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. 1987 Apr 1;104(4):805–815. doi: 10.1083/jcb.104.4.805

A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones

PMCID: PMC2114441  PMID: 3558482

Abstract

We have detected and begun to characterize a 17-kD centromere-specific protein, CENP-A (Earnshaw, W. C., and N. Rothfield, 1985, Chromosoma., 91:313-321). Sera from several humans with CREST scleroderma autoimmune disease (CREST: calcinosis, Raynaud's phenomenon, esophageal dsymotility, sclerodactyly, and telangiectasia) bind this protein in immunoblot assays of HeLa whole cell or nuclear extracts. We have affinity purified the anti-17-kD centromere protein (anti-CENP-A) specific antibodies from immunoblots of HeLa nuclear protein. The antibodies react with epitopes present on CENP-A derived from humans but apparently do not recognize specific epitopes in either rat or chicken nuclei. Only human nuclear protein is CENP-A positive by immunoblot. Furthermore, human cells show localization of anti-CENP-A antibody to centromeres by immunofluorescence microscopy, whereas rat cells do not. On extraction from the nucleus, CENP-A copurifies with core histones and with nucleosome core particles. We conclude that this centromere-specific protein is a histone-like component of chromatin. The data suggest that CENP-A functions as a centromere-specific core histone.

