Sequence of centromere separation: kinetochore formation and DNA replication in dicentric chromosomes showing premature centromere separation in rat cerebral cells

A subpopulation of rat cerebral endothelial cells, designated B1, exhibits an array of multicentric chromosomes. Because of the formation of bridges at anaphase, this cell population produced new types of multicentrics at every cell division. These chromosomes showed kinetochore proteins at every centromeric site and all centromeric regions replicated their DNA at the end of the S phase, more or less simultaneously. A new subpopulation of cells, designated B2, obtained from the original sample frozen at Wayne State University displayed several dicentrics. In contrast to B1 these chromosomes exhibit premature centromere separation as reported for mouse and human cell lines. These B2 dicentrics show only one site of kinetochore protein deposition. The timing of DNA replication around the centric region of prematurely separating centromere is also changed similar to the earlier reported premature DNA synthesis for mouse dicentrics. These observations suggest a universality of relationship between premature centromere separation, a lack of kinetochore formation, and early replication of the centric/pericentric DNA associated with these centromeres. The cause of sudden change from activity to inactivity of these chromosomes, though interesting, is not clear.     

Sequence of centromere separation: a mechanism for orderly separation of dicentrics

Stable dicentric chromosomes from three mouse cell lines (viz., SEWA Rec4, brain tumor, and L-cells), as well as a human t(9;11) line were analyzed for the sequence in which the two centromeres separate. At prometaphase, as well as in many cells at midmetaphase, the dicentrics express the two centromeres in the form of two primary constrictions. As the cell advances to late metaphase, one of the constrictions loosens the two chromatids so that eventually there is no connection between them. The other centromere stays intact during this period and separates into two units at the metaanaphase junction along with the rest of the genome. The centromere that separates prematurely (out-of-phase) usually is the same in a given dicentric. It is proposed that such a prematurely separating centromere does not function as active element during chromatid migration. Apparently, in dicentrics some sort of control is exerted to eliminate the functioning of one centromere. The nature of such control is not understood at this time. The mouse dicentrics "synthesize" only one kinetochore as definable by antikinetochore antibody studies.     

Nature of anaphase laggards and micronuclei in female cytokinesis-blocked lymphocytes

We used pancentromeric fluorescence in situ hybridization and X chromosome painting to characterize late anaphase aberrations in cultured (72 h) female lymphocytes in the presence of cytochalasin B (Cyt-B). Aberrant cells, mostly containing laggards, were very common (34.5%) among multipolar anaphases but fewer (5.4%) among bipolar anaphases. Characterization of the laggards showed that 75% were autosomes, 15% autosomal fragments and 10% X chromosomes in bipolar divisions; similar figures were obtained in multipolar cells. The X chromosome lagged behind more often than would be expected by chance (1/23), representing 12 and 7% of all lagging chromosomes in bipolar and multipolar divisions, respectively. Bipolar divisions contained more lagging autosomes but fewer lagging fragments and X chromosomes with Cyt-B than without it. Comparison of the frequencies of anaphase laggards and interphase micronuclei (MN) showed that lagging autosomes seldom form MN in bipolar divisions, 11% being micronucleated without Cyt-B and 8% with Cyt-B. In multipolar divisions, autosome laggards produced MN more often (35%) and were mainly responsible for the excessive MN frequency of multinucleate cells. Lagging acentric fragments frequently formed MN, with a higher efficiency in the presence of Cyt-B (65% bipolar, 58% multipolar) than in its absence (41%). X chromosome laggards were very easily micronucleated, half of them forming MN in untreated cells and seemingly all after Cyt-B treatment. Our findings suggest that most autosome laggards are merely delayed in their poleward movement, eventually being engulfed by the nucleus. Lagging fragments and X chromosomes are probably detached from the spindle and, therefore, preferentially form MN. X laggards are particularly efficiently micronucleated in Cyt-B-treated cells, perhaps because they stay further away from the poles in round cytokinesis-blocked anaphases than in normally elongated non-blocked anaphases.     

