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PMID: 1730770 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Centromere protein B assembles human centromeric alpha-satellite DNA at the 17-bp sequence, CENP-B box.

The Journal of cell biology ·Vol. 116 ·No. 3 ·1992-02-00 ·Pages 585-96

Muro Y, Masumoto H, Yoda K, Nozaki N, Ohashi M, Okazaki T

Abstract

We purified 15,000-fold from HeLa cell nuclear extract the centromere antigen that reacts specifically with the 17-bp sequence, designated previously as CENP-B box, in human centromeric alpha-satellite (alphoid) DNA by a two-step procedure including an oligonucleotide affinity column. The purified protein was identified as the centromere protein B (CENP-B) by its mobility on SDS-PAGE (80 kD), and reactivities to a monoclonal antibody raised to CENP-B (bacterial fusion protein) and to anticentromere sera from patients with autoimmune diseases. Direct binding by CENP-B of the CENP-B box sequence in the alphoid DNA has been proved using the purified CENP-B by DNA mobility-shift assay, Southwestern blotting, and DNase I protection analysis. The binding constant of the antigen to the CENP-B box sequence is 6 x 10(8) M-1. DNA mobility-shift assays indicated that the major complex formed between the CENP-B and the DNA contains two DNA molecules, suggesting the importance of the CENP-B/CENP-B box interaction in organization of higher ordered chromatin structures in the centromere and/or kinetochore. Location of DNA binding and dimerization domains in CENP-B was discussed based on the DNA mobility-shift assays performed with a protein fraction containing intact and partial cleavage products of CENP-B.

MeSH Terms
Autoantigens Base Sequence Centromere/metabolism Centromere Protein B Chromosomal Proteins, Non-Histone/isolation & purification,metabolism DNA, Satellite/chemistry,metabolism DNA-Binding Proteins HeLa Cells Humans Molecular Sequence Data
Chemicals
Autoantigens CENPB protein, human Centromere Protein B Chromosomal Proteins, Non-Histone DNA, Satellite DNA-Binding Proteins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Muro Y
Department of Dermatology, Nagoya University School of Medicine, Japan.
Masumoto H
Yoda K
Nozaki N
Ohashi M
Okazaki T
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.116.3.585
Published
1992-02-00
Pages
585-96
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2289308
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.116.3.585 PMC Full Text View journal details
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