Histone H3 phosphorylation of mammalian chromosomes.

Inferences about the role and location of phosphorylated histone H3 are derived primarily from biochemical studies. A few direct observations at chromosome level have shown that phosphorylation begins at the site of heterochromatin and spreads throughout the chromosome. However, a comparative study of chromosomes of mouse (L929 cells), Chinese hamster (CHO 9 cells) and the Indian muntjac (male cells) reveals some distinguishable details among mammalian species. Whereas the L929 cells exhibit the typical pattern of phosphorylation at the region of centromeric heterochromatin associated with the active centromere, the heterochromatin blocks associated with the inactive centromeres also show label of about equivalent intensity. Throughout the cell cycle, heterochromatin exhibits sharper (denser) and better defined label than does euchromatin which expresses somewhat diffuse label. The centromere constriction on biarmed chromosomes, originating in Robertsonian translocations, appears phosphorylated in some, if not all chromosomes. A similar situation was found for the CHO 9 cells indicating that phosphorylation does include the region in which H3 is supposedly replaced by CENP-A. An interesting feature of the CHO cell line was the dense label at and near the telomeres; this feature was not observed in either the mouse or the Indian muntjac. The centromere regions of the Indian muntjac chromosomes showed three sites of label in the multicentric X chromosome and two each on chromosome pair number 1 and Y2; the sites coinciding with the reaction sites of antikinetochore antibodies. Also, the X and Y, chromosomes of Indian muntjac show intense phosphorylation at the sites of secondary constrictions. The chromosomes of all three species were phosphorylated throughout the cell cycle. As the chromosomes started to decondense during anaphase, heavy phosphorylation was observed in the form of discontinuous beaded structures indicating partial despiralization of the chromosome. Interestingly, when cells had completed karyokinesis and resolved into two independent nuclei, the phosphorylation was observed at the midbody. At this stage, the cytoplasm appeared to be again phosphorylated.

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 (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.

Visualization of prekinetochore locus on the centromeric region of highly extended chromatin fibers: does kinetochore autoantigen CENP-C constitute a kinetochore organizing center?

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

Recognition of mammalian centromeres by anti-dynein and anti-dynactin components.

Antibodies against one component of dynein, namely IC, and two components of dynactin, namely p50 and p150, were studied for their activity against the centromeres of three mammalian species, human, mouse and Indian muntjac. Ordinarily human and mouse chromosomes carry one dot-like centromere, whereas Indian muntjac has two or three tandemly organized centromeres per chromosome. Dynein and dynactin antigens can be detected on all active centromeres. However, inactive centromeres (which do not bind microtubules and lack a trilamellar kinetochore) in human MDA 435 cells and mouse L cells do not respond to any of the three antibodies. In parallel with their response to CREST serum every Indian muntjac chromosome exhibits more than one anti-dynein or anti-dynactin binding site. The area reacted upon by any of these three antibodies is very precise and apparently associated with the kinetochore at the site of the active centromere. This is in contrast to the label observed with CREST serum, which 'stains' both the active as well as inactive centromeres and spans a considerable distance, including some heterochromatin. The label on colcemid-arrested mouse and human chromosomes was more intense than that observed on untreated chromosomes. In preparations not treated with colcemid metaphase chromosomes in the ring configuration exhibited label with all of the three antibodies. This observation is contrary to the belief that the dynein motor proteins do not persist on centromeres at metaphase.

Aneuploidy in male Indian muntjac cells is limited to the Y2 chromosome.

In untreated cell cultures of Indian muntjac (Munticus muntiacus vaginalis; 2n = 6 female, 7 male) we observed that spontaneous aneuploidy is limited primarily to the Y2 chromosome. We therefore treated the cells with aneugenic agents to determine if induced aneuploidy follows the same pattern and, hence, if there are limitations on the generation of aneuploidy or survival of cells lacking certain chromosomes. Exposing the cells to benomyl (8-100 micrograms/ml for 1 h), caffeine (5 x 10(-5)-2 x 10(-4) M for 2, 24 and 72 h) and colchicine (2 x 10(-4) and 5 x 10(-5) M for 1, 24, 48 and 72 h) resulted in cells primarily aneuploid for the Y2 chromosome. The frequency of cells lacking Y2 was far higher than those having an extra Y2 chromosome. The frequency of cells aneuploid for all other chromosomes combined was much lower than that for Y2. The data imply that the Indian muntjac genome can tolerate loss of the Y2 chromosome only and that aneuploidy for other chromosomes might cause lethality. This might be because the small number of chromosomes in the genome predisposes aneuploid cells to an imbalance of genes carrying out the basic activities required for cell division and cell survival. Because of the small chromosome number, the large size of the chromosomes and the ease of distinguishing every chromosome without banding or any other special treatment, e.g. FISH, this system could be useful and convenient in the study of induction of aneuploidy. This simple and inexpensive system can be utilized as a screening system for preliminary studies dealing with induced aneuploidy.

