Formation of primary constriction and heterochromatin in mouse does not require minor satellite DNA.

Whereas the major satellite fraction in mouse extends its domain from the centromere to the distal end of the pericentric heterochromatin, the minor satellite DNA is present specifically in the centromere or primary constriction. We hybridized the biotinylated minor satellite sequence to L929 cells of mouse origin. The sequence hybridized to all chromosomes. Whereas hybridization was detected on all active centromeres, the inactive centromeres in certain dicentrics did not show any signal. This satellite, however, was detected in all inactive centromeres in a heptacentric chromosome. The intensity of fluorescence on the inactive centromeres of the heptacentric was similar to that present on the active centromeres. Several heterochromatin blocks, which were not associated with any centromere, were also found to lack hybridization with the minor satellite. The inactive centromeres, whether carrying the minor satellite DNA fraction or not, generally do not react with the antikinetochore antibodies present in the scleroderma serum. These studies are interpreted to show that (1) the primary constriction in mouse can be formed without the participation of minor satellite, (2) heterochromatin in mouse may constitute without this fraction, (3) the major and minor satellite may not be interspersed but are joined at some defined boundary, and (4) the binding of CENP-B does not depend upon the quantity of minor satellite or the number of CENP boxes present in the inactive centromeres.     

5-Azacytidine- and Hoechst-induced aneuploidy in Indian muntjac

Hoechst 33258 (bis-benzimidazole) and 5-azacytidine (5-AC) cause decondensation of the pericentric heterochromatin in mouse and aberrations in the sequence of centromere separation apparently by different mechanisms. We treated the male Indian muntjac cells (2n=7), which do not undergo decondensation of the pericentric heterochromatin, to study if these chemicals would result in induction of aneuploidy limited to the Y(2) chromosome. This paper reports that both agents result in aneuploidy primarily limited to one chromosome, the Y(2). It is likely that other chromosomes are not tolerated in aneuploid condition because every chromosome carries some household genes including those essential for mitotic progression. The loss/gain of the Y(2) chromosome is tolerated because it is the smallest chromosome and is almost entirely composed of constitutive heterochromatin. Since Indian muntjac has only three pairs of large chromosomes comprising its basic genome, which can be clearly viewed under high dry objective, these cells are very suitable for the preliminary analysis of aneuploidy-inducing ability of various chemicals.     

Heterochromatin diversity and cyclic responses to selective silver staining in Aedes aegypti (L.)

In interphase cells of Aedes aegypti (L.) (2n=4+ XX/XY), only the nucleolus responded to selective silver staining. The secondary constriction on chromosome 3 remained unresponsive at all times but the six centromeres were identified throughout mitosis from early prophase as well as those stages of meiosis subsequent to diplotene. The centromeric blocks were not synonymous with the pericentric heterochromatin revealed by C-banding. X chromosomes without an intercalary C-band were newly discovered in Ae. aegypti in the Bangalore strain. Sequential Q-or Hoechst 33258/C-banding in this and the Trinidad-30 strain revealed intercalary heterochromatin diversity within and between strains and also differences between intercalary and pericentric heterochromatin.     

Sequence of centromere separation: influence of pericentromeric heterochromatin (repetitive DNA) inMus

A relationship between the sequence of centromere separation and quantity of pericentromeric constitutive heterochromatin was studied using bone marrow cells ofMus musculus molossinus and three cell lines, viz., SEWA-Rec 4, brain tumor and L-cells, ofM. m. domesticus origin. The timing of separation of a centromere into two daughter centromeres is related to the quantity of pericentromeric heterochromatin. In these genomes, having qualitatively uniform DNA in their heterochromatin fraction, the chromosomes with none or small quantities of heterochromatin separate first. These are followed by those chromosomes which have increasingly larger quantities of heterochromatin. It appears that one function of repetitive DNA (pericentromeric heterochromatin) is to regulate the timing of separation of centromeres.     

