Molecular Cloning of cDNA for CENP-B, the Major Human Centromere Autoantigen

• Published in: The Journal of cell biology 1987-04, Vol.104 (4), p.817-829
• Main Authors: Earnshaw, William C., Sullivan, Kevin F., Machlin, Paula S., Cooke, Carol A., Kaiser, Donald A., Pollard, Thomas D., Rothfield, Naomi F., Cleveland, Don W.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Antibodies; Autoantigens - genetics; Base Sequence; Biological and medical sciences; Centromere Protein B; Centromeres; Chromosomal Proteins, Non-Histone; Chromosomes; Chromosomes, Human - ultrastructure; Cloning, Molecular; Complementary DNA; DNA; DNA - metabolism; DNA-Binding Proteins; Epitopes; Epitopes - analysis; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Generally accepted auditing standards; Genes. Genome; Histones; Humans; Kinetochores; Messenger RNA; Molecular and cellular biology; Molecular genetics; Rheumatic Diseases - genetics; Rheumatic Diseases - immunology; RNA, Messenger - genetics
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.104.4.817

We have isolated a series of overlapping cDNA clones for ∼95% of the mRNA that encodes CENP-B, the 80-kD human centromere autoantigen recognized by patients with anticentromere antibodies. The cloned sequences encode a polypeptide with an apparent molecular mass appropriate for CENP-B. This polypeptide and CENP-B share three non-overlapping epitopes. The first two are defined by monoclonal antibodies elicited by injection of cloned fusion protein. Epitope 1 corresponds to a major antigenic site recognized by the anticentromere autoantibody used to obtain the original clone. Epitope 2 is a novel one not recognized by the autoantibody. These epitopes were shown to be distinct both by competitive binding experiments and by their presence or absence on different subcloned portions of the fusion protein. The third independent epitope, recognized by a subset of anticentromere-positive patient sera, maps to a region substantially closer to the amino terminus of the fusion protein. DNA and RNA blot analyses indicate that CENP-B is unrelated to CENP-C, a 140-kD centromere antigen also recognized by these antisera. CENP-B is the product of a 2.9-kb mRNA that is encoded by a single genetic locus. This mRNA is far too short to encode a polypeptide the size of CENP-C. The carboxy terminus of CENP-B contains two long domains comprised almost entirely of glutamic and aspartic acid residues. These domains may be responsible for anomalous migration of CENP-B on SDS-polyacrylamide gels, since the true molecular mass of CENP-B is ∼65 kD, 15 kD less than the apparent molecular mass deduced from gel electrophoresis. Quite unexpectedly, immunofluorescence analysis using antibodies specific for CENP-B reveals that the levels of antigen vary widely between chromosomes.