Chromosoma (Berl.) 50, 313--326 (1975) 9 by Springer-Verlag 1975
Non-random Chromosome Distribution in Radial Metaphases from the Chinese Hamster I. U n c u l t u r e d Cells D i a n e K. J u r i c e k Department of Biology, Emory University, Atlanta, Georgia Abstract. Chromosome distribution was analyzed in uncultured radial rectaphase cells (corneal epithelium, testicular mitotic cells, cells in diakinesis, and cells in metaphase II) from the Chinese hamster. The hypothesis of random distribution was rejected at the 0.001 level (Z~ = 31.6). - - Homologous association was observed for two pairs of chromosomes (3 and 10) in corneal epithelial cells. It was observed for all chromosomes in the testicular mitotic cells. Acroeentrie association was observed in all four cell types. The chromosomes associated in four groups of similarly sized and shaped chromosomes. While group membership did not appear to vary, position within the group was highly variable. - - An elevenpoint model of chromosome relationships was constructed from the data.
Introduction W h i l e it was n o t e d quite e a r l y (Stevens, 1907, 1908; Metz, 1916) t h a t some organisms e x h i b i t c o n s t a n t c h r o m o s o m e o r i e n t a t i o n in t h e nucleus, it was n o t u n t i l t h e w o r k of Miller et al. (1963a, 1963b) t h a t a d e t a i l e d analysis of c h r o m o s o m e d i s t r i b u t i o n was a t t e m p t e d in cells which d i d n o t h a v e a n obvious n o n - r a n d o m d i s t r i b u t i o n of chromosomes. Since then, a large n u m b e r of studies h a v e a p p e a r e d (for p a r t i a l reviews see Comings, 1968, 1972) on t h e question of a n o n - r a n d o m c h r o m o s o m e a r r a n g e m e n t in cells. Most of this w o r k was c o n d u c t e d w i t h c u l t u r e d h u m a n leukoeytes, a n d t h e results were c o n t r a d i c t o r y a t best. H e n e e n a n d Nichols (1972) clearly d e m o n s t r a t e d one source of t h e c o n t r a d i c t i o n s when t h e y showed t h a t a n t i - m i t o t i c agents, such as colchicine a n d Colcemid, a n d h y p o t o n i e t r e a t m e n t d e s t r o y e d c h r o m o s o m e o r i e n t a t i o n in t h e cell (comp a r e w i t h Cohen et al., 1972). I n d i r e c t l y , t h e findings of H e n d e r s o n et al. (1973) s u p p o r t t h i s conclusion. The w o r k of H e n e e n a n d Nichols was p e r f o r m e d on a n a d a p t e d cell line of t h e m u n t j a e . W h i l e t h e y d i d find t h a t c h r o m o s o m e l o c a t i o n was d e f i n i t e l y n o n - r a n d o m in t h e s e cells, t h e o b s e r v a t i o n of a large a m o u n t of v a r i a b i l i t y of c h r o m o s o m e p o s i t i o n raised t h e question of t h e effect of culturing on t h e p h e n o m e n o n . Boss (1954) d e m o n s t r a t e d t h a t homologous p a i r i n g of c h r o m o s o m e s t e n d s to increase in c u l t u r e d cells. Since H e n e e n
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and Nichols observed an increase in homologous pairing over t h a t expected b y chance, their significant results might have been d u e to culture artifact. Alternatively, the variability which t h e y observed might have been the result o f culturing. Recently, N u r (1973) has questioned some of their statistical assumptions and, if he is correct, IIeneen and Nichols actually observed a r a n d o m chromosome arrangement. F o r these reasons, it was t h o u g h t desirable to s t u d y chromosome positioning in uncultured cells not subjected to either anti-mitotic agents or excessive hypotonic treatment.