Sequence of centromere separation of mitotic chromosomes in Chinese hamster

Chromosome preparations in late metaphase cells from bone marrow of colcemid treated male Chinese hamsters were used to analyse the sequence of separation of sister centromeres. Chromatids of chromosomes 2 and 1 are the first ones to separate at centromeres, followed by members of group B, D and C. Some acrocentric chromosome is always the last one to separate at the centromere. The data point to a possible correlation between the position of a centromere in the separation sequence in the genome and the amount of centromeric heterochromatin as well as relation to the phenomenon of non-disjunction.     

Sequence of centromere separation: differential replication of pericentric heterochromatin in multicentric chromosomes

The dicentric and multicentric chromosomes in L cells and a brain tumor cell line of mouse display only one site of kinetochore formation associated with the active centromere. The accessory or inactive centromeres show premature separation. These cell lines were treated with 10^–6 M 5-bromodeoxyuridine (BrdUrd) followed by anti-BrdUrd antibody to study the pattern of replication of pericentric heterochromatin flanking the active vs inactive centromeres. Regardless of its quantity, heterochromatin around the inactive centromere replicates earlier than that associated with the active centromere. There appears to be a relationship between the timing of separation of a centromere and the timing of replication of pericentric heterochromatin. The premature replication of heterochromatin associated with an inactive centromere may be responsible for its premature separation and, hence, inactivity.     

Discontinuous undercondensation of centromeric heterochromatin in mouse chromosomes: evidence in Hoechst 33258-treated cells.

Mouse chromosomes from the L929 cell line have been treated with Hoechst 33258 to induce undercondensation of centromeric heterochromatin. The morphological changes induced by this fluorochrome were analyzed in electron micrographs of whole-mounted chromosomes. Results show that the condensation inhibition of centromeric heterochromatin caused by Hoechst 33258 is not produced homogeneously and suggest compositional differences within an individual centromere.     

Sequence of Centromere Separation: Role of Centromeric Heterochromatin

The late metaphase-early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late replicating X chromosome appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separation of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of constitutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the genome separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quantitative differences in controlling the separation of centromeres.     

Sequence of centromere separation: characterization of multicentric chromosomes in a rat cell line

The B₁ cell line of rat cerebral endothelium origin exhibits several dicentric and multicentric chromosomes. These chromosomes, unlike multicentrics in mouse (Vig and Zinkowski 1986) do not show premature centromere separation. All centromeres deposit kinetochore proteins and appear to be functional. Even the centromeres which fail to migrate to the poles during anaphase and make side arm bridges bind to spindle microtubules. Some multicentric chromosomes show kinetochores spaced apart with intervening stretches of euchromatin while others are located adjacent to each other thus exhibiting tandem repeats and forming a compound kinetochore (Brinkeley et al. 1984). Also, unlike mouse multicentric chromosomes in which different pericentric regions and the centromeres replicate at different times, the rat chromosomes appear to replicate all pericentric and centric regions in a given multicentric simultaneously. The present studies indicate that centromeres in rat and mouse replicate during the last part of the S-phase and in continuation with the pericentric heterochromatin.     

Analysis of centromere size in human chromosomes 1, 9, 15, and 16 by electron microscopy.

Human chromosomes were treated with 5-azacytidine and analyzed by whole-mount electron microscopy. This base analogue produces undercondensation of heterochromatin and separation of the centromere from the bulk of pericentromeric heterochromatin in chromosomes 1, 9, 15, and 16, which allows clear delimitation of the centromere regions. A quantitative analysis of centromeres showed that chromosomes 1, 9, and 16 have centromeres of different size. The centromere of chromosome 15 is similar in size to that of chromosome 9 and different from those of chromosomes 1 and 16. No interindividual variation for centromere size was found. A positive correlation between centromere and chromosome size was found for the chromosomes analyzed.     