Sequence of centromere separation: effect of 5-azacytidine-induced epigenetic alteration

The factors which control the sequential separation of the various chromosomes in a genome at the meta-anaphase junction are not well understood. In genomes in which separation is correlated with the quantity of pericentric heterochromatin one factor appears to be the epigenetic nature, namely condensation, of pericentric heterochromatin. When we induced decondensation of pericentric heterochromatin in mouse cells with 10(-6), 4x 10(-6) and 6x10(-6) M 5-azacytidine (5-AC) for 8 h, it resulted in alteration of the sequence of centromere separation. The centromeres which lacked pericentric heterochromatin appeared not to have been affected because there could not be an epigenetic alteration induced by 5-AC. The major effect was on chromosomes with the largest quantity of pericentric heterochromatin. These chromosomes separated at significantly higher frequency than in the untreated population. We also treated human cells, in which separation does not depend upon the quantity of heterochromatin, with 2x10(-5) and 6x10(-6) M 5-AC for 5 and 8 h. Compared with the control, 5-AC treatment resulted in an increased frequency of separated centromeres of acrocentric chromosomes in relation to those of non-acrocentric chromosomes. In the control the acrocentric chromosomes are the last to separate; in the treated population there was almost random separation of the two types of chromosomes. This epigenetic alteration might be another factor which results in genesis of aneuploidy.     

Sequence of centromere separation: Occurrence,possible significance,and control

This review describes the existence of a phenomenon, sequential separation of centromeres, in mitotic cells of various species including both animals and plants. Critical observations at metaanaphase show that the centromeres of chromosomes in a given genome do not separate into two sister units randomly, but that there is a genetically controlled, nonrandom, species-specific sequence which is independent of the length of the chromosome or the position of the centromere. A stricter control appears to exist for late-separating than for early-separating chromosomes. At early stages of metaanaphase several chromosomes initiate onset of separation simultaneously or in rapid succession, but late-separating chromosomes are better defined in their sequential position. The effect of Colcemid on the sequence of separation is minimal. It is proposed that aneuploidy in humans and other organisms may result from out-of-phase separation of a given chromosome. With the exception of chromosome No. 16, it appears that very early- or very late-separating centromeres are involved in human trisomies more often than those in between.Perhaps one function of centromeric heterochromatin is the control of centromere separation. The amount of such chromatin shows a positive correlation with the timing of separation of the centromeres. Superimposed upon this quantitative influence is the qualitative aspect, as discussed for various genomes. This suggestion explains a lack of extremely large quantities of heterochromatin near the centromere. Its existence in the form of homogeneously staining regions distal to the centromere, as in some cancer cells or in sex chromosomes, seemingly has no influence on the separation of centromeres.A brief discussion of centromere separation errors in human disease is provided, and suggestions for further studies are made.     

Sequence of centromere separation: a possible role for repetitive DNA

The centromeres of a mitotic cell at the meta-anaphase junctionseparate in a non-random, genetically controlled sequence beforeanaphase migration ensues. In several, but not all, of the organismsstudied so far it appears that the timing of separation of acentromere into two visible units depends upon the quantityof pericentric heterochromatin. A critical analysis of thisparameter of cell cycle suggests that the sequence of centromereseparation is influenced by repetitive DNA present in the pericentricregion. In those cases with qualitatively uniform repetitiveDNA in centromeric regions, the chromosomes carrying lesserquantities separate earlier than those with greater amounts.However, the overall pattern of separation may be determinedby interactions between both quantitative and qualitative parameteresof the repetitive DNA. It has been suggested that repetitiveDNA has no transcrip-tional properties. It is probably onlya structural component which acts as a site for the accumulationof protein molecules synthesized by some locus not present inthe centromeric region. These proteins accumulate to saturatethe centromeric repetitive DNA resulting in a (trilaminar) structurecalled the kinetochore. The longer the stretch to be saturated,the larger would be the kinetochore-like structure. Once saturated,the centromere splits into two subunits. Premature separationresults in a lack of saturation and formation of an ‘immature’kinetochore. This may lead to chromosome malsegregation. Thus,indirectly, one property of repetitive DNA in the centromericregion appears to be the maintenance of diploidy.     

