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1.
Neocentromeres are functional centromeres formed in chromosome regions outside the normal centromere domains and are found in an increasing number of mitotically stable human marker chromosomes in both neoplastic and non-neoplastic cells. We describe here the formation of a neocentromere in a previously undescribed chromosomal region at 1p32-->p36.1 in an oligospermic patient. Cytogenetic GTL banding analysis and the absence of detectable fluorescence in situ hybridisation (FISH) signals using telomeric probes indicate the marker to be a ring chromosome. The chromosome is negative for CBG banding and is devoid of detectable centromeric alpha satellite and its associated centromere protein CENP-B, suggesting activation of a neocentromere within the 1p32-36.1 region. Functional activity of the neocentromere is shown by the retention of the ring chromosome in 97% of the patient's lymphocytes and 100% of his cultured fibroblasts, as well as by the presence of key centromere binding proteins CENP-E, CENP-F, and INCENP. These results indicate that in addition to CENP-A, CENP-C, and CENP-E described in earlier studies, neocentromere activity can further be defined by CENP-F and INCENP binding. Our evidence suggests that neocentromere formation constitutes a viable mechanism for the mitotic stabilisation of acentric ring chromosomes.  相似文献   

2.
Normal human centromeres contain large tandem arrays of alpha-satellite DNA of varying composition and complexity. However, a new class of mitotically stable marker chromosomes which contain neocentromeres formed from genomic regions previously devoid of centromere activity was described recently. These neocentromeres are fully functional yet lack the repeat sequences traditionally associated with normal centromere function. We report here a supernumerary marker chromosome derived from the short arm of chromosome 20 in a patient with manifestations of dup(20p) syndrome. Detailed cytogenetic, FISH, and polymorphic microsatellite analyses indicate the de novo formation of the marker chromosome during meiosis or early postzygotically, involving an initial chromosome breakage at 20p11.2, followed by an inverted duplication of the distal 20p segment due to rejoining of sister chromatids and the activation of a neocentromere within 20p12. This inv dup(20p) marker chromosome lacks detectable centromeric alpha-satellite and pericentric satellite III sequences, or centromere protein CENP-B. Functional activity of the neocentromere is evidenced by its association with 5 different, functionally critical centromere proteins: CENP-A, CENP-C, CENP-E, CENP-F, and INCENP. Formation of a neocentromere on human chromosome 20 has not been reported previously and in this context represents a new mechanism for the origin of dup(20p) syndrome.  相似文献   

3.
Centromere and kinetochore proteins have a pivotal role in centromere structure, kinetochore formation and sister chromatid separation. However, the molecular architecture and the precise dynamic function of the centromere-kinetochore complex during mitosis remain poorly understood. Here we report the isolation and characterization of human CENP-H. Confocal microscopic analyses of HeLa cells with anti-human CENP-H-specific antibody demonstrated that CENP-H colocalizes with inner kinetochore plate proteins CENP-A and CENP-C in both interphase and metaphase. CENP-H was present outside centromeric heterochromatin, where CENP-B is localized, and inside the kinetochore corona, where CENP-E is localized during prometaphase. Furthermore, CENP-H was detected at neocentromeres, but not at inactive centromeres in stable dicentric chromosomes. In vitro binding assays of human CENP-H with centromere-kinetochore proteins suggest that the CENP-H binds to itself and MCAK, but not to CENP-A, CENP-B or CENP-C. CENP-H multimers were observed in cells in which both FLAG-tagged CENP-H and hemagglutinin-tagged CENP-H were expressed. These results suggest that CENP-H multimers localize constitutively to the inner kinetochore plate and play an important fundamental role in organization and function of the active human centromere-kinetochore complex.  相似文献   