INCENP loss from an inactive centromere correlates with the loss of sister chromatid cohesion

Inactive centromeres of stable dicentric chromosomes provide a unique opportunity to examine the resolution of sister chromatid cohesion in mitosis. Here we show for the first time that inactive centromeres are composed of heterochromatin, as defined by the presence of heterochromatin protein HP1(Hs alpha). We then show that both the inner centromere protein (INCENP) and its binding partner Aurora-B/AIM-1 kinase can also be detected at the inactive centromere. Thus, targeting of the chromosomal passengers is not dependent upon the presence of an active centromere/kinetochore. Furthermore, we show that the association of INCENP with the inactive centromere correlates strictly with the state of cohesion between sister chromatids: loss of cohesion is accompanied by loss of detectable INCENP. These results are consistent with recent suggestions that one function of the chromosomal passenger proteins may be to regulate sister chromatid separation in mitosis.     

DNA loops generate intracentromere tension in mitosis

The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.     

Asymmetric decondensation of the L cell heterochromatin by Hoechst 33258

A benzimidazole derivative, Hoechst 33258 can induce decondensation of constitutive heterochromatin in the mouse derived L cell chromosomes when the compound is given in sufficiently high concentration (40 micrograms/ml) to the L cell culture. Hoechst 33258 at low concentration (1 micrograms/ml, 16 h) cannot produce this effect on L cell chromosomes. Bromodeoxyuridine (BUdR) incorporation for one cell cycle simultaneous with the Hoechst 33258 treatment at low concentration could decondense heterochromatin segments in metaphase chromosomes. The heterochromatin decondensation, however, was asymmetric; it was observed only on one chromatid and the other of a chromosome remained in condensed state. The observation of asymmetric decondensation of heterochromatin by Hoechst 33258 after BUdR incorporation for one cell cycle, the association of A-T rich satellite DNA to mouse heterochromatin, and available data on the specific binding of Hoechst 33258 to A-T base pairs of DNA and on the higher affinity of the compound to BUdR substituted DNA than to ordinary DNA implied that the binding of Hoechst 33258 molecules to A-T rich satellite DNA is the cause of heterochromatin decondensation.     

Cytogenetic and molecular evaluation of centromere-associated DNA sequences from a marsupial (Macropodidae: Macropus rufogriseus) X chromosome

The constitution of the centromeric portions of the sex chromosomes of the red-necked wallaby, Macropus rufogriseus (family Macropodidae, subfamily Macropodinae), was investigated to develop an overview of the sequence composition of centromeres in a marsupial genome that harbors large amounts of centric and pericentric heterochromatin. The large, C-band-positive centromeric region of the X chromosome was microdissected and the isolated DNA was microcloned. Further sequence and cytogenetic analyses of three representative clones show that all chromosomes in this species carry a 178-bp satellite sequence containing a CENP-B DNA binding domain (CENP-B box) shown herein to selectively bind marsupial CENP-B protein. Two other repeats isolated in this study localize specifically to the sex chromosomes yet differ in copy number and intrachromosomal distribution. Immunocytohistochemistry assays with anti-CENP-E, anti-CREST, anti-CENP-B, and anti-trimethyl-H3K9 antibodies defined a restricted point localization of the outer kinetochore at the functional centromere within an enlarged pericentric and heterochromatic region. The distribution of these repeated sequences within the karyotype of this species, coupled with the apparent high copy number of these sequences, indicates a capacity for retention of large amounts of centromere-associated DNA in the genome of M. rufogriseus.     

There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology.

There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle.     

Plasmodium falciparum centromeres display a unique epigenetic makeup and cluster prior to and during schizogony

Centromeres are essential for the faithful transmission of chromosomes to the next generation, therefore being essential in all eukaryotic organisms. The centromeres of Plasmodium falciparum, the causative agent of the most severe form of malaria, have been broadly mapped on most chromosomes, but their epigenetic composition remained undefined. Here, we reveal that the centromeric histone variant PfCENH3 occupies a 4–4.5 kb region on each P. falciparum chromosome, which is devoid of pericentric heterochromatin but harbours another histone variant, PfH2A.Z. These CENH3 covered regions pinpoint the exact position of the centromere on all chromosomes and revealed that all centromeric regions have similar size and sequence composition. Immunofluorescence assay of PfCENH3 strongly suggests that P. falciparum centromerescluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion. In summary, we reveal a dynamic association of Plasmodium centromeres, which bear a unique epigenetic signature and conform to a strict structure. These findings suggest that DNA‐associated and epigenetic elements play an important role in centromere establishment in this important human pathogen.     

