Fine structure of kinetochore in Indian muntjac

Experimental

FINE

STRUCTURE

Cell Research 67 (1971) 97-l 10

OF KINETOCHORE D. E. COMINGS

IN INDIAN

MUNTJAC

and T. A. OKADA

Department of Medical Genetics, City of Hope National Medical Center, Duarte, Cal& 91010, USA

SUMMARY The large size of Indian muntjac (miniature deer) chromosomes and the presence of an unusually long centromere region on an X-autosome fusion chromosome provided an ideal subject for the study of the fine structure of the kinetochore. Whole mount electron microscopy demonstrated a marked restriction in the lateral looping of chromatin fibers in the region of the centromere. By thin section electron microscopy it was possible to distinguish longitudinal from transverse sections of the kinetochore. In longitudinal sections the outer layer was long (up to 1.45 pm) and straight and subcomponents consisting of two parallel or seemingly intertwined 90 8, lines were frequently seen. In transverse sections the outer layer was shorter, averaging 0.45 pm. It curved in a semicircle around part of the chromosome, and the subcomponents frequently appeared as a series of tubule-like structures. The inner layer of the kinetochore was intimately associated with and was probably composed of chromatin fibers. Transverse and serial longitudinal sections showed that the outer layer was an oval plate-like structure and was not composed of two kinetochore filaments. The structural features of the outer layer suggested that it does not contain chromatin fibers. The spindle fibers pass through the outer layer and usually disappear from view in the middle layer. In some sections they can be seen to pass through the middle and inner layer, and in other sections they appear to turn within the body of the chromosome and re-emerge through the inner and outer layer.

Acting on the premise that studiesof extremes are frequently informative, we report here whole mount and thin section electron microscopy studies of the centromere region and kinetochore of Indian muntjac chromosomes. The Indian muntjac has the lowest chromosome number yet reported for any vertebrate and as a consequencethe chromosomesare large [25]. In addition, there is an unusually long centromere region on an X-autosome fusion chromosome. This region is heterochromatic, replicates during a short period of time in the latter portion of the S period, shows lesscondensation in response to Colcemid treatment than the remaining portions of the chromosome, stains heavily following denaturation and renaturation, and 7-

711811

fluoresces poorly after staining with quinacrine mustard [5]. This combination of large chromosomes plus an unusually large centromere region with well defined properties provided an ideal setting for studies of the fine structure of the kinetochore. MATERIALS

AND METHODS

Fibroblasts from a male Indian muntjac (kindly provided by K. Benirschke) were grown in McCoy media supplemented with 10% fetal calf serum and 1 % essential and non-essential amino acids. The cells were seeded into 250 ml elastic flasks. During the log phase of growth the‘ flasks were shake; vigorously to remove the cells in mitosis. These were collected by centrifugation. For whole mount

studiesa concentratedsuspension of thesecellswas

allowed to run down a trough onto a surface of distilled water and the surface was then touched with copper grids that had been coated with Formvar and Exptl

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D. E. Comings & T. A. Odada

carbon. The grids were placed on a solution of 2 % uranyl acetate for 10 min, dehydrated through a series of ethanol washes, washed twice with amyl acetate and then either air dried or dried in the critical point apparatus of Anderson [I]. For thin section studies cell susnensions were fixed in 3 % glutaraldehyde both at 4°C and 22°C for 1 h, then in 1 % osmic acid for 1 h, washed, dehydrated through a series of ethanol washes at room temperature, placed sequentially in propylene oxide, propylene oxide Epon-Araldite 1: 1, then Epon-Araldite mixture. The samples were sectioned at 800 A, stained with uranyl acetate and lead citrate and examined in an Hitachi HS-8-l electron microscope at 50 KV.

region that is an intimate part of the chromosome itself, the inner layer [14]; and the region between the inner and outer layer, the middle layer [14]. All of these areas, together constitute the kinetochore. The chromatin fibers in the region of the kinetochore will be referred to as the centromere region and the two sites (in a metacentric chromosome) where the chromatin fibers

RESULTS

with each other will be termed the area of chromatid association.