4.
Normal human centromeres contain large tandem arrays of α-satellite DNA of varying composition and complexity. However, a new class of mitotically stable marker chromosomes which contain neocentromeres formed from genomic regions previously devoid of centromere activity was described recently. These neocentromeres are fully functional yet lack the repeat sequences traditionally associated with normal centromere function. We report here a supernumerary marker chromosome derived from the short arm of chromosome 20 in a patient with manifestations of dup(20p) syndrome. Detailed cytogenetic, FISH, and polymorphic microsatellite analyses indicate the de novo formation of the marker chromosome during meiosis or early postzygotically, involving an initial chromosome breakage at 20p11.2, followed by an inverted duplication of the distal 20p segment due to rejoining of sister chromatids and the activation of a neocentromere within 20p12. This inv dup(20p) marker chromosome lacks detectable centromeric α-satellite and pericentric satellite III sequences, or centromere protein CENP-B. Functional activity of the neocentromere is evidenced by its association with 5 different, functionally critical centromere proteins: CENP-A, CENP-C, CENP-E, CENP-F, and INCENP. Formation of a neocentromere on human chromosome 20 has not been reported previously and in this context represents a new mechanism for the origin of dup(20p) syndrome. Am. J. Med. Genet. 85:403–408, 1999. © 1999 Wiley-Liss, Inc.  相似文献   

5.
Human centromeres contain large arrays of alpha-satellite DNA that are thought to provide centromere function. The arrays show size and sequence variation, but the extent to which extremely low levels of this DNA can occur on normal centromeres is unclear. Using a set of chromosome-specific alpha-satellite probes for each of the human chromosomes, we performed interphase fluorescence in situ hybridization (FISH) in a population-screening study. Our results demonstrate that extreme reduction of chromosome-specific alpha satellite is unusually common in chromosome 21 (screened with the alphaRI probe), with a prevalence of 3.70%, compared to < or =0.12% for each of chromosomes 13 and 17, and 0% for the other chromosomes. No analphoid centromere was identified in >17,000 morphologically normal chromosomes studied. All of the low-alphoid centromeres are fully functional as indicated by their mitotic stability and binding to centromere proteins CENP-B, CENP-C, and CENP-E. Sensitive metaphase FISH analysis of the low-alphoid chromosome 21 centromeres established the presence of residual alphaRI as well as other non-alphaRI alpha-satellite DNA suggesting that centromere function may be provided by (1) the residual alphaRI DNA, (2) other non-alphaRI alpha-satellite sequences, (3) a combination of 1 and 2, or (4) an activated neocentromere DNA. The low-alphoid centromeres, in particular those of chromosome 21, should provide unique opportunities for the study of the evolution and the minimal DNA requirement of the human centromere.  相似文献   

6.
Using combined immunofluorescence and fluorescence in situ hybridization (FISH) analysis we have extensively characterized the proteins associating with two different homologue human neocentromeres at interphase and prometaphase/metaphase, and compared these directly with those found with normal human centromeres. Antisera to CENP-A, CENP-B, CENP-C, CENP-E, CENP-F, INCENP, CLIP-170, dynein, dynactin subunits p150 (Glued) and Arp1, MCAK, Tsg24, p55CDC, HZW10, HBUB1, HBUBR1, BUB3, MAD2, ERK1, 3F3/2, topoisomerase II and a murine HP1 homologue, M31, were used in immuno-fluorescence experiments in conjunction with FISH employing specific DNA probes to clearly identify neocentromeric DNA. We found that except for the total absence of CENP-B binding, neocentromeres are indistinguishable from their alpha satellite-containing counterparts in terms of protein composition and distribution. This suggests that the DNA base of a potential centromeric locus is of minimal importance in determining the overall structure of a functional kinetochore and that, once seeded, the events leading to functional kinetochore formation occur independently of primary DNA sequence.  相似文献   

7.
CENP-A, a centromere-specific histone H3, is conserved throughout eukaryotes, and formation of CENP-A chromatin defines the active centromere region. Here, we report the isolation of CENP-A chromatin from HeLa interphase nuclei by chromatin immunoprecipitation using anti-CENP-A monoclonal antibody, and systematic identification of its components by mass spectrometric analyses. The isolated chromatin contained CENP-B, CENP-C, CENP-H, CENP-I/hMis 6 and hMis 12 as well as CENP-A, suggesting that the isolated chromatin may represent the centromere complex (CEN-complex). Mass spectrometric analyses of the CEN-complex identified approximately 40 proteins, including the previously reported centromere proteins and the proteins of unknown function. In addition, we unexpectedly identified a series of proteins previously reported to be related to functions other than chromosome segregation, such as uvDDB-1, XAP8, hSNF2H, FACTp180, FACTp80/SSRP1, polycomb group proteins (BMI-1, RING1, RNF2, HPC3 and PHP2), KNL5 and racGAP. We found that uvDDB-1 was actually localized to the centromeric region throughout cell cycle, while BMI-1 was transiently co-localized with the centromeres in interphase. These results give us new insights into the architecture, dynamics and function of centromeric chromatin in interphase nuclei, which might reflect regulation of cell proliferation and differentiation.  相似文献   