Materials and Methods Uncultured corneal cells from tile Chinese hamster (2N~22) were obtained by the method of Fredga (1964). Uncultured testicular cells, mitotic and meiotic, were obtained by a method essentially the same as that published by Stock et al. (1972). Hypotonic treatment (0.07 % sodium citrate) for periods of up to 5 minutes was utilized to obtain cells with sufficient spreading of chromosomes to allow for recognition of chromosomes. In no case, however, were the cells which were studied distorted out of the radial configuration. Within each meiotic cell, the neighbors of each of the 11 bivalents or dyads were ranked from nearest to farthest measuring centromere-to-centromere distances. For corneal and testicular mitotic ceils, only the closest 3 neighbors were recorded and ranked for each chromosome. Results Cytological Observations Mitotic metaphase cells from the cornea had a radial distribution of chromosomes such as t h a t cited b y N u r (1973) as indicative of nondistortion of chromosome position. The six smallest chromosomes of the complement were invariably within a circle formed b y the centromeres of the remaining 16 chromosomes (Fig. 1 a). Identification of specific chromosomes was difficult due to their tight packing on the metaphase plate. This was also observed to be a problem in the m u n t j a c (Heneen and Nichols, 1972). Stronger or extended hypotonic treatment, while making chromosome identification easier, destroyed the radial chromosome arrangement (Juricek, 1973). N o indication of homologous association in the corneal cells was observed other t h a n the central location of the six smallest chromosomes. B o t h mitotic and meiotic cells from the testis had a radial distribution of chromosomes identical to t h a t observed in corneal cells (Figs. 1 and 2). The mitotic cells consisted of two populations: (1) cells with no homologous pairing (Fig. 1 b) ; and (2) cells in which homologous pairing was evident. In the latter case, the range of pairing was from very few homologously paired chromosomes per cell (Fig. I c, e) to cells in which there was complete homologous pairing of chromosomes
Fig. 11. (a) Radial metaphase cell from corneal epithelium. The chromosomes do not lie in one focal plane. Note central location of small chromosomes (b) Testicular mitotic metaphase cell with no pre-meiotie pairing. (c) Testicular mitotic metaphase cell with incomplete pre-meiotic pairing. (d) Testicular mitotic metaphase ceils. Cell to left has complete pre-meiotic pairing. Cell to right has complete pairing except for chromosomes number 2. The homologues of pair 1 separate the homologues of pair 2. (e) Interpretive drawing of e. (f ~nd g) Interpretive drawings of (d)
(Fig. l d , f). I n all cases, t h e smaller c h r o m o s o m e s were p a i r e d before t h e larger chromosomes. No cell was o b s e r v e d in which chromosomes 1 a n d 2 were p a i r e d in t h e a b s e n c e of p a i r i n g b y all o t h e r chromosomes. 1 Bars on the figures represent 5 lzm.
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Fig. 2. (a) Cell in diakinesis. Scale indicates 10 microns. (b) Cell in metaphase of meiosis II Thus pre-meiotic pairing (see review by Brown, 1972) appears to be a gradual process occurring over several cell cycles, and there seems to be a sequence observed by the chromosomes during this process.