from

the two

sister

chromatids

interdigitate

Light microscopy Detailed light microscopy studies, including the effect of Colcemid on chromosome condensation and investigation of the DNA replication patterns have been reported in a companion paper [5]. The chromosome complement of the male muntjac consists of two large metacentric autosomes, two sub-telocentric autosomes, a single telocentric autosome, an X-autosome fusion chromosome, and a small Y chromosome (fig. 1). In an earlier whole mount electron microscopy study of chromosomes from a number of mammals [lo] it was apparent that the centromere region of metacentric chromosomes consisted of two regions of chromatid association separated by a clear area. This is shown particularly well in the large metacentric chromosome (double arrows). The region of fusion of the X chromosome and the telocentric autosome can also be seen (long arrow).

Whole mount electron microscopy

In whole mount studies all but the inner layer or chromosomal portion of the kinetochore is eluted away so that what is seen is the centromere region. In some cell lines, when special precautions are taken, the spindle fibers are retained intact [8]. However, they were not seen in the present preparations. The most striking feature of the low magnification whole mount preparations was the tendency for the chromatin fibers in the immediate vicinity of the kinetochore to take on a sharply curved, scimitar-like configuration (figs 2, 3, 5, 6). In this region none of the chromatin fibers tended to loop out laterally as they did in the chromosome arms. It is unlikely that this was due entirely to the tension placed on this region by the chromosome arms since a similar restriction on the lateral looping of fibers was noted in the long centromere region of the Xautosome fusion chromosome (fig. 4). In Definition of terms most preparations there was a clear area Before progressing further it is necessary to in the immediate vicinity of the region where clarify the terms that will be used in this the kinetochore had been (fig. 6, dotted paper. They are illustrated in fig. 29. The line). This was comparable to a similar clear plate-like structure that is set somewhat area at the centromere region seen by light away from the chromosome and to which microscopy [19]. A higher magnification of the microtubules tend to converge, will be this scimitar-like region of chromatin fiber termed the outer layer [14]; the clear region compaction is shown in fig. 7. The area lateral to it the corona [14]; the plate-like where the kinetochore had been is clear of Exptl

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Kinetochore fine structure

99

Fig. I. Light microscopy of the metaphase chromosomes of the male muntjac. There are two large metacentric chromosomes, two smaller sub-telocentric chromosomes, two telocentric chromosomes, one of which is fused to the X chromosome (nrrow), and a small Y chromosome (short arrow). The two regions of chromatid association in the large metacentric are shown by the double arrows. x 1 300. Fig. 2. A whole mount preparation of the large autosomal metacentric chromosome. The scimitar-like configuration of the chromatin fibers in the region of the centromere is illustrated by the double arrows. x 3 240. Fig. 3. A higher magnification of the centromere region of the chromosome shown in fig. 2. The chromatin fibers in the region of the centromere in the left upper portion of the figure (urrows) describe a sharply outlined curving configuration. x 7 400. Fig. 4. A whole mount preparation of the X-autosome fusion chromosome. There is an extended area of narrowing along the centromere region of this chromosome. The distance between the two arrows is 2.4 pm. x 6 600. Fig. 5. A metacentric chromosome. The chromatin fibers of both arms are free to diffuse in a lateral direction while there is considerable restriction on the lateral diffusion of fibers in the region of the centromere. x 4 400. Fig. 6. A metacentric chromosome showing a relatively clear area (dots) around the region of the centromere. X6600.

chromatin fibers (lower left). The chromatin fibers in this region showed the same tangled net-like appearance typical of the fibers of the chromosome arms [Ill. There were

frequent areas where the chromatin fibers tended to converge (arrow, fig. 8). These sites of convergence may be fortuitous or they may represent sites where the chromatin Exptl

