Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen

Abstract

Abstract. 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 SDSpolyacrylamide 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.T HE structure and function of the centromere (CEN) t regions of mitotic chromosomes have recently become the focal point of investigations by workers in three diverse disciplines. First, cell biologists have known for a number of years that the centromere is both the site of sister chromatid pairing (the mechanism of which is currently unknown) and the site of mitotic spindle attachment. The latter site, the kinetochore, has been well-characterized at the ultrastructural level (5,(40)(41)(42). The kinetochore is a trilaminar plaque structure embedded in the chromatin at the surface of the chromosome. It is solidly anchored in the chromosomal infrastructure and is found in the chromosome scaffold fraction (13).Second, geneticists have long been interested in the role of cis-acting DNA sequences in the control of chromosomal K. E Sullivan's present address is

Clinical features of anti-chromo antibodies associated with anti-centromere antibodies

“…ACA commonly recognize the three chromosomal autoantigens 17 kD (CENP-A), 80 kD (CENP-B), and 140 kD (CENP-C) in immunoblotting analysis [6,7]. cDNA for these three antigens have been cloned [8][9][10][11], and of these, autoimmune responses against CENP-B have been studied most extensively [12][13][14][15][16].…”

“…Autoantibodies against these proteins did not cross-react to centromere antigens [8,20], were inactive in IIF to intact human cell, and failed to bind to centromere [17,18,20]. Therefore these antigens were classified as non-centromeric.…”

Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T

“…The HORs of all but the human Y chromosome contain a 17-bp motif called the CENP-B box for the specific recruitment of the centromere protein B, CENP-B (Fig. 1A) (7,8). Despite the accumulating knowledge of HOR organization, the repetitive nature of the α-satellite has hindered assembly of a contiguous centromere sequence for each chromosome (9) and has made it difficult to understand how the centromeric DNAs are organized at the primary constriction.…”

Host genome surveillance for retrotransposons by transposon-derived proteins

“…Human CENP-B protein, which facilitates centromere formation by binding to short repeats within centromeric alpha satellite DNA 14,15 , is derived from transposases of pogo DNA transposons 16,17 . CENP-B, highly conserved in mammals, has homologues in other systems 13,18 .…”