Statistical Analysis o/Neighbor Ranking Four cell types were examined for chromosome patterning: (1) five mitotic cells from the cornea; (2) three testicular mitotic cells with complete homologous pairing; (3) ten cells in diakinesis; and (4) ten cells in metaphase of meiosis II. A total of four animals provided the cells which were examined. Table 1 shows the frequency of ranked associations of all chromosomes with respect to chromosome 1 in ten cells in diakinesis. Note that in seven of the ten cells, chromosome 11 was one of the three chromosomes closest to chromosome 1. Because of the number of tables generated by neighbor analysis, eleven per cell type, only this sample table is included in this report. The rest of the data can be found in Juricek (1973). For each chromosome of each cell type, the mean rank value was calculated for each of its neighbors. This appears as the last column of Table 1. Since only the first three neighbors of each mitotic chromosome were recorded, it was necessary to assume that the remaining 18 ranks were randomly filled for this calculation. Table 2 is a summary table of the mean ranks for cells in diakinesis. If chromosomes are randomly distributed in the nucleus, the expected value of each mean rank would be 5.50 for meiotic cells and 6.00 for mitotic cells. If one specific chromosome is invariably the closest neighbor
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Table 1. Chinese hamster diakinesis: frequency of ranked association for reference chromosome 1 Neighbor chromo-
Ranks
somes
1
2
3
4
5
6
7
8
9
10
0 0 0 2 0 3 0 1 0 4
2 1 1 0 0 1 1 1 3 0
1 0 0 1 2 1 1 0 1 3
1 0 1 1 3 1 2 0 1 0
2 2 0 0 1 0 0 3 1 1
1 2 3 0 0 0 1 1 0 2
0 1 0 2 2 2 0 1 2 0
0 1 3 0 1 0 2 1 2 0
0 2 0 2 1 2 1 2 0 0
3 1 2 2 0 0 2 0 0 0
Mean rank
I
2 3 4 5 6 7 8 9 10 11
5.7 6.7 6.8 6.1 5.4 4.4 6.4 5.7 4.8 3.0
Table 2. Chinese hamster diakinesis : summary of mean ranks l~eler-
Neighbor chromosomes
ence
chromo-
1
2
3
4:
5
6
7
8
9
10
11
--
5.7 -8.0 6.8 6.4 7.0 5.8 6.4 7.8 6.2 7.2
6.7 7.6 -5.2 4.7 7.1 5.9 6.5 5.5 7.5 6.2
6.8 5.2 4.4
6.1 6.2 3.5 3.7 -6.0 6.0 6.4 5.7 6.2 6.8
5.4 5.7 5.6 4.9 6.2
4.4 3.9 4.6 4.4 5.4 5.0 -5.6 4.5 4.6 4.9
6.4 6.0 6.3 5.9 6.3 5.7 6.1 -5.3 3.8 5.4
5.7 5.5 3.9 4.7 4.6 3.2 4.1 4.9 -4.1 3.7
4.8 3.4 5.9 5.2 4.8 4.8 4.0 2.4 4.3 -3.3
3.0 4.6 4.9 4.8 5.7 4.4 4.3 4.2 3.6 3.3 --
somes
1
2 3 4 5 6 7 8 9 10 11
6.9 7.9 9.4 6.9 7.3 7.9 8.2 8.0 7.9 6.0
4.0 4.5 5.0 5.3 5.4 5.9 5.8
5.9 5.1 4.9 5.5 5.7
t o a n o t h e r , t h e m e a n r a n k w o u l d b e 1.00. I n T a b l e 2, n e i g h b o r b i v a l e n t 5 h a s a m e a n r a n k of 3.7 w i t h r e f e r e n c e b i v a l e n t 4. T h e r e c i p r o c a l a s s o c i a t i o n h a s a m e a n r a n k of 4.0. B o t h v a l u e s i n d i c a t e t h a t t h e s e c h r o m o s o m e s are closer to each o t h e r t h a n would be e x p e c t e d u n d e r a r a n d o m h y p o t h e s i s . I n the same table, the m e a n ranks for t h e association of b i v a l e n t s 2 a n d 3 (7.6, 8.0) i n d i c a t e t h a t t h e s e t w o c h r o m o s o m e s are farther from each other than would be expected. The mean rank values within each table were in turn ranked from l o w e s t t o h i g h e s t a c r o s s t h e r o w s . T h i s a p p e a r s i n T a b l e 3 f o r cells i n
318
Diane K. Juricek Table 3. Chinese hamster meiosis II: ranking of mean ranks
ReferNeighbor chromosomes ence chromo- 1 2 3 4
5
6
7
8
9
10
11
2.0 2.0 3.0 1.0 3.0 1.0 3.0 5.0 2.0 1.0 --
somcs
1 2 3 4 5 6 7 8 9 10 11
-10.0 10.0 10.0 8.0 5.0 7.0 10.0 9.0 7.0 6.0
9.0 -9.0 8.0 9.0 9.0 9.0 9.0 8.0 10.0 7.5
7.0 4.0 -4.0 5.0 6.0 4.0 4.0 5.0 4.5 4.0
8.0 3.0 7.0 -7.0 4.0 10.0 8.0 7.0 9.0 5.0
6.0 7.0 7.0 7.0 -10.0 5.0 7.0 4.0 8.0 9.5
4.0 9.0 7.0 2.0 10.0 -8.0 6.0 3.0 3.0 3.0
5.0 8.0 5.0 9.0 4.0 7.5 -2.0 10.0 4.5 7.5
10.0 6.0 2.0 6.0 6.0 7.5 2.0 -6.0 6.0 9.5
3.0 1.0 4.0 4.0 1.0 3.0 6.0 3.0 -2.0 2.0
1.0 5.0 1.0 4.0 2.0 2.0 1.0 1.0 1.0 -1.0
Aa Bb
82.0 93.0
87.5 98.5
47.5 58.5
68.0 79.0
70.5 81.5
55.0 66.0
62.5 73.5
61.0 72.0
29.0 40.0
19.0 23.0 30.0 34.0
a Column sum of row ranks, b Corrected column sum of row ranks.
