow; those with exact matches to cDNAs are blue. Mouse cDNA sequences with no homology to human genomic sequence are not included. Mouse genes are denoted as described in Fig. 2.

In addition to the known genes, other putative genes were identified because sequences within this region matched 15 existing EST clusters (Mm.23825, Mm.23492, NM_025338, Mm.28978, NM_024472, Mm.22021, Mm.56803, Mm.182484, Mm.133906, Mm.172945, Mm.32248, NM_026125, Mm.102764, Mm.42006, and Mm.159514) and 11 single cDNA clones (AA546397, AA435261, AA967911, BG243209, BF181401, BF120370, AA218417, AW743506, AI225779, BF319334, AW987045; Table 4). The function of these genes, other than that suggested by expression patterns inferred from tissue libraries from which they were derived, are unknown.

To further confirm that putative genes were actual genes, the EST sequences were aligned with genomic DNA and the intron/exon junctions identified (Table 4). The location of each exon for each gene or EST was determined, as well as the acceptor and donor sites. If we assume that all exons identified by alignment of cDNA with genomic DNA are true exons, then Genscan accurately predicted 58% of the exons. If we assume that all EST clusters or single, non-overlapping ESTs are genes, Genscan predicted at least one exon correctly for 41% of genes.

Characterization of genes within this region

Several genes were selected for further study because they were located within a critical region for the taste-related QTL, Sac (Bachmanov et al. 2001). The intron/exon structure of these genes was determined by PCR amplification of genomic and tongue cDNA, using primers that spanned intronic sequence. The PCR experiments were designed to confirm that the predicted genes were present as mRNA, to assess the accuracy of predicted intron/exon junctions, and to determine whether these genes were expressed in tongue. Tongue is an un-derrepresented tissue as a source of template for publicly available EST sequencing data.

Of the seven genes tested, all were expressed in mouse tongue (primers are given in Table 5; results are given in Tables 4 and 5). Of the 16 intron/exon junctions tested by PCR, two junctions predicted by the computer algorithm did not match the junction obtained from sequencing of the cDNA (12.5%; Table 5). The results of this experiment confirm gene expression in tongue cDNA.

Analysis of single nucleotide polymorphisms within genomic and cDNA

Genes within the critical Sac region were sequenced by using genomic and cDNA derived from the B6 and 129 strains. The purpose of this experiment was two-fold: to discover allelic genes that could be considered candidate genes for the Sac locus, and to identify single nucleotide polymorphisms (SNPs) useful for genetic mapping.

SNPs were detected, on average, every 100–200 bp. Most genes were polymorphic between the two strains, and the frequency of nucleotide variants did not differ appreciably from gene to gene. In addition to nucleotide substitutions, small insertions and deletions were a frequent source of polymorphisms between strains. All sequence variants detected in coding regions were either silent or missense mutations (Table 5); no nonsense or frameshift mutations were found, nor were any variants detected that would alter an intron/exon splice junction.

There were several sequence variants within the 129 strain. The possibility that these variants were due to sequencing artifacts was excluded by re-sequencing of the areas, which confirmed the heterozygosity of the 129 strain in all cases.

Comparative map between mouse and human

To construct a human–mouse comparative map (Fig. 2), the sequences in the human that align with the 1.2-Mb mouse contig were identified. To do this, mouse genes were submitted to multiple BLAST searches of the human nr, est, htgs, and gss databases. For comparative mapping, we used human sequences that fit all three of the following criteria: (1) they were highly homologous to mouse genes; (2) they were located on human Chr 1p36; and (3) they were contained within a BAC or PAC clone.

The selected positive human BACs were then assembled, a minimum tiling path was selected, and the mouse genes and their human homologs were aligned (Fig. 2). This human sequence was compared with the mouse sequence, by using the PIP algorithm that plots percentage identity between two sequences (Schwartz et al. 2000). Almost all named genes in mouse were present in the human sequence, and they were arranged in the same order on the chromosome (Fig. 3). In many areas outside of the predicted exons, there were highly conserved sequences between mouse and humans (e.g., 6–8 kb, 121–126 kb; Fig. 3). These highly conserved regions may represent undetected exons or important and highly conserved regulatory elements.

Conclusions

We have described the genetic and physical map of the subtelomeric region of mouse Chr 4, and a comparative map of the region of conserved synteny in humans. Several interesting features of this region were found. First, gene densities near the mouse and human telomeres are high. More than 20 genes were confirmed within the 284-kb sequence in the mouse. High gene density of the human telomeric region has been demonstrated (Saccone et al. 1992), and the current report suggests this is true in mice, to the extent that Chr 4 is representative.

Second, the human-mouse conservation between gene order and number was strong. Almost all named genes were present in both mouse and human, and in the same order along the chromosome. There is, however, a caveat to the interpretation of the mouse-human conserved synteny map: the human BAC clones were derived from the htgs database, and are unfinished. Although the PIP algorithm we used takes into account the possibility of broken clones and creates the most likely alignment, some adjustment of gene order may occur as finished sequence is available. The region of strict conserved synteny extends over at least 1.2 Mb (the size of the mouse–human contig and homology map). Mapping data from other sources indicate that the homology may extend as far as 1p31 (http://genome.ucsc.edu).

Third, the recombination frequency in this region was very high. The 1.2-Mb region represented in the BAC contig corresponded to a genetic distance of over 5 cM as measured in an F2 mapping panel. This high rate of recombination has been observed in mouse telomeric regions (de Boer and Groen 1974). This terminal map expansion is fortunate for those mapping quantitative traits or genes, because more informative meioses are obtained than in less recombinogenic regions of the genome. The high rate of recombination, and its beneficial effects on gene mapping, partially offset the difficulties encountered in sequencing telomeric regions.

