ࡱ> ܥhWedhhhhhhhbbbbbb l@bg1(3IXtghFG ghh^hhϡ|VҢhhhh8Cytogenetic integrado traam de cromossomo brao 4S de A. thaliana: organizao estrutural de boto de heterochromatic e regio de centromere. Fransz PF, Armstrong S, de Jong JH, Parnell LD, furgo Drunen C, Decano C, Zabel P, Bisseling T, Jones GH, Escola de Cincias Biolgicas, Universidade de Birmingham, Reino Unido. fransz@ipk-gatersleben.de [Medline registram em processo] Ns construmos um cytogenetic integrado traa de cromossomo brao 4S de thaliana de Arabidopsis. O mapa mostra as posies detalhadas de vrios multicopy e sucesses sem igual relativo para euchromatin e segmentos de heterochromatin. Uma anlise quantitativa do mapa posiciona em fases de meiotic subseqentes revelou um padro notvel de variao de espao e temporal em condensao de chromatin para euchromatin e heterochromatin. Por exemplo, a regio de centromere consiste em trs domnios com propriedades estruturais, moleculares, e funcionais distinguveis. Ns tambm caracterizamos um boto de heterochromatic distinto de aproximadamente 700 kb que acomodam um tandem repita e alguns dispersaram pericentromere-especfico repete. Alm disso, nossos dados provem evidncia para um evento de inverso que localizou de novo sucesses de pericentromeric para uma posio intersticial e resulta no boto de heterochromatic. PMID: 10676818, UI: 20139988 Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres. Aagaard L, Schmid M, Warburton P, Jenuwein T Research Institute of Molecular Pathology (IMP), The Vienna Biocenter, Dr Bohrgasse 7, A-1030 Vienna, Austria. jenuwein@nt.imp.univie.ac.at [Record supplied by publisher] Centromeres of eukaryotes are frequently associated with constitutive heterochromatin and their activity appears to be coregulated by epigenetic modification of higher order chromatin. Recently, we isolated murine (Suv39h1) and human (SUV39H1) homologues of the dominant Drosophila suppressor of position effect variegation Su(var)3-9, which is also related to the S. pombe silencing factor Clr4. We have shown that mammalian Su(var)3-9 homologues encode novel centromeric proteins on metaphase-arrested chromosomes. Here, we describe a detailed analysis of the chromatin distribution of human SUV39H1 during the cell cycle. Although there is significant heterochromatic overlap between SUV39H1 and M31 (HP1(&bgr;)) during interphase, mitotic SUV39H1 displays a more restricted spatial and temporal association pattern with metaphase chromosomes than M31 (HP1(&bgr;)), or the related HP1(&agr;) gene product. SUV39H1 specifically accumulates at the centromere during prometaphase but dissociates from centromeric positions at the meta- to anaphase transition. In addition, SUV39H1 selectively associates with the active centromere of a dicentric chromosome and also with a neocentromere. Interestingly, SUV39H1 is shown to be a phosphoprotein with modifications at serine and, to a lesser degree, also at threonine residues. Whereas SUV39H1 steady-state protein levels appear constant during the cell cycle, two additional phosphorylated isoforms are detected in mitotic extracts. This intriguing localisation and modification pattern would be consistent with a regulatory role(s) for SUV39H1 in participating in higher order chromatin organisation at mammalian centromeres. PMID: 10671371 A polymorphic L1 retroposon insertion in the centromere of the human Y chromosome. Santos FR, Pandya A, Kayser M, Mitchell RJ, Liu A, Singh L, Destro-Bisol G, Novelletto A, Qamar R, Mehdi SQ, Adhikari R, Knijff P, Tyler-Smith C Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK, [Record supplied by publisher] We have identified a novel polymorphic L1 retroposon insertion, designated LY1, in the centromeric alphoid array of the human Y chromosome. The element belongs to the transpositionally active Ta subset and its presence is compatible with normal centromere function. It was found at highest frequency in China, where it accounts for 23% of the Han sample, and was present at low frequencies in the surrounding areas, but was not found at all outside Asia. Chromosomes carrying LY1 show considerable microsatellite diversity, suggesting an ancient origin for the lineage at approximately 10 000 years ago (with