Role of the mouse ank gene in control of tissue calcification and arthritis

Science

Volumn 289, Issue 5477, 2000, Pages 265-270

  Ho, Andrew M ^a   Johnson, Michelle D ^a   Kingsley, David M ^a,b  

^a STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b STANFORD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

HYDROXYAPATITE; MEMBRANE PROTEIN; PYROPHOSPHATE;

ANKYLOSING SPONDYLITIS; ARTHRITIS; ARTICLE; CALCIFICATION; CELL CULTURE; DISEASE PREDISPOSITION; EXPRESSED SEQUENCE TAG; GENE LOCUS; GENE MAPPING; GENE MUTATION; MOUSE; NONHUMAN; NUCLEOTIDE SEQUENCE; PHENOTYPE; PRIORITY JOURNAL; SEQUENCE HOMOLOGY;

ANIMALS; ARTHRITIS; BASE SEQUENCE; BIOLOGICAL TRANSPORT; CALCINOSIS; CHROMOSOME MAPPING; CLONING, MOLECULAR; COS CELLS; DIPHOSPHATES; DNA; DURAPATITE; GENE EXPRESSION; GENETIC COMPLEMENTATION TEST; HUMANS; MEMBRANE PROTEINS; MICE; MICE, TRANSGENIC; MOLECULAR SEQUENCE DATA; MUTATION; PHENOTYPE; PHOSPHATE TRANSPORT PROTEINS; PHYSICAL CHROMOSOME MAPPING; SEQUENCE HOMOLOGY, NUCLEIC ACID; TISSUE DISTRIBUTION;

ANIMALIA; VERTEBRATA;

EID: 0034647482     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.289.5477.265     Document Type: Article

References (52)

