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PMID: 3897244 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes.

The Journal of cell biology ·Vol. 101 ·No. 3 ·1985-09-00 ·Pages 1124-34

Valdivia MM, Brinkley BR

Abstract

We have partially isolated the kinetochore and associated centromeric structures from mammalian metaphase chromosomes. Human autoantibodies from scleroderma CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) patients were used as immunofluorescent probes to monitor fractionation. The procedure includes digestion of total chromosomal DNA with micrococcal nuclease, dehistonization with heparin, and dissociation of the remaining material with detergent and urea. We used a density gradient (metrizamide) to obtain an enriched fraction of stained material (kinetochore). When examined by electron microscopy, the kinetochore fraction is seen to contain numerous small immunoperoxidase-positive masses which are morphologically similar to the centromere/kinetochore region of intact metaphase chromosomes. The particulate fraction that contains kinetochore components represents less than 5% of total chromosomal proteins and contains less than 1% of total DNA. Two polypeptides of 18 and 80 kD were identified as kinetochore antigens by immunoblotting with CREST antiserum. In this paper we discuss the distribution of these kinetochore polypeptides with the associated centromeric chromatin.

MeSH Terms
Animals Autoantibodies/immunology Cell Fractionation Cell Line Centromere/analysis,ultrastructure Chromosomes/analysis,ultrastructure Cricetinae Female Fluorescent Antibody Technique Humans Metaphase Microscopy, Electron Molecular Weight Scleroderma, Systemic/immunology
Chemicals
Autoantibodies
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Valdivia M M
Brinkley B R
References (34)
34 references, click to expand
  1. Microtubule initiation at kinetochores and centrosomes in lysed mitotic cells. Inhibition of site-specific nucleation by tubulin antibody.
    J Cell Biol. 1979 Aug;82(2):585-91 PMID: 479317
  2. Ribonucleoprotein staining of centrioles and kinetochores in newt lung cell spindles.
    J Cell Biol. 1979 Jan;80(1):1-9 PMID: 84814
  3. Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro.
    J Cell Biol. 1980 Jan;84(1):151-9 PMID: 7350167
  4. Acid-soluble nuclear proteins of the testis during spermatogenesis in the winter flounder. Loss of the high mobility group proteins.
    J Biol Chem. 1980 Mar 25;255(6):2533-9 PMID: 7358686
  5. Autoantibody to centromere (kinetochore) in scleroderma sera.
    Proc Natl Acad Sci U S A. 1980 Mar;77(3):1627-31 PMID: 6966403
  6. Structure of the mammalian kinetochore.
    Chromosoma. 1981;82(2):153-70 PMID: 7227036
  7. Two-dimensional hybridization mapping of nucleosomes. comparison of DNA and protein patterns.
    J Mol Biol. 1981 Mar 5;146(3):287-304 PMID: 7265232
  8. Kinetochore structure, duplication, and distribution in mammalian cells: analysis by human autoantibodies from scleroderma patients.
    J Cell Biol. 1981 Oct;91(1):95-102 PMID: 7298727
  9. Autoantibodies to nucleosomal proteins: antibodies to HMG-17 in autoimmune diseases.
    Science. 1982 Mar 5;215(4537):1245-7 PMID: 6460317
  10. Yeast centromere DNA is in a unique and highly ordered structure in chromosomes and small circular minichromosomes.
    Cell. 1982 Jun;29(2):305-17 PMID: 6288253
  11. Tubulin nucleation and assembly in mitotic cells: evidence for nucleic acids in kinetochores and centrosomes.
    Cell Motil. 1980;1(1):1-15 PMID: 6184168
  12. The nuclear matrix: three-dimensional architecture and protein composition.
    Cell. 1982 Jul;29(3):847-58 PMID: 7151171
  13. Histone deacetylase. Association with a nuclease resistant, high molecular weight fraction of HeLa cell chromatin.
    J Biol Chem. 1983 Mar 25;258(6):3726-34 PMID: 6833227
  14. Chinese hamster metaphase chromosomes isolated under physiological conditions. A partial characterization of associated non-histone proteins and protein cores.
    Exp Cell Res. 1983 Mar;144(1):1-14 PMID: 6840197
  15. Human anticentromere antibodies: distribution, characterization of antigens, and effect on microtubule organization.
    Cell. 1983 Nov;35(1):331-9 PMID: 6605200
  16. The kinetochore is part of the metaphase chromosome scaffold.
    J Cell Biol. 1984 Jan;98(1):352-7 PMID: 6707096
  17. 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 PMID: 6383665
  18. Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes.
    Cell. 1984 Nov;39(1):111-22 PMID: 6386181
  19. Protein measurement with the Folin phenol reagent.
    J Biol Chem. 1951 Nov;193(1):265-75 PMID: 14907713
  20. The structure of the kinetochore in meiosis and mitosis in Urechis eggs.
    Exp Cell Res. 1965 Sep;39(2):643-57 PMID: 5891318
  21. The fine structure of the kinetochore of a mammalian cell in vitro.
    Chromosoma. 1966;19(1):28-43 PMID: 5912064
  22. The ultrastructure and spatial organization of the metaphase kinetochore in mitotic rat cells.
    J Ultrastruct Res. 1967 Jul;19(1):19-44 PMID: 5339062
  23. A new method for the rapid isolation of chromosomes, mitotic apparatus, or nuclei from mammalian fibroblasts at near neutral pH.
    Exp Cell Res. 1970 Mar;59(3):469-78 PMID: 5461579
  24. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
    Nature. 1970 Aug 15;227(5259):680-5 PMID: 5432063
  25. Fine structure of kinetochore in Indian muntjac.
    Exp Cell Res. 1971 Jul;67(1):97-110 PMID: 5569205
  26. The mechanism of C- and G-banding of chromosomes.
    Exp Cell Res. 1973 Mar 15;77(1):469-83 PMID: 4120442
  27. Presence of actin during chromosomal movement.
    Proc Natl Acad Sci U S A. 1975 Jun;72(6):2451-5 PMID: 1094471
  28. Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.
    Proc Natl Acad Sci U S A. 1975 Oct;72(10):4023-7 PMID: 1060085
  29. Localization of tubulin in the mitotic apparatus of mammalian cells by immunofluorescence and immunoelectron microscopy.
    Chromosoma. 1977 Apr 19;60(3):223-35 PMID: 322970
  30. Solubilization of chromatin with heparin and the isolation of nuclear membranes.
    Methods Cell Biol. 1977;15:163-75 PMID: 327198
  31. Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: nucleosomal proteins associated with a highly repetitive mammalian DNA.
    Proc Natl Acad Sci U S A. 1977 Aug;74(8):3297-301 PMID: 269392
  32. Isolation of a protein scaffold from mitotic HeLa cell chromosomes.
    Proc Natl Acad Sci U S A. 1977 Nov;74(11):4937-41 PMID: 270727
  33. Selective digestion of mouse metaphase chromosomes.
    Chromosoma. 1978 Apr 25;66(3):259-68 PMID: 648280
  34. 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):4350-4 PMID: 388439
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.101.3.1124
Published
1985-09-00
Pages
1124-34
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2113728
Subset
IM
Grants
NCI NIH HHS · CA-23022 · United States
External Links
PubMed source DOI: 10.1083/jcb.101.3.1124 PMC Full Text View journal details
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