wide confidence limits), but only limited subsequent migration. PMID: 10655552 Proc Natl Acad Sci U S A 2000 Feb 1;97(3):1148-53 Early disruption of centromeric chromatin organization in centromere protein A (Cenpa) null mice. Howman EV, Fowler KJ, Newson AJ, Redward S, MacDonald AC, Kalitsis P, Choo KH The Murdoch Institute, Royal Children's Hospital, Flemington Road, Parkville 3052, Australia. [Medline record in process] Centromere protein A (Cenpa for mouse, CENP-A for other species) is a histone H3-like protein that is thought to be involved in the nucleosomal packaging of centromeric DNA. Using gene targeting, we have disrupted the mouse Cenpa gene and demonstrated that the gene is essential. Heterozygous mice are healthy and fertile whereas null mutants fail to survive beyond 6.5 days postconception. Affected embryos show severe mitotic problems, including micronuclei and macronuclei formation, nuclear bridging and blebbing, and chromatin fragmentation and hypercondensation. Immunofluorescence analysis of interphase cells at day 5.5 reveals complete Cenpa depletion, diffuse Cenpb foci, absence of discrete Cenpc signal on centromeres, and dispersion of Cenpb and Cenpc throughout the nucleus. These results suggest that Cenpa is essential for kinetochore targeting of Cenpc and plays an early role in organizing centromeric chromatin at interphase. The evidence is consistent with the proposal of a critical epigenetic function for CENP-A in marking a chromosomal region for centromere formation. PMID: 10655499, UI: 20122586 Chromosoma 1999 Dec;108(7):401-11 The mitotic centromeric protein MEI-S332 and its role in sister-chromatid cohesion. LeBlanc HN, Tang TT, Wu JS, Orr-Weaver TL Whitehead Institute for Biomedical Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02142, USA. [Medline record in process] Faithful segregation of sister chromatids during cell division requires properly regulated cohesion between the sister centromeres. The sister chromatids are attached along their lengths, but particularly tightly in the centromeric regions. Therefore specific cohesion proteins may be needed at the centromere. Here we show that Drosophila MEI-S332 protein localizes to mitotic metaphase centromeres. Both overexpression and mutation of MEI-S332 increase the number of apoptotic cells. In mei-S332 mutants the ratio of metaphase to anaphase figures is lower than wild type, but it is higher if MEI-S332 is overexpressed. In chromosomal squashes centromeric attachments appear weaker in mei-S332 mutants than wild type and tighter when MEI-S332 is overexpressed. These results are consistent with MEI-S332 contributing to centromeric sister-chromatid cohesion in a dose-dependent manner. MEI-S332 is the first member identified of a predicted class of centromeric proteins that maintain centromeric cohesion. PMID: 10654079, UI: 20119575 J Cell Biol 2000 Jan 24;148(2):233-8 Polyploidy induces centromere association. Martinez-Perez E, Shaw PJ, Moore G John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom. [Medline record in process] Many species exhibit polyploidy. The presence of more than one diploid set of similar chromosomes in polyploids can affect the assortment of homologous chromosomes, resulting in unbalanced gametes. Therefore, a mechanism is required to ensure the correct assortment and segregation of chromosomes for gamete formation. Ploidy has been shown to affect gene expression. We present in this study an example of a major effect on a phenotype induced by ploidy within the Triticeae. We demonstrate that centromeres associate early during anther development in polyploid species. In contrast, centromeres in diploid species only associate at the onset of meiotic prophase. We propose that this mechanism provides a potential route by which chromosomes can start to be sorted before meiosis in polyploids. This explains previous reports indicating that meiotic prophase is shorter in polyploids than in their diploid progenitors. Even artificial polyploids exhibit this phenotype, suggesting that the mechanism must be present in diploids, but only expressed in the presence of more than one diploid set of chromosomes. J Rheumatol 2000 Jan;27(1):142-8 Circulating