1. E. Yelin and L. F. Callahan, Arthritis Rheum. 38, 1351 (1995).
2. C. G. Helmick et al., Arthritis Care Res. 8, 203 (1995).
3. T. D. Spector et al., Br. Med. J. 312, 940 (1996).
4. M. A. Brown et al., Arthritis Rheum. 40, 1823 (1997).
5. A. J. MacGregor et al., Arthritis Rheum. 43, 30 (2000).
6. P. Wordsworth, Br. Med. Bull. 51, 249 (1995).
7. H. O. Sweet and M. C. Green, J. Hered. 72, 87 (1981).
8. F. T. Hakim et al., Arthritis Rheum. 27, 1411 (1984).
9. F. T. Hakim, K. S. Brown, J. J. Oppenheim, Arthritis Rheum. 29, 114 (1986).
10. H. W. Sampson and J. S. Davis, Spine 13, 650 (1988).
11. M. L. Mahowald, H. Krug, P. Halverson, J. Rheumatol. 16, 60 (1989).
12. H. E. Krug et al., J. Rheumatol. 24, 115 (1997).
13. 2 progeny were subsequently typed with D15Mit251 and D15Mit55. Seventy-seven mice with recombination between these markers were then typed with 26 additional microsatellite markers. D15Mit20 and D15Mit164 showed no recombination with the ank trait in the high-resolution mapping panel.
14. The CITB Release II library (Research Genetics) was screened with D15Mit20. BAC end probes were isolated with bubble polymerase chain reaction (PCR) [J. Riley et al., Nucleic Acids Res. 18, 2887 (1990)] and were used for additional rounds of chromosome walking. Contigs were assembled by pulsed-field gel mapping and cross-hybridization of BAC end probes. BACs B to H correspond to clones 307D1, 282M13, 456B18, 110P8, 477E24, 284120, and 144P21, respectively. BAC A corresponds to clone 104F8 (Genome Systems, St. Louis, MO).
15. Circular BAC DNA (0.3 to 1.0 ng/μl) was microinjected into fertilized FVB mouse oocytes at the Stanford Transgenic Research Facility. Founder mice were mated to ank mutants, and the ank/+, tg+ progeny were intercrossed. The ank/ank progeny were identified by homozygosity of alleles at the D15Mit180 locus, and transgenic carriers were identified by Southern blot analysis with a BAC vector probe. Mouse limbs were partially decalcified for 12 to 24 hours in Calex II (Fisher, Pittsburgh, PA), fixed in 10% formalin, dehydrated in ethanol, cleared in xylene, and embedded in paraffin wax. Adjacent 6-μm sections were collected and stained with hematoxylin and eosin or with von Kossa's method.
16. DNA from BAC C was sheared mechanically to 2 to 3 kb and subcloned into the Eco RV site of pBluescript SK(+) (Stratagene, La Jolla, CA) by blunt end ligation. Clones were sequenced with both T3 and T7 primers and the ABI Prism Dye Terminator Kit (PE Applied Biosystems, Foster City, CA). Candidate genes were amplified from total brain RNA of wild-type and mutant mice with gene-specific primers and Thermoscript reverse transcriptase (Gibco-BRL, Grant Island, NY). Initial cDNA products were treated with 2 U ribonuclease H (RNase H) at 37°C (30 min) and amplified with forward and reverse primers. Secondary PCR products were purified on a 1% low-melt agarose gel (FMC Bioproducts, Rockland, ME), extracted with the Ultraclean Gelspin DNA purification kit (Doc Frugal Scientific, San Diego, CA), and sequenced with gene-specific primers. The genomic region surrounding the ank mutation was amplified from ank/ank, JGBF, 129/J, A/J, AKR/J, Balb/cByJ, BTBR(+/T), C3H/HeJ, C57BL/6J, C57BR/cdJ, C57L/J, CAST/Ei, CBA/J, CZECHII/Ei, DBA/2J, FVB/NJ, NZB/ BINJ, P/J, and SPRET/Ei strains (DNA Resource, The Jackson Laboratory) with GGCTCCCTTCTAGCAGGGTT and AGCATGCTGCAAGGGCAACC primers, digested with Hinf I, and analyzed by electrophoresis in a 4% agarose gel.
17. GenBank accession numbers AF001532 and AF001533.
18. M. G. Claros and G. von Heijne, Comput. Appl. Biosci. 10, 685 (1994).