Analysis of three whole-arm translocations in a mouse sarcoma cell line

Three apparently whole-arm translocation chromosomes in the mouse sarcoma 180 ascites cell line have been studied by G- and C-banding and by Hoechst staining. All three chromosomes appear to have two centromeres. Both centromeres of one, which are very close together, are likely to be active and to produce parallel separation of the chromatids. The centromeres of the other two chromosomes are well separated. One of these two centromeres may be inactive either because the kinetochore organizer has been inactivated or because the kinetochore plate has been deleted, leaving the AT-rich centromeric constituative heterochromatin intact. The possibility that these whole arm translocations arose by telomeric fusion and the molecular basis of such fusions are discussed.     

Editor's choice: Heterochromatin Blocks Constituting the Entire Short Arms of Acrocentric Chromosomes of Azara's Owl Monkey: Formation Processes Inferred From Chromosomal Locations

Centromeres and telomeres of higher eukaryotes generally contain repetitive sequences, which often form pericentric or subtelomeric heterochromatin blocks. C-banding analysis of chromosomes of Azara''s owl monkey, a primate species, showed that the short arms of acrocentric chromosomes consist mostly or solely of constitutive heterochromatin. The purpose of the present study was to determine which category, pericentric, or subtelomeric is most appropriate for this heterochromatin, and to infer its formation processes. We cloned and sequenced its DNA component, finding it to be a tandem repeat sequence comprising 187-bp repeat units, which we named OwlRep. Subsequent hybridization analyses revealed that OwlRep resides in the pericentric regions of a small number of metacentric chromosomes, in addition to the short arms of acrocentric chromosomes. Further, in the pericentric regions of the acrocentric chromosomes, OwlRep was observed on the short-arm side only. This distribution pattern of OwlRep among chromosomes can be simply and sufficiently explained by assuming (i) OwlRep was transferred from chromosome to chromosome by the interaction of pericentric heterochromatin, and (ii) it was amplified there as subtelomeric heterochromatin. OwlRep carries several direct and inverted repeats within its repeat units. This complex structure may lead to a higher frequency of chromosome scission and may thus be a factor in the unique distribution pattern among chromosomes. Neither OwlRep nor similar sequences were found in the genomes of the other New World monkey species we examined, suggesting that OwlRep underwent rapid amplification after the divergence of the owl monkey lineage from lineages of the other species.     

Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa

We describe the morphology and molecular organization of heterochromatin domains in the interphase nuclei, and mitotic and meiotic chromosomes, of Brassica rapa, using DAPI staining and fluorescence in situ hybridization (FISH) of rDNA and pericentromere tandem repeats. We have developed a simple method to distinguish the centromeric regions of mitotic metaphase chromosomes by prolonged irradiation with UV light at the DAPI excitation wavelength. Application of this bleached DAPI band (BDB) karyotyping method to the 45S and 5S rDNAs and 176 bp centromere satellite repeats distinguished the 10 B. rapa chromosomes. We further characterized the centromeric repeat sequences in BAC end sequences. These fell into two classes, CentBr1 and CentBr2, occupying the centromeres of eight and two chromosomes, respectively. The centromere satellites encompassed about 30% of the total chromosomes, particularly in the core centromere blocks of all the chromosomes. Interestingly, centromere length was inversely correlated with chromosome length. The morphology and molecular organization of heterochromatin domains in interphase nuclei, and in mitotic and meiotic chromosomes, were further characterized by DAPI staining and FISH of rDNA and CentBr. The DAPI fluorescence of interphase nuclei revealed ten to twenty conspicuous chromocenters, each composed of the heterochromatin of up to four chromosomes and/or nucleolar organizing regions.