Cd bands and centromeric function in dicentric chromosomes

Summary The Cd technique was applied to two cases of dicentric attached X chromosomes (XpXp and XqXq) and to cells from an established cell line of tumor origin (MaNo9) in which dicentrics with two active centromeres were present and dicentrics with one active and one inactive centromere. It was confirmed that the Cd technique discriminates between active and latent centromeres, and it was demonstrated that true dicentrics and dicentrics with one latent centromere can co-exist in the same cells. This indicates that the mechanism of centromere inactivation is a phenomenon that is specific to each chromosome and not generalized at the level of the individual cell.     

Telomere Disruption Results in Non-Random Formation of De Novo Dicentric Chromosomes Involving Acrocentric Human Chromosomes

Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the α-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extra-chromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same α-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.     

A tdic(5;15)(p31;p11) chromosome showing variation for constriction in the centromeric regions in a patient with the cri du chat syndrome.

Some dicentric chromosomes show only one primary constriction at metaphase and behave in cell division as if they are monocentric. The few previous reports of tdic (translocation dicentric) chromosomes showing one morphologic indicate that among the cells of an individual the same centromere consistently shows the primary constriction. The present case deals with a tdic(5;15)(p13;p11) chromosome that is an exception to this pattern. Scoring 98 GTG-, C-, and QFQ-banded metaphases specifically for primary constrictions revealed 15 (15%) containing a tdic chromosome with a single primary constriction. Among these chromosomes, 8 (8%) were at the chromosome 15 centromere and 7 (7%) were at the chromosome 5 centromere. The remaining 83 (85%) tdic chromosomes showed two primary constrictions. We analyzed a total of 172 metaphases from peripheral blood, and all except 3 (1.7%) contained the tdic chromosome. Among these three cells, the tdic chromosome was broken in two and absent in one, which indicates that there was some unstable separation of this dicentric in cell division. In two metaphases, there was a chromatid gap at the site of one centromere. Possibly, the absence of certain primary constrictions was associated with deletion of centromeres. This mechanism may be a continual source for additional centromere inactivation during the life of this patient. This case demonstrates that for some dicentrics either centromere may become nonfunctional and inactivation can occur more than once within an individual. The karyotype of this patient was 45,XX,tdic(5;15)(p31;p11). Thus, she was monosomic for about 3/4 of the chromosome 5 short arm. Clinically, this infant had a shrill catlike cry and facies of the cri du chat syndrome.     

CENP-E, a novel human centromere-associated protein required for progression from metaphase to anaphase.

We have identified a novel human centromere-associated protein by preparing monoclonal antibodies against a fraction of HeLa chromosome scaffold proteins enriched for centromere/kinetochore components. One monoclonal antibody (mAb177) specifically stains the centromere region of mitotic human chromosomes and binds to a novel, approximately 250-300 kd chromosome scaffold associated protein named CENP-E. In cells progressing through different parts of the cell cycle, the localization of CENP-E differed markedly from that observed for the previously identified centromere proteins CENP-A, CENP-B, CENP-C and CENP-D. In contrast to these antigens, no mAb177 staining is detected during interphase, and staining first appears at the centromere region of chromosomes during prometaphase. This association with chromosomes remains throughout metaphase but is redistributed to the midplate at or just after the onset of anaphase. By telophase, the staining is localized exclusively to the midbody. Microinjection of the mAb177 into metaphase cells blocks or significantly delays progression into anaphase, although the morphology of the spindle and the configuration of the metaphase chromosomes appear normal in these metaphase arrested cells. This demonstrates that CENP-E function is required for the transition from metaphase to anaphase.     

Chromosome aberrations induced in human lymphocytes by 3.45 MeV alpha particles analyzed by premature chromosome condensation.

Premature chromosome condensation (PCC) experiments using human lymphocytes with centromere staining have shown that after exposure to 3.45 MeV alpha-particle radiation, the full number of dicentric chromosomes appears when the cell fusion protocol is applied immediately after irradiation. In this case, the time available for repair and misrepair of DNA damage is only about 30 min. The number of dicentrics does not change with a further increase in the time available for chromatin rearrangement. This fast response confirms the expectation based on our previous experiments using PCC with 150 kV X rays in which the alpha component of the yield of dicentrics was found to appear when the cell fusion protocol was applied immediately after irradiation, whereas the beta component was delayed by several hours. The time constant for rejoining of the excess acentric chromosome fragments is found to be donor-specific and not to differ for alpha particles and X rays, but alpha-particle radiation leaves a larger fraction of the excess acentric fragments unrejoined. The RBEs of the 3.45 MeV alpha-particle radiation compared to 150 kV X rays, evaluated for the alpha component for the yield of dicentrics and for the yield of unrepaired acentric fragments, have almost equal values of about 4. This is consistent with data in the literature on chromosome aberrations observed in metaphase that show the equality of the RBE values for production of dicentrics and acentric fragments. Our experimental results concerning the fast kinetics of the alpha component of the yield of exchange-type chromosome aberrations are not consistent with Lea's pairwise lesion interaction model, and they support the proposed alternative mechanism of lesion-nonlesion interaction between chromatin regions carrying clustered DNA damage and intact chromatin regions.     

