Evidence for a relatively random array of human chromosomes on the mitotic ring.

We used fluorescence in situ hybridization (FISH) to study the positions of human chromosomes on the mitotic rings of cultured human lymphocytes, MRC-5 fibroblasts, and CCD-34Lu fibroblasts. The homologous chromosomes of all three cell types had relatively random positions with respect to each other on the mitotic rings of prometaphase rosettes and anaphase cells. Also, the positions of the X and Y chromosomes, colocalized with the somatic homologues in male cells, were highly variable from one mitotic ring to another. Although random chromosomal positions were found in different pairs of CCD-34Lu and MRC-5 late-anaphases, the separations between the same homologous chromosomes in paired late-anaphase and telophase chromosomal masses were highly correlated. Thus, although some loose spatial associations of chromosomes secondary to interphase positioning may exist on the mitotic rings of some cells, a fixed order of human chromosomes and/or a rigorous separation of homologous chromosomes on the mitotic ring are not necessary for normal mitosis. Furthermore, the relative chromosomal positions on each individual metaphase plate are most likely carried through anaphase into telophase.

It is still not known whether the positions of the chromosomes relative to each other, when the mitotic ring of the metaphase plate is viewed head-on as a flat disc, are rigorously fixed, have loose preferences for associating with favored neighbors, or are entirely random. Early studies of this question gave widely differing results. The chromosomes of Dipteran insects showed pairing of homologous chromosomes during prophase and on the mitotic ring (Metz, 1916). However, the chromosomal order on the metaphase rings of grasshopper (Melanoplus femorrubrum) spermatocytes was found to be random (Nur, 1976). In hexaploid wheat, Triticum aestivum, the homologous chromosomes were close to each other and possibly adjacent on the mitotic ring (Feldman et al., 1966). A study of the grasses Hordeum vulgaris and Hordeum bullosum suggested that chromosomal arms of similar lengths were adjacent, and possibly in a fixed order, on the mitotic ring (Heslop-Harrison and Bennett, 1984). One study of the plant Crepis capillaris showed homologue association on the mitotic rings (Ferrer and Lacadena, 1977), whereas another study of this plant showed a random chromosome order except for clustering of the two chromosomes involved in nucleolus formation (Tanaka, 1981).

Early studies of mammalian cells also showed adjacent homologous chromosomes on the mitotic rings of human (Schneiderman and Smith, 1962), Muntjac deer (Heneen and Nichols, 1972), and Chinese hamster cells (Juricek, 1975), whereas later studies showed largely random, or widely separated, homologous chromosomes for these cell types (Hens, 1976; Korf and Daicumakos, 1977; Nagele et al., 1995). In a recent fluorescence in situ hybridization (FISH)1 study of the chromosomal positions in the prometaphase rosettes of four human cell lines, the investigators concluded that homologous chromosomes were always separated from each other by at least 90° and were most likely to be arrayed in a fixed order on the mitotic ring (Nagele et al., 1995). Only a small proportion of the rosettes was suitable for analysis in this study, however, leaving open the possibility that selection may have influenced these results.

We now report the FISH localization of the relative positions of human chromosomes in prometaphase rosettes, early, mid-, and late-anaphases, and telophases of cultured human lymphocytes, MRC-5 cells, and CCD-34Lu cells. A new method was developed for measuring chromosomal positions in virtually all anaphases to ensure sampling of the entire mitotic segment. The results of this study were somewhat surprising in that we found largely random chromosomal positions.

Materials and Methods

Cells

Fibroblasts of the diploid MRC-5 line (a gift of Dr. J. Willey, Medical College of Ohio) and the diploid CCD-34Lu cell line (American Type Culture Collection), both derived from human lung tissue, were grown as monolayers directly on glass slides in RPMI 1640 or EMEM containing l-glutamine (GIBCO BRL), 10% FBS (GIBCO BRL), penicillin, gentamicin, and sodium bicarbonate (Amersham Life Sciences), respectively. The cells were fixed in situ with Carnoy's solution just before confluence. Human lymphocytes were grown in RPMI 1640 with the addition of phytohemagglutinin (Amersham) for 72 h, fixed in Carnoy's, and dropped onto glass slides from 10 cm. The slides were not flamed, but were allowed to air-dry and were stored until hybridization. In some experiments, CCD-34Lu cells were fixed in 4% paraformaldehyde in PBS and stored without drying in 95% alcohol at −20°C until hybridization (Nagele et al., 1995).