anticentromere CENP-A and CENP-B antibodies in patients with diffuse and limited systemic sclerosis, systemic lupus erythematosus, and rheumatoid arthritis. Russo K, Hoch S, Dima C, Varga J, Teodorescu M Department of Microbiology/Immunology, College of Medicine, University of Illinois at Chicago, USA. [Medline record in process] OBJECTIVE: To determine the disease sensitivity and specificity of testing for autoantibodies against 2 of the 3 main human centromere antigenic components, CENP-A and CENP-B (recombinant, expressed in baculovirus). METHODS: ELISA with CENP-A and CENP-B antigens were used to test 45 sera showing a centromere pattern by immunofluorescence (IFA) and sera from 96 patients with systemic sclerosis (SSc), subdivided into diffuse (dSSc) and limited (lSSc) forms. For controls, the same tests were performed on sera from 100 patients with rheumatoid arthritis (RA), 100 with systemic lupus erythematosus (SLE), and 50 random blood donors. Sera from all the patients with SSc were also tested for the presence of anti-Scl70 antibody by ELISA (bovine antigen), and for pattern and titer by IFA (HEp-2 cells). RESULTS: Of the 45 IFA positive sera, 93% were positive for anti-CENP-A and 91% for anti-CENP-B. There was a very good quantitative correlation between the antibody levels against these 2 centromere components (r = 0.597; p<0.001). Anti-CENP-A and B were found in 48% of patients with lSSc, and in 11% and 9%, respectively, of those with dSSc. The difference in the frequency of anti-CENP-A between the 2 patient groups was significant (chi-squared, p<0.001). Similar levels of anticentromere staining pattern by IFA were observed for these 2 groups. Anti-Scl70 was elevated in 8% of lSSc and 25% of dSSc patients; this difference was also significant (chi-squared, p = 0.02). Neither CENP-A nor CENP-B reacted with IgG from SSc patients containing anti-Scl70. The frequency of abnormal levels in patients with SLE and RA was, respectively, 11% and 3% for anti-CENP-A and 4% and 3% for anti-CENP-B. The reaction of IgG from SLE and RA patients with CENP-A was not inhibited by histone H3, i.e., it was not due to recognition of the histone-like domain in CENP-A. Thus, when 96 SSc patients were compared to 200 patients with RA and SLE, the disease specificity of anti-CENP-A and B was 93% and 96.5%, respectively. CONCLUSION: In addition to IFA, ELISA tests for CENP-A and CENP-B yield results with similar sensitivity and specificity for the diagnosis of SSc. CENP-A and CENP-B are primarily associated with lSSc. In SSc the autoantibody response is directed simultaneously and with similar amplitude against these 2 components of the centromere structure, whereas in other autoimmune diseases the response is directed mainly against one of the 2 components. Arthritis Rheum 1996 Aug;39(8):1355-61 The centromere kinesin-like protein, CENP-E. An autoantigen in systemic sclerosis. Rattner JB, Rees J, Arnett FC, Reveille JD, Goldstein R, Fritzler MJ Department of Anatomy, University of Calgary, Alberta, Canada. OBJECTIVE. Autoantibodies directed against centromere proteins (CENPs) are a serologic feature in some patients with systemic sclerosis (SSc). Previous studies have focused on autoantibodies to CENPs A, B, and C. CENP-E is a recently described 312-kd protein that also localizes to the centromere. Therefore, we studied the presence of autoantibodies to recombinant CENP-E in patients with SSc. METHODS. Sixty sera from patients with the SSc spectrum of diseases were screened for the presence of autoantibodies against CENP-E, by indirect immunofluorescence and immunoblotting using recombinant CENP-E protein. HLA class II alleles were determined by DNA oligotyping. RESULTS. Among the SSc sera, 15 of 60 (25%) demonstrated antibody reactivity with recombinant CENP-E, and 14 of these 15 sera (93%) had antibodies directed against another CENP. Anti-CENP-E was seen in 13 of 30 sera with anti-CENP (43%). All patients with anti-CENP-E had a limited form of SSc, known as the CREST variant (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). When patients with anti-CENPs A, B, or C were compared with patients with anti-CENP-E, no unique clinical features in the anti-CENP-E