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.