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.

Lack of recognition of a mouse centromere by anti-centromere proteins.

In studies dealing with cancer as well as mutagenesis, the centromere region is being looked at critically. Generally, this region of chromosomes reacts to certain antibodies present in the sera of scleroderma patients. Some chromosomes in the Cl1D cell culture of mouse origin fail to respond to these antibodies even though, in a sub-metacentric chromosome, they do react at a site distal to the centromere. This site of reaction, though, coincides with the presence of minor satellite DNA. Is it that neither the minor satellite nor the so-called CENPs constitute an essential component of a functional centromere?

The centromeres of the Indian muntjac: evidence for the existence of multiple centromeres?

Unlike the centromeres of other species, the "compound' centromeres of the Indian muntjac span over exceptionally extended regions (Brinkley et al., 1984). We extend this concept and show that some of these centromeres are divisible into several chromomeres in which the light staining regions alternate with the dark staining C-band positive segments. Unlike the centromeres of other species where the centromere replicates as one unit, the replication of the sub-units constituting the centromere of the X-chromosome in the muntjac occurs at different times as at least three independent segments. The CREST staining of the centromere regions of even the smallest (Y2) chromosome is interrupted by non-staining segments. Electron microscopy shows similar interruptions in the continuity of the trilamellar kinetochore. Sister chromatid exchanges occur in the region of the centromeres and chromatid breaks within the centromere region occur in the non-fluorescent segments. We interpret these data to suggest that the centromere regions of the Indian muntjac are made up of independent multiple centromeres interrupted by non-centromeric chromatin. Relevance of these parameters in mutagenesis is briefly discussed.

Localization of anti-CENP antibodies and alphoid sequences in acentric heterochromatin in a breast cancer cell line.

Karyotype alterations are a hallmark of cancer cells. Of particular interest to our laboratory are the inactive centromeres and blocks of heterochromatin devoid of the accompanying centromere. When purified or monospecies anticentromere proteins (CENP) antibodies or the whole serum from scleroderma (crest) patients are applied to human chromosomes, the centromere region exhibits the label. When we treated MDA 435 cells with the anti-CENP-A, anti-CENP-B, or the whole serum, the label was apparent on heterochromatin pericentric to the active and inactive centromeres. Moreover, blocks of heterochromatin not associated with any centromere also exhibited the label. Anti-CENP-C, however, is more strictly confined to the centromere in discrete dots and is not detected on any region except the sites of active centromeres. Distribution of alpha sequences also shows a pattern compatible with its distribution in the heterochromatin. Apparently, the use of anti-CENP-A and anti-CENP-B antibodies or alphoid DNA may not detect either the centromere (primary constriction) or the kinetochore; CENP-C may be an exception.

Satellite I DNA in transformed rat cells.

P93-50, a 93-basepair (bp) repetitive DNA sequence from rats, was hybridized to transformed sublines of rat endothelial origin. The sequence hybridized at or near the centromeres of most but not all chromosomes in two transformed cell lines and three single-cell derived cultures. The hybridization signal was also frequently present at the telomeres. These cell lines have a highly aberrant karyotype including dicentric and multicentric chromosomes; however, even though this sequence labeled the centromere regions of some chromosomes, it did not hybridize with the telomere regions of the cell line XC, which rarely shows any dicentrics. Apparently, the telomere signals represent prematurely separating, inactive, terminal centromeres. The p93-50 sequence does not influence the timing of centromere separation, nor it is necessary for formation of heterochromatin.

The centromere: kinetochore complex.