8.
The centromere is an essential functional domain responsible for the correct inheritance of eukaryotic chromosomes during cell division. Eukaryotic centromeres include the highly conserved centromere-specific histone H3 variant, CENP-A, which has provided a powerful tool for investigating the recruitment of centromere components. However, the trigger that targets CENP-A to a specific genomic locus during centromere assembly remains unknown. Although, on rare occasions, CENP-A chromatin may assemble at non-centromeric DNA, all normal human centromeres are assembled and maintained on alpha-satellite (alphoid) DNA. The importance of alphoid DNA and CENP-B binding sites (CENP-B boxes), typical of normal human centromere DNA configurations, has been demonstrated through their requirement in de novo centromere assembly and Human Artificial Chromosome (HAC) assays. Mechanisms to link the centromere tightly to specific genomic sequences exist in humans and the two yeast species.  相似文献   

9.
Centromeres are essential cis-elements on chromosomes that are crucial for the stable transmission of genetic information during mitotic and meiotic cell divisions. Different species employ a variety of centromere configurations, from small genetically defined centromeres in budding yeast to holocentric centromeres that occupy entire chromosomes in Caenorhabditis, yet the incorporation of nucleosomes containing the essential centromere-specific histone H3 variant CENP-A is a common feature of centromeres in all eukaryotes. In vertebrates and fungi, CENP-A is specifically deposited at centromeres by a conserved chaperone, called HJURP or Scm3, respectively. Surprisingly, homologs of these proteins have not been identified in Drosophila, Caenorhabditis, or plants. How CENP-A is targeted to centromeres in these organisms is not known. The Drosophila centromeric protein CAL1, found only in the Diptera genus, is essential for CENP-A localization, is recruited to centromeres at a similar time as CENP-A, and interacts with CENP-A in both chromatin and pre-nucleosomal complexes, making it a strong candidate for a CENP-A chaperone in this lineage. Here, we discuss the conservation and evolution of this essential centromere factor and report the identification of a "Scm3-domain"-like region with similarity to the corresponding region of fungal Scm3 as well as a shared predicted alpha-helical structure. Given the lack of common ancestry between Scm3 and CAL1, we propose that an optimal CENP-A binding region was independently acquired by CAL1, which caused the loss of an ancestral Scm3 protein from the Diptera lineage.  相似文献   

10.
Robertsonian translocations are the most common structural dicentricrearrangements in humans. The stability of these dicentricsis attributed to the inactivation of one centromere by mechanismswhich are currently unknown. The presence and amounts of centromericproteins (CENPs) differ between the centromeres of the few dicentricswhich have been studied, providing a limited understanding ofthe protein components necessary for centromeric function. However,CENP-C previously has been observed only at the active centromeresin two dicentric chromosomes. In the present investigation,the presence and localizations of several centromeric antigens,CENP-B, -C and -E, have been determined in 12 dicentric Roberisoniantranslocations. Each translocation was studied initially usingin situ hybridization with  相似文献   

11.
We have expressed an EGFP-CENP-A fusion protein in human cells in order to quantitate the level of CENP-A incorporated into normal and variant human centromeres. The results revealed a 3.2-fold difference in the level of CENP-A incorporation into α-satellite repeat DNA-based centromeres, with the Y centromere showing the lowest level of all normal human chromosomes. Identification of individual chromosomes revealed a statistically significant, though not absolute, correlation between chromosome size and CENP-A incorporation. Analysis of three independent neocentromeres revealed a significantly reduced level of CENP-A compared to normal centromeres. Truncation of a neocentric marker chromosome to produce a minichromosome further reduced CENP-A levels, indicating a remodelling of centromeric chromatin. These results suggest a role for increased CENP-A incorporation in the faithful segregation of larger chromosomes and support a model of centromere evolution in which neocentromeres represent ancestral centromeres that, through adaptive evolution, acquire satellite repeats to facilitate the incorporation of higher numbers of CENP-A containing nucleosomes, thereby facilitating the assembly of larger kinetochore structures.  相似文献   