FISH

Centromere-specific probes, directly labeled with FluorX (green fluorescence) or Cy3 (red-orange fluorescence), were used when available (Amersham). For the remaining chromosomes, chromosome “paints,” labeled with Spectrum orange or Spectrum green, were used (Vysis), and the brightest point on each “paint image” was used as the location of the centromere. For FISH, slides were incubated in a 2× SSC solution (pH 7.0) for 30 min, followed by dehydration. The centromeric probe mixtures consisted of 2 μl of Cy3-labeled centromeric probe, 2 μl of FluorX-labeled centromeric probe, and 10 μl of hybridization solution (50% formamide/ 2× SSC/10% dextran sulfate). The centromeric-paint probe mixtures consisted of 1 μl of Spectrum orange or green paint probe, 2 μl of FluorX or Cy3 centromeric probe, 1 μl of ddH2O, and 7 μl of hybridization solution. The probe mixtures were denatured at 70°C for 5 min and placed at 4°C until use. Cells hybridized to the centromeric and the paint-centromeric probe mixtures were denatured for 2 and 5 min, respectively, in 70% formamide/2× SSC solution at pH 7.0. The slides were incubated overnight with probe solution in a humidified chamber at 43°C. The slides incubated with the paint-centromeric and the centromeric probes were washed in 50–65% formamide/2× SSC solution (pH 7.0), 2× SSC, and 2× SSC with NP-40 or PBD (pH 8.0), respectively, and counterstained with DAPI. The appropriate number of centromeres were always clearly localized in the Carnoy-fixed mitotic and interphase cells (Fig. 1 A). The paraformaldehyde-fixed CCD-34Lu cells (Nagele et al., 1995) gave relatively dim probe localization under a variety of denaturation times (2–6 min) when compared with the Carnoy-fixed cells. However, treatment of the paraformaldehyde-fixed cells with a weak solution of HCl (200 mM in PBS) for 20 min at room temperature before a 3-min denaturation allowed detection of the appropriate number of fluorescence signals in the majority of rosettes.

Figure 1

(A) FISH-localized homologues of the X chromosomes in female human lymphocytes doubly hybridized with a FluorX-labeled whole chromosome paint probe (yellow) and a Spectrum orange–labeled centromeric probe (blue). The colocalization of both probes in interphase and mitotic cells is apparent. (B) FISH-localized homologues of MRC-5 chromosomes 11 (yellow) and 17 (blue) in prophase (a); the X (yellow) and 17 (blue) chromosomes in a prometaphase rosette (b); FISH-localized homologues of lymphocyte chromosomes 11 (yellow) and 17 (blue) in a metaphase with undivided centromeres (c); and an early anaphase (d). (C) A grid used to measure rosette chromosomal positions (a); placed over the rosette (b); a mid-anaphase cell with the separating chromosomal masses (c); and a transform of image c in which both mitotic rings are given an identical diameter set on the x-axis, with positive directions on the y-axis being toward the nuclear pole (d). (D) Male rosette with two homologues of chromosome 17 positioned at 90° and 270° and the X chromosome at 45°. (E) Ratios of two independent sets of measurements of angular separation between the same chromosomes in MRC-5 and lymphocyte rosettes (n = 1,011), showing increasing variability as the angular separations decrease. Bars, 20 μm.

Microscopy and Image Processing

The Cy3 and Spectrum orange fluorochromes were localized with a rhodamine-specific filter cube, BP510-560, FT580, LP590, in a Zeiss microscope under epifluorescence optics with a Neofluar 100× oil immersion lens (NA 1.30; Carl Zeiss, Inc.). The FluorX and Spectrum green fluorochromes were visualized with filter cube BP450-490, FT510, LP520, and a G365, FT395, LP420 filter cube was used for the DAPI stain. Analogue images from a CCD camera mounted on the microscope were digitized and processed for removal of extraneous background fluorescence by Probevision software (Applied Imaging Corp. [AI]). The early and mid-anaphase mitotic rings are perpendicular to the slide surface, and FISH-localized chromosomes in these cells were often in slightly different focal planes. When this occurred, the objective was set at an intermediate focal plane between the two probes, which appeared as slightly larger and less bright spots of light than perfectly focused probes. AI image analysis transforms were used to select the brightest points in each of the defocused spots as the location of probe fluorescence. The AI fluorescence microscopy system separately acquires three black and white images at the emission wavelength of the fluorochrome being localized. The black and white images are combined into one pseudocolor image without any movement or alignment changes. Each image was converted into a color graphic overlay (AI) and further processed with Adobe Photoshop (Adobe Systems Inc.) and Probe Ratio software (JVB Imaging). Data were stored and analyzed with the Quatro Pro spreadsheet (Borland) and the SPSS statistical programs (SPSS Inc.).

The emitted light from the contrasting fluorochromes has different refractive indices in the microscope objective. To test whether the varying focal planes and emission spectra caused significant shifts in image positions, we hybridized female lymphocytes with the FluorX paint probe and the Cy3 centromeric probe for the X chromosome. The two probes showed a perfect positional correspondence for all cells measured (Fig. 1 A), ruling out significant spectral aberrations and alignment problems.

Results

Experimental Approach

After S-phase, the newly replicated sister chromatids condense in prophase (Fig. 1 B, panel a), and many, if not all, prophase cells form a tight ring of chromosomes parallel to the slide surface called the prometaphase rosette (Chaly and Brown, 1988; Nagele et al., 1995) (Fig. 1 B, panel b). The prometaphase rosettes progress directly to less compact metaphases (Fig. 1 B, panel c), followed shortly by anaphase (Chaley and Brown, 1988; Nagele et al., 1995). The early (Fig. 1 B, panel d) and mid- (Fig. 1 C, panel c) anaphase mitotic rings are perpendicular to the slide surface. We found, similar to Nagele et al. (1995), that it was difficult to determine the positions of FISH-localized chromosomes in metaphase figures, which often have partially broken or folded mitotic rings (Fig. 1 B, panel c). This was not the case for the more compact rosettes and anaphases (Fig. 1 B, panels b and d, and Fig. 1 C, panel c). The symmetry of chromosomal positions in >99% of the daughter early and mid-anaphases (Fig. 1 B, panel d, and Fig. 1 C, panel c) established that the relative chromosomal positions in the living early and mid-anaphases were maintained after fixation. The mitotic rings of late-anaphases were often parallel to the slide surface (Fig. 1 A, bottom right-hand corner).

