Orientation and segregation of a micromanipulated multivalent: Familiar principles,divergent outcomes

We studied the orientation and segregation of a particular quadrivalent in living grasshopper spermatocytes. Quadrivalents were detached from the spindle by micromanipulation, then placed and bent as desired. The detached quadrivalents reattach and orient on the spindle. Their orientation is determined by the same principles that apply to ordinary chromosomes in mitosis and meiosis, but the outcome is different. Certain characteristics of the quadrivalent lead to a variety of orientations rather than the single one typical of ordinary chromosomes. Two kinetochores in the quadrivalent are linked to the others by unusually long, flexible chromosome arms. These kinetochores may face either the same pole or opposite poles and tend to orient initially to the pole toward which they face. Consequently, the initial orientation of the flexibly linked kinetochores is variable, and, moreover, they frequently reorient. In contrast, the other two kinetochores are as rigidly connected as those in a small bivalent and so display the typical back-to-back arrangement. Usually, this arrangement leads quickly to a stable orientation of the two kinetochores to opposite poles. Sometimes, however, the back-to-back arrangement changes to a side-by-side arrangement so that the orientation of both kinetochores to the same pole is favored. The combined effect of this diverse behavior is that the quadrivalent has four stable orientations, each leading to a different distribution of chromosomes in anaphase. The result is genetic chaos. Ironically, this chaos is produced by the same mechanisms that, in ordinary bivalents and mitotic chromosomes, produce a single stable orientation and genetically appropriate chromosome distribution.by P.B. Moens     

Bivalent orientation and behavior in crane-fly spermatocytes recovering from cold exposure

At metaphase in crane-fly primary spermatocytes, the two sister kinetochores at the centromere of each homologue in a bivalent normally are adjacent and face the same pole; one homologue has all its kinetochore microtubules (kMTs) extending toward one pole and its partner has all its kMTs extending toward the opposite pole. In contrast, during recovery from exposure to 2 degrees C, one or both homologues in many metaphase bivalents had bipolar malorientations: all kMTs of one kinetochore extended toward one pole and some or all those of its sister extended toward the other. Metaphase sister kinetochores that had most of their kMTs extending toward the same pole were adjacent, and those with most extending toward opposite poles were separated from each other. Distances between homologous centromeres were similar to those in properly oriented bivalents. Maloriented bivalents were tilted relative to the spindle axis, and analysis of living cells showed that tilted configurations were rare during prometaphase in untreated cells but frequently arose in cold-recovering cells as initial configurations, then persisted through metaphase. This was in contrast to unipolar configurations of bivalents (configurations suggesting orientation of both homologous centromeres toward the same pole), which always reoriented shortly after the configuration arose. We conclude that in cold-recovering cells, bipolar malorientations are more stable than unipolar malorientations, and the orientation process is affected such that bipolar malorientations arise in bivalents upon initial interaction with the spindle and persist through metaphase.     

Tension,microtubule rearrangements,and the proper distribution of chromosomes in mitosis

The basis for stable versus unstable kinetochore orientation was investigated by a correlated living-cell/ultrastructural study of grasshopper spermatocytes. Mal-oriented bivalents having both kinetochores oriented to one spindle pole were induced by micromanipulation. Such malorientations are stable while the bivalent is subject to tension applied by micromanipulation but unstable after tension is released. Unstable bivalents always reorient with movement of one kinetochore toward the opposite pole. Microtubules associated with stably oriented bivalents, whether they are mal-oriented or in normal bipolar orientation, are arranged in orderly parallel bundles running from each kinetochore toward the pole. Similar orderly kinetochore microtubule arrangements characterize mal-oriented bivalents fixed just after release of tension. A significantly different microtubule arrangement is found only some time after tension release, when kinetochore movement is evident. The microtubules of a reorienting kinetochore always include a small number of microtubules running toward the pole toward which the kinetochore was moving at the time of fixation. All other microtubules associated with such a moving kinetochore appear to have lost their anchorage to the original pole and to be dragged passively as the kinetochore proceeds to the other pole. Thus, the stable anchorage of kinetochore microtubules to the spindle is associated with tension force and unstable anchorage with the absence of tension. The effect of tension is readily explained if force production and anchorage are both produced by mitotic motors, which link microtubules to the spindle as they generate tension forces.     