Sequence of centromere separation of mitotic chromosomes during human cellular aging

The sequence of centromere separation of mitotic chromosomes derived from lymphocytes of 17 individuals of various ages was studied. A comparison of the mean CSI (centromeric separation index) values for individual chromosomes in the complement indicated a significant difference between the older and the younger subjects only with regard to the chromosome nos. 6, 11, and 19 in both sexes and the X chromosome exclusively in the female. With age, chromosome no. 6 in both males and females and the X chromosome in females showed premature centromeric separation whereas chromosome nos. 11 and 19 in both males and females displayed delayed centromeric separation. Our study, therefore, indicates that changes in the sequence of centromeric separation of certain chromosomes do occur in lymphocytes during chronological aging in humans.     

Characterization of internal DNA-binding and C-terminal dimerization domains of human centromere/kinetochore autoantigen CENP-C in vitro: role of DNA-binding and self-associating activities in kinetochore organization

Human centromere protein C (CENP-C), a chromosomal component of the inner plate of kinetochores, was originally identified as one of the centromere auto-antigens. In a previous study, we showed that it possesses DNA-binding activity in vitro. Recently, centromere-binding activity was suggested at the C-terminal region in vivo. However, little is known about the role of CENP-C in kinetochore organization. Here, to characterize its biochemical properties, three separate antigenic regions of human CENP-C were expressed in Escherichia coli, affinity purified and used in South-western blotting and chemical cross-linking analyses. We found that the internal DNA-binding domain was composed of two kinds of elements: the core and two flanking stabilizing elements that support the activity. When cross-linked with disuccinimidyl suberate (DSS), the N-terminal region produced the ladder bands of dimerand tetramer: the C-terminal region exclusively produced the dimer band, whereas the internal region was not affected at all. Dimer formation at the C-terminus in the native state was also indicated by gel filtration and the presence of conformation-specific autoantibodies in the patient's sera. These results suggest that human CENP-C consists of three functional units required for kinetochore assembly: a putative N-terminal oligomerization domain, an internal DNA-binding domain and a C-terminal dimerization domain.This revised version was published online in November 2005 with corrections to the Cover Date.     

Sequence analysis of a functional Drosophila centromere

Centromeres are the site for kinetochore formation and spindle attachment and are embedded in heterochromatin in most eukaryotes. The repeat-rich nature of heterochromatin has hindered obtaining a detailed understanding of the composition and organization of heterochromatic and centromeric DNA sequences. Here, we report the results of extensive sequence analysis of a fully functional centromere present in the Drosophila Dp1187 minichromosome. Approximately 8.4% (31 kb) of the highly repeated satellite DNA (AATAT and TTCTC) was sequenced, representing the largest data set of Drosophila satellite DNA sequence to date. Sequence analysis revealed that the orientation of the arrays is uniform and that individual repeats within the arrays mostly differ by rare, single-base polymorphisms. The entire complex DNA component of this centromere (69.7 kb) was sequenced and assembled. The 39-kb "complex island" Maupiti contains long stretches of a complex A+T rich repeat interspersed with transposon fragments, and most of these elements are organized as direct repeats. Surprisingly, five single, intact transposons are directly inserted at different locations in the AATAT satellite arrays. We find no evidence for centromere-specific sequences within this centromere, providing further evidence for sequence-independent, epigenetic determination of centromere identity and function in higher eukaryotes. Our results also demonstrate that the sequence composition and organization of large regions of centric heterochromatin can be determined, despite the presence of repeated DNA.     