Assay of Prometaphase Rosettes, Late-Anaphases, and Telophases.

The proportion of prometaphases forming flat rosette rings was graded in consecutive lymphocytes and MRC-5 cells. Because it had been reported that only “perfect” rosettes were suitable for analysis of chromosomal positions (Nagele et al., 1995), MRC-5 and lymphocyte rosettes were further classified as being perfect (compact, even, and unbroken mitotic rings), “slightly spread” (slight separation of some chromosomes and/or some central asymmetry), or “gap” (<10% broken area in the ring) rosettes. Perfect rosettes were found for 38% (101/261) and 9% (48/551) of the lymphocyte and MRC-5 prometaphases, respectively. However, no differences in the angular separations in perfect, slightly spread, or gap rosettes were found for any of the cell lines (data not shown), and all three rosette types were subsequently measured, giving estimated sampling frequencies of 90% (234/261) and 29% (162/557) of the lymphocyte and MRC-5 prometaphases, respectively.

Fig. 1 D shows a prometaphase or late-anaphase mitotic ring parallel to the slide surface with the two homologues of chromosome 17 separated by 180°. A change of position of one homologue leads to two separation angles between these chromosomes, one <180° and one >180°. The lower angle was measured, allowing a 0–180° separation range between two rosette chromosomes. However, it was necessary arbitrarily to select a center point to place a measuring grid (Fig. 1 C, panel a) over the ring (Fig. 1 C, panel b). To test the reproducibility of this step, we performed two sets of measurements of the same prometaphase rosettes, with the second measurement set performed without knowledge of the prior location of each rosette's center point (Fig. 1 C, panel b). In Fig. 1 E, the ratios of the first to second angular measurements for each rosette are plotted on the y-axis against the mean value of the two measurements on the x-axis. There was considerable variability between the two measurement sets, especially for measurements of smaller angular separations (Fig. 1 E). The variability in our study seemed random, because the ratios were both above and below the value of one (Fig. 1 E).

Consecutive, widely separated CCD-34Lu and MRC-5 chromosomal masses were graded as being late-anaphases (flat rings, Fig. 2 D, panel a, and Fig. 2 E, panel a), telophases (flat, elliptical areas without a ring structure, Fig. 2 D, panel b, and Fig. 2 E, panel b), or of indeterminate morphology (not shown), leading to the following classifications: both chromosomal masses being late-anaphases (CCD-34Lu, n = 18 pairs; MRC-5, n = 18 pairs); both being telophases (CCD-34Lu, n = 14 pairs; MRC-5, n = 30 pairs); and being of mixed/indeterminate morphology (CCD-34Lu, n = 40 pairs; MRC-5, n = 22 pairs). The angular separations in nonpaired, i.e., individual, coded images of these chromosomal masses were measured one at a time, using the geometric centers of each chromosomal mass to center the measuring grid (Fig. 1 C, panel b).

Figure 2

(A) Male lymphocyte rosettes with the FISH-localized homologues of chromosome 17 (blue) and X (yellow) showing widely varying positions. (B) MRC-5 rosettes with the FISH-localized homologues of chromosome 17 (blue) and X (yellow) showing widely varying positions. (C) CCD-34Lu rosettes with the FISH-localized homologues of chromosomes X (yellow) and 7 (blue) showing widely varying positions. (D) CCD-34Lu late-anaphase (a) and telophase (b) pairs with the FISH-localized homologues of chromosomes X (yellow) and 7 (blue). (E) MRC-5 late-anaphase (a) and telophase (b) pairs with the FISH-localized homologues of chromosome 7 (yellow) and X (blue). Bars, 20 μm.

Early and Mid-Anaphase Assay.

The angular chromosomal separations cannot be measured directly in early and mid-anaphase mitotic rings, which are perpendicular to the slide surface (Fig. 1 B, panel d, and Fig. 1 C, panel c). Linear distances were measured between the anaphase chromosomes and then analyzed to gain an estimate of the native chromosome sequence as detailed in the Appendix.

Experimental Results

Rosette Results.

The angular separations measured between the homologues of chromosomes 11 (n = 103) and 17 (n = 203) in MRC-5 rosettes, chromosome 17 in male lymphocyte rosettes (n = 100), chromosome 7 in female lymphocyte rosettes (n = 104), and chromosomes X and 7 in the CCD-34Lu rosettes (n = 156) were highly variable (Figs. 2, A–C, and Fig. 3, A–F). No evidence was found for fixed ranges of separation between these homologues on the mitotic ring, as equal numbers of homologues were separated by <90° and by >90° (Fig. 3, A–F). If the chromosomes are in fixed positions in male cells, the angular separations between the X chromosome and the same two somatic homologues should be identical for every rosette (Fig. 1 D). This was not the case for measurements of the X and 17 chromosome homologues made on male MRC-5 and lymphocyte rosettes, where widely variable angles of separation were found (Fig. 2, A and B, and Fig. 3, G and H).