The rate of polymorphisms detected between the two mouse strains, B6 and 129, suggests that many of the genes may have altered function because of numerous amino acid substitutions. The B6 and 129 strains are only distantly related, and the large number of polymorphisms makes these strains good choices for gene mapping studies. Compared with data collected in a large-scale effort to map SNPs among mouse strains (Lindblad-Toh et al. 2000), the frequency of SNPs in distal Chr 4 was more than threefold higher than elsewhere in the mouse genome for comparable strains.

Polymophisms between substrains of the 129 strains have been well characterized (Simpson et al. 1997), but the rate of heterozygosity within the 129P3/J strain was surprising: inbred strains are supposed to be homozygous at all loci. This finding is consistent with our observations during genotyping with microsatellite markers, which identified some polymorphisms within the 129P3/J substrain (Bachmanov et al., unpublished observations). The rate of within substrain polymorphisms could account for some of the phenotypical within-strain variation of the 129 substrain, such as variable agenesis of the corpus callosum (Wahlsten 1982). Several explanations are possible for this variability within this inbred strain. The 129P3/J substrain is maintained using a forced heterozygosity mating scheme: in each generation, heterozygotes at the albino locus Tyrc/Tyrc-ch are mated with homozygotes, Tyrc/Tyrc. This mating scheme has been conducted at The Jackson Laboratory since 1948 (Withham 1990). The forced heterozygosity may account for variation in the albino region on Chr7, but should not increase the level of heterozygosity of other regions. Alternatively, the increased heterozygosity might be due to the later initiation of inbreeding of the 129P3/J strain compared with other inbred strains. Another possibility might be that the strains could have been contaminated, like other substrains of the 129 strain (Simpson et al. 1997). Finally, the 129P3/J strain could be unusually prone to de novo mutations. Further study of the heterogeneity of the 129P3/J strain should be conducted, because this heterozygosity could have impact upon QTL mapping or other phenotypic studies.

There are two ways to detect genes within genomic DNA sequence: prediction of genes using computer algorithms, and similarity searches. Both work well at identifying most genes, but neither method identifies all genes with certainty. Ab initio methods, implemented with Genscan, detected more than half of the genes in our study, but the genes detected were not necessarily assembled with the same intron/exon boundaries found in the cDNA. These Genscan assemblies could be errors, or they could reflect alternatively spliced forms of a gene that are undetected because they are expressed in tissues not represented in cDNA libraries, or have a low abundance. Comparisons of the genomic sequence using sequence similarity methods also provided additional information about the presence of genes and their structures, many of which were not identified through the Genscan analysis. Still, other genes could exist in this region, and the regions of high sequence similarity identified through the PIP analysis would be good candidates for the undetected genes. The publicly available human draft sequence was poorly annotated in this region and reflected less than half of the gene content of the current analysis (http://genome.ucsc.edu/). Therefore, it is still important for investigators to annotate genomic regions rather than to rely on the annotation available through public and private databases.

Annotation of genomic sequence and predicting genes through areas of conserved synteny with other species should be approached cautiously. For example, regions of high human–mouse homology existed, and in some cases the sequence encompassed a known gene in one species, but not in the other species, and no cDNAs in the other species were found. A case in point is the Ubc6p gene in the mouse: there is a high sequence homology between mouse and human genomic sequence, but without the presence of human cDNAs, further work would be needed to name the human sequence appropriately. Another challenge was the large number of orphan mouse cDNAs in the region; many of these cDNAs could represent novel genes, but it was difficult to determine this accurately without additional work.

Examination of this region led to the identification of many genes that were polymorphic between B6 and 129 mice, near the peak linkage interval for Sac, and that were expressed in the tongue. Of these genes, Tas1r3 is the best candidate for the Sac locus (Bachmanov et al. 2001; Kitagawa et al. 2001; Max et al. 2001; Montmayeur et al. 2001; Nelson et al. 2001; Sainz et al. 2001).

The detailed genetic, physical, and comparative maps created here will assist in identification of loci that influence development, behavior, and disease susceptibility, and the sequence-ready BAC contig will be useful for the public Mouse Sequencing consortium. While drafts of the homologous regions are now available from human DNA (Human Genome Sequencing Consortium 2001; Venter et al. 2001), comparison of the maps described herein with the recently released human draft suggests that gaps still remain in the human sequence, particularly in areas closest to the telomere. Identification of all genes in human and mouse subtelomeric regions and the closing of telomeric gaps will involve more effort than that taken to close gaps in other regions of the genome.

Acknowledgments

We thank Tim Wiltshire and Maja Bucán for advice about physical mapping, and R. Arlen Price for help with initial genotyping of F2 hybrids. We also thank Joe Catanese, Dulce San Juan, and Al Cairo for help with the physical mapping. The excellent technical support of Sarah Tobias and Pfizer Global Research and Development-Alameda genotyping and sequencing core is acknowledged. This work was supported by National Institutes of Health grants R01DC00882 (G.K. Beauchamp), R03DC03509, R01DC04188 and R01DK55853 (D.R. Reed), R01AA11028 (M.G. Tordoff) and R03DC03853 (A.A. Bachmanov), and by a special grant from the Ambrose Monell Foundation.

Footnotes

The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers: G67732–G67781 (STS D4Mon1-D4Mon68), AF311386 (Tas1r3 gene) and AF389853 (BAC RPCI-23-118E21).

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