positive group were identified. Ninety-three percent of the patients with anti-CENP-E had HLA-DQB1 alleles that had polar amino acids at position 26 (primarily DQB1*05), similar to patients with other CENP autoantibodies. CONCLUSION. Antibodies to CENP-E are common in patients with SSc, and are seen in higher frequency in sera from patients with a limited form, or CREST variant, of the disease. PMID: 8702444, UI: 96320238 Clin Immunol 1999 Feb;90(2):182-9 Anticentromere autoantibodies in patients without Raynaud's disease or systemic sclerosis. Vazquez-Abad D, Grodzicky T, Senecal JL Department of Medicine, University of Connecticut School of Medicine, Farmington, Connecticut, 06030-1310, USA. Anticentromere autoantibodies (ACA) are associated with Raynaud's disease and systemic sclerosis (SSc). ACA usually bind at least one of three major centromere proteins (CENPs), particularly CENP-B. We identified 16 patients with ACA who do not have Raynaud's disease or SSc. The objective of this study was to determine whether these 16 ACA differ in antigenic specificity from the ACA found in patients with Raynaud's disease or SSc. Binding of these serum ACA was tested using competition experiments with recombinant CENP-B, and native centromere proteins from HEp-2 cells and HeLa nuclear extracts in ELISAs, immunoblots, and indirect immunofluorescence assays. The ACA from these 16 patients are strikingly different from those obtained from patients who have Raynaud's disease or SSc. Only 5 of the 16 index sera (31.25%) bound CENP-B from two or more different sources by at least two methods. Six of these 16 sera (37.5%) did not bind CENP-B on ELISA, and 8 of 16 (43.75%) did not bind CENP-B on immunoblots. Three sera did not bind CENP-B either by ELISA or immunoblots. Of the 13 sera that bound CENP-B, their patterns of binding to CENP-B strong Cytogenet Cell Genet 1993;63(3):169-75 Cytogenetic survey in systemic sclerosis: correlation of aneuploidy with the presence of anticentromere antibodies. Jabs EW, Tuck-Muller CM, Anhalt GJ, Earnshaw W, Wise RA, Wigley F Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD 21287-3914. Previous cytogenetic studies of patients with systemic sclerosis have obtained conflicting results regarding the presence of chromosomal anomalies. We studied 38 patients and 15 controls to determine whether these inconsistencies were due to differences in the subgroups of patients who were studied. Because many patients with systemic sclerosis produce autoantibodies to protein antigens that have been implicated in chromosome structure and function, we further hypothesized that the presence of these autoantibodies might correlate with the presence of chromosomal anomalies. Patients were classified into clinical subgroups based on the extent of their disease. Their sera were assayed for autoantibodies to topoisomerase I and centromere proteins (CENP-A, CENP-B, and CENP-C) by immunoblotting. Cytogenetic analyses for aneuploidy and chromosome breaks were performed. Anticentromere antibody positive (ACA+) patients had significantly more aneuploidy than either ACA negative (ACA-) patients or controls (P = 0.041). Although the patient group, when considered as a whole, had significantly greater aneuploidy than the control group (P < 0.005), patients who were ACA--did not have more aneuploidy than the controls had. Patients with Type I disease (sclerodactyly), the majority of whom were ACA+, also had significantly more aneuploidy than did either the controls or patients with Type III (diffuse) disease, most of whom were ACA- (P < 0.005). ACA+ patients also had more chromatid breaks than the controls had (P < 0.05). The correlation between the presence of ACAs and chromosomal aneuploidy suggests that aneuploidy may be the result of nondisjunction secondary to centromeric dysfunction. In support of this hypothesis, the ACA+ patients who had antibodies to CENP-C exhibited more chromosomal aneuploidy than did either anti-CENP-A or anti-CENP-B positive patients (P < 0.048). Unlike CENP-A and CENP-B, which are present at both functional and inactivated centromeres, CENP-C is present at the kinetochore of functional centromeres. PMID: 8387418, UI: 93251825 J Invest