Figure 3

Angular separations of FISH-localized chromosomes in MRC-5, lymphocyte, and CCD-34Lu rosettes. (A) MRC-5 chromosome 11 homologues (n = 103). (B) MRC-5 chromosome 17 homologues (n = 203). (C) Male lymphocyte chromosome 17 homologues (n = 100). (D) Female lymphocyte chromosome 7 (n = 104). (E) CCD-34Lu chromosome X homologues (n = 156). (F) CCD-34Lu chromosome 7 homologues (n = 156). (G and H) Nearest (black bars) and farthest (striped bars) angular separations between the 17 homologues and the X chromosome in male MRC-5 rosettes (n = 100, G) and male lymphocyte rosettes (n = 100, H).

Random separations of homologous rosette chromosomes were also found for all of the individual lymphocyte chromosomes, MRC-5 chromosomes 11 and 17, and CCD-34Lu chromosomes X and 7 (see Appendix, Table I). Furthermore, the distributions of the nearest angular separations between the somatic chromosomes to either the X or Y chromosome in male lymphocytes rosettes were also highly variable (see Appendix, Table II).

Table I

Separation of the Individual Homologues in Prometaphase Rosettes of All Lymphocyte Chromosomes, MRC-5 Chromosomes 11 and 17, CCD-34Lu Chromosomes X and 7; and the Separation of the Homologues of CCD-34Lu Chromosomes X and 7 and MRC-5 Chromosome 7 in Late-Anaphase Mitotic Rings

Prometaphase rosettes
Average homologue separation
Lymphocytes (male)										Lymphocytes (female)
Chr.		n		Mean		Range*		SD‡		Chr.		n		Mean		Range		SD
				°		°		°						°		°		°
1		10		114		35–177		54		X-X		104		88		4–179		53
2		15		97		23–180		60		7		104		89		5–179		52
3		11		90		23–174		55
4		12		99		17–173		55		MRC-5 (male)
5		10		103		21–175		49		11		103		68		2–171		44
6		25		91		6–179		55		17		203		61		0–175		42
7		23		95		1–180		56
8		10		106		34–157		47		CCD-34Lu (female)
9		10		133		52–178		42		X-X		156		88		2–180		52
10		10		68		18–158		44		7		156		90		1–179		53
11		12		66		4–149		49		X-X§		26		64		5–163		52
12		11		122		32–170		47		7§		33		93		9–172		53
13		11		72		15–178		55
14		12		91		14–149		48
15		12		107		36–169		48
16		15		96		21–150		43
17		108		83		1–173		47
18		12		85		13–168		49
19		10		86		15–150		47
20		10		87		19–155		48
21		10		81		26–137		38
22		10		105		1–172		59
X-Y		15		97		12–179		58
Late-anaphase rings
CCD-34Lu										MRC-5
Chr.		n		Mean		Range*		SD‡		Chr.		n		Mean		Range		SD
				°		°		°						°		°		°
X-X		36		94		10–176		52		7		33		101		10–173		51
7		36		94		0–172		50

 Range of angular separations measured between the individual homologues in rosette or late-anaphase rings.  

 Standard deviation.  

 Paraformaldehyde-fixed cells.  

Table II

The Nearest Separation of the Individual Homologues of Chromosomes 1–22 to the X or Y Chromosomes in Male Lymphocyte Rosettes and between the Chromosome 7 Homologues to the X Chromosome in Male MRC-5 Late-Anaphases (Fig. 2 E, panel a)

Nearest angle in lymphocyte prometaphase rosettes to the sex chromosomes (male)
Chr.		X chromosome								Y chromosome
		n		Mean		Range*		SD‡		n		Mean		Range		SD
				°		°		°				°		°		°
1		5		61		6–122		41		5		60		2–140		51
2		5		59		9–118		39		10		78		9–140		42
3		5		62		31–117		36		6		106		58–141		34
4		5		53		5–168		67		7		48		13–99		26
5		5		56		10–75		26		5		50		2–123		49
6		5		87		29–156		52		20		64		9–120		38
7		5		88		24–155		61		18		61		9–178		44
8		5		44		20–106		36		5		35		3–85		31
9		5		50		9–102		40		5		47		1–145		61
10		5		52		21–142		51		5		69		11–135		58
11		6		94		17–158		53		6		44		5–97		34
12		5		52		4–79		31		6		68		33–107		25
13		5		109		73–137		25		6		65		40–109		25
14		6		53		4–102		33		6		65		9–129		41
15		5		69		25–124		37		7		82		33–104		24
16		5		52		10–145		55		10		57		3–164		50
17		103		59		0–162		42		5		73		10–116		43
18		5		43		9–77		31		5		52		5–88		30
19		5		26		2–66		27		5		99		68–139		30
20		5		55		2–105		38		5		67		19–150		52
21		5		53		5–105		47		5		75		17–161		54
22		5		50		1–105		40		5		75		31–127		39
Nearest angle between the chromosome 7 homologues and the X chromosome in MRC-5 late-anaphases
		n		Mean		Range*		SD‡
				°		°		°
		33		66		1–158		40

 Range of the nearest angular separations measured between the individual homologues in rosette or late-anaphase rings.  