meiosis I I . L o w r a n k values i n d i c a t e close association while a high r a n k v a l u e indicates non-association. D a t a for t h e r e m a i n i n g cell t y p e s can be f o u n d in J u r i c e k (1973). T h e c o l u m n sums of t h e r o w r a n k s were c a l c u l a t e d w i t h i n each table. F o r purposes of c o m p a r i s o n b e t w e e n m e i o t i c a n d m i t o t i c cell types, 11 was a d d e d t o t h e t o t a l s of each c o l u m n sum for m ei o t i c cells (Table3). A modified Z 2 t e s t ( F r i e d m a n , 1937) of t h e c o l u m n sums was p e r f o r m e d for each cell t y p e . F o r b o t h m e i o t ic cell t y p e s t h e chi-square v a l u e (Z~0 ~ 4 4 . 3 for diakinesis; Z~0 ~ 4 4 . 1 for meiosis I I ) i n d i c a t e d a proba b i l i t y of less t h a n 1 in a t h o u s a n d t h a t t h e o b s e r v a t i o n s came f r o m a p o p u l a t i o n of cells which h a v e a r a n d o m c h r o m o s o m e distribution. T h e r a n d o m h y p o t h e s i s was n o t r e j e c t e d for either m i t o t i c cell type. H o w e v e r , such a result was e x p e c t e d since 18 of t h e 21 r an k s were assumed to be r a n d o m in order t o complete t h e r a n k i n g s to allow comparisons a m o n g all four cell types. Th e c o m p a r i s o n a m o n g t h e four cell t y p e s was accomplished b y r a n k i n g t h e c o l u m n sums of t h e r o w r a n k s w i t h i n each cell t y p e (Table 4) A Z ~ t e s t of these four rankings r e j e c t e d i n d e p e n d e n c e (g~ = 3 1 . 6 , p ~ 0.001). So, a l t h o u g h t h e results f r o m corneal an d t e s t i c u l a r m i t o t i c cells were non-significant in t e r m s of c o l u m n sums of r o w ranks, t h e r a n k i n g of t h e c o l u m n sums conforms to t h e r a n k i n g of c o l u m n sums in t w o s t at i s t i c a ll y n o n - r a n d o m populations.
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Table 4. Ranking of column sums for the four cell types in the Chinese hamster Cell type
Chromosome 1
2
3
4
5
6
7
8
9
10
11
Cornea Testicular mitosis Diakinesis
7.0 11.0 10.0 11.0 11.0 10.0
8.0 5.0 9.0
9.0 10.0 9.0 4.0 5.0 7.0
6.0 7.5 6.0
5.0 7.5 4.0
3.0 6.0 8.0
4.0 2.0 1.0 3.0 2.0 3.0
1.0 2.0 1.0
Meiosis II
10.0 11.0
4.0
8.0
5.0
7.0
6.0
3.0 1.0
2.0
Total
38.0 43.0 26.0 31.0 30.0 24.5 23.5 23.0 10.0 9.0
6.0
9.0
It must be concluded that there is definitely a non-random distribution of chromosomes in the dividing cell of the Chinese hamster and that the pattern of chromosome distribution is consistent among cell types.
Discussion While the results of neighbor analysis indicated that there is a consistent pattern of centromere position among four cell types from the Chinese hamster, a large amount of variability in chromosome association was observed. How much of the variability was due to the process of preparing cells for study, how much was due to errors in chromosome identification, and how much was due to an inherent variability of chromosome positioning, perhaps reflecting the dynamic nature of the structures responsible for maintaining chromosome position, cannot be determined precisely. However, the relative contribution of each of these factors can be approximated. I n addition, an assessment can be made of the extent of homologous association in the two mitotic cell types, the degree of acrocentric association during mitosis and meiosis, and the overall pattern of chromosome distribution in the four cell types.
Chromosome Identi]ication The use of non-distorted strict metaphase chromosome figures precluded the use of banding techniques for chromosome identification due to the highly condensed state of the chromosomes at this stage. Thus, chromosome size and shape were the only criteria which could be applied. While these characteristics are sufficient to determine group membership upon casual inspection, they are often not sufficient for the specific identification of each chromosome in a given cell. I n particular, the acrocentric chromosomes of pairs 7 and 8 are difficult to identify, as are the metacentric chromosomes of pairs 10 and 11 (Kate and Yosida, 1972; Rodman and Biedler, 1973). I t was this difficulty in distinguishing 21 Chromosoma(Berl.), Bd. 50
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Diane K. Juricek
among these chromosomes which led directly to the small sample size reported here; if there was any doubt about the specific identification of the chromosomes of a cell, t h a t cell was excluded from the study. In order to check the consistency of chromosome identification, five of the original meiotic chromosome figures were chosen at random and re-karyotyped after an interval of approximately ten months. A difference in identification was observed in one cell where a previously labelled 10 was called chromosome 11 in the second karyotype. Of the total of 55 chromosomes from these 5 cells, the identification of 53 was consistent. This suggests only that the criteria were consistently applied within a cell. Differential variations in chromosome condensation between cells could lead to mis-identification of chromosomes. However, the variation associated with the acrocentric or small metacentric chromosomes, which are difficult to identify, was no greater t h a n t h a t associated with chromosomes 1 and 2, which can be readily identified. I f error in identification was a major component of the observed variability, this would not be expected. Also, it should be pointed out t h a t Heneen and Nichols (1972) observed a large amount of variation in chromosome association in the muntjac. This animal has a low number of chromosomes (2n = 6 in females, 7 in males), each of which is rather easy to identify. Thus, although mis-identification of chromosomes probably occurred, the contribution of error in identification to the observed variability is considered to be minimal.
