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Mammalian Genome 8, 530–532 (1997). © Springer-Verlag New York Inc. 1997 Comparative mapping in cattle of genes located on human Chromosome 18 Morris K. Agaba,1 Stephen J. Kemp,2 William Barendse,3 Alan Teale1 1 International Livestock Research Institute, P.O. Box 30709, Nairobi, Kenya School of Biological Sciences, Donnan Labs, University of Liverpool, Liverpool L69 3BX, UK 3 CSIRO, Division of Tropical Animal Production, Molecular Animal Genetics Centre, Gehrmann Laboratories, Research Rd., University of Queensland, St. Lucia 4072, Australia 2 Received: 7 December 1996 / Accepted: 25 February 1997 Human Chromosome (Chr) 18 (HSA18) contains approximately 85 million base pairs of DNA (Morton 1991). Thirty genes and pseudogenes have been mapped to HSA18 by somatic cell genetics or in situ hybridization (Le Beau et al. 1993; Overhauser et al. 1993; van Kessel et al. 1994). They include pituitary adenylcyclase activating peptide (ADCYAP1), laminin a chain (LAMA1), and mitochondrial NADH-ubiquinone reductase, 24 kDa subunit (NDUFV2) on the short arm, and transthyretin (TTR), B-cell lymphoma 2 (BCL2), plasminogen activator inhibitor type 2 (PAI2), n-cadherin (CDH2), desmocollin type 2 (DSC2), ferrochelatase (FECH), cytochrome b5 (CYB5) and myelin basic protein (MBP) on the long arm (Fig. 1a). The murine homologs of HSA18 genes have been mapped to at least four chromosomes as follows: Bcl-2 and Planh2 genes are on MMU1, thymidylate synthase (Ts1) gene is on MMU5, Lama1 is on MMU17, and the rest are on MMU18 (Mouse Genome Database 3.1, 1996). Two bovine genes—Yamaguchi sarcoma viral oncogene homolog 1 (YES1) and desmocollin type 1 (DSC1)—homologous to HSA18 loci have been mapped by in situ hybridization to Chr 24 (Chowdhary et al. 1996), and ZOO-FISH analyses show that HSA18 loci are located entirely on BTA24 (Hayes 1995; Toldo et al. 1995; Chowdhary et al. 1996). We report here the genomic localization of eight bovine genes homologous to HSA18 genes Correspondence to: A. Teale ADCYAP1, CDH2, CYB5, DSC2, FECH, NDUFV2, PAI2, and TTR, using somatic cell genetics and linkage analysis in the International Bovine Reference Panel (IBRP). Oligonucleotide primers for PCR were designed on the basis of the published nucleotide sequences of sheep or cattle genes ADCYAP1, CDH2, CYB5, DSC2, FECH, NDUFV2 and TTR, and were used to amplify the corresponding DNA fragments from bovine total genomic DNA. Primers for PAI2 gene fragments were designed on the basis of a consensus sequence of the human and murine genes. PCR amplification and single-strand conformation polymorphism (SSCP) analysis were performed as described previously (Agaba et al. 1996) except that the annealing and gel temperatures were optimized for each DNA fragment as shown in Table 1. The primers for PAI2 were allelic; that is, under optimum PCR conditions amplification of one allele from some individuals was favored over the alternative (null) allele, and, as expected, dominant inheritance was observed in informative IBRP families. Somatic cell hybrid mapping was achieved by PCR amplification of bovine sequences in a panel of 27 bovine × rodent somatic cell hybrids kindly provided by J. Womack of Texas A & M University. In the case of the PAI2 gene, bovine fragments were differentiated from rodent fragments by SSCP analysis. Seven genes, ADCYAP1, CDH2, CYB5, DSC2, FECH, PAI2 and TTR, segregated 100% concordantly with each other and with Chr 24, while the NDUFV2 gene segregated 100% concordantly with BTA5. Fig. 1. (A) Location of some of the genes on human Chr 18 determined by in situ hybridization (from Le Beau et al. 1993; van Kessel et al. 1994); (B) assignment of eight of the genes to bovine Chrs 5 and 24 by somatic cell hybrid analysis; (C) location of the genes on the linkage map of Chr 24. Some loci are at the bottom of the multipoint map because they cannot be ordered with odds