Sequence organization and evolutionary dynamics of Brachypodium-specific centromere retrotransposons

Brachypodium distachyon is a wild annual grass belonging to the Pooideae, more closely related to wheat, barley, and forage grasses than rice and maize. As an experimental model, the completed genome sequence of B. distachyon provides a unique opportunity to study centromere evolution during the speciation of grasses. Centromeric satellite sequences have been identified in B. distachyon, but little is known about centromeric retrotransposons in this species. In the present study, bacterial artificial chromosome (BAC)-fluorescence in situ hybridization was conducted in maize, rice, barley, wheat, and rye using B. distachyon (Bd) centromere-specific BAC clones. Eight Bd centromeric BAC clones gave no detectable fluorescence in situ hybridization (FISH) signals on the chromosomes of rice and maize, and three of them also did not yield any FISH signals in barley, wheat, and rye. In addition, four of five Triticeae centromeric BAC clones did not hybridize to the B. distachyon centromeres, implying certain unique features of Brachypodium centromeres. Analysis of Brachypodium centromeric BAC sequences identified a long terminal repeat (LTR)-centromere retrotransposon of B. distachyon (CRBd1). This element was found in high copy number accounting for 1.6 % of the B. distachyon genome, and is enriched in Brachypodium centromeric regions. CRBd1 accumulated in active centromeres, but was lost from inactive ones. The LTR of CRBd1 appears to be specific to B. distachyon centromeres. These results reveal different evolutionary events of this retrotransposon family across grass species.     

Induction of kinetochore positive and negative micronuclei in V79 cells by the alkylating agent diethylsulphate

The induction by diethylsulphate of micronuclei derived fromacentric fragments or from whole chromosomes was studied inChinese hamster V79 cells using autoantibodies from the serumof a scieroderma patient (CREST-syndrome) to detect centromere-kinetochorestructures. Centromere-contaimng micronuclei appeared earlyafter treatment and plateaued both earlier and at lower levelthan centromere-lacking micro-nuclei. The frequency of centromere-containingmicronuclei was in good agreement with that of mitotic chromosomedisplacement, suggesting that a high proportion of displacedchromosomes were transmitted to the cytoplasm of one of thetwo daughter cells, where they gave rise to micronuclei. Onthe contrary, centromere-lacking micronuclei were more frequentthan what could be expected from chromosome fragments observedin mitotic stages.     

Molecular cytogenetic evaluation of the mechanism of micronuclei formation induced by camptothecin,topotecan, and irinotecan

We used the conventional bone marrow micronucleus test complemented with the fluorescent in situ hybridization with the minor satellite DNA probe to investigate the mechanisms of induction of micronuclei in mice treated with camptothecin and its clinical antineoplastic analogues topotecan and irinotecan. All experiments were performed with male Swiss albino mice. Single doses of 1 mg/kg camptothecin or 0.6 mg/kg topotecan were injected intraperitoneally and bone marrow was sampled at 30 hr (camptothecin) or 24 hr (topotecan) after treatment. A dose of 60 mg/kg irinotecan was injected intravenously, once every fourth day for 13 days and bone marrow was sampled 24 hr after the last treatment. In animals treated with camptothecin, a total of 1.07% micronuclei were found and 70% of them were centromere‐negative, indicating their formation by DNA strand breaks and reflecting the predominant clastogenic activity of camptothecin. Exposure to topotecan and irinotecan yielded 1.71 and 0.83% micronuclei, respectively. About 52.7 and 48.8% of the induced micronuclei, respectively, were centromere‐positive, indicating their formation by whole chromosomes and reflecting the aneugenic activity of both compounds. Correspondingly, about 47.3 and 51.2% of the induced micronuclei, respectively were centromere‐negative, demonstrating that topotecan and irinotecan not only induce chromosome loss but also DNA strand breaks. Both the clastogenic and aneugenic potential of these drugs can lead to the development of secondary tumors and abnormal reproductive outcomes. Therefore, the clinical use of these agents must be weighed against the risks of secondary malignancies in cured patients and persistent genetic damage of their potential offspring. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Condensin function at centromere chromatin facilitates proper kinetochore tension and ensures correct mitotic segregation of sister chromatids

