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PMID: 8756673 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Surprising deficiency of CENP-B binding sites in African green monkey alpha-satellite DNA: implications for CENP-B function at centromeres.

Molecular and cellular biology ·Vol. 16 ·No. 9 ·1996-09-00 ·Pages 5156-68

Goldberg IG, Sawhney H, Pluta AF, Warburton PE, Earnshaw WC

Abstract

Centromeres of mammalian chromosomes are rich in repetitive DNAs that are packaged into specialized nucleoprotein structures called heterochromatin. In humans, the major centromeric repetitive DNA, alpha-satellite DNA, has been extensively sequenced and shown to contain binding sites for CENP-B, an 80-kDa centromeric autoantigen. The present report reveals that African green monkey (AGM) cells, which contain extensive alpha-satellite arrays at centromeres, appear to lack the well-characterized CENP-B binding site (the CENP-B box). We show that AGM cells express a functional CENP-B homolog that binds to the CENP-B box and is recognized by several independent anti-CENP-B antibodies. However, three independent assays fail to reveal CENP-B binding sites in AGM DNA. Methods used include a gel mobility shift competition assay using purified AGM alpha-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencing of 76 AGM alpha-satellite monomers. Immunofluorescence studies reveal the presence of significant levels of CENP-B antigen dispersed diffusely throughout the nuclei of interphase cells. These experiments reveal a paradox. CENP-B is highly conserved among mammals, yet its DNA binding site is conserved in human and mouse genomes but not in the AGM genome. One interpretation of these findings is that the role of CENP-B may be in the maintenance and/or organization of centromeric satellite DNA arrays rather than a more direct involvement in centromere structure.

MeSH Terms
Animals Autoantigens Base Sequence Binding Sites Cell Line Centromere/metabolism,ultrastructure Centromere Protein B Chlorocebus aethiops/genetics Chromosomal Proteins, Non-Histone/metabolism,physiology DNA, Neoplasm/metabolism DNA, Satellite/genetics DNA-Binding Proteins/metabolism,physiology Fibroblasts/metabolism,ultrastructure HeLa Cells/metabolism Humans In Situ Hybridization, Fluorescence Mammals/genetics Mice Molecular Sequence Data Recombinant Fusion Proteins/metabolism Regulatory Sequences, Nucleic Acid Species Specificity Transfection
Chemicals
Autoantigens CENPB protein, human Cenpb protein, mouse Centromere Protein B Chromosomal Proteins, Non-Histone DNA, Neoplasm DNA, Satellite DNA-Binding Proteins Recombinant Fusion Proteins
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Goldberg I G
Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Sawhney H
Pluta A F
Warburton P E
Earnshaw W C
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.16.9.5156
Published
1996-09-00
Pages
5156-68
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC231516
Subset
IM
Grants
NIGMS NIH HHS · GM35212 · United States
External Links
PubMed source DOI: 10.1128/MCB.16.9.5156 PMC Full Text View journal details
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