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D. E. Comings & T. A. Okada

fibers converge to attach to the nuclear membrane during interphase [9]. Thin section electron microscopy The unusual length of the muntjac centromere provided an opportunity of distinguishing whether a given section of the kinetochore was passing longitudinally (parallel to the long axis of the chromosome) or transversely (perpendicular to the long axis) Longitudinal

sections

The sections which probably represent longitudinal cuts had the following characteristics. (a) The kinetochore was straight. A typical straight kinetochore is shown in figs 9-11 which represent three of six serial sections through what is probably a kinetochore of the X-autosome chromosome. This is suggested by the unusual length of this kinetochore (1.1 pm) and by the fact that the secondary constriction came into view in the 5th (fig. 14) and 6th serial section. (b) The kinetochore was long. This is demonstrated in fig. 28 which shows measurements of the length of the outer layer in 46 sections in which the kinetochore did not appear to be cut obliquely. The straight kinetochores are in white, the curved ones in black. There was a wide variation in the length of straight kinetochores ranging from 0.55 to 1.4 pm. Those which were in the range of 1.O to 1.4pm probably represent the kinetochores of the X-autosome chromosomes. Those less than 1.0 pm probably represent predominantly those of the nonX-autosome chromosomes. (c) The subcomponents of the outer layer frequently appeared as two lines which were either parallel or gave the illusion of being relationally coiled about each other (fig. 13). These lines were approx. 90 A in width. In the longitudinal sections a given segment of the outer layer persisted through four to Exptl

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Fig. 7. A whole mount preparation of a moderately well dispersed metacentric chromosome illustrating the double scimitar-like arrangement of the fibers making up the lateral portions of the centromere region. x 10 500. Fig. 8. A higher magnification of the well dispersed centromere region of an autosomal chromosome. There are a number of areas where the chromatin fibers appear to converge (arrow). x 17 000.

five 800 A serial sections, suggesting that the width of the kinetochore ranged from 0.32 to 0.40 ,um. This was confirmed by the transverse sections (see p. 101).

Kinetochore fine structure

101

Fig. 9. A thin section electron microscopy of the kinetochore of what is probably the long centromere region of the X-autosome fusion chromosome. The distance between the arrows measures 1.2 ,um. x 18 500. Figs 10, II. Successive serial sections of the same chromosome illustrated in fig. 9. x 18 500. Fig. 12. A higher magnification of the kinetochore region from fig. 9. The outer layer of the kinetochore is paler than the chromatin fibers and is surrounded by a less dense region on both sides. x 31 000.

Transversesections The sections which probably represent transverse cuts had the following characteristics. (a) The kinetochore was curved. This is shown in figs 2425. Both the outer and inner layers formed a semi-circle. There was occasionally a decreasein the density of the chromatin fibers at the center of the semicircle formed by the inner layer (large arrow). (6) The kinetochore was short. This is demonstrated in fig. 28. The measurements of the length of the curved kinetochores showed that they tended to represent a distinct class with lengths less than 0.65 pm

and averaging 0.44 pm. This mean width of 0.44 is somewhat wider than that suggested by the serial longitudinal sections. However, the two measurements are actually quite comparable since the serial sections measured the diameter of the semi-circle while the transverse sections measured the circumference. (c) The subcomponents of the outer layer frequently appeared as a row of individual dots (figs 24, 25). These may represent a series of tubule-like structures cut transversely. There were about lo-14 such dots in the outer layer. Exptl

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D. E. Comings & T. A. Okada

Fig. 13. A high magnification of fig. 10 showing the substructure of the outer layer as consisting of two fine lines, 90 A in width, which in some areas give the appearance of being plectonemically entwined. x 62 700. Fig. 14. Section 5 of the 6 serial sections through the kinetochore region shown in figs 9-11. The arrow denotes the narrowed region of the chromosome which probably corresponds to the secondary constriction of the X-autosome chromosome. > 16 500. Fin. 15. An enlargement of fig. 14 showing numerous ribosome-like particles in the region of the secondary constriction. y 40400. -

Outer layer In both the longitudinal and transverse sections the thickness of the outer layer ranged from 300 to 450 A and the thickness of the middle layer ranged from 200 to 380 8. Serial longitudinal sections of the kinetochore (figs 9-11) gave no evidence for the existence of two portions or kinetochore filaments [4]. This could be confirmed by the transverse sections which also showed that the outer layer was a single continuous semi-circular structure. Inner layer The inner layer of the kinetochore is intimately associated with and is probably ExptI