19. Full-length ank cDNA was amplified from total brain RNA with forward and reverse primers that flank the ank ORF (CGCGGATCCACCATGGTGAAATTCCCGGCGC, CGCGGATCCTTACTCATTTTCTTCTCTCATCTCT). Amplified products were digested with Bam HI, subcloned into the Bam HI site of pCDNA3 (Invitrogen, Carlsbad, CA), and sequenced to verify the structure of the insert. Rabbit polyclonal antibodies were generated against keyhole limpet hemocyanin (KLH)-conjugated peptides containing amino acids 118 to 130 (Ab1), 304 to 317 (Ab2), and 477 to 492 (Ab3) of murine ANK by Zymed Laboratories (San Francisco, CA). COS-7 cells were plated onto two-chamber glass slides and transfected with 0.8-μg DNA and 2-μl FuGENE 6 reagent (Boehringer Mannheim, Indianapolis, IN). After 40 hours, cells were fixed in 3% paraformaldehyde, washed in phosphate-buffered saline (PBS), and blocked with 10% goat serum (Jackson Immunoresearch, West Grove, PA) and 5% fetal bovine serum (FBS) (Hyclone, Logan, UT) in PBS, with or without 0.1% Triton X-100. Blocked cells were incubated with rabbit antibody to mouse ANK primary antibodies diluted 1:50 (Ab1), 1:200 (Ab2), or 1:500 (Ab3) in blocking solution, incubated with fluorescein isothiocyanate (FITC)-conjugated Affinipure goat antibody to rabbit immunoglobulin G (IgG) secondary antibodies (Jackson Immunoresearch) diluted 1:143 in blocking solution, and washed in PBS. Nuclei were counterstained with 10 μg/ml 7-AAD (Molecular Probes, Eugene, OR). Confocal images were captured on an MRC 1000 Confocal Imaging Microscope (Biorad, Richmond, CA).
20. Y. Gluzman, Cell 23, 175 (1981).
21. A. M. Ho, M. D. Johnson, D. M. Kingsley, data not shown.
22. Other topologies are possible, including one in which the first hydrophobic stretch serves as a signal sequence instead of a transmembrane region.
23. 32P-labeled probe containing the complete ank ORF. Forelimbs from E16.0 mouse embryos were frozen, sectioned on a cryostat microtome, and hybridized with a 380 base pair (bp) digoxygenin-labeled cRNA probe from the 3′ untranslated region (UTR) of ank as described [E. E. Storm and D. M. Kingsley, Development 122, 3969 (1996)].
24. A. M. Ho, M. D. Johnson, D. M. Kingsley, unpublished data.
25. Detailed methods available on request.
26. Marker Hs. 11974 in P. Deloukas et al., Science 282, 744 (1999).
27. A. E. Hughes et al., Hum. Mol. Genet. 4, 1225 (1995).
28. L. J. Andrew et al., Am. J. Hum. Genet. 64, 136 (1999).
29. K. Rojas et al., Genomics 62, 177 (1999).
30. G. Lust, G. Faure, P. Netter, J. E. Seegmiller, Science 214, 809 (1981).
31. 4 precipitate, and assayed for pyrophosphate as above. Sample means were compared with the use of the Student's t test.
32. H. Fleisch, Metab. Bone Dis. Relat. Res. 3, 279 (1981).
33. _ and S. Bisaz, Nature 195, 911 (1962).
34. 6) were plated in 100-mm dishes, grown for 6 to 12 hours, and incubated for 4 to 6 hours in serum-free medium with DNA (5 μg), Plus reagent (18 μl), and Lipofectamine (18 μl)(Gibco-BRL). Following transfection, cells were grown in fresh antibiotic-free medium for 36 to 48 hours before assay. Control experiments with a pCMV-lacZ plasmid showed about fivefold higher expression levels in COS-7 cells than in mouse fibroblasts under these conditions.
35. S. E. Guggino, G. J. Martin, P. S. Aronson, Am. J. Physiol. 244, F612 (1983).
36. A. K. Rosenthal and L. M. Ryan, J. Rheumatol. 21, 896 (1994).
37. H. Fleisch, D. Schibler, J. Maerki, I. Frossard, Nature 207, 1300 (1965).
38. H. Fleisch et al., Am. J. Physiol. 211, 821 (1966).
39. A. M. Caswell, M. P. Whyte, R. G. Russell, Crit. Rev. Clin. Lab. Sci. 28, 175 (1991).
40. A. Okawa et al., Nature Genet. 19, 271 (1998).
41. I. Nakamura et al., Hum. Genet. 104, 492 (1999).
42. D. J. White et al., J. Clin. Dent. 7, 46 (1996).
43. W. P. Tew et al., Biochemistry 19, 1983 (1980).
44. H. E. Krug et al., Arthritis Rheum. 36, 1603 (1993).
45. P. Dieppe and I. Watt, Clinics Rheum. Dis. 11, 367 (1985).
46. D. T. Felson et al., J. Rheumatol. 16, 1241 (1989).
47. P. A. Gibilisco, H. R. Schumacher, Jr., J. L. Hollander, K. A. Soper, Arthritis Rheum. 28, 511 (1985).
48. A. Swan et al., Ann. Rheum. Dis. 53, 467 (1994).
49. M. Doherty, I. Watt, P. A. Dieppe, Lancet i, 1207 (1982).
50. A. L. Concoff and K. C. Kalunian, Curr. Opin. Rheumatof. 11, 436 (1999).
51. L. M. Ryan and H. S. Cheung, Rheum. Dis. Clin. North Am. 25, 257 (1999).
52. We thank M. Wagener for excellent assistance with animal experiments and G. Barsh and members of the Kingsley lab for helpful comments on the manuscript. A.M.H. is a trainee of the Medical Scientist Training Program at Stanford, supported by NIH training grant 5T32GM07365 (NIGMS). The initial stages of the genetic cross were supported by a Stanford-SmithKline Beecham research award. D.M.K. is an HHMI assistant investigator.