Effect of storage duration on cell cycle kinetics and yield of chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes.

Phytohaemagglutinin (PHA)-responsive lymphocytes from human peripheral blood samples, either irradiated or un-irradiated, showed increased frequency of first division metaphase cells (detected by fluorescence plus Giemsa (FPG) staining) as a function of duration of storage. Irradiated and subsequently stored samples showed small but significant increase for the yield of dicentrics. The yield of aberrant metaphases and deletions (excess acentrics) remained unchanged. Increasing Bromodeoxyuridine (BrdU) concentrations slowed down the cell cycle progression but did not influence the yield of aberrations including that of dicentrics.     

Indirect immunofluorescence of inactive centromeres as indicator of centromeric function

Summary Two previous single case reports from the literature showed the presence or absence of centromeric antigens at the site of the inactive centromeres in one (X;X) and in one (9;11) dicentric chromosome. We studied nine different dicentric chromosomes using anticentromeric antibodies and immunofluorescence techniques. In the four autosomal dicentrics the inactive centromere was consistently positive while the dicentrics composed of two X chromosomes were either positive or negative; one case of (X;Y) dicentric was negative. The results indicate that the X chromosome mode of replication may be involved in the suppression of immunofluorescence at the site of the inactive centromere and that one centromere of the dicentric chromosome may lose its function but conserve some of its antigenic properties. This indicates that not all these antigens play a rôle in the microtubules-centromere interaction.     

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G₁ phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.     

Observations on dicentrics in living cells

Summary In previously irradiated endosperm cells of Haemanthus katherinae studied in vitro by means of micro-cinematography, two-kinetochore chromatids and dicentric chromosomes have been observed. Breaking of such dicentric chromatids and chromosomes has been analysed. Behaviour of some of the dicentric chromosomes during anaphase deserves special attention: interlocking dicentrics cut one through another and rejoin in a few minutes. In this way from a metaphase interlocking dicentric, two sister anaphase dicentrics are formed. Interlocked dicentrics can also uncoil and not break at all. In this case no activity was observed in one kinetochore of one dicentric in later stages of anaphase (two kinetochores were active in one dicentric and only one in its sister). Analysis of chromosome movements in two-kinetochore chromatids and dicentrics is also presented.     

A tightly bound chromosome antigen is detected by monoclonal antibodies in a ring-like structure on human centromeres

Monoclonal antibodies (Mabs) were raised against isolated Chinese hamster protein-depleted chromosomes Chromosome scaffolds) in order to probe for components involved in the higher-order structure of mammalian chromosomes. One of the Mabs detected a ring-like structure in metaphase at the centromere, which is conserved between Chinese hamster and human cells. Additionally, the Mab stained the centrioles in interphase cells in these two species. The antigen was enriched in chromosomal protein preparations by comparison with nuclear protein samples, and has an apparent Mr=170,000. The centromere antigen remained present in chromosome scaffold preparations, indicating that it was tightly associated with DNA. The antigen was distinct in its centromeric localisation from any of the centromere antigens reported to date. A possible role of the antigen in stabilising the centromere, by holding the sister chromatids together until their separation at the metaphase-anaphase transition is presented.     

The effect of hyper- and hypothermia on induced telomeric chromosome fusion]

After administration of colcemid and 5-BrdU in the cell culture, the cells pass through the first interphase to delay in mitosis. Then the cells overcome the colcemid blockade, and polykaryocytes with micronuclei are formed. The second interphase in followed by the second mitosis, during which dicentric chromosomes are observed. These dicentrics are the result of telomeric chromosome fusion. The action of hyperthermia (40 degrees C) during the whole period of colcemid and 5-BrdU treatment or that of the hyperthermia (40 degrees C) only during the first 17 hours (the first interphase and the first mitosis) lead to the increased frequency of dicentrics. Under condition of hypothermia (34 degrees C) the frequency of dicentric formation decreases. Changes in cultivation temperature during the latest 25 hr of colcemid and 5-BrdU action (the second interphase and the second mitosis) exert no influence on the dicentric formation frequency. Because there are no dicentrics in cells during the metaphase of the first mitosis, it is supposed that the temperature--sensitive period may be the latest steps of colcemid blockade, i.e. the period of formation of micronuclei.     

Centromere dynamics in the primitive red alga Cyanidioschyzon merolae

Centromeres are universally conserved functional units in eukaryotic linear chromosomes, but little is known about the structure and dynamics of the centromere in lower photosynthetic eukaryotes. Here we report the identification of a centromere marker protein CENH3 and visualization of centromere dynamics in the ultra-small primitive red alga Cyanidioschyzon merolae. Immunoblotting and immunofluorescence microscopy showed that CENH3 increased rapidly during S phase, followed by a drastic reconstitution into two discrete foci adjacent to the spindle poles at metaphase, suggesting the cell-cycle-regulated expression of CENH3. Immunoelectron microscopy revealed that the CENH3 signals were associated with the nuclear envelope, implying interplay between the kinetochore complex and the nuclear envelope. These results demonstrate dynamic centromere reconstitution during the cell cycle in an organism in which the chromosomes do not condense at metaphase.     