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composed largely of the chromatin fibers themselves. This layer is almost impossible to see when the chromatin fibers of the kinetochore region are highly compacted as they are in the sections shown in figs 9-12. In some sections, however, it was easy to discern the inner layer (figs 16-21). Figs 16 and 17 are two of a seriesof serial sections. In fig. 16 the inner layer of the lower kinetochore can be seen and in fig. 17 the inner layer of the upper kinetochore has become obvious. Occasionally the inner layer was partially (figs 19, 20) or completely (figs 21) pulled away from the chromosome proper and in some sections it appeared to be composed of a series of granule-like regions

Kinetochore fine structure

103

Figs 16-21. The arrows point to the inner layer of the kinetochore. In figs 16-18 the inner layer is in intimate contact with the chromatin fibers of the centromere region. In fig. 17 there is an area of decreased chromatin density between the two sister kinetochores which probably represents a separation of the two chromatids. In figs 19 and 20 the inner layer has pulled away somewhat from the centromere region, and in fig. 20 a number of small granule-like areas can be seen. These may represent sites of convergence of chromatin fibers shown in fig. 8. In fig. 21 the inner layer has almost completely pulled away from the centromere region. Figure magnification: 16, x 26 600; 17, x 28 500; 18, x 38 500; 19, x 33 400; 20, x 50 500; 21, x 31 600.

(fig. 20, arrows). These granules might represent the sites of chromatin convergence shown in fig. 8. Although someof the sections suggested the presence of a double kineto-

chore filament [4] one of the pair was always intimately associated with the chromatin fibers, indicating it was actually an inner layer [14] rather than a double outer layer. Exptl

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D. E. Comings & T. A. Okada

The fact that the inner layer could be pulled away from the chromosome (fig. 21) does not mean it is necessarily distinct from the chromosome itself, but it does indicate that it is a sufficient of an entity that it can be broken away from the rest of the chromatin fibers. Spindle fibers and the kinetochore The spatial relationship between the spindle fibers and the kinetochore proved to be one of the most intriguing and at the same time one of the most difficult questions to investigate. In most of the preparations in which the spindle fibers were clearly visualized they could be seen to pass through the outer layer. They usually disappeared from view in the middle layer (fig. 22). In occasional preparations some of the fibers could be seen to pass through both the middle and inner layer (fig. 23). A feature of the spindle fiber arrangement that was sometimes so subtle as to approach the level of a subliminal impression was the appearance that the fibers, after passing through the outer and inner layers, seemed to curve back on themselves. This was seen primarily in the transverse sections. For example, in fig. 24 the arrow points to an area inside the semi-circle formed by the inner layer, where there is a decrease in the density of chromatin fibers. Two spindle fibers can be seen to traverse this area. In the higher magnification view (fig. 25) several additional spindle fibers can be seen which appear to pass through the upper portion of the outer layer, through the upper portion of the inner layer, and bend in a semi-circle to pass through the area of decreased chromatin fiber density, Fig. 22. The spindle fibers kinetochore. x 60 000. Fig. 2.3. A spindle fiber can extend into the centromere an abrupt 90” change in the ExptI

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pass back out through the lower portion of the inner layer, and then either pass through or stop at the lower portion of the outer layer. The smaller arrows point to several dot-like structures which may represent cross-sections of microtubule-like structures which may be an integral part of the outer layer. A second example of a section in which the spindle fibers appear to pass through both the outer and inner layers and then make a sharp bend in the body of the chromosome, is shown in fig. 27. The bottom arrow in this figure points to a microtubule which seems to be making a complete U-turn. A further example of a curving microtubule is shown by the small arrows in fig. 23. Another interesting aspect of kinetochore structure is shown in fig. 26. Here a double layered section of the outer layer appears to be making a sharp turn away from the chromosome toward the centriole. This may represent a longitudinal section of one of the tubulelike structures within the outer layer. Nucleolus organizer Fig. 14 is the 5th of six serial sections through what is presumably the centromere region of the X-autosome chromosome. The constriction occurring in the proximal one-half of the long arm most likely represents the secondary constriction on the X-autosome fusion chromosome. Higher magnification of the area shows the presence of a high concentration of ribosome-like bodies. DISCUSSION In fine provides

structure studies, the kinetochore an oasis of complexity compared

(arrow) can be seen to pass through to the inner side of the outer layer of the be seen to pass through both the outer and inner layers of the kinetochore and region (long arrow). The small arrows show a spindle fiber which is undergoing direction of its course. x 50 000.