greater than 1000:1; instead, they are listed as being at a distance relative to an ordered locus. M.K. Agaba et al.: Human Chr 18 genes located in cattle 531 Table 1. List of the HSA18 genes mapped in cattle showing the primer sequences, SSCP analysis conditions, number of alleles, and meioses informative for linkage analysis. Gene symbol Primer sequences Ta (°C)a Product size (bp) SSCP conditions (power)(Tg)b Alleles Meioses Reference for nucleotide sequence ADCYAP1 acctctgtaataaccaccgg aaaagtttgacgacgctcgg tgactatgaaggcagtggc attggatcaccgatattccc aagataaccaagccttcgg agaagacattgacagacacc ctcaagactatgtcctgacc gctatttacagcatccctcg ggttggcccgatgccctggc tcactggctagaacttgag agttgaatgtttaggggcc ccactgacatatttacagcg actcaacaagtggaccagc tctgctacaaactgtgggc gctggtgaatccaagtgtcc tccctgaggcaaatggctcc 56 349 5W, 4°C 2 110 Kimura et al. 1990 55 320 5W, 4°C 2 83 Liaw et al. 1990 53 388 5W, 4°C 4 138 Cristiano et al. 1993 56 326 5W, 4°C 2 85 Koch et al. 1992 56 165 5W, 4°C 3 243 Shibuya et al. 1995 53 260 5W, 4°C or 25°C 2 162 Pilkington and Walker 1989 56 303 — 2 68 Belin et al. 1989; Ahern et al. 1989 56 130 5W, 4°C or 25°C 1 0 Tu et al. 1989 CDH2 CYB5 DCS2 FECH NDUFV2 PAI2c TTR a b c Ta 4 annealing temperature. Tg 4 gel temperature. PAI2 was genotyped by an allele-specific amplification assay. Table 2. Two-point linkage analyses showing recombination fractions and the Lod scores. FECH FECH PAI2 TGLA351 CSSM31 CDH2 CYB5 ILSTS101 DSC2 ADCYAP1 CSSM23 INRA90 BM315 PAI2 TGLA351 CSSM31 CDH2 CYB5 ILSTS101 DSC2 ADCYAP1 CSSM23 INRA90 — — — — 0.19; 4.87 0.20; 9.02 — — — 0.05; 10.60 — — 0.05; 6.47 0.06; 5.45 — 0.21; — — 0.11; 0.05; 0.18; — — 0.04; 0.00; — 0.13; 0.06; — — — 0.17; 3.75 0.04; 4.54 — 0.09; 4.67 — — 0.13; 0.18; 0.10; 0.18; — 0.20; — — 0.13; 13.98 — — — — — 0.18; 7.61 0.19; 3.84 — — The PCR-generated fragments were screened for polymorphism in the IBRP by SSCP analysis, and informative families were genotyped (Table 1). Mendelian inheritance was demonstrated and genotype data were analyzed for linkage against the Cattle Genotypic Database (CGD) markers with CRI-MAP v2.4 SunOs as described by Barendse and associates (1994). Two-point analyses revealed genetic linkage as shown in Table 2. The loci CSSM31 and CSSM23 have previously been mapped to BTA24 (Barendse et al. 1994), and BM315 to BTA5 (Bishop et al. 1994). The order of three loci FECH–ADCYAP1—CYB5 on BTA24 is rearranged compared with the order ADCYAP1-FECH-CYB5 on HSA18 (Fig. 1c). These data provide further information on the bovine equivalents of HSA18 and reveal extensive conservation of synteny between human and cattle similar to that previously observed for other genomic regions (Threadgill et al. 1990, 1994; Zhang et al. 1992). Unlike the situation in the mouse genome, synteny of the PAI2 gene with other HSA18 homologous loci is conserved in cattle. However, syntenic conservation between BTA24 and HSA18 loci was disrupted by the translocation of the NDUFV2 locus to BTA5. The size of the HSA18 homolog represented by NDUFV2 that is located on BTA5 will become clearer as additional type I loci in the vicinity of NDUFV2 on HSA18 are mapped in cattle. Since ZOO-FISH analyses of the bovine chromosomes do not show homology between HSA18 and BTA5 (Hayes 1995; Toldo et al. 1995; Chowdhary et al. 1996), we conclude that the HSA18 homolog represented by NDUFV2 on BTA5 is probably smaller than 7 million base pairs, the lower detection limit of ZOO-FISH (Scherthan et al. 1994). 4.51 3.07 8.83 3.50 4.23 11.14 4.90 9.77 5.69 7.45 22.49 5.01 5.36 NDUFV2 0.09; 20.98 References Agaba MK, Kemp SJ, Barendse W, Teale AJ (1996) Polymorphism at the bovine tumor necrosis factor alpha locus and assignment to BTA23. Mamm Genome 7, 186–187 Ahern SM, Le Beau MM, Lebo RV, Sadler JE (1989) Structure of the gene for human plasminogen activator inhibitor-2: the nearest mammalian homologue of chicken ovalbumin. 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