Localization of kinetochore fragments isolated from single chromatids in mitotic CHO cells

Summary Chinese hamster ovary (CHO) cells are treated with hydroxurea followed by a caffeine treatment to form detached kinetochore fragments in the absence of sister chromatids. Detached kinetochores in mitotic CHO cells display a functional association with MTs initiated from one or both centrosomes such that these association(s) have a significant influence on the location and orientation of detached kinetochores and/or their fragments. Kinetochore fragments which are amphitelically oriented are positioned approximately midway between the two centrosomes. Thus, a kinetochore isolated from a single chromatid can capture MTs from both poles. Monotelic orientation of these fragments is more frequently observed with kinetochore fragments located an average distance of 2.5 m from the nearest centrosome, compared to an average distance of 4.4 m in amphitelically oriented fragments. In cells treated with the potent MT poison, nocodazole, kinetochore isolation also occurs and therefore is not dependent on the presence of MTs. CHO cells treated to produce isolated kinetochores or kinetochore fragments then subsequently hyperosmotically shocked show no MTs directly inserted into kinetochore lamina, similar to the response of sucrose-treated metapbase PtK₁ cells. This treatment shows circular kinetochores tangentially associated with bundles of MTs that are located an average of 1.5 m from the centrosome. Our results suggest that a single kinetochore fragment can attach to MTs initiated from one or both centrosomes and that their specific association to MT fibers defines orientation of detached kinetochores within the spindle domain.     

Chromosome micromanipulation

The relationship of kinetochore orientation and reorientation to orderly chromosome distribution in anaphase has been studied experimentally by micromanipulation of living grasshopper spermatocytes. Bivalents or the X chromosome at prometaphase or metaphase I can be detached from the spindle with a microneedle and moved to any desired location within the cell. Following a pause of variable duration the detached chromosome invariably moved, kinetochores foremost, back to the spindle, reassumed its characteristic metaphase position, and, with one exception, segregated normally at anaphase I. Detachment from the spindle is demonstrated unequivocally (1) by manipulation evidence for the absence of the firm spindle connections seen both before detachment and after reattachment and (2) by a functional criterion: a given kinetochore, oriented to one pole before detachment, often orients to the opposite pole after detachment. The segregation in anaphase was always as expected from the final, post-operation, orientation. Reorientation and prometaphase and anaphase movement after detachment cannot be distinguished from their counterparts in control cells. Kinetochore position after detachment is the primary determinant of the pole to which that kinetochore will orient. Therefore, since the experimenter determines kinetochore position, he can cause any given half-bivalent to segregate to a predetermined pole at anaphase I. Similarly, orientation of both half-bivalents to the same pole can be induced. These mal-oriented bivalents invariably reorient and normal anaphase segregation ensues. Non-disjunction can, however, be produced directly in late anaphase. These experiments are based upon current views of orderly chromosome distribution; their success confirms our understanding of the fundamental orientation process.     

Anaphase spindle mechanics prevent mis-segregation of merotelically oriented chromosomes

Merotelic kinetochore orientation is a kinetochore misattachment in which a single kinetochore is attached to microtubules from both spindle poles instead of just one. It can be favored in specific circumstances, is not detected by the mitotic checkpoint, and induces lagging chromosomes in anaphase. In mammalian cells, it occurs at high frequency in early mitosis, but few anaphase cells show lagging chromosomes. We developed live-cell imaging methods to determine whether and how the mitotic spindle prevents merotelic kinetochores from producing lagging chromosomes. We found that merotelic kinetochores entering anaphase never lost attachment to the spindle poles; they remained attached to both microtubule bundles, but this did not prevent them from segregating correctly. The two microtubule bundles usually showed different fluorescence intensities, the brighter bundle connecting the merotelic kinetochore to the correct pole. During anaphase, the dimmer bundle lengthened much more than the brighter bundle as spindle elongation occurred. This resulted in correct segregation of the merotelically oriented chromosome. We propose a model based on the ratios of microtubules to the correct versus incorrect pole for how anaphase spindle dynamics and microtubule polymerization at kinetochores prevent potential segregation errors deriving from merotelic kinetochore orientation.     