106 D. E. Comings & T. A. Okada

Fig. 24. A transverse section of the muntjac kinetochore. The semi-circular outer and inner layers can be seen and there is an area of decreased density of the chromatin fibers within the inner layer (nrrow). Two spindle fibers can be seen to be traversing this area. x 32 000. Fig. 25. A higher magnification of fig. 24. Again, the two spindle fibers traversing the area of decreased chromatin fiber density within the inner layer can be seen (long arrow). If one follows the spindle fibers coming in from the upper left portion of the figure they appear to pass through the upper portion of the outer and inner layers, then curve around to pass out again through the lower portion of the inner layer. The outer layer also appears to contain within it a series of small dot-like structures which are partially or completely surrounded x 56 200. by fine 90 A lines (shout arrows). Fig. 26. A tubule-like portion of the outer layer appears to be bending out toward the centriole. x 54 600. Fig. 27. The spindle fibers pass through the outer and inner layers and then appear to turn abruptly and run parallel to the kinetochore within the region of the chromosome (arrows). The lower arrow points to a spindle fiber which appears to be undergoing a complete U-turn. x 73 500.

Exptl

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Kinetochore fine structure 75 3 1 I -1-1 - ~1 r 0 01 0.2 03

0.4 0.5 0.6

07

0.8

0.9 1.0

IL 1.1 1.2 1 3 1.L 1.5

Fig. 28. Abscissa: pm; ordinate: number. n , Semicircular; 7, straight kinetochores. Measurements in microns of the size of the outer layer in sections in which the kinetochore was straight (presumptive longitudinal sections) or semi-circular (presumptive transverse sections).

to the tedious uniformity of the rest of the chromosome. A number of recent investigations have probed the structure of this region [2-4, 14-16, 20-211. There is general agreement on the major aspects of kinetochore structure but significant variation on some details. For example, all investigators agree on the presence of the outer layer [14] or kinetochore filament [4.]. This structure averages 300 to 400 8, in thickness, stains less intensely than the chromatin fibers and is set away from the chromosome 150 to 350 A [14]. There is a clear area surrounding the outer layer on all sides. Jonkalin has termed the outer portion of this area the corona, and the inner portion the middle layer. Brinkley & Stubblefield [2-41 have

107

shown that this clear area around the outer layer is fibrillar in nature and have termed these lateral fibrils. Some of the questions that remain to be clarified are the following: Is the outer layer a plate-like structure [14] or is it composed of two individual portions, the kinetochore filaments [4]? Does the outer layer contain chromatin fibers which are possibly genetically active and contribute to the synthesis of the spindle fibers [4]? Do the microtubules stop in the outer layer [4] or do they pass through outer layer [14] and inner layer? If the latter is true, what is the fate of the microtubules in the centromere region? The outer layer Brinkley & Stubblefield [4] suggestedthat the outer layer is composed of two kinetochore filaments laying parallel to each other in each sister kinetochore. This was based on serial sections and a few instanceswhen both filaments appeared to be present in one section. They further suggestedthat a genetically active chromatin fiber traversed the kinetochore filament and that the presence of two filaments was a reflection of the

AREA OFCHROMATIC ASSOCIATION

Fig. 29. Components of the centromere region. The kinetochore is depicted as a structure composed of a curved plate-like outer layer surrounded on both sides by light staining material consisting of an oiter portion(corona) and an inne; portion (middle layer), and an inner layer which is in intimate -contact with anh probably composed of chromatin material. In acetic methanol fixed or water-spread chromosomes only the chromatin of the centromere region is seen. In metacentric chromosomes this is composed of two areas of chromatid association with a halo-like region between them [IO]. Exptl