12.
Kinetochore is morphologically defined as a trilaminated, highly differentiated structure at the primary constriction of mitotic chromosomes. This subcellular organella is assumed to be composed of DNA and proteins. Immunoelectron microscopy has shown that centromere autoantigens CENP-C and CENP-B localize to the kinetochore inner plate and the underlying centromeric region respectively. We previously indicated that both are DNA-binding proteins that constitute centromeric heterochromatin throughout the cell cycle. Here, we tried to elucidate how these molecules are involved in the kinetochore/centromere organization in vivo by analyzing their morphological behavior in nuclei. Using immunofluorescence microscopy, we found that CENP-C remained as round discrete dots, whereas CENP-B displayed larger surrounding materials. To examine the CENP-C-binding locus on the genome, we prepared highly extended chromatin fibers and performed simultaneous immunofluorescence and fluorescence in situ hybridization. We obsreved that centromeric alphoid DNA, targeted by CENP-B, was highly dispersed, whereas the CENP-C antigen persisted as small dots well situated on the fibers. These features reminded us of the ‘ball and cup’ structure that had been presented for ‘prekinetochore’. We propose here that CENP-C constitutes a ‘kinetochore organizing center’ tightly associating with DNA, whereas CENP-B heterochromatin offers the solid support during kinetochore maturation. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

13.
Autoantibody reactivity to centromere proteins CENP-A, CENP-B and CENP-C was examined in 58 patients with systemic sclerosis (SSc). 218 first degree relatives and 22 spouses, HLA class II typing for HLA-DRB1 and HLA-DQA1 was performed by restriction fragment length polymorphism (RFLP) analysis in 50 families, and HLA-DRB1, HLA-DQA1 and HLA-DQB1 typing was performed by olignucleolitde typing in 44 families. Eleven probands and two relatives had ACA. The two relatives with ACA also had SSc. One relative was an identical twin sister of a pro band with ACA and the other relative was a sister of a proband with ACA. All ACA-positive probands and relatives were female, and all recognized CENP-A, CENP-B and CENP-C. The presence of at least one HLA-DQB1 allele not coding for leueine at position 26 of the first domain appeared necessary, although not sufficient for the generation of ACA, Therefore within SSc families ACA is strongly associated with female gender and disease phenotype, and is at least in part genetically determined.  相似文献   

14.
While the formation of a dicentric chromosome often leads to chromosome instability, human dicentric Robertsonian translocations usually remain stable. To investigate the basis for this stability, we have examined the centromeres of 15 structurally dicentric rob(13q14q) Robertsonian translocations using immunofluorescence and fluorescence in situ hybridization (FISH). The immunofluorescence detection of centromere protein C (CENP-C) was used as a marker for centromere function as CENP-C seems to play an essential role in kinetochore structure and stability and was previously shown to be absent from inactive centromeres. In all 15 translocation-containing cell lines, CENP-C was confined to only one of the centromeres of the translocation in a fraction of the cells analyzed. This suggests that centromere inactivation commonly occurs on dicentric Robertsonian translocations and may serve as one mechanism allowing for their stability. However, in the majority of the translocations (12 out of 15), a portion of the cells analyzed displayed CENP-C immunofluorescence at both centromeres, suggesting that both centromeres were active and that the translocation was functionally dicentric. The percentage of cells with CENP-C at both centromeres ranged from 2% to 82%. These results support the hypothesis that the close proximity of two functional centromeres on Robertsonian translocations allows them to remain stable.  相似文献   

15.
Multicentric chromosomes are often found in tumor cells and certain cell lines. How they are generated is not fully understood, though their stability suggests that they are non-functional during chromosome segregation. Growing evidence has implicated microtubule motor proteins in attachment of chromosomes to the mitotic spindle and in chromosome movement. To better understand the molecular basis for the inactivity of centromeres associated with secondary constrictions, we have tested these structures by immunofluorescence microscopy for the presence of motor complexes and associated proteins. We find strong immunoreactivity at the active, but not inactive, centromeres of prometaphase multicentric chromosomes using antibodies to the cytoplasmic dynein intermediate chains, three components of the dynactin complex (dynamitin, Arp1 and p150 Glued ), the kinesin-related proteins CENP-E and MCAK and the proposed structural and checkpoint proteins HZW10, CENP-F and Mad2p. These results offer new insight into the assembly and composition of both primary and secondary constrictions and provide a molecular basis for the apparent inactivity of the latter during chromosome segregation.   相似文献   

