Sequence Of Centromere Separation Research Articles

Chromatid segregation analysis in native human lymphocyte anaphases using sequential fluorescence in situ hybridization.

A sequential multiprobe fluorescence in situ hybridization technique was developed to study the 13, 18, 21, X and Y chromatid segregation in human lymphocytes anaphases cultures without antimitotic treatment. This method was used to know if exist any different chromosomes segregation in lymphocytes from Down syndrome patients and compared it with controls. The results show that the prevalent sequence of centromere separation was X, 13, 21, Y and 18 in Down syndrome patients and Y, 13, X, 21 and 18 in controls. Chromatid segregation in early anaphase was asynchronic for all chromosome pairs studied. Late anaphase showed a frequency of non-disjunction of 4.5% in the controls, affecting only chromosomes 18 and Y; in the Down syndrome patients, the frequency was higher (20.3%) and affected all chromosomes studied. This technique could be applicated to know the incidence of non disjunction in couples with repetitive abortions or in cases with different aneuploidies in the offspring.

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.

Separation vs. Replication of Inactive and Active Centromeres in Neoplastic Cells

The inactive centromeres in neoplastic and transformed cells exhibit premature separation at prophase or pro-metaphase. The factor(s) that control this behavior are not known. Using a human breast cancer cell line, MDA 435, and a transformed mouse cell line (L929), we studied the relationship between the sequence of centromere separation and the replication of centromeric region associated with the active and inactive centromeres. Whereas the inactive centromeres in L929 cells replicate their pericentric heterochromatin earlier than that associated with the active centromeres, those in MDA 435 cells exhibited no strong correlation between early separation and replication. A comparison between the intragenomic patterns of separation with replication of only active centromeres showed that the former is not dependent upon the latter in either L929 cells or MDA 435 cells. These studies indicate that, whereas inactive centromeres in neoplastic cells separate prematurely in different species, there is no uniformity in the control for replication nor does the timing of separation depend upon the timing of replication of the centric region.

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 (2 n=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.

Decondensation of pericentric heterochromatin alters the sequence of centromere separation in mouse cells.

The centromeres of a genome separate in a sequential, nonrandom manner that is apparently dependent upon the quantity and quality of pericentric heterochromatin. It is becoming increasingly clear that the biological properties of a centromere depend upon its physicochemical makeup, such as its tertiary structure, and not necessarily on its particular nucleotide sequence. To test this idea we altered the physical state of the AT-rich pericentric heterochromatin of mouse with Hoechst 33258 (bis-benzimidazole) and studied a biological parameter, viz., sequence of separation. We report that an alteration in the physical state of heterochromatin, i.e., decondensation, is accompanied by aberrations in the pattern of centromere separation. The most dramatic effect seems to be on chromosomes with large blocks of heterochromatin. Many chromosomes with large blocks of heterochromatin that, in untreated cells, separate late tend to separate early. Decondensation with Hoechst 33258 does not seem to alter the sequence of separation of inactive centromeres relative to that of active centromeres. These data indicate that alteration in the physical parameters of the pericentric heterochromatin might dispose the centromeres to errors. It is likely that this aberration results from early replication of the pericentric heterochromatin associated with active centromeres.

Objective analysis of centromere separation.

Centromere separation sequence and premature centromere division have gained increasing interest; however, inaccuracy and subjectivity in their investigation have often been criticized. We describe a simple computerized image analysis system that makes an objective and exact staging of centromere division possible.

Sequence of centromere separation: Minor satellite DNA does not influence separation of inactive centromeres in transformed cells of mouse

Neoplastic cells may carry inactive centromeres on some multicentric, yet stable, chromosomes. We report that some inactive centromeres in L929 mouse cells do not contain minor satellite DNA, the DNA fraction which has been suggested to constitute the centromere. We compared the sequence of separation of inactive centromeres carrying the minor satellite with those lacking this fraction. The sequence of separation appears to be independent of whether or not the inactive centromeres carry the minor satellite DNA. The timing of replication of the inactive centromeres is also independent of this DNA. Hence, minor satellite of mouse is not a factor in holding together the subunits of inactive centromeres. Extension of these results to active centromeres might suggest that the minor satellite DNA is not a factor responsible for adhesion of the two centromere sub-units up until late meta-anaphase.

A possible mosaic form of delayed centromere separation and aneuploidy

On average, a "normal" centromere separation sequence was seen in 3 neonates with trisomy 18 and in their parents. When evaluating individual mitoses, unusually late separating chromosomes 18 were found in a few cells of one of the parents in each family. A possible germline mosaicism of delayed separation in the parent may account for trisomy in the offspring.

Sequence of centromere separation: separation in a quasi-stable mouse-human somatic cell hybrid.

A quasi-stable mouse-human hybrid cell line, HR61, containing between one and ten human chromosomes was analyzed for the sequence of centromere separation. The purpose was to determine which genome of the two initiates centromere separation first. The data clearly indicate that the separation of centromeres of the human genome is not only initiated but is completed before any centromeres from the mouse chromosomes start splitting into daughter units. The information on whether uniparental chromosome loss results from a lack of deposition of kinetochore proteins was equivocal. The human genome also completes its DNA replication before the mouse genome does. Our studies, therefore, show that the timing of centromere separation is tightly linked to the completion of replication of DNA. At least in this cell line the segregant genome is not the one which exhibits delayed DNA replication.