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Selected References

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  1. Batteiger B., Newhall W. J., 5th, Jones R. B. The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods. 1982 Dec 30;55(3):297–307. doi: 10.1016/0022-1759(82)90089-8. [DOI] [PubMed] [Google Scholar]
  2. Bloom K. S., Anderson J. N. Fractionation and characterization of chromosomal proteins by the hydroxyapatite dissociation method. J Biol Chem. 1978 Jun 25;253(12):4446–4450. [PubMed] [Google Scholar]
  3. Bloom K. S., Carbon J. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes. Cell. 1982 Jun;29(2):305–317. doi: 10.1016/0092-8674(82)90147-7. [DOI] [PubMed] [Google Scholar]
  4. Brakke M. K., Van Pelt N. Linear-log sucrose gradients for estimating sedimentation coefficients of plant viruses and nucleic acids. Anal Biochem. 1970 Nov;38(1):56–64. doi: 10.1016/0003-2697(70)90155-7. [DOI] [PubMed] [Google Scholar]
  5. Brenner S., Pepper D., Berns M. W., Tan E., Brinkley B. R. Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients. J Cell Biol. 1981 Oct;91(1):95–102. doi: 10.1083/jcb.91.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brinkley B. R., Cartwright J., Jr Cold-labile and cold-stable microtubules in the mitotic spindle of mammalian cells. Ann N Y Acad Sci. 1975 Jun 30;253:428–439. doi: 10.1111/j.1749-6632.1975.tb19218.x. [DOI] [PubMed] [Google Scholar]
  7. Brinkley B. R., Cox S. M., Pepper D. A. Structure of the mitotic apparatus and chromosomes after hypotonic treatment of mammalian cells in vitro. Cytogenet Cell Genet. 1980;26(2-4):165–174. doi: 10.1159/000131438. [DOI] [PubMed] [Google Scholar]
  8. Donahue P. R., Palmer D. K., Condie J. M., Sabatini L. M., Blumenfeld M. Drosophila histone H2A.2 is associated with the interbands of polytene chromosomes. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4744–4748. doi: 10.1073/pnas.83.13.4744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Earnshaw W. C., Halligan N., Cooke C., Rothfield N. The kinetochore is part of the metaphase chromosome scaffold. J Cell Biol. 1984 Jan;98(1):352–357. doi: 10.1083/jcb.98.1.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Earnshaw W. C., Rothfield N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma. Chromosoma. 1985;91(3-4):313–321. doi: 10.1007/BF00328227. [DOI] [PubMed] [Google Scholar]
  11. Euteneuer U., McIntosh J. R. Structural polarity of kinetochore microtubules in PtK1 cells. J Cell Biol. 1981 May;89(2):338–345. doi: 10.1083/jcb.89.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fritzler M. J., Kinsella T. D. The CREST syndrome: a distinct serologic entity with anticentromere antibodies. Am J Med. 1980 Oct;69(4):520–526. doi: 10.1016/0002-9343(80)90462-3. [DOI] [PubMed] [Google Scholar]
  13. Gershoni J. M., Palade G. E. Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal Biochem. 1982 Aug;124(2):396–405. doi: 10.1016/0003-2697(82)90056-2. [DOI] [PubMed] [Google Scholar]
  14. Guldner H. H., Lakomek H. J., Bautz F. A. Human anti-centromere sera recognise a 19.5 kD non-histone chromosomal protein from HeLa cells. Clin Exp Immunol. 1984 Oct;58(1):13–20. [PMC free article] [PubMed] [Google Scholar]
  15. Isenberg I. Histones. Annu Rev Biochem. 1979;48:159–191. doi: 10.1146/annurev.bi.48.070179.001111. [DOI] [PubMed] [Google Scholar]
  16. Lutter L. C. Kinetic analysis of deoxyribonuclease I cleavages in the nucleosome core: evidence for a DNA superhelix. J Mol Biol. 1978 Sep 15;124(2):391–420. doi: 10.1016/0022-2836(78)90306-6. [DOI] [PubMed] [Google Scholar]
  17. Margolis R. L., Job D., Pabion M., Rauch C. T. Sliding of STOP proteins on microtubules: a model system for diffusion-dependent microtubule motility. Ann N Y Acad Sci. 1986;466:306–321. doi: 10.1111/j.1749-6632.1986.tb38402.x. [DOI] [PubMed] [Google Scholar]
  18. McCarty G. A., Valencia D. W., Fritzler M. J. Antibody to the mitotic spindle apparatus: immunologic characteristics and cytologic studies. J Rheumatol. 1984 Apr;11(2):213–218. [PubMed] [Google Scholar]
  19. McNeilage L. J., Whittingham S., McHugh N., Barnett A. J. A highly conserved 72,000 dalton centromeric antigen reactive with autoantibodies from patients with progressive systemic sclerosis. J Immunol. 1986 Oct 15;137(8):2541–2547. [PubMed] [Google Scholar]
  20. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding. J Cell Biol. 1985 Sep;101(3):755–765. doi: 10.1083/jcb.101.3.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mitchison T. J., Kirschner M. W. Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol. 1985 Sep;101(3):766–777. doi: 10.1083/jcb.101.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mitchison T., Evans L., Schulze E., Kirschner M. Sites of microtubule assembly and disassembly in the mitotic spindle. Cell. 1986 May 23;45(4):515–527. doi: 10.1016/0092-8674(86)90283-7. [DOI] [PubMed] [Google Scholar]
  23. Moroi Y., Hartman A. L., Nakane P. K., Tan E. M. Distribution of kinetochore (centromere) antigen in mammalian cell nuclei. J Cell Biol. 1981 Jul;90(1):254–259. doi: 10.1083/jcb.90.1.254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moroi Y., Peebles C., Fritzler M. J., Steigerwald J., Tan E. M. Autoantibody to centromere (kinetochore) in scleroderma sera. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1627–1631. doi: 10.1073/pnas.77.3.1627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  26. Pabion M., Job D., Margolis R. L. Sliding of STOP proteins on microtubules. Biochemistry. 1984 Dec 18;23(26):6642–6648. doi: 10.1021/bi00321a055. [DOI] [PubMed] [Google Scholar]
  27. Palmer D. K., Margolis R. L. Kinetochore components recognized by human autoantibodies are present on mononucleosomes. Mol Cell Biol. 1985 Jan;5(1):173–186. doi: 10.1128/mcb.5.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pepper D. A., Brinkley B. R. Tubulin nucleation and assembly in mitotic cells: evidence for nucleic acids in kinetochores and centrosomes. Cell Motil. 1980;1(1):1–15. doi: 10.1002/cm.970010102. [DOI] [PubMed] [Google Scholar]
  29. Pettijohn D. E., Henzl M., Price C. Nuclear proteins that become part of the mitotic apparatus: a role in nuclear assembly? J Cell Sci Suppl. 1984;1:187–201. doi: 10.1242/jcs.1984.supplement_1.12. [DOI] [PubMed] [Google Scholar]
  30. Philip M., Jamaluddin M., Sastry R. V., Chandra H. S. Nucleosome core histone complex isolated gently and rapidly in 2 M NaCl is octameric. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5178–5182. doi: 10.1073/pnas.76.10.5178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pickett-Heaps J. D., Tippit D. H., Porter K. R. Rethinking mitosis. Cell. 1982 Jul;29(3):729–744. doi: 10.1016/0092-8674(82)90435-4. [DOI] [PubMed] [Google Scholar]
  32. Rhodes D., Klug A. An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. Cell. 1986 Jul 4;46(1):123–132. doi: 10.1016/0092-8674(86)90866-4. [DOI] [PubMed] [Google Scholar]
  33. Rieder C. L. The formation, structure, and composition of the mammalian kinetochore and kinetochore fiber. Int Rev Cytol. 1982;79:1–58. doi: 10.1016/s0074-7696(08)61672-1. [DOI] [PubMed] [Google Scholar]
  34. Ris H., Witt P. L. Structure of the mammalian kinetochore. Chromosoma. 1981;82(2):153–170. doi: 10.1007/BF00286101. [DOI] [PubMed] [Google Scholar]
  35. Rosok M. J., Rohrschneider L. R. Increased phosphorylation of vinculin on tyrosine does not occur during the release of stress fibers before mitosis in normal cells. Mol Cell Biol. 1983 Mar;3(3):475–479. doi: 10.1128/mcb.3.3.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tahourdin C. S., Bustin M. Chromatin subunits elicit species-specific antibodies against nucleoprotein antigenic determinants. Biochemistry. 1980 Sep 16;19(19):4387–4394. doi: 10.1021/bi00560a002. [DOI] [PubMed] [Google Scholar]
  37. Telzer B. R., Haimo L. T. Decoration of spindle microtubules with Dynein: evidence for uniform polarity. J Cell Biol. 1981 May;89(2):373–378. doi: 10.1083/jcb.89.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thomas J. O., Kornberg R. D. An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2626–2630. doi: 10.1073/pnas.72.7.2626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Valdivia M. M., Brinkley B. R. Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes. J Cell Biol. 1985 Sep;101(3):1124–1134. doi: 10.1083/jcb.101.3.1124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. West M. H., Bonner W. M. Histone 2A, a heteromorphous family of eight protein species. Biochemistry. 1980 Jul 8;19(14):3238–3245. doi: 10.1021/bi00555a022. [DOI] [PubMed] [Google Scholar]
  42. West M. H., Bonner W. M. Histone 2B can be modified by the attachment of ubiquitin. Nucleic Acids Res. 1980 Oct 24;8(20):4671–4680. doi: 10.1093/nar/8.20.4671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zieve G. W., Turnbull D., Mullins J. M., McIntosh J. R. Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole. Nocodazole accumulated mitotic cells. Exp Cell Res. 1980 Apr;126(2):397–405. doi: 10.1016/0014-4827(80)90279-7. [DOI] [PubMed] [Google Scholar]

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