The condensin complex is essential for sister chromatid segregation in eukaryotic mitosis. Nevertheless, in budding yeast, condensin mutations result in massive mis-segregation of chromosomes containing the nucleolar organizer, while other chromosomes, which also contain condensin binding sites, remain genetically stable. To investigate this phenomenon we analyzed the mechanism of the cell-cycle arrest elicited by condensin mutations. Under restrictive conditions, the majority of condensin-deficient cells arrest in metaphase. This metaphase arrest is mediated by the spindle checkpoint, particularly by the spindle-kinetochore tension-controlling pathway. Inactivation of the spindle checkpoint in condensin mutants resulted in frequent chromosome non-disjunction, eliminating the bias in chromosome mis-segregation towards rDNA-containing chromosomes. The spindle tension defect in condensin-impaired cells is likely mediated by structural defects in centromere chromatin reflected by the partial loss of the centromere histone Cse4p. These findings show that, in addition to its essential role in rDNA segregation, condensin mediates segregation of the whole genome by maintaining the centromere structure in Saccharomyces cerevisiae.     

Parental genome separation and asynchronous centromere division in interspecific F1 hybrids inLathyrus

Chromosomes were studied in root-tip metaphase cells of several F₁ interspecificLathyrus hybrids including:L. hirsutus × L. cassius (H × C),L. cassius × L. hirsutus (C × H),L. cassius × L. odoratus (C × O), and their parents, all with 2n = 2x = 14. Two types of morphologically distinct centromeres were identified in the hybrids on the basis of the degree of contraction of the primary constriction. At least 12 well-defined centromeres were seen in all cells ofL. hirsutus, L. cassius andL. odoratus, and about 80% of cells had 14. The hybrids were more variable than the species. H × O contained between six and 14 welldefined centromeres, while cells of H × C, C × H and C × O all had seven well-defined and seven weakly defined centromeres. These were used as markers to plot their spatial disposition in two dimensions on metaphase spreads. In H × C, C × H and C × O the two types of centromeres showed a significant tendency to occupy two spatially distinct and concentrically arranged domains on the metaphase plate (P < 0.005). Owing to shortage of material subsequent work was restricted to H × C and C × H. Six or seven chromosomes of one parental genome were selectively labelled byin situ hybridization using biotinylated total genomic DNA from either parent as a probe. Moreover, there was a very strong correlation between centromere type and genomic origin (P < 0.001). Clearly the tendency for spatial segregation of parental genomes into concentrically arranged domains previously noted in several F₁ interspecific sexual hybrids between grasses can occur strongly in dicots. The present work onLathyrus also included the first comparative study of the spatial arrangement of parental genomes in reciprocal hybrids. Significantly,L. cassius chromosomes tended to surroundL. hirsutus chromosomes in both H × C and C × H. Thus, the polarity of concentric parental genome separation was not determined as a maternal effect.     

Coumarin inhibits micronuclei formation induced by benzo(a)pyrene in male but not female ICR mice.

Coumarin has been shown to be an effective inhibitor of carcinogenesis in rodents if given before and during the carcinogen treatment. We investigated the possibility that pretreatment with coumarin would inhibit the genotoxicity of benzo(a)pyrene (BP) in ICR mice as indicated by the bone marrow micronucleus test, a widely used in vivo test for genotoxicity. Our studies showed that pretreatment of male mice with doses of coumarin at 65 or 130 mg/kg/day for 1 week (with 1 day of no treatment at midweek) partially inhibited the genotoxicity of BP at a single intraperitoneal dose of 150 mg/kg. Time course experiments showed a decrease in induced micronuclei in the bone marrow at several time points after the BP treatment, thus indicating a true inhibition and not a lag in the induction of micronuclei. However, no inhibition in micronuclei formation was seen in female mice pretreated with the same doses of coumarin. Coumarin treatment alone did not induce micronuclei in either sex. Future studies are needed to analyze the mechanisms responsible for the difference noted between the sexes.     