Homologous Association I n the mitoses from the corneal epithelium, there was only slight indication of homologous pairing. Chromosomes pairs 3 (X and Y) and 10 seem to have strong homologous pairing (taken as row ranks of 2.5 or less), and chromosomes 4, 5 and 11 have somewhat weaker homologous association (taken as row ranks of 4 or less). The other six chromosome pairs are definitely non-associated. I n contrast, homologous association in mitotic cells from the testis was found for every chromosome pair (row rank of 1.0 for chromosomes 3 through 11, 2.0 for chromosome 1, and 3.0 for chromosome 2). Indeed, such association was obvious simply from looking at the cells. I t was expected that the central location of the small metacentric chromosomes, 9-11, would result in the observation of homologous association for them, as defined b y neighbor analysis. While this was true for testicular mitotic cells selected because of their homologous pairing, in corneal cells these six chromosomes were found to associate more closely with other chromosomes t h a n with their respective homolo-
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321
gues. I t appears that size of the chromosomes is very important in determining their general location in the dividing cell. The smallest chromosomes are found in the center of chromosome figures in the Chinese hamster whether or not homologous pairing exists in the cell. Acrocentric Association There was good evidence of non-homologous acrocentric association (chromosomes 6, 7 and 8) in all four cell types. Neighbor analysis indicated association (row ranks less than 4) of 6 with 7, 6 with 8, and 7 with 8 in the corneal cells. In the testicular mitotic cells, a close association was observed only between chromosomes 7 and 8. This was also the only association for cells in meiosis I I (Table 3). In diakinesis, acrocentric association was observed between chromosomes 6 and 8. It is commonly thought that the association of acrocentric chromosomes is due to the nucleolar organizing regions located on the small arms of these chromosomes (Cooke, 1972; Ohno et al., 1961). In a number of organisms the region responsible for organizing the nucleolus is, in fact, located near the satellites of acroccntric chromosomes (see Brown, 1972, for review), and Henderson et al. (1973) have recently demonstrated that the aerocentric chromosomes in cultured human lymphocytes are frequently connected by rDNA. However, Phillips and Phillips (1969) have clearly demonstrated that all the chromosomes in the Chinese hamster can, and do, form functional nucleoli. The typical localized nucleolar organizing region does not appear to exist in this organism except, possibly, on the X and u chromosomes (pair 3) (Yerganian et al., 1960; also observed in this study). If acrocentric chromosomes associate in the absence of localized nucleolar organizing regions, then that association must be due to factors other than the presence of nucleo]ar organizing regions. Furthermore, if all the chromosomes contain regions responsible for the organization of nucleoli, then acrocentric association cannot be explained on the basis of nucleolar organizing regions. The obvious similarity in size and shape between the two acrocentrie chromosomes (7 and 8) which were observed to associate closely in three cell types in the Chinese hamster suggests that size and shape of chromosomes may be the most important factors in determining chromosome distribution in the Chinese hamster. The Relative Location o] Chromosomes in the Chinese Hamster Cell The ranking of chromosome associations in neighbor analysis can be used to construct an l 1-point mode] of chromosome relations in the Chinese hamster cell. The model is suggested in part by the statistical 21.*
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Diane K. Juricek
Fig. 3. (a) Eleven-point model of chromosome relationships in metaphase II from the Chinese hamster. (b) Eleven-point composite model of chromosome relationships in the Chinese hamster. (a, b) Chromosomes drawn to scale
analysis. I n all cell types, chromosomes 9, 10 and 11 were very close to each other and to the remaining eight chromosomes. This suggests a circle of eight chromosomes with 9, 10 and 11 in the center. I t is also what was observed in each cell. In Table 3, certain strong associations (taken as row ranks of 2.5 or less) can be observed: 7-8, 3-8, 4-6, 5-9, etc. Other weaker but also important associations (row ranks of 4 or less) can also be observed: 5-7, 2-4, 8-9 etc. By considering these associations and the 8 -k 3 model suggested by both statistical analysis and biological observation, a model (Fig. 3 a), which represents the most likely position of the eleven dyads, can be constructed. I n a similar way, models were constructed for the other cell types. I n order to compare these models, the sequence of the peripheral chromosomes was recorded, starting with the 7-8 complex and moving away from the 1-2 complex. The center chromosomes were recorded by listing the one closest to the first member of the peripheral sequence and moving around the models in the same direction followed for the peripheral sequence. These results appear in Table 5. By assuming that the validity of the four models is equal, a composite model (Fig. 3 h) was constructed from Table 5. If a given chromosome appeared in a specific position two or more times in Table 5, it occupied that position in the composite model. Only chromosomes 3 and 4 could not be definitely located by this :_.process. Chromosomes 9-11 were arranged as an inner circle which can revolve with respect to the outer circle. What relationship does the composite model have with reality ? I t was constructed by democratic vote from four models, each of which was
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Table 5. Chromosome sequence lor models of chromosome relationships in the Chinese hamster MeiosisII Cornea Testicular mitotic Diakinesis
8 7 8 8
7 8 7 7
Composite
8 7
5 6 3 4
2 3 4 6
4 5 5 5
6 4 6 3
4 3 5 6
1 2 1 1
3 1 2 2
1 2
10 9 11 11 10 9 11 10 9 10 9 11 (11 10
9) a
a Circularly permutated.