CENP-C is necessary but not sufficient to induce formation of a functional centromere.

CENP-C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP-C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP-C with those of inducibly overexpressing the protein. Removing CENP-C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase-anaphase junction. Overexpressed CENP-C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP-C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP-C changes during the cell cycle. In metaphase, the excess CENP-C coats the chromosome arms. At the metaphase-anaphase transition, the excess CENP-C clusters, and during interphase it is present in large bodies which form around pre-existing centromeres which are also clustered. These results indicate that CENP-C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP-C may be regulated during the cell cycle.     

Dicentric chromosomes and the inactivation of the centromere

Summary The origin and behavior of human dicentric chromosomes are reviewed. Most dicentrics between two non-homologous or two homologous chromosomes (isodicentrics), which are permanent members of a chromosome complement, probably originate from segregation of an adjacent quadriradial; such configurations are the result of a chromatid translocation between two nonhomologous chromosomes, or they represent an adjacent counterpart of a mitotic chiasma. The segregation of such a quadriradial may also give rise to a cell line monosomic for the chromosome concerned (e.g., a 45,X line). Contrary to the generally held opinion, isodicentrics rarely result from an isolocal break in two chromatids followed by rejoining of sister chromatids. In this case the daughter centromeres go to opposite poles in the next anaphase, and the resulting bridge breaks at a random point. This mechanism, therefore, leads to the formation of an isodicentric chromosome only if the two centromeres are close together, or if one centromere is immediately inactivated. Observations on the origin of dicentrics in Bloom syndrome support these conclusions. One centromere is permanently inactivated in most dicentric chromosomes, and even when the dicentric breaks into two chromosomes, the centromere is not reactivated. The appearance and behavior of the acentric X chromosomes show that their centromeres are similarly inactivated and not prematurely divided. Two Bloom syndrome lymphocytes, one with an extra chromosome 2 and the other with an extra chromosome 7, each having an inactivated centromere, show that this can also happen in monocentric autosomes.     

On fully automatic feature measurement for banded chromosome classification

Procedures for fully automatic location of chromosome axis and centromere in metaphase chromosomes are described for a practical interactive chromosome analysis system that omits the usual stages of interactive axis and centromere correction. Accuracy of centromere finding and consequential determination of a chromosome's polarity, i.e., which end is which, is measured experimentally. The saving in interaction by not correcting centromeres is compared to the increase in errors at the classification stage and the consequent increase in interaction needed to correct these errors. Some previously unreported features for banded chromosome classification are described, and in particular a set of global shape features is introduced. The discrimination capability of the feature measurements is evaluated by use of simple statistics and by reference to the performance of classifiers trained with various feature subsets. Class discrimination capability of the global shape feature set is shown to be comparable to that of centromere position, a widely used local shape feature. The variability of feature measurements that might occur in data from different laboratories on account of differing tissue, preparation methods, and digitiser hardware is assessed using three data bases of G-banded human metaphase cells. It is shown that the differences can be considerable and that appropriate feature selection and classifier training substantially improve classification performance.     

A stable dicentric chromosome: Both centromeres develop kinetochores and attach to the spindle in monocentric and dicentric configuration

A stable, dicentric human chromosome, which is known from light microscopy to show a 50:50 distribution between monocentric/dicentric appearance, was examined by conventional electron microscopy and after labelling the centromere with anticentromere antibodies from CREST serum. Both centromeres of the chromosome developed kinetochores whether in monocentric or dicentric configuration. The eight monocentrics observed had all developed kinetochores at the centromere outside the constriction; at least six of them also had kinetochores at the centromere in the constriction. The dicentrics from glutaraldehyde fixed cells had spindle microtubules attached to both kinetochore sets irrespective of monocentric/dicentric configuration. The chromosome thus appeared to use both centromeres, either equally or with one serving a chromatid adhesion function while the second was used for transport along the spindle.     

The spatial localization of homologous chromosomes in human fibroblasts at mitosis

Chromosomes from ten human male fibroblast metaphases were completely reconstructed from electron micrographs of serially sectioned material. Chromosome centromere positions were determined by finding the three-dimensional coordinates of the centromere midpoint. The data set showed the identity of nine chromosome types (chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and the Y chromosome) preserved as they are positioned in vivo. The results indicate that there is (1) no significant association of the homologous chromosomes examined, (2) a significant tendency for a central location of the Y chromosome and of chromosome 18, (3) a significant tendency for a peripheral location of chromosome 6, (4) no significant tendency for homologous chromosomes to reorganize as metaphase advances and (5) no significant differential condensation across the metaphase plate. Therefore, the only organization pattern observed for the centromeres of the homologous chromosomes studied is some sorting by size across the metaphase plate. These results may be typical of dividing cell types. Different chromosome arrangements are found in some non-dividing cell types (e.g. mammalian brain cells). The different distributions of chromosomes in different cell types can be considered as forms of nuclear differentiation. It is postulated that nuclear differentiation may be related to cell differentiation.