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108 D. E. Comings & T. A. Okada supposed presence of two “half-chromatids”. There are reasons, however, to suspect that chromosomes are actually single-stranded [6, 7, 17, 18, 231, and that half-chromatids do not actually represent two independent portions of the chromatid [ll]. Because of this we were interested in testing an alternative proposal that the two kinetochore filaments in metacentric chromosomes might have arisen as a result of the centric fusion of two telocentric chromosomes [lo] each with only a single kinetochore filament and that following fusion the two single filaments might slide into a parallel alignment with each other. We thought that the long centromere region of the X-autosomal fusion chromosome in the muntjac might represent a situation in which the two kinetochore filaments had fused end-to-end rather than sliding into a parallel position with each other. Initially it seemed this interpretation might be valid since serial sections on the X-autosome kinetochores failed to show the presence of two kinetochore filaments. At first we interpreted configurations such as those shown in figs 16-21 as evidence for a pair of kinetochore filaments in non-Xautosome chromosomes. However, it was disturbing that one of the pair was always much more intimately associated with the chromosome than the other. Examination of a large series of sections led us to conclude that instead of seeing two kinetochore filaments, the inner one was actually the inner layer described by Jonkalin and the other was the outer layer of a single kinetochore. This also seems to be a reasonable interpretation of the figures presented by Brinkley & Stubblefield [4] (their figs 7 and 22). It must be agreed that their fig. 17 cannot be explained on the basis of a single rod-like kinetochore filament. Such a configuration could, however, be easily produced by an appropriate section through the kinetochore Exptl

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if instead of being a rod the outer layer was more like a plate or disc, as suggested by Jonkalin. Strong support for this interpretation came from the present studies of the kinetochore in the muntjac. The unusually long kinetochores in this species provided an opportunity to distinguish transverse from longitudinal sections (see Results). These transverse sections show that the kinetochore is an elongated plate-like structure that curves around part of the chromosome of the centromere region. Similar conclusions based on longitudinal and transverse sections of Chinese hamster cells have recently been presented by Journey & Whaley 1151. They also show that the outer layer is single, with no evidence for two portions or two kinetochore filaments. For this reason the term outer layer [14] seems more appropriate than kinetochore filament [4].

The subcomponents of the outer layer High magnification views of the outer layer frequently show the presence of what appears to be two tiny filaments about 90 to 100 A in thickness [4, 16 and fig. 131. These may run parallel to each other [16] or they may appear to be plectonemically coiled (b) [4, 16, fig. 131.

0

b

In some tranverse sections the outer layer appears to be composed of a semicircular array of dot-like structures (figs 24, 25), which are partially ( * ) or completely 0 surrounded by lines which have the same appearance as those seen in longitudinal section. This suggests that the outer layer may contain within it a series of tubulelike elements. This could account for its appearance both on longitudinal and transverse sections. Alternatively, if the kineto-

Kinetochore fine structure chore was originally derived from the nuclear membrane, a possibility suggested by studies of the evolution of the mitotic apparatus [22], the 90 A lines might represent modified membranous material. Relationship of the spindle fibers to the kinetochore Investigation of this facet of kinetochore structure is aided by an appreciation of the fact that a slight change in direction of a microtubule seen in thin section electron microscopy will cause it to disappear from view. Thus, when a microtubule appears to stop, has it really stopped or has it merely changed direction? To answer this question requires a section that is in exactly the right plane to catch the entire curve of the microtubule. Thus, only a section of a few degrees out of a potential 360 degrees would provide evidence on the curving of a single microtubule. The question of the relationship of spindle fibers to the kinetochore can be broken down into several sub-questions. (1) Do the spindle fibers pass through the outer layer? (2) Do they pass through the inner layer? (3) Do they pass completely through the centromere region and emerge through the opposite sister kinetochore? (4) Do they change direction in the centromere region? In almost all preparations in which the spindle fibers are sectioned longitudinally it can be seen that they pass through the outer layer and then usually disappear from view in the middle layer (fig. 22). In some preparations some of the fibers can be seen to pass through the outer, middle and inner layers and extend into the chromatin of the centromere region (fig. 23). Although we have frequently observed that microtubules immediately adjacent to the kinetochore will pass entirely through both chromatids we have yet to find any tubules that clearly pass through both sister kinetochores. Care-

109

ful examination of some sections gave the impression that some of the spindle fibers passed through the outer (a) and inner layers (b), then curved around in the chromatin fibers of the centromere region (c) and passed out again through the inner and outer layer of the same kinetochore (figs 24,25,27).