 Standard deviation.  

Early and Mid-Anaphase Results.

We measured the relative chromosomal positions in early and mid-anaphases of all three cell types. Virtually all anaphases were measured, allowing a complete sampling of the mitotic segment. The x-axis distances measured between the early and mid-anaphase chromosomes were compared with different theoretical models of chromosomal separation on the mitotic ring (see Appendix). The pooled x-axis distances measured between all of the homologous chromosomes in the lymphocyte, MRC-5, and CCD-34Lu early and mid-anaphases strongly fit the theoretical model for a random, but no other, distribution (see Appendix, Fig. 7 and Table III). The x-axis distances between the individual early and mid-anaphase chromosomes of these cell types predominately fit the random model, although some heterogeneity among these data sets was observed (see Appendix, Table IV).

Figure 7

Theoretical and measured distributions of the x-axis distances between homologous chromosomes in early and mid-anaphases. (A–C) Theoretical models of the x-axis distance distributions for two chromosomes separated within the ranges of 0–90° (A), 90–180° (B), and 0–180° (random distribution, C). (D) Pooled x-axis distances between 5,304 homologous pairs of lymphocyte chromosomes 1–22, XX, and XY. (E) Pooled x-axis distances measured between 1,042 pairs of homologous MRC-5 chromosomes 11 and 17. (F) Pooled x-axis distances measured between 816 pairs of homologous CCD-34Lu chromosomes X and 7.

Table III

χ 2 Analysis for Fit between the Theoretical Models of X-Axis Distances Measured on the Slide Surface for Different Chromosomal Separations on the Native Mitotic Ring and Pooled Anaphase X-Axis Distances Actually Measured between Homologous Lymphocyte (1–22, XX, XY), MRC-5 (11, 17), and CCD-34Lu (X, 7) Chromosomes

Theoretical models						Analysis of fit
Chr. separation position		Mean x-axis dist.*		Range		Lymph, 1–22, XX, XY (male & female) n = 5,304 P ‡		Lymph, 1–22, XY (male) n = 3,524 P		Lymph, 3, 6, 7, 8, 11, 15, 17, XX (female) n =1,780 P		MRC-5, 11, 17 (male) n = 1,042 P		CCD-34Lu, XX, 7 (female) n = 816 P
°		%		%
1 (8°)		4		0–11		0.000		0.000		0.000		0.000		0.000
2 (16°)		9		0–17		0.000		0.000		0.000		0.000		0.000
3 (25°)		12		0–22		0.000		0.000		0.000		0.000		0.000
4 (33°)		15		0–26		0.000		0.000		0.000		0.000		0.000
5 (41°)		18		0–30		0.000		0.000		0.000		0.000		0.000
6 (49°)		21		0–33		0.000		0.000		0.000		0.000		0.000
7 (57°)		24		0–37		0.000		0.000		0.000		0.000		0.000
8 (65°)		26		0–41		0.000		0.000		0.000		0.000		0.000
9 (74°)		28		0–44		0.000		0.000		0.000		0.000		0.000
10 (82°)		30		0–48		0.000		0.000		0.000		0.000		0.000
11 (90°)		32		0–52		0.000		0.000		0.000		0.000		0.000
12 (98°)		34		0–56		0.000		0.000		0.000		0.000		0.000
13 (106°)		36		0–59		0.002		0.002		0.009		0.004		0.002
14 (115°)		37		0–63		0.001		0.001		0.002		0.001		0.001
15 (123°)		38		0–67		0.008		0.008		0.031		0.002		0.008
16 (131°)		40		0–69		0.003		0.003		0.001		0.001		0.080
17 (139°)		41		0–72		0.005		0.005		0.002		0.002		0.057
18 (147°)		42		0–75		0.001		0.001		0.000		0.000		0.018
19 (155°)		42		0–78		0.003		0.003		0.001		0.001		0.030
20 (164°)		43		0–83		0.005		0.005		0.003		0.004		0.086
21 (172°)		43		0–89		0.006		0.006		0.003		0.005		0.023
22 (180°)		44		0–100		0.005		0.005		0.003		0.004		0.066
1–11 (0–90°)§		20		0–52		0.000		0.000		0.000		0.000		0.000
12–22 (90–180°)‖		40		0–100		0.005		0.005		0.003		0.004		0.069
1–22 (0–180°) ¶		30		0–100		0.935		0.935		0.821		0.877		0.907

 Expressed as percentage of the total range of anaphase centromeric x-axis positions (Fig. 6 B).  

 A P value of >0.05 means that the hypothesis that the theoretical model and the measured x-axis distances are identical cannot be rejected.  

 Model in which the two chromosomes can be in any position within 90° of each other (Fig. 7 A).  

 Model in which one chromosome is at 0° and the second chromosome can be in any position within 90–180° from it (Fig. 7 B).  

 Model in which the two chromosomes can be in any position (0–180°, random) to each other (Fig. 7 C).  