Malorientation in half-bivalents at anaphase in crane fly spermatocytes following Colcemid treatment

Addition of Colcemid to the medium in which larvae of the crane fly Nephrotoma suturalis are cultivated induces a number of anomalous patterns of chromosome segregation. One of these is the anaphase lagging of autosomal half-bivalents. To investigate the cause of anaphase lagging, the orientation of sister kinetochores in Colcemidtreated spermatocytes having lagging half-bivalents was analyzed in serial sections. In contrast to nonlaggard halfbivalents that had pure syntelic orientation (sister kinetochores having all of their kinetochores microtubules (KMTs) extending to the same pole), six of the seven autosomal laggards that were selected for analysis had kinetochores with either amphitelic orientation (sister kinetochores each with a bundle of KMTs extending to opposite poles) or merotelic orientation (a single kinetochore having KMTs extending toward both poles). An additional laggard had syntelic orientation but two of the microtubules that were in its kinetochore fiber passed through the kinetochore and extended beyond it toward the equator. The bipolar malorientations observed in anaphase half-bivalents are interpreted to be a cause of the anaphase lagging induced by Colcemid treatment. Furthermore, it is hypothesized that such bipolar malorientations also may be stabilized at metaphase and thus explain the unusual tilting of metaphase bivalents commonly observed in Colcemid-treated cells.     

The segregation pattern of a translocation quadrivalent

The segregation pattern of a translocation quadrivalent was studied in three hybrid families of the tetraploid (2n=28) oat, of the Avena strigosa polyploid complex. The rate of IV formation in F₁ was high and the fertility was normal. The adjacent-alternate orientation of this quadrivalent was very variable. Conspicuous variation in this frequency was found between anthers of the same floret and between florets. The alternate type was usually more common toward the end of MI. The adjacent-alternate ratio was found to be an unreliable measure for calculating the type of gametes produced by the F₁ and the F₂ plants derived from them. An attempt was made to determine the type of viable gametes produced on the F₁ and their frequency by examining the cytology of F₂ plants. The various combinations of the chromosomes of the translocation complex expected in the F₂ plants were derived and their expected frequencies calculated by taking into account chiasma formation at the chromosome ends and various restrictions of gamete viability. In none of the three hybrid families F₂ individuals were found to produce trivalents as the most complex chromosome configuration, indicating that one type of gamete derived from adjacent separation was not formed. The 11 ratio between F₂ plants having only bivalents (2II) and those with F ₁-like quadrivalent configuration R(c), expected when gametes resulting from alternate separation are fully functional, with the exclusion of other types, was not found. That ratio 2II/R(c), was 2-1/3 in the various families. The cytology of selected F₃ individuals basically followed the predictions based on chromosome association in the F₂.     

A transmissible dicentric chromosome in Drosophila melanogaster

A transmissible dicentric chromosome was recovered in Drosophila melanogaster. The radiation-induced secondary chromosome rearrangement consists essentially of the entire Y and fourth chromosomes joined by 2R heterochromatin. The Y ^S · Y ^L 2Rh4 · chromosome pairs with the X and the free fourth chromosome to form a trivalent in meiosis that is unusual because it forms few chromosome bridges in primary spermatocytes and is transmitted at high frequency. We suggest that the orientation of the weaker fourth chromosome kinetochore eventually fails when opposing the stronger Y kinetochore so that the Y ^S · Y ^L 2Rh4 · moves to the pole to which the Y kinetochore is oriented. There is however an increased frequency of sex chromosome nondisjunction (14%) and of chromosome laggards (6%) in primary spermatocytes; the frequency of exceptional progeny of males containing the Y ^S · Y ^L 2Rh4 · was 7.44% compared with 0.25% in the controls. Disruption of normal sex chromosome disjunction also occurs in females containing the Y ^S · Y ^L 2Rh4 · and a compound X chromosome; the frequency of exceptional progeny was 2.55% versus 0.91% in the controls. Chromosome nondisjunction appears to occur when orientation of the X and Y kinetochores to the same pole is stabilized through tension by the orientation of one or both fourth chromosome kinetochores to the opposite pole. During anaphase, the orientation of the fourth chromosome kinetochore of the Y ^S · Y ^L 2Rh4 · appears to fail and the X and Y ^S · Y ^L 2Rh4 · chromosomes move to the same pole. Y ^S · Y ^L 2Rh4 · chromosome laggards occur with both the Y and fourth chromosome kinetochores amphitelically oriented. This orientation appears to be stable as a result of equal opposing forces toward opposite poles.     