16.
Two cases of marker chromosomes derived from a non-centromeric location were studied to determine the characteristics of these markers with respect to the presence of functional centromeres and whether an associated phenotype could be described. The markers were characterized by fluorescence in situ hybridization and centromeric protein studies. Assessments were done to identify clinical features. Case 1 is a girl referred at age 1.5 years with swirly areas of hyperpigmentation, bilateral preauricular pits, hypotonia, developmental delay, and seizures. Case 2 is a male first evaluated as a newborn and then later during the first year of life. He had streaky hypopigmentation, right preauricular pit, accessory nipples, postaxial polydactyly, asymmetric cerebral ventricles, duplicated right kidney, a right pulmonary artery stenosis, and seizures. Mosaicism for an extra marker from the 3qter region was present in both cases. Both markers had a constriction near one end and were C-band negative. Centromeric protein studies indicated absence of CENP-B, presence of CENP-C (data for case 1 only), and presence of CENP-E. Marker chromosomes were thus identified with a chromosomal origin far from their usual centromeric region and yet appeared to have functional centromeres. These two cases did not permit a specific clinical phenotype to be ascribed to the presence of tetrasomy for 3q26.2 approximately 3q27.2-->3qter.  相似文献   

17.
The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.  相似文献   

18.
Anticentromere antibodies (ACA) present in a high percentage of patients with complete or incomplete CREST scleroderma, and which are presently used in the diagnosis of this disease, also appear in some primary Raynaud's phenomenon patients. Three principal centromeric antigens, CENP-A, CENP-B and CENP-C, have been described as reacting with the sera of these individuals. We attempt to determine whether or not a correlation between the presence of ACA and serum reactivity against one or more of these peptides could be established, and have observed that CENP-A, but not CENP-B or CENP-C, is specifically recognized by all patients sera tested. The fact that this reactivity is clearly detectable at very high serum dilutions, thus eliminating other non-specific interference, suggests that anti-CENP-A activity might be useful in the diagnosis of patients with CREST-associated Raynaud's phenomenon.  相似文献   

19.
哺乳动物的几种培养细胞的动粒蛋白的研究   总被引:6,自引:1,他引:5  
杨新林  李时 《解剖学报》1994,25(4):385-389,T010
用来自人类硬皮病中一种称为CREST综合征的自身免疫性疾病病人的抗动粒抗体血清和蛋白质免疫印迹技术,研究了来源于人(HL60细胞,HeLa细胞以及外周血淋巴细胞)和小鼠(NIH3T3细胞,转化的LA90细胞,TC3H10T1/2细胞)的6种培养细胞的动粒蛋白。用间接免疫荧光法显示此种ACA血清对哺乳动物培养细胞的动粒有特异的染色反应。此种血清在上述细胞中共识别出6种动粒蛋白,我们命名为AA1(17  相似文献   

20.
We observed an analphoid marker chromosome stable through cell division in a 16-year-old girl with developmental delay, short stature, limb contractures, and ovaries containing multiple cysts. She also developed myasthenia gravis at 15 years. The marker chromosome, present in 75% of metaphases (and in 90% of transformed lymphoblastoid cells), was C-band negative, and had no pan alpha-satellite sequences detectable by fluorescence in situ hybridization (FISH). The 8q origin of the marker was determined by use of subtelomeric probes and was confirmed by chromosome 8 painting probes. The marker was shown to be an inversion duplication of 8q when subtelomeric, telomeric, and c-myc FISH probes hybridized to both ends of the marker. The karyotype was 47,XX,+inv dup(8)(qter--> q23.3::q23.3-->[neocen]-->qter), resulting in tetrasomy for 8q23.3qter. The parents had normal karyotypes. Centromeric proteins CENP-C and CENP-E were present, but alpha associated centromere protein CENP-B was absent at a position defining a neocentromere.  相似文献   

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