Table IV

Analysis of the Fit of the X-Axis Distances between the Individual Early and Mid-Anaphase Chromosomes of All Cell Types to the 0–90°, 90–180°, and 0–180° (Random) Theoretical Models, and of the Shortest Anaphase X-Axis Distances between the Somatic and Sex Chromosomes in Male Lymphocytes to the Two-Position Model of Chromosomal Separation (Table I)

Theoretical models										Sex chromosomes
				Compared						X						Y
Lymph				0–90°		90–180°		0–180° (Random)		Shortest x-axis distance						Shortest x-axis distance
Chr.		n		P *		P		P		n		Range‡		P § 2-Pos fit		n		Range		P 2-Pos fit
												%						%
1		150		0.000		0.667		0.150		46		0–74		0.000		46		0–62		0.000
2		160		0.000		0.527		0.410		46		0–54		0.000		60		1–96		0.000
3		172		0.000		0.197		0.304		48		0–48		0.000		48		0–46		0.000
4		158		0.000		0.002		0.688		50		1–59		0.000		48		0–52		0.000
5		174		0.000		0.180		0.552		46		0–36		0.150		48		0–60		0.000
6		334		0.002		0.002		0.775		48		3–87		0.000		52		0–40		0.371
7		570		0.000		0.009		0.803		50		1–63		0.000		50		0–55		0.136
8		254		0.002		0.000		0.205		48		1–44		0.000		48		0–71		0.000
9		140		0.000		0.006		0.036		46		1–71		0.000		46		0–48		0.000
10		140		0.000		0.394		0.002		56		1–67		0.010		46		1–42		0.000
11		358		0.003		0.000		0.646		48		0–54		0.006		48		1–36		0.011
12		154		0.000		0.015		0.571		46		0–47		0.000		48		0–50		0.000
13		208		0.000		0.168		0.911		56		1–54		0.000		56		1–33		0.020
14		168		0.000		0.150		0.374		52		0–63		0.000		46		1–37		0.000
15		316		0.099		0.000		0.360		52		1–50		0.013		60		1–58		0.000
16		164		0.054		0.001		0.590		46		0–62		0.000		60		1–60		0.006
17		446		0.497		0.000		0.155		48		0–69		0.000		48		0–40		0.000
18		142		0.024		0.000		0.000		46		1–47		0.000		50		1–36		0.002
19		148		0.939		0.000		0.010		48		0–59		0.000		48		0–39		0.485
20		142		0.043		0.000		0.033		50		1–71		0.000		52		0–34		0.000
21		160		0.244		0.000		0.412		46		0–54		0.094		62		1–43		0.000
22		154		0.000		0.001		0.628		50		1–42		0.000		46		1–34		0.000
X-Y		92		0.000		0.027		0.754
X-X		400		0.000		0.060		0.897
MRC-5
11		496		0.000		0.014		0.935
17		546		0.000		0.007		0.657
CCD-34Lu
7		408		0.000		0.069		0.994
X		408		0.000		0.080		0.930

 A P value of >0.05 means that the hypothesis that the theoretical model and the measured values are identical cannot be rejected.  

 Range of x-axis distances is 0–100% of all possible centromeric x-axis positions (Fig. 6).  

  P 2-Pos fit, the probability of fit between the set of shortest x-axis distances measured between each somatic homologue and the sex chromosomes with the theoretical model for a 2-position chromosomal separation (Table III).  

Late-Anaphase and Telophase Results.

The chromosomal separations measured in widely separated, late-anaphase rings between the homologues of the CCD-34Lu chromosomes X and 7 and MRC-5 chromosome 7 were random (see Appendix, Table I), similar to the prometaphase rosettes. The nearest angles between the homologues of chromosome 7 and the X chromosome measured in MRC-5 (male) late-anaphase rings were highly variable, a finding inconsistent with fixed positions of these chromosomes on the late-anaphase rings (see Appendix, Table II). Also, symmetrical positions were found for the same chromosomes in each daughter of the widely separated chromosomal masses, regardless of chromosomal mass morphology (Fig. 1 A, bottom right-hand corner, and Fig. 2, D and E). This symmetry is quantified in Fig. 4, which shows the correlation between angles measured in each daughter of 142 unselected, and consecutively measured, pairs of widely separated CCD-34Lu (n = 72) and MRC-5 (n = 70) chromosomal masses. In the figure, the x-axis coordinate of every point is the angle between two homologues measured in one chromosomal mass. The y-axis coordinate is the same angle measured in either the other daughter chromosomal mass of the pair (daughter-paired, Fig. 4 A) or in a randomly selected chromosomal mass of the same cell type (randomly paired, Fig. 4 B). The 214 pairs of angular measurements made in the daughter-paired chromosomal masses (Fig. 4 A) were highly correlated with each other (correlation coefficient = 0.788), whereas the randomly paired angles (Fig. 4 B) were not correlated (correlation coefficient = −0.087). The daughter-paired angular separations remained highly correlated after the morphologic separation into late-anaphase (54 pairs of measurements), telophase (58 pairs), and mixed/indeterminate (102 pairs) subgroups, with correlation coefficients of 0.856, 0.791, and 0.749, respectively. The randomly paired angles in these morphologic subgroups remained uncorrelated, with correlation coefficients of 0.010, −0.189, and −0.078, respectively.