Centromeric dots in crane-fly spermatocytes: meiotic maturation and malorientation

During the first meiotic division in crane-fly spermatocytes, the two homologs of a metaphase bivalent each bear two sister kinetochores oriented toward the same pole. We have previously reported treatments that increase the percentage of metaphase bivalents in which one or both homologs have bipolar malorientations: kinetochore microtubules] extending from a homolog toward both poles. The maloriented homologs lag at anaphase. Treatments that induce this behavior include: (a) recoverey from exposure to low temperatures or Colcemid or Nocodazole concentrations that prevent spindle formation but allow nuclear membrane breakdown, and (b) exposure to 6° C, a temperature that permits spindle assembly but slows progression through meiosis. Giemsa staining methods reveal two 0.5 m diameter dots at the centromeric region of each metaphase homolog; these often are more separated in maloriented homologs. This investigation was undertaken to assess whether this separation precedes the establishment of bipolar malorientation, and hence may be a cause of it, or is only a consequence of forces resulting from bipolar malorientation. Analysis showed that, in untreated cells, the average center-to-center distance between sister centromeric dots increases during the course of meiosis I. After the above-mentioned treatments, center-to-center distances similar to those normally seen in untreated half-bivalents at anaphase I were seen in bivalents, both after and before nuclear membrane breakdown. Longer exposure to temperatures that arrested meiosis increased the degree of dot separation. Based on our data, we conclude that normal orientation during the first meiotic division is aided by the close apposition of centromeric dots, and that a time-dependent maturation occurs causing centromeric dots to separate for the second meiotic division and facilitating orientation of sister kinetochores to opposite poles. If centromeric maturation occurs either prior to or during early stages of the first meiotic division, then it may contribute to persisting bipolar malorientation.     

Location of satellite DNAs in the chromosomes of the kangaroo rat (Dipodomys ordii)

The location of satellite DNA sequences in metaphase chromosomes has been studied in the kangaroo rat by the in situ hybridization technique, staining techniques and phase contrast microscopy. The HS- satellite DNA is located at the kinetochores of all but three chromosome pairs. The HD satellite is located predominantly in the short arms of the chromosomes containing HS- and in the kinetochores of chromosome pairs that lack HS-. The regions that contain the satellite DNA sequences can also be identified by the Giemsa staining technique, and can be visualized with phase contrast microscopy or following Feulgen staining of fixed chromosome preparations.     

Centromere orientation,reorientation, and segregation in an interchange quadrivalent during anther development in pearl millet

Prometaphase I orientation, reorientation and anaphase I segregational behaviour of a chain-forming interchange quadrivalent involving one of the long chromosomes and the long arm of the seventh (nucleolar) chromosome was studied during anther development in pearl millet. The data obtained from 34 anthers showed that by early prometaphase I about 90% of the bivalents have attained stable bipolar orientation but about 48% of the quadrivalents are mal-oriented. There seems to be an interaction between bivalents and quadrivalents during mal-orientation and reorientation. The mal-oriented bivalents reoriented before the quadrivalents. For quadrivalent mal-orientation four types, 4/0, 3/1, 2/1/1/1 and 2/2 (adjacent 1), were distinguished in addition to the regular types, adjacent 2 and alternate. Based on their potential to reorient, the order of the mal-oriented quadrivalent types was 4/0 > 3/1 > 2/1/1; 2/2 led to anaphase I disjunction as for an adjacent 1 segregation. The data from 36 anthers at anaphase I showed alternate segregation of chromosomes in nearly 50% of pollen mother cells (PMCs) up to a developmental index of about 65. In late anthers about 35% PMCs showed alternate segregation. This suggests that the PMCs that reached metaphase I later had more adjacent 2 orientations since mal-oriented configurations delay meiotic development, and implies preferential reorientation behaviour of the maloriented quadrivalent types.     

MITOSIS IN THE FUNGUS THRAUSTOTHECA CLAVATA

The ultrastructure of mitosis is described in Thraustotheca clavata, an oömycete fungus. An intranuclear spindle develops between differentiated regions of the nuclear envelope which move apart, each associated with 180° oriented centriole pairs. The spindle contains low numbers of continuous and interdigitating microtubules in addition to chromosomal microtubules. Each kinetochore is attached to only one microtubule. Serial section analysis shows that at meiosis there are probably 12 chromosomes in the diploid nucleus, yet at mitosis the methods utilized in the present study suggest that there may be less than 12 kinetochores connected to each pole. At mitosis many of the kinetochores within a given spindle are not arranged in opposite pairs. The behavior of the spindle microtubules during mitosis is comparable to that of higher organisms but the rarity of short intertubular distances appears to preclude significant force generation by means of intertubular bridge mechanisms. Evidence is presented for a nuclear envelope-microtubule interaction which is capable of generating shear forces during both mitosis and interphase nuclear movements.     