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.

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 [7]. 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 [1]. 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 [8]. 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 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.

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.

Sequence of centromere separation: generation of unstable multicentric chromosomes in a rat cell line.

A transformed cell line, B1, of cerebral endothelial origin from the Wistar-Kyoto male rat has chromatid and chromosome type bridges in virtually every cell. It exhibits various dicentric and polycentric chromosomes. Most dicentrics are symmetric isochromosomes. Certain isodicentrics are present in a fair segment of the cell population; however, almost all cells have some newly arising isodicentrics. The live cells show a lengthened prometaphase. Anaphase is also retarded possibly due to the occurrence of bridges. At anaphase some multicentrics split at only one centromere. When pulled to the two poles the unsplit centromeres and the distal chromosome segment form a side arm bridge. Another mechanism appears to be a total lack of separation of daughter centromeres at meta-anaphase ('meiotic-like' behavior of mitotic chromosomes). This is realized by the pulling of each of the two unsplit centromeres to opposite poles and results in bridges with both sister chromatids running parallel to each other. A break at corresponding weak points in the two sister chromatids followed by rejoining can form a dicentric isochromosome. A third mechanism, the breakage-fusion-bridge cycle, is also operative but would not produce isodicentrics. In the case of the first two mechanisms some or all centromeres apparently split between telophase and onset of the following DNA synthesis rather than at the usual time at late metaphase. These observations may suggest some previously unknown behavior of multicentric chromosomes during mitosis.

Parental centromere separation sequence and aneuploidy in the offspring.

We have studied the centromere separation sequence in lymphocyte mitoses of the parents of four infants with trisomy 18, five patients with trisomy 21, and five children with normal karyotype. "Late separation" of chromosome 18 was found in both parents of a neonate with trisomy 18, "early separation" of chromosome 21 in three mothers and in one father of four children with trisomy 21. No "out-of-phase" separation occurred in the mitoses of the parents of normal children. The findings provide further evidence for the correlation between alteration of the parental centromere separation sequence and aneuploidy of the offspring.

Sequence of centromere separation: a possible role for repetitive DNA.

The centromeres of a mitotic cell at the meta-anaphase junction separate in a non-random, genetically controlled sequence before anaphase migration ensues. In several, but not all, of the organisms studied so far it appears that the timing of separation of a centromere into two visible units depends upon the quantity of pericentric heterochromatin. A critical analysis of this parameter of cell cycle suggests that the sequence of centromere separation is influenced by repetitive DNA present in the pericentric region. In those cases with qualitatively uniform repetitive DNA in centromeric regions, the chromosomes carrying lesser quantities separate earlier than those with greater amounts. However, the overall pattern of separation may be determined by interactions between both quantitative and qualitative parameters of the repetitive DNA. It has been suggested that repetitive DNA has no transcriptional properties. It is probably only a structural component which acts as a site for the accumulation of protein molecules synthesized by some locus not present in the centromeric region. These proteins accumulate to saturate the centromeric repetitive DNA resulting in a (trilaminar) structure called 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 separation results 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 centromeric region appears to be the maintenance of diploidy.

Sequence of centromere separation in rat (Rattus norvegicus) and guinea pig (Cavia porcellus) bone marrow cells and the influence of Herbal drugs in rat.

Sequence of centromere separation in rat (Rattus norvegicus) and guinea pig (Cavia porcellus) bone marrow cells and the influence of Herbal drugs in rat.

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.

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.

Sequence of centromere separation: kinetochore formation in induced laggards and micronuclei

Mouse L-cells were treated with bis-benzimidazole derivative (Hoechst 33258), caffeine and bleomycin in order to study genesis of laggards and micronuclei and formation of kinetochores as revealed by antikinetochore antibody staining. Apparently, the Hoechst 33258-induced decondensation experienced by the A:T-rich pericentric heterochromatin does not extend into the centromeric region and does not affect formation, physical appearance or function of kinetochores. The laggards induced by Hoechst 33258 are generally whole chromosome laggards which have antikinetochore antibody binding sites. These kinetochore-carrying laggards were seen to lie outside the spindle region in cells untreated with spindle inhibitors or hypotonic and stained differentially for spindle and chromosomes. Some micronuclei did not show kinetochore dots indicating their origin in acentric chromosome fragments. When cells were treated with caffeine or bleomycin, both types of micronuclei, namely those carrying kinetochores and those generated by acentric fragments, were seen. These results are interesting in that caffeine prevents the rejoining of chromosome breaks and one would expect only kinetochore-less micronuclei in caffeine-treated cells. It may mean that caffeine also induces aneuploidy. The L-cells carry minichromosomes which are no more than a pair of kinetochore dots. Such chromosomes, though detectable by antikinetochore antibody staining, may be missed in routine, acid-fixed, Giemsa-stained preparations.