Figure 4

Correlation of the angles between the homologues of CCD-34Lu chromosomes X and 7 and MRC-5 chromosome 7 consecutively measured in 142 widely separated pairs of CCD-34Lu (n = 72) and MRC-5 (n = 70) chromosomal masses (214 pairs of angular measurements compared). The y-axis coordinate of each point plotted is the angle measured between a pair of homologues in one chromosomal mass with the x-axis coordinate being either the same angle measured in the other chromosomal mass of a pair (daughter-paired) or a randomly selected, nondaughter chromosomal mass of the same cell type (randomly paired). It can be seen that the daughter-paired measurements (A) are highly correlated (correlation coefficient = 0.788), whereas the randomly paired measurements (B) are not correlated (correlation coefficient = −0.087).

Discussion

We have found several lines of evidence for a largely random assortment of chromosomal positions on the mitotic rings of three human cell types. The first question addressed was whether or not the rigorous >90° separation for all human homologous chromosomes reported by Nagele et al. (1995) for several cell lines could be confirmed and extended to other nontransformed human cells. We were unable to confirm this finding of >90° separation of homologous chromosomes in rosettes of the CCD-34Lu line, a cell type in which this phenomenon had been reported previously to occur (Nagele et al., 1995), or in lymphocyte or MRC-5 rosettes. For all three cell types in our study, an equal number of rosette homologues were separated by <90° as by >90° (Fig. 2, A–C, and Fig. 3; see Appendix, Tables I and II). Also, the pooled x-axis distances between homologous early and mid-anaphase chromosomes of the lymphocytes, MRC-5, and CCD-34Lu cells strongly fit the random separation model and only weakly fit, or rejected, all other theoretical models of chromosomal separation (see Appendix, Fig. 7 and Tables III and IV). Finally, the individual angular separations measured in different pairs of late-anaphase rings between the homologues of CCD-34Lu chromosomes X and 7 and MRC-5 chromosome 7 were highly variable and thus incompatible with fixed chromosomal positions on the mitotic ring (see Appendix, Tables I and II). These differing results between our study and Nagele's study (1995) are not due to variations in fixation, as the CCD-34Lu chromosomes 7 and X have random positions in both Carnoy- and paraformaldehyde-fixed rosettes (see Appendix, Table I).

The previously reported finding of widely separated homologous chromosomes led to the speculation that all human chromosomes were in the same fixed order on the mitotic ring and in interphase (Nagele et al., 1995). In addition to our direct experimental evidence against widely separated and fixed chromosomal positions on the mitotic ring (Figs. 1–3; see Appendix, Fig. 7 and Tables I II III IV), there are strong theoretical arguments against Nagele's model of rigorously connected chromosomal positions being carried through interphase into subsequent mitotic and meiotic divisions (Nagele et al., 1995). This model requires permanent interconnections between chromosomes, or some other mechanism, to maintain chromosomal spatial order. Although interphase chromosomes are connected to each other, if not by nucleotide strands (Korf and Diacumakos, 1980), then by DNA–protein complexes (Maniotis et al., 1997), there is no evidence that such connections are permanent. The interphase positions of mammalian chromosomes are not static: Barr and Bertram (1949) showed that the position of the X chromosome shifted with electrical stimulation in postmitotic neurons. Shifts in interphase chromosomal positions have also been found in neurons from human epileptic cortex (Borden and Manuelidis, 1988), in lymphocytes during different phases of the cell cycle (Ferguson and Ward, 1992), and in other cells with differentiation (Manuelidis, 1984; Park and De Boni, 1992; Choh and De Boni, 1996). Finally, although Dipteran homologues are paired in adult flies (Metz, 1916), histone gene repeats on the Dipteran chromosome 2 are randomly positioned in the nucleus during the first 13 embryonic cell cycles, and only subsequently pair in late embryos (Hiraoka et al., 1993). It is difficult to imagine how such freedom of interphase chromosome movement, observed for a wide variety of cell types, can be reconciled with fixed and permanent connections between the chromosomes during interphase and on the mitotic ring. Also, if the fixed order of the relative positions of chromosomes on the mitotic ring was maintained from the initial fusing of parental haploid genomes into the next meiotic division, the random, Mendelian segregation of chromosomes could not occur.

A simple mechanism can reconcile many of the conflicting results reported for relative chromosomal positions on the mitotic ring: some have shown loosely organized, or even random, chromosomal positions (Hens, 1976; Nur, 1976; Korf and Diacumakos, 1977; Tanaka, 1981); and others have shown nonrandom positions on the ring (Schneiderman and Smith, 1962; Feldman et al., 1966; Heneen and Nichols, 1972; Juricek, 1975; Hens, 1976; Ferrer and Lacadena, 1977; Heslop-Harrison and Bennett, 1984; Nagele et al., 1995). Different chromosomes have discrete domains within the interphase nucleus (Boveri, 1909; Wilson, 1925; Zorn et al., 1979; Vogel and Krüger, 1983; Fussell, 1984; Hubert and Bourgeois, 1986; Ferguson and Ward, 1992; Cremer et al., 1993; Spector, 1993; Carmo-Fonseca et al., 1996; Choh and De Boni, 1996). In 1885, Rabl suggested that the radial chromosomal positions on the mitotic ring during mitosis were a reflection of the relative chromosomal positions in the preceding interphase (Rabl, 1885; Wilson, 1925). The prophase movements of chromosomes support this view, as there are no wide shifts in the positions of the prophase chromosomes relative to each other as they move to the metaphase plate (Bajer and Molè-Bajer, 1956, 1981; Tanaka, 1981; Fussell, 1984; Chaly and Brown, 1988; Hiraoka et al., 1990).