Role of plasma membrane proteins and cell wall in meridional arrangement of cortical microtubules inBoodlea coacta

Summary The effects of 2,6-dichlorobenzonitrile (DCB, an agent which inhibits cellulose synthesis) and cycloheximide (CHI, a known inhibitor of protein synthesis) on the construction and stability of the cortical microtubule (MT) cytoskeleton in two kinds of protoplasts (smaller protoplasts and larger ones) prepared fromBoodlea coacta (Dickie) Murray et De Toni were examined by immunofluorescence microscopy. In smaller protoplasts which develop from released protoplasmic masses in culture media, parental cortical MTs assume a convoluted configuration, but new cortical MTs appear following disassembly of convoluted MTs. New cortical MTs initially have a random arrangement but later, a rough meridional arrangement following development of cell polarity and finally, a high density meridional arrangement. In larger protoplasts which are formed within cell wall cylinders of thalli cut at 500 m length, longitudinally oriented parental cortical MTs are preserved. Each exhibits a curving configuration just after protoplast formation, but a straight configuration after 3 h of culture. In smaller protoplasts, cortical MT orientation changes from random to rough meridional orientation but never to a high density meridional orientation following treatment with 10 M CHI, and MT density decreases after 12 h. However, rough meridional and high density meridional arrangements of MTs ceased to be formed and MT density decreased following treatment with 10 M DCB. In larger protoplasts, high density meridional arrangements of MTs were noted not to be affected by treatment with CHI; instead, they continued to remain oriented meridionally, but the length and density were decreased after treatment with DCB for 3–4 h. After 10 h, the MTs became fragmented and orientation was random. From these findings it is summarized that: (1) There are no putative anchors in the plasma membrane of nascent smaller protoplasts, but the meridional orientation of cortical MTs requires anchors which may be distributed in the plasma membrane following the establishment of cell polarity. (2) Plasma membranes in larger protoplasts contain parental anchors oriented meridionally. Anchors stabilize cortical MTs via their close relation to cell walls (especially to cellulose). Anchors are detached from the plasma membrane when cellulose is not formed. (3) Cellulose regeneration may be indispensable to the formation and stabilization of the MT cytoskeleton inBoodlea.Abbreviations CHI cycloheximide - DCB 2,6-dichlorobenzonitrile - DMSO dimethylsulfoxide - MT microtubule     

Orientation of nonrandomly segregating sex chromosomes in spermatocytes of the flea beetle, Alagoasa bicolor L.

In males of the flea beetle, Alagoasa bicolor L., spermatocytes have two achiasmate sex chromosomes, X and Y, each of which is approximately five times larger than the ten pairs of chiasmate autosomes. At metaphase I, these univalent sex chromosomes are located on a spindle domain separated from the autosomal spindle domain by a sheath of mitochondria. A single centriole pair is located at each pole of the spindle. In prometaphase I, each sex chromosome appears to maintain an attachment to both spindle poles via kinetochore microtubules (i.e., amphitelic orientation). Before anaphase I, this orientation changes to the syntelic orientation (both sister kinetochores connected to the same pole), perhaps by the release of microtubule attachments from the more distant pole by each of the chromosomes. The syntelic orientation just prior to anaphase I leaves each sex chromosome attached to the nearest pole via kinetochore microtubules, ensuring nonrandom segregation. As the sex chromosomes reorient, the autosomes follow in a sequential manner, starting with the bivalent closest to the sex spindle domain. We report here data that shed new light on the mechanism of this exceptional meiotic chromosome behavior.     

Chromosomes selectively detach at one pole and quickly move towards the opposite pole when kinetochore microtubules are depolymerized in <Emphasis Type="Italic">Mesostoma ehrenbergii</Emphasis> spermatocytes

In a typical cell division, chromosomes align at the metaphase plate before anaphase commences. This is not the case in Mesostoma spermatocytes. Throughout prometaphase, the three bivalents persistently oscillate towards and away from either pole, at average speeds of 5–6 μm/min, without ever aligning at a metaphase plate. In our experiments, nocodazole (NOC) was added to prometaphase spermatocytes to depolymerize the microtubules. Traditional theories state that microtubules are the producers of force in the spindle, either by tubulin depolymerizing at the kinetochore (PacMan) or at the pole (Flux). Accordingly, if microtubules are quickly depolymerized, the chromosomes should arrest at the metaphase plate and not move. However, in 57/59 cells, at least one chromosome moved to a pole after NOC treatment, and in 52 of these cells, all three bivalents moved to the same pole. Thus, the movements are not random to one pole or other. After treatment with NOC, chromosome movement followed a consistent pattern. Bivalents stretched out towards both poles, paused, detached at one pole, and then the detached kinetochores quickly moved towards the other pole, reaching initial speeds up to more than 200 μm/min, much greater than anything previously recorded in this cell. As the NOC concentration increased, the average speeds increased and the microtubules disappeared faster. As the kinetochores approached the pole, they slowed down and eventually stopped. Similar results were obtained with colcemid treatment. Confocal immunofluorescence microscopy confirms that microtubules are not associated with moving chromosomes. Thus, these rapid chromosome movements may be due to non-microtubule spindle components such as actin-myosin or the spindle matrix.     