Dermatol 1991 Aug;97(2):378-80 Purification of a human centromere antigen (CENP-B) and application of DNA immunoprecipitation to quantitative assay for anti-CENP-B antibodies. Muro Y, Masumoto H, Okazaki T, Ohashi M Department of Dermatology, Nagoya University School of Medicine, Japan. Centromere protein-B (CENP-B) was purified from HeLa nuclear extract by using a combination of Q-sepharose ion change and oligonucleotide sepharose column chromatography. CENP-B was purified more than 10,000 times and was analyzed by immunoblotting and DNA immunoprecipitation. Purified CENP-B was used as an antigen to develop DNA immunoprecipitation for rapid and specific detection of anti-CENP-B antibody in human sera. In this analysis, none of the tested sera immunoprecipitated DNA alone. All of the 40 anticentromere antibody (ACA)-positive sera immunoprecipitated CENP-B-alphoid DNA fragment complex, whereas 10 healthy control sera and 10 other autoantibody positive sera did not. Five of the ACA-positive sera, which did not show reactivity of CENP-B in immunoblotting analysis, immunoprecipitated CENP-B-DNA complex. In some sera antigenicity to CENP-B may be weakened by denaturation. ly suggested that they bind different epitopes within the CENP-B antigen. Independently of their binding to CENP-B, these sera reacted mainly with minor CENP antigens detected by HeLa nuclear extracts. We have identified unusual ACA not associated with Raynaud's disease or SSc. Copyright 1999 Academic Press. PMID: 10080829, UI: 99180789 Clin Invest Med 1997 Oct;20(5):308-19 High frequency of neoplasia in patients with autoantibodies to centromere protein CENP-F. Rattner JB, Rees J, Whitehead CM, Casiano CA, Tan EM, Humbel RL, Conrad K, Fritzler MJ Department of Anatomy, University of Calgary, Alta. rattner@acs.ucalgary.ca OBJECTIVE: To study the clinical features of patients with autoantibodies to centromere protein CENP-F and the frequency of CENP-F autoantibodies in patients with various diseases. DESIGN: Retrospective clinical and serologic study. METHODS: Thirty-six patients with anti-CENP-F were identified by a characteristic pattern of indirect immunofluorescence (IIF) on HEp-2 cells. Fifty patients with melanoma, 50 with breast cancer, 10 with lung cancer, 354 with systemic sclerosis, 120 with systemic lupus erythematosus and 50 with rheumatoid arthritis were also studied. Recombinant proteins were produced from 5 CENP-F cDNA clones representing amino acids 2192-3317 (p-F1), 5561-7126 (p-F2), 5892-6883 (p-F3), 7538-10,116 (p-F4) and 9242-10,096 (p-F5). The presence of CENP-F antigen was studied in a breast carcinoma cell line, cryosections of breast carcinoma, normal breast tissue and tonsils. RESULTS: Twenty-two of 36 patients with CENP-F antibodies had neoplasms; breast (9/22) and lung (5/22) cancer were the most common diagnoses. Thirty-three sera were available for further study; when tested for reactivity to the recombinant peptides, the sera of 21 of 21 patients with neoplasms and 5 of 12 patients with other diseases bound the C-terminal p-F4 peptide. When the terminal third of the p-F4 peptide (p-F5) was studied, a significant difference in pattern of reactivity was not detected. By comparison, the frequency of reactivity with peptides representing other domains of CENP-F was less than that with p-F4 (p-F2 > p-F3 > p-F1). CENP-F autoantibodies were not found in any of the control sera from patients with systemic lupus erythematosus, rheumatoid arthritis or systemic sclerosis or in unselected sera from various malignancies. CENP-F antigens were identified in breast carcinoma tissue but were rarely observed in normal tissues. CONCLUSIONS: A high proportion of individuals with CENP-F antibodies have neoplasia, and there is a bias among their sera for reactivity with determinants in the carboxy terminal domain of CENP-F. CENP-F antigens appear to be highly expressed in malignant tissues. PMID: 9336656, UI: 97477723 An antigenic determinant on human centromere protein B (CENP-B) available for production of human-specific anticentromere antibodies in mouse. Sugimoto K, Migita H, Hagishita Y, Yata H, Himeno M Department of Agricultural Chemistry, University of Osaka Prefecture, Japan. Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromere heterochromatin throughout the cell cycles. Some components of mammalian centromeres including CENP-B are target antigens for autoimmune disease patients, often those with scleroderma. Recent isolations of CENP-B genes from human and mouse suggested that CENP-B was highly conserved among mammals. From the previous analysis of the reactivity of patient anticentromere sera, two autoepitopes have been located on the DNA binding domain at the amino-terminal region. The amino acid sequences for both the epitopes are perfectly conserved in the two species, human and mouse. In this study, to identify a human-specific antigenic determinant, the remaining two epitopes were further located in separate carboxyl-terminal regions of human CENP-B. Although the amino acid sequence of one epitope is identical to that of the corresponding region in mouse CENP-B, the other has a less homologous sequence. To confirm that the latter epitope was available for production of human-specific anticentromere antibodies, mice were immunized with the recombinant human CENP-B product. One serum that exclusively stained human centromere structure, but not that of other mammals, was identified in the immunofluorescence microscopic observation. The epitope analysis showed that the less conserved one was recognized by this serum. These results suggested that the corresponding region defines the antigenic determinants for the species specificity. PMID: 1376639, UI: 92298401 Cytogenet Cell Genet 1993;63(1):54-8 Published erratum appears in Cytogenet Cell Genet 1994;65(1-2):135 Anticentromere antibody specific to human cells directed against the CENP-B autoantigen. Bejarano LA, Bolivar J, Valdivia MM Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad de Cadiz, Puerto Real, Spain. We describe the generation of a new antipeptide antibody that binds to the centromeric region of human mitotic chromosomes. This antibody was raised against a synthetic peptide corresponding to the 481-493 amino acid sequence of the human CENP-B autoantigen. Immunofluorescence analysis revealed that this anti-CENP-B serum showed an identical pattern to the human CREST anticentromere autoantibody in both mitotic cells and interphase nuclei. Immunoblotting showed that this antibody reacts with the recombinant human CENP-B autoantigen, indicating that it is directed to the 80-kDa centromere polypeptide. We have used this serum to determine, by indirect immunofluorescence, whether CENP-B is conserved in different mammalian species. Surprisingly, the human antipeptide antibody does not react with the centromeric proteins of cultured mouse, hamster, or Indian muntjac cells. Because the CENP-B gene has been cloned in human and mouse, our results suggest that the CENP-B epitope used as an immunogen in this study is not ubiquitous in mammalian cells, and that we have most probably established a monospecific antibody to the human centromere. PMID: 7680607, UI: 93193478 J Rheumatol 1998 Jul;25(7):1419-24 Anti-centromere autoantibody in a patient evolving from a lupus/Sjogren's overlap to the CREST variant of scleroderma. Ford AL, Kurien BT, Harley JB, Scofield RH Department of Medicine, University of Oklahoma Health Science Center, Department of Veterans Affairs Medical Center, Oklahoma City, USA. We characterized the development of the anti-centromere antibody in a patient prior to the development of CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) symptoms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting (IgG and IgM) of cellular extracts enriched for centromere antigens and indirect immunofluorescence were used to study the anti-centromere immune response. The sera recognized 3 centromere antigens with molecular masses 18,000 (CENP-A), 50,000 (CENP-D), and 80,000 (CENP-B). For CENP-A, IgM was present before the appearance of the IgG response. Anti-CENP-D revealed an IgM response that decreased over time but no IgG, while CENP-B showed an IgG response that strengthened and then weakened over time. The appearance of an anti-centromere nuclear fluorescence pattern correlated with the appearance of IgG anti-CENP-A. Signs and symptoms typical of CREST began