The relative positions of the chromosomes to each other may vary in different interphase cells due to heterogeneity of nucleolus formation from cell to cell (Hens, 1976; Tanaka, 1981; Hubert and Bourgeois, 1986; Borden and Manuelidis, 1988; Park and De Boni, 1992; Lawrence et al., 1993; Carmo-Foneseca et al., 1996), specific transcription patterns induced in response to local differentiation signals (Manuelidis, 1984; Park and De Boni, 1992; Choh and De Boni, 1996), random drift, and possibly other types of chromatin–nuclear envelope interactions (Lamond and Earnshaw, 1998). The shifts in chromosomal positions due to differentiation or in response to external signals may be related to the coupling of actively induced genes to the mRNA processing machinery. Pre-mRNA transcription sites are preferentially associated with discrete pre-mRNA splicing domains (Lawrence et al., 1993; Spector, 1993; Xing et al., 1995; Carmo-Fonseca et al., 1996). It is not clear whether the splicing domains are induced where transcription occurs, and/or whether actively transcribed genes move to these splicing domains (Lawrence et al., 1993; Spector, 1993; Xing et al., 1995; Carmo-Fonseca et al., 1996). If the latter were true, differentiated or induced gene activity would determine gene, and possibly chromosome, location. In support of this occurring, γ-amino butyric acid, a powerful inducer of specific gene expression in pheochromocytoma cells, induces chromatin movement and kinetochore rearrangements in cultured mouse neurons (Holowacz and De Boni, 1991). Also, estrogen induction of the vitellogenin gene family in male Xenopus laevis hepatocytes is associated with kinetochore rearrangements (Janevski et al., 1995).

In addition to our finding that seemingly all possible chromosomal arrangements may occur on the mitotic ring (Figs. 1–3; see Appendix, Fig. 7 and Tables I II III IV), several lines of evidence in our study also suggested that the relative positions of the chromosomes to each other on a given metaphase plate are transmitted into telophase with remarkable fidelity. First, the homologous centromeres clearly had symmetrical positions in the separating early and mid-anaphase chromosomal masses (Fig. 1 B, panel d, and Fig. 1 C, panel c), ruling out chaotic shifts of chromosomal positions during early karyokinesis. Second, rings similar to those of the prometaphase rosettes (Fig. 1 B, panel b) and metaphases (Fig. 1 B, panel c) are present in many late-anaphases (Fig. 1 A, bottom right-hand corner, Fig. 2 D, panel a, and Fig. 2 E, panel a); suggesting that the ring structure remains intact throughout karyokinesis. Finally, the centromeric positions measured in unselected, individual pairs of late-anaphase and telophase chromosomal masses are highly correlated (Fig. 2, D and E, and Fig. 4), confirming earlier claims of symmetrical chromosomal positions in nonmammalian late-anaphases (Rabl, 1885; Metz, 1916; Tanaka, 1981). All of these findings are consistent with the chromosomal positions on the mitotic plate being carried through anaphase into telophase.

This finding of a permissive mitotic ring which transmits its relative chromosomal order into both daughter telophases suggests a mechanism by which the chromosomal organization of a given interphase nucleus is reestablished in its progeny. Specifically, the nonrandom chromosomal positions of a given interphase cell, induced by nucleolus formation, gene activation, differentiation, or other factors, may lead to similar, nonrandom chromosomal positions on the mitotic ring. This is strongly supported by the results of UV radiation experiments which showed that irradiation of small parts of Go/G1 nuclei caused damage to only a few, usually nonhomologous, chromosomes that are later adjacent to each other on the mitotic ring (Zorn et al., 1979; Cremer et al., 1993), a finding consistent with adjacent interphase chromosomes injured by the irradiation ending up in close proximity to each other during mitosis. The symmetrical homologous chromosomes found in the daughter late-anaphase and telophase pairs in our (Fig. 2, D and E, and Fig. 4) and earlier (Rabl, 1885; Metz, 1916; Tanaka, 1981) studies can be simply explained by the carrying over of the relative chromosomal positions on the mitotic ring through anaphase into telophase. Taken together, these results suggest that the spatial chromosomal organization of the interphase nucleus is maintained from one generation to the next.

In summary, there is a relatively random organization of chromosomal positions on the mitotic rings of human MRC-5 cells, CCD-34Lu cells, and lymphocytes, in contrast to a previous report of an invariable >90° separation of homologous human chromosomes on the mitotic ring (Nagele et al., 1995). We also speculate that nonrandom chromosomal associations on the mitotic ring reported for other cell types may be due to the carrying over of nonrandom interphase chromosomal positions to the mitotic ring, and not to the mitotic ring apparatus selecting out a preferred radial chromosomal order before karyokinesis. Thus, our results show that a fixed order of chromosomal positions on the mitotic ring is not fundamental to, or necessary for, the mitotic segregation of human chromosomes, because human MRC-5 cells, CCD-34Lu cells, and lymphocytes go through mitosis quite smoothly. We also found that the relative positions of chromosomes on each metaphase ring seem to be carried through anaphase into telophase.