Structure of kinetochore fibers: Microtubule continuity and inter-microtubule bridges

To understand how microtubules interact in forming the mitotic apparatus and orienting and moving chromosomes, the precise arrangement of microtubules in kinetochore fibers in Chinese hamster ovary cells was examined. Individual microtubules were traced, using high voltage electron microscopy of serial 0.25 m sections, from the kinetochore toward the pole. Microtubule arrangement in kinetochore fibers in untreated mitotic cells and in cells recovering from Colcemid arrest were similar in two respects: the number of microtubules per kinetochore (mean 14 and 12, respectively) and the nearest neighbor intermicrotubule distance (mean90 nm). In Colcemid recovered cells, over 90% of the microtubules in kinetochore fibers were attached to the kinetochore (i.e. kinetochore microtubules) and extended most or all of the distance to the pole. Few free microtubules were present in the kinetochore fibers; most non-kinetochore microtubles terminated in the pole. Since kinetochores in this Colcemid-recovered system have been demonstrated to nucleate microtubules (Witt et al., 1980), it seems likely that most if not all of these kinetochore microtubules originated at the kinetochore. Some of the reconstructed kinetochore fibers were attached to chromosomes with bipolar orientation, suggesting that kinetochore microtubules need not interact with many polar microtubules for orientation to occur. In Colcemid recovered cells lysed to reduce cytoplasmic background, microtubules in kinetochore fibers were preferentially preserved. The parallel and near-hexagonal order typical of microtubules in kinetochore fibers was maintained, as was the number of kinetochore microtubules (mean, 13). The intermicrotubule distance was slightly reduced in lysed cells (mean, 60 nm). Crossbridges about 5 nm wide and 30–40 nm long were visible in kinetochore fibers of lysed cells. Such crossbridges probably contribute to the stabilization and parallel order of microtubules in kinetochore fibers, and may have a functional role as well.     

Chromosome segregation: clamping down on deviant orientations

Chromosome segregation depends on proper orientation of sister kinetochores. The protein Csm1 is required for mono-orientation of sister kinetochores at meiosis I in budding yeast. Surprisingly, its homologue in fission yeast appears instead of clamp micro-tubule binding sites together on single mitotic kinetochores so that they all face one spindle pole.     

Compound kinetochores of the Indian muntjac

The chromosomes of the Indian muntjac (Muntiacus muntjak vaginalis) are unique among mammals due to their low diploid number (2N=6, 7) and large size. It has been proposed that the karyotype of this small Asiatic deer evolved from a related deer the Chinese muntjac (Muntiacus reevesi) with a diploid chromosome number of 2n= 46 consisting of small telocentric chromosomes. In this study we utilized a kinetochore-specific antiserum derived from human patients with the autoimmune disease scleroderma CREST as an immunofluorescent probe to examine kinetochores of the two muntjac species. Since CREST antiserum binds to kinetochores of mitotic chromosomes as well as prekinetochores in interphase nuclei, it was possible to identify and compare kinetochore morphology throughout the cell cycle. Our observations indicated that the kinetochores of the Indian muntjac are composed of a linear beadlike array of smaller subunits that become revealed during interphase. The kinetochores of the Chinese muntjac consisted of minute fluorescent dots located at the tips of the 46 telocentric chromosomes. During interphase, however, the kinetochores of the Chinese muntjac clustered into small aggregates reminiscent of the beadlike arrays seen in the Indian muntjac. Morphometric measurements of fluorescence indicated an equivalent amount of stained material in the two species. Our observations indicate that the kinetochores of the Indian muntjac are compound structures composed of linear arrays of smaller units the size of the individual kinetochores seen on metaphase chromosomes of the Chinese muntjac. Our study supports the notion that the kinetochores of the Indian muntjac evolved by linear fusion of unit kinetochores of the Chinese muntjac. Moreover, it is concluded that the evolution of compound kinetochores may have been facilitated by the nonrandom aggregation of interphase kinetochores in the nuclei of the ancestral species.