At the meta-anaphase transition the centromeres in a genome separate in non-random sequential manner. This sequential separation depends upon the timing of replication of DNA located in the pericentric and centromeric region. Cells in long term cultures as well as some newborn humans carry dicentric chromosomes. The inactive centromeres in these dicentric chromosomes do not show any sequence of separation. Whether or not a dicentric chromosome would segregate equationally depends upon if only one centromere binds to microtubules or both are functional. In man and other higher apes, a 171 base pair long DNA repeat (the alphoid sequence) is present on all centromeres. In mouse, the minor satellite fraction is said to constitute the centromere. These two DNAs also carry a 17 bp long sequence, the CENP-B 'box' to which the CENP-B antigen is bound. Other species-eg, rat, pig, fish, Chinese hamster-exhibit still different sequences at the centromere and do not carry the CENP-B 'box' even though the antigen is ubiquitously present in all species. It is not clear why so many diverse sequences constitute the centromere when all centromeres look alike and perform the same function. I propose that the primary constriction owes its property not necessarily to its DNA composition but to some stereophysical property, eg the curvature and that the region is held together till late metaphase-anaphase due to a specific proteinaceous factor. The mammalian centromeres bind a complex of several proteins dubbed as CENtromere Proteins (CENP's). This complex, however, is not what constitutes the trilamellar kinetochore structure as see under the electron microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of minor satellite from the centromere in mouse.

The minor satellite DNA of mouse is believed to constitute the centromere. We report that centromeres of some chromosomes in the Cl1D cells of mouse are not associated with this DNA even though the latter is present on these chromosomes. The satellite DNA was detected distally from the centromere and could not be mistaken as a component of the centromere. We also report that the site of the primary constriction may not always coincide with the site of the anti-kinetochore antibody reaction. Whereas the regions containing the major satellite decondense upon treatment with bisbenzimidazole (Hoechst 33258), the sites carrying minor satellite resist decondensing.

Do specific nucleotide bases constitute the centromere?

It is becoming increasingly clear that the underlying base composition of the DNA located in the centromere region is vastly different in different organisms. The chromosomes of related species or even the various chromosomes of the same species differ widely in their so-called centromeric DNA. Yet all centromeres appear physically alike and perform similar functions. The present communication proposes that the physical properties of the centromere are not due to its base composition but due to stereophysical make-up of the DNA segment constituting the centromere. This unique make up might reflect some physical parameter, like curvature, of the DNA present in the centromere constriction. It is further proposed that a proteinaceous factor, centromerase, is responsible for holding the centromeres as one unit until meta-anaphase.

Chromosomes lacking kinetochore proteins: their meta-anaphase location suggests potential malsegregation.

We have previously reported that a rare chromosome may not carry the kinetochore protein complex--the CENtromere Proteins or CENPs. These chromosomes should not bind to spindle microtubules and, hence, should be found peripheral to the meta-anaphase arrangement exhibited by the chromosomes which do carry CENPs. This communication shows that this actually is the case. When 3T3 mouse cells were not treated with colcemid or hypotonic, the kinetochore-lacking (K-) as well as kinetochore-bearing (K+) chromosomes were found off the spindle zone. When the spindle is disrupted with mild hypotonic treatment or by colcemid, the frequency of K- chromosomes remains unchanged. However, even mild disruption of the spindle with hypotonic treatment increases the frequency of off-lying K+ chromosomes significantly. These data indicate that K- chromosomes do not bind to the spindle and, hence, are a factor in the genesis of aneuploidy. A considerable proportion of K- chromosomes carry the putative centromere DNA indicating that these are not acentric fragments. Since the CREST serum used recognizes all essential kinetochore proteins, the K- centromeres must also lack all essential CENPs.

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.

Formation of dicentrics with variable lengths in transformed cells.

Several dicentric chromosomes in transformed cells exhibit drastic variations in the length of the region between the two centromeres. In these dicentric chromosomes, the position of the centromeres from the proximal end remains unaltered. The increase or decrease in the length of the intercentromeric segment is attributed to interchromatid reorganization so that one chromatid gains a piece of the sister chromatid. This is achieved simply through two chromatid breaks in the interchromosomal loop, followed by nonreciprocal rejoining. These observations provide new credence to Revell's hypothesis.

Cytogenetic changes in primary, immortalized and malignant mammalian cells.

Some chromosomes in transformed rat cells and somatic cell hybrids fail to display the presence of kinetochore proteins as detected by antikinetochore antibodies. Such chromosomes (K- chromosomes) may constitute a novel mechanism for the genesis of aneuploidy. We have analyzed primary, immortalized and malignant mammalian cells for the presence of kinetochore proteins and micronuclei. Our results suggest a correlation of the K- chromosome and micronucleus frequency with the variability in chromosome number. Upon in situ hybridization with the minor satellite and alpha satellite sequences some K- chromosomes showed a signal. This indicates that the observed lack of kinetochores is not necessarily due to a lack of centromeric DNA. We conclude that dislocated K- chromosomes may become incorporated into micronuclei which are prone to loss. Such events would be associated with the generation of aneuploidy.