This sudden change in direction of the fibers once they were past the outer layer could account for the usual disappearance of the microtubules in the middle layer. Thus, most sections of the outer layer would appear like this

while a few sections would show that the apparent cessation of the fibers on the inner side of the outer layer is actually due to the fact that they abruptly change direction in the middle layer.

These are only general outlines of possible interrelationships between the spindle fibers Exptl

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110 D. E. Comings & T. A. Okada and the kinetochore which are compatible with the present observations. There are, of course, many permutations on this theme. It is possible, for example, that some of the spindle fibers may pass from the kinetochore of one chromosome to the kinetochore of another in this manner.

sequences in the centromeric DNA may act as a kinetochore recognition site. However, it is equally possible that none of these is true. There are many situations in morphogenesis in which two different structures join together at specific points even though neither contains DNA. This work was supported by NIH grant no. GM15886 and the Charles and Henrietta Detov Research Fellowship.

REFERENCES

Right angle microtubules that could be interpreted in this way have been reported in other species (fig. 7 in ref. [24]) Such a configuration might explain why chromosomes seem to dance on a string when the mitotic apparatus is removed by microsurgery and could play a role in maintaining chromosomes in a specific order on the spindle [12]. It must be admitted, however, that the data at present are far too meager to choose among the many possible permutations on the interrelationship between the spindle fibers and the kinetochores. If the outer layer is plate-like rather than being composed of two kinetochore filaments, it is rather difficult to envision one (or two) chromatin fibers playing a major role in its structure. This does not, however, rule out the possibility that the chromatin of the inner layer may play a role in the formation of (a) the kinetochore, (6) the kinetochore microtubules, or (c) that specific

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I. Anderson, T F, Trans NY acad sci 13 (1951) 130 (ser II). 2. Brinkley, B R & Stubblefield, E, Chromosoma 19 (1966) 28. 3. Brinkley, B R, Stubblefield, E & Hsu, T C, J ultrastruct res 18 (1967) 1. 4. Brinkley, B R & Stubblefield, E, Advances in cell biology 1 (1970) 119. 5. Comings,% E; Exptl cell res. In press. 6. - Canad j genet & cytol 12 (1970) 960. 7. - New biol 229 (1971) 24. 8. Comings, D E & Okada, T A. Unpublished observations. 9. - Exptl cell res 62 (1970) 293. 10. - Cvtoaenetics 9 (1970) 436. 11. - ldid 19(1970) 450. ’ 12. Costello. D P. Proc natl acad sci US 67 (1970) 1951. ’ ’ 13. Hoskins, G C, Nature 217 (1968) 748. 14. Jokelainen, P T, J ultrastruct res 19 (1967) 19. 15. Journey, L J & Whaley, Z A, J cell sci 7 (1970) 49. 16. Krishan, A, Exptl cell res 23 (1968) 134. 17. Laird, C D. In preparation. 18. Lindahl-Kiesslina. K. Santesson. B & Book. 1 J A.I J A, Chromosoma 31 (1970) 286. 19. Lubs. H A & Blitman. S L. Exnerientia 23 (1967), 1067.’ 20. Luvkx. P. Exotl cell res 39 (1965) 643. 21. --Ibid 39 (1965) 658. ~ ’ 22. Pickett-Heaps, J D, Cytobios 3 (1969) 257 23. Prescott, D M, Advances in cell biology 1 (1970) 57. 24. Roth, L E, Wilson, H J & Chakraborty, J, J ultrastruct res 14 (1966) 460. 25. Wurster, D H & Benirschke, K, Science 168 (1970) 1364. I

Received February 11, 1971

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