constructed by ignoring the high degree of variability observed and concentrating instead on an underlying pattern to the variability. As such, it represents a picture of the average position of each chromosome, which may have no correspondence with any particular meiotic cell and which definitely cannot correspond to a mitotic cell unless complete homologous pairing is present. Despite this objection to the model, it is worth considering for it highlights certain possibly fundamental relationships in the cell. The chromosomes appear to be grouped by size and shape in the composite model. Only chromosome 6, the largest acrocentrie, violates this correspondence since it is closest to chromosomes 7 and 8 in terms of size and centromere position. Furthermore, there appears to be balancing of chromosomal mass about the periphery. Both a balanced metaphase plate and the size, shape, position correlation have been noted before. The size dependency for regular segregation of the fourth chromosomes in Drosophila melanogaster led the Grells to propose the theory of distributive pairing in meiosis (E. H. Grell, 1963; R. F. Grell, 1964; R. F. Grell and Day, 1970). Also, Heneen and Nichols (1972) observed associations based on proximity in size along with a balancing of the chromosomal mass on the metaphase plate in the muntjac. Lawrence (1931) reported secondary associations in over 100 plant species. I n all cases, the associations were between chromosomes of similar size and shape. The results, then, suggest strongly that chromosome position in the cell is determined by size and configuration.
Maintenance o/Chromosome Patter~ : Interaction o] Genetic In/ormation and Physical Forces In four cell types of the Chinese hamster, a consistent pattern of chromosome associations was observed to underlie a great deal of variability in position. The general chromosome distribution, which was observed repeatedly, was that of a circle of large chromosomes formed about the smaller chromosomes. I t was suggested that the
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Diane K. Juricek
association of acrocentric chromosomes in this organism was due to the size and configuration of the chromosomes rather than to homologous function on non-homologous chromosomes. The composite model of chromosome relationships constructed from the four cell types also appeared to reflect a correspondence between position and size and shape. Theoretically, there are two extremes which might exist with regard to chromosome distribution in the cell: (1) the chromosomes are completely autonomous units possessing total freedom of movement in response to physical forces acting upon them; and (2) the chromosomes are completely subject to position specification, the information for which resides in the DNA. Apparently, neither extreme is realized in actuality. If chromosomes are autonomous units, then a cyclic stochastic process, in which all probabilities closely approach unity, would have to be postulated in order to account for chromosome localization in the nucleus, centromere restriction to one portion of the nucleus in sperm (Macgregor and Walker, 1973), the observations in this report, and the m a n y other indications of non-randomness of chromosome position which have been cited. I n addition, this process would have to account for the changes which occur during pre-meiotic pairing, the unique meiotic divisions, and the continuance of the chromosomal distribution pattern between generations. While physical components such as chromosome mass and the "pulling force" generated b y the division apparatus are most likely constant (Mazia, 1961), the other characteristics of the physical environment of chromosomes tend to respond to internal and external changes in a homeostatic manner. Clearly, the tendency of the cell to maintain homeostasis over a short period of time contradicts the possibility of long-term cyclic changes being achieved solely b y interacting components of the physical environment. Thus, it is highly unlikely t h a t chromosome position is maintained b y the response of autonomous chromosomes to a cyclically changing summation of physical forces acting upon them. While chromosomes are not purely autonomous, neither are they totally without autonomy. If chromosomes were distributed in the cell according to some highly structured plan contained in the DNA, then no variation of chromosome associations would be observed. The fact t h a t a good deal of variation was observed in indicative of a lack of total genetic control or, stated in another way, proof of some freedom of movement on the part of the chromosomes in response to varying physical conditions. The observations reported here can only be regarded as a beginning study of chromosome positioning in the cell. With further work, the observed variability m a y come to have deeper significance than was suggested. Individual organisms or cell types m a y have subtle differences in chromosome patterning which were not detected in this study.