about 4 years after antibodies to centromere antigens were found. The development of the CREST syndrome in our patient was preceded by the appearance of anti-centromere autoantibodies. For at least one of the antigens (CENP-A), there was an immunoglobulin class switch from IgM to IgG. Chromosoma 1989 Jun;98(1):1-12 Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Earnshaw WC, Ratrie H 3d, Stetten G Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205. We have screened for the presence of two centromere autoantigens, CENP-B (80 kDa) and CENP-C (140 kDa) at the inactive centromere of a naturally occurring stable dicentric chromosome using specific antibodies that do not cross-react with any other chromosomal proteins. In order to discriminate between the active and inactive centromeres on this chromosome we have developed a modification of the standard methanol/acetic acid fixation procedure that allows us to obtain high-quality cytological spreads that retain antigenicity with the anti-centromere antibodies. We have noted three differences in the immunostaining patterns with specific anti-CENP-B and CENP-C antibodies. (1) The amount of detectable CENP-B varies from chromosome to chromosome. The amount of CENP-C appears to be more or less the same on all chromosomes. (2) CENP-B is present at both active and inactive centromeres of stable dicentric autosomes. CENP-C is not detectable at the inactive centromeres. (3) While immunofluorescence with anti-CENP-C antibodies typically gives two discrete spots, staining with anti-CENP-B often appears as a single bright bar connecting both sister centromeres. This suggests that while CENP-C may be confined to the outer centromere in the kinetochore region, CENP-B may be distributed throughout the entire centromere. Our data suggest that CENP-C is likely to be a component of some invariant chromosomal substructure, such as the kinetochore. CENP-B may be involved in some other aspect of centromere function, such as chromosome movement or DNA packaging. PMID: 2475307, UI: 89356164 J Rheumatol 1990 Aug;17(8):1042-7 The heterogeneity of anticentromere antibodies in immunoblotting analysis. Muro Y, Sugimoto K, Okazaki T, Ohashi M Department of Dermatology, Nagoya University School of Medicine, Aichi, Japan. We tested anticentromere antibody positive sera from 37 patients by immunoblotting techniques. Three antigenic polypeptides were recognized when immunoblotted against protein extracts from HeLa cell nuclei or from chromosomal segments enriched with centromere region. These were a 17 kDa (CENP-A recognized by 34 sera), an 80 kDa (CENP-B recognized by 33 sera), and a 140 kDa polypeptide (CENP-C recognized by 26 sera). There was no disease specific pattern of antigenic polypeptides, although Raynaud's phenomenon was frequent in patients with anti-CENP-B reactivities (p less than 0.01). The heterogeneity of the anticentromere antibody response in Japanese patients shows anticentromere antibody may not be a disease specific autoantibody and diagnostic marker. PMID: 2213779, UI: 91012435 J Dermatol 1992 Oct;19(10):584-91 The clinical expression in anticentromere antibody-positive patients is not specified by the epitope recognition of CENP-B antigen. Muro Y, Sugimoto K, Himeno M, Ohashi M Department of Dermatology, Nagoya University School of Medicine, Japan. Centromere protein B (CENP-B), which is an alphoid DNA binding protein, is the target antigen in autoimmune disease patients (often with scleroderma). From our previous analysis of the reactivity of anticentromere sera, four independent epitopes were identified on recombinant CENP-B. The anticentromere sera displayed heterogeneity in their patterns of reactivity to the four epitopes. We have investigated to what extent this heterogeneity of the target autoepitope on CENP-B accounts for the clinical diversity of anticentromere antibody (ACA)-positive patients. A major autoepitope, epitope I, was recognized by all 40 ACA-positive sera; however, the other three epitopes were recognized differently from case to case. We could not find any significant correlation between the reactivity to CENP-B autoepitopes and the clinical presentation of ACA-positive patients. There