Cargando…

Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes

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 pr...

Descripción completa

Detalles Bibliográficos
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1985
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2113728/
https://www.ncbi.nlm.nih.gov/pubmed/3897244
_version_ 1782140253285384192
collection PubMed
description 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.
format Text
id pubmed-2113728
institution National Center for Biotechnology Information
language English
publishDate 1985
publisher The Rockefeller University Press
record_format MEDLINE/PubMed
spelling pubmed-21137282008-05-01 Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes J Cell Biol Articles 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. The Rockefeller University Press 1985-09-01 /pmc/articles/PMC2113728/ /pubmed/3897244 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes
title Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes
title_full Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes
title_fullStr Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes
title_full_unstemmed Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes
title_short Fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes
title_sort fractionation and initial characterization of the kinetochore from mammalian metaphase chromosomes
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2113728/
https://www.ncbi.nlm.nih.gov/pubmed/3897244