Arsenic-induced micronuclei formation in mammalian cells and its counteraction by tea.

The Gangetic plain of West Bengal, India, has been engulfed by a disastrous environmental calamity of arsenic contamination of the ground water. Chronic arsenic toxicity caused by drinking arsenic-contaminated water has been one of the worst health hazards gradually affecting nine districts of West Bengal since the early 1980s. Over and above hyperpigmentation and keratosis,weakness, burning sensation of the eyes, swelling of the legs, liver fibrosis, chronic lung disease, gangrene of the toes, neuropathy, and skin cancer are other manifestations. Induction of cancer is frequently associated with DNA damage, changes in ploidy of cells, and non-random chromosome aberrations. Counteraction of these genotoxic and cytogenetic abnormalities with natural dietary polyphenols could be a useful strategy to combat arsenic-induced DNA damage and thereby cancer. A review of the literature showed that it is the antioxidant property of tea polyphenols that affords protection against various types of cancer. The present study was conducted to investigate whether the extracts of green tea and black tea (Darjeeling and Assam) as well as their polyphenols could ameliorate this arsenic-induced genotoxicity. The normal mammalian cell culture derived from male Chinese hamster lung fibroblast cells (V79) was used as the test system to assess the genotoxicity by micronucleus assay. The results showed that both green tea and black tea extracts have equal potential in modulating the arsenic-induced genotoxicity. This effect was perhaps induced by the constituent polyphenols present in green and black tea. In addition, the repair activity of the damaged cells was enhanced when treated with these tea extracts and their polyphenols. Thus, tea and its polyphenols may have a promising role in counteracting the devastating effects of arsenic.     

Polyurethane scaffold formation via a combination of salt leaching and thermally induced phase separation

Porous scaffolds have been made from two polyurethanes based on thermally induced phase separation of polymer dissolved in a DMSO/water mixture in combination with salt leaching. It is possible to obtain very porous foams with a very high interconnectivity. A major advantage of this method is that variables like porosity, pore size, and interconnectivity can be independently adjusted with the absence of toxic materials in the production process. The obtained compression moduli were between 200 kPa and 1 MPa with a variation in porosity between 76 and 84%. Currently the biological and medical aspects are under evaluation.     

Sequence of centromere division in bone marrow cells of patients with chronic myelocytic leukemia

An abnormal and highly variable centromere separation sequence was found in bone marrow mitoses of 20 patients with chronic myelocytic leukemia (CML). The most conspicuous alterations were the less frequent early separation of chromosome #3 in Ph-negative cases and the relatively late division of the X chromosome in the leukemic patients. The findings did not confirm an out-of-phase centromere division of chromosome #22 in CML.     

Inactivation of a centromere during the formation of a translocation in maize

Fluorescence in situ hybridization analysis of a reciprocal translocation in maize between chromosomes 1 and 5 that has been used extensively in maize genetics revealed the presence of an inactive centromere at or near the breakpoints of the two chromosomes. This centromere contains both the satellite repeat, CentC, and the centromeric retrotransposon family, CRM, that are typical of centromere regions in maize. This site does not exhibit any of the tested biochemical features of active centromeres such as association with CENP-C and phosphorylation of serine-10 on histone H3. The most likely scenario for this chromosome arrangement is that a centromere was included in the repair process that formed the translocation but became inactive and has been inherited in this state for several decades. The documentation of an inactive A chromosome centromere in maize extends the evidence for an epigenetic component to centromere function in plants. This case provides an experimental example of how karyotype evolution might proceed via changes in centromere inactivation.