was considerable clinical diversity, even among the nine patients showing specificity for the single major autoepitope. In conclusion, we found that, although ACA-positive patients were both clinically and immunologically heterogeneous, in most respects the clinical expression appeared to be independent of the reactivity to the CENP-B autoepitope, a finding which suggests that identification of the target epitope of CENP-B is unlikely to assist in the clinical classification of the disease in ACA-positive patients. The identification of multiple B cell epitopes on CENP-B is consistent with the concept that the self-antigen drives the antibody response. However, factors other than CENP-B autoepitope specificity must determine the clinical expression of ACA responses. PMID: 1283396, UI: 93147348 J Cell Biol 2000 Jan 24;148(2):233-8 Polyploidy induces centromere association. Martinez-Perez E, Shaw PJ, Moore G John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom. Many species exhibit polyploidy. The presence of more than one diploid set of similar chromosomes in polyploids can affect the assortment of homologous chromosomes, resulting in unbalanced gametes. Therefore, a mechanism is required to ensure the correct assortment and segregation of chromosomes for gamete formation. Ploidy has been shown to affect gene expression. We present in this study an example of a major effect on a phenotype induced by ploidy within the Triticeae. We demonstrate that centromeres associate early during anther development in polyploid species. In contrast, centromeres in diploid species only associate at the onset of meiotic prophase. We propose that this mechanism provides a potential route by which chromosomes can start to be sorted before meiosis in polyploids. This explains previous reports indicating that meiotic prophase is shorter in polyploids than in their diploid progenitors. Even artificial polyploids exhibit this phenotype, suggesting that the mechanism must be present in diploids, but only expressed in the presence of more than one diploid set of chromosomes. Blood 2000 Mar 1;95(5):1608-1615 Spatial associations of centromeres in the nuclei of hematopoietic cells: evidence for cell-type-specific organizational patterns. Alcobia I, Dilao R, Parreira L Institute of Histology and Embryology, Lisbon Medical School, and the Nonlinear Dynamics Group, IST Department of Physics, Lisbon, Portugal. [Record supplied by publisher] It is believed that the 3-dimensional organization of centromeric heterochromatin in interphase may be of functional relevance as an epigenetic mechanism for the regulation of gene expression. Accordingly, a likely possibility is that the centromeres that spatially associate into the heterochromatic structures (chromocenters) observed in the G1 phase of the cell cycle will differ in different cells. We sought to address this issue using, as a model, the chromocenters observed in quiescent normal human hematopoietic cells and primary fibroblasts. To do this, we analyzed the spatial relationships among different human centromeres in 3-D preserved cells using nonisotopic in situ hybridization and confocal microscopy. We showed quantitatively that chromocenters in all cell types do indeed represent nonrandom spatial associations of certain centromeres. Furthermore, the observed patterns of centromere association indicate that the chromocenters in these cell types are made of different combinations of specific centromeres, that hematopoietic cells are strikingly different from fibroblasts as to the composition of their chromocenters and that centromeres in peripheral blood cells appear to aggregate into distinct "myeloid" (present in monocytes and granulocytes) and "lymphoid" (present in lymphocytes) spatial patterns. These findings support the idea that the chromocenters formed in the nucleus of quiescent hematopoietic cells might represent heterochromatic nuclear compartments involved in the regulation of cell-type-specific gene expression, further suggesting that the spatial arrangement of centromeric heterochromatin in interphase is ontogenically determined during hematopoietic differentiation. 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