Localization of the centromere protein CENP-B using scleroderma sera and evidence for a role in centromere survival.
OBJECTIVE
To determine whether centromeric CENP A, B and C proteins play a role in centromere survival.


METHODS
Sixteen anti-centromere sera from scleroderma patients were used. The most common reactivity demonstrated by Western blot was anti-CENP-A, followed by anti-CENP-B and -C, in that order. The reactivity of these sera with HEp-2 cells was studied using an indirect immunofluorescence assay with and without prior digestion by a DNase, Aspergillus nuclease and the restriction endonucleases Bam HI, Hind III, and Eco RI. CENP-B was purified using affinity chromatography and anti-CENP-B antibody. The interaction between CENP-B and the CENP-B box was evaluated using immunoprecipitation. Precipitates containing alphaDNA were amplified using a PCR method with specific primers for the CENP-B box.


RESULTS
None of the nucleases altered the fluorescence pattern. PCR amplification showed that CENP-B adsorbed on a Sepharose-4B/anti-CENP-B antibody column retained alphaDNA satellites. No retention was seen in the absence of CENP-B.


CONCLUSIONS
CENP-B protects alphaDNA from digestion by nucleases and prevents DNase or restriction enzyme digestion from affecting the morphology and location of centromeres. CENP-B may promote and maintain joining of DNA satellites in the centromere.
Barbosa-Cisneros
O
O
Department of Immunology, CBE, Universidad Autónoma de Zacatecas, México.
Avalos-Diaz
E
E
Vera
O
O
Moreno
J
J
Herrera-Esparza
R
R
D000974:Antibodies, Antinuclear / Q000032:analysis; D001324:Autoantigens; D002503:Centromere / Q000502:physiology*; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone / Q000097:blood* / Q000494:pharmacology; D004247:DNA / Q000187:drug effects; D004268:DNA-Binding Proteins; D004347:Drug Interactions; D004950:Esterases / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D012595:Scleroderma, Systemic / Q000097:blood*; D014018:Tissue Distribution
D000974:Antibodies, Antinuclear; D001324:Autoantigens; C497926:CENPB protein, human; D051936:Centromere Protein B; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D004247:DNA; D004950:Esterases