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I n four cell types from the Chinese hamster, there was a n o n - r a n d o m d i s t r i b u t i o n of chromosomes which was consistent for the four cells types. This n o n - r a n d o m n e s s was n o t e v i d e n t from casual inspection due to the large a m o u n t of v a r i a b i l i t y associated with it. The n o n - r a n d o m n e s s , the variability, a n d the size-shape-position correlation were also f o u n d b y H e n e e n a n d Nichols (1972) in their s t u d y of m u n t j a c cells. I t is quite likely t h a t a detailed analysis of chromosome position will reveal the same characteristics i n other organisms.
Acknowledgments. This work was supported by the Department of Biology of Emory University and the National Science Foundation Predoctoral Fellowship Program. I thank A. Alan Humphries, Jr., William Murdy, Charles Ray, Jr., Martin Shapiro, all of Emory University, and l~obert Fetner, of the Georgia Institute of Technology, for their guidance. Re~erences Boss, J. M.N.: Mitosis in cultures of newt tissue. II. Chromosome pairing in anaphase. Exp. Cell Res. 7, 225-231 (1954) Brown, W. V. : Textbook of Cytogenetics. Saint Louis: C. V. Mosby Comp. 1972 Cohen, M. M., Enis, •., Pfeifer, C. G. : An investigation of somatic pairing in the muntjac (Muntiacus muntjak). Cytogenetics 11, 145-152 (1972) Comings, D. E. : The rationale for an ordered arrangement of chromatin in the interphase nucleus. Amer. J. hum. Genet. 29, 440-460 (1968) Comings, D. E. : The structure and function of chromatin. Advanc. human Genet. a, 237436 (1972) Cooke, P. : Patterns of secondary association between tile acrocentric autosomes of man. Chromosoma (Berl.) 36, 221-240 (1972) Fredga, K. : A simple technique for demonstration of the chromosomes and mitotic stages in a mammal: chromosomes from cornea. Hereditas (Lund.) 51, 268-273 (1964:) Friedman, M. : The use of ranks to avoid the assumption of normality. J. Amer. Statist. Ass. 32, 675-701 (1937) Grell, E. I-I.: Distributive pairing of compound chromosomes in females of Drosophila melanogaster. Genetics 48, 1217-1229 (1963) Grell, R . F . : Distributive pairing: the size-dependent mechanism for regular segregation of the fourth chromosomes in Drosophila melanogaster. Genetics 52, 226-232 (1964:) Grell, 1%.F., Day, J. W. : Chromosome pairing in the oogonial cells of Drosophila melanogaster. Chromosoma (Berl.) 31, 434:44:5 (1970) Henderson, A. S., Warburton, D., Atwood, K. C.: Ribosomal DNA connectives between human aerocentrie chromosomes. Nature (Lond.) 245, 95-97 (1973) Heneen, W. K., Nichols, W. W.: Nonrandom arrangement of metaphase chromosomes in cultured cells of the Indian deer, Muntiacus muntjak. Cytogenetics l l , 153-164: (1972) Juricek, D. K. : Non-random chromosome distribution in selected cell types from the Chinese hamster. UnpublishedPh.D. dissertation, Emory University, Atlanta, Georgia, 1973 Kato, H., u T . H . : Banding patterns of Chinese hamster chromosomes revealed by new techniques. Chromosoma (Berl.) 36, 272-280 (1972)
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Lawrence, W. J. C. : The secondary association of chromosomes. Cytologia (Tokyo) 2, 352-384 (1931) Macgregor, H. C., Walker, M. H. : The arrangement of chromosomes in nuclei of sperm from the plethodontid salamanders. Chromosoma (Berl.) 40, 243-262 (1973) Mazia, D.: Mitosis and the physiology of cell division. In: The Cell (J. Brachet and A. E. Mirsky, eds.), vol. 3, p. 77412. New York-London: Academic Press 1961 Metz, C. W.: Chromosome studies on the Diptera. II. The paired association of chromosomes in the Diptera, and its significance. J. exp. Zool. 21, 213-279 (1916) Miller, O. J., Breg, W. R., Mukherjee, B. B., Gamble, A. V. N., Christakos, A. C. : Non-random distribution of chromosomes in metaphase figures from cultured human leucocytes. II. The peripheral location of chromosomes 13, 17-18 and 21. Cytogenetics 2, 152-167 (1963b) Miller, O. J., Mukherjee, B. B., Breg, W. R., Gamble, A. V. N. : Non-random distribution of chromosomes in metaphase figures from cultured human leucocytes. I. The peripheral location of the Y chromosome. Cytogeneties 2, 1-14 (1963a) Nur, U. : Random arrangement of chromosomes in a radial metaphase configuration. Chromosoma (Berl.) 40, 263-267 (1973) Ohno, S., Trujilla, J. M. : Kaplan, W. D., Kinosita, P. : Nucleolus-organisers in the causation of chromosomal anomalies in man. Lancet 1961 II, 123-125 Phillips, S. G., Phillips, D. M.: Sites of nucleolus production in cultured Chinese hamster cells. J. Cell Biol. 40, 248-268 (1969) Rodman, T. C., Biedler, J. L. : Specificity of compaction in meiotic chromosomes of the female Chinese hamster. Chromosoma (Berl.) 42, 229-246 (1973) Stevens, N. M. : The chromosomes of Drosophila ampelophila. Proc. VIIth Int. Congr. Zool. 380-381 (1907) Stevens, N. M.: A study of the germ cells of certain Diptera, with reference to the heterochromosomes and the phenomenon of synapsis. J. exp. Zool. 5, 359-374 (1908) Stock, A. D., Burnham, D. B., Hsu, T. C.: Giemsa banding of meiotic chromosomes with description of a procedure for cytological preparations from solid tissues. Cytogeneties l l , 534-539 (1972) Yerganian, G., Kato, R., Leonard, M.L., Gagnon, H . J . , Grodzins, M.S.: Sex chromosomes in malignancy, transplantability of growths, and aberrant sex determination. In: Cell physiology of neoplasia, p. 49-93. Austin: University of Texas Press 1960 Received May 29, 1974 - - January 18, 1975 / Accepted by J. H. Taylor Ready for press January 20, 1975 Diane K. Juricek Department of Biology Emory University Atlanta, Georgia 30322 U.S.A.
Non-random Chromosome Distribution in Radial Metaphases from the Chinese Hamster I. U n c u l t u r e d Cells D i a n e K. J u r i c e k Department of Biology, Emory University, Atlanta, Georgia Abstract. Chromosome distribution was analyzed in uncultured radial rectaphase cells (corneal epithelium, testicular mitotic cells, cells in diakinesis, and cells in metaphase II) from the Chinese hamster. The hypothesis of random distribution was rejected at the 0.001 level (Z~ = 31.6). - - Homologous association was observed for two pairs of chromosomes (3 and 10) in corneal epithelial cells. It was observed for all chromosomes in the testicular mitotic cells. Acroeentrie association was observed in all four cell types. The chromosomes associated in four groups of similarly sized and shaped chromosomes. While group membership did not appear to vary, position within the group was highly variable. - - An elevenpoint model of chromosome relationships was constructed from the data.
Introduction W h i l e it was n o t e d quite e a r l y (Stevens, 1907, 1908; Metz, 1916) t h a t some organisms e x h i b i t c o n s t a n t c h r o m o s o m e o r i e n t a t i o n in t h e nucleus, it was n o t u n t i l t h e w o r k of Miller et al. (1963a, 1963b) t h a t a d e t a i l e d analysis of c h r o m o s o m e d i s t r i b u t i o n was a t t e m p t e d in cells which d i d n o t h a v e a n obvious n o n - r a n d o m d i s t r i b u t i o n of chromosomes. Since then, a large n u m b e r of studies h a v e a p p e a r e d (for p a r t i a l reviews see Comings, 1968, 1972) on t h e question of a n o n - r a n d o m c h r o m o s o m e a r r a n g e m e n t in cells. Most of this w o r k was c o n d u c t e d w i t h c u l t u r e d h u m a n leukoeytes, a n d t h e results were c o n t r a d i c t o r y a t best. H e n e e n a n d Nichols (1972) clearly d e m o n s t r a t e d one source of t h e c o n t r a d i c t i o n s when t h e y showed t h a t a n t i - m i t o t i c agents, such as colchicine a n d Colcemid, a n d h y p o t o n i e t r e a t m e n t d e s t r o y e d c h r o m o s o m e o r i e n t a t i o n in t h e cell (comp a r e w i t h Cohen et al., 1972). I n d i r e c t l y , t h e findings of H e n d e r s o n et al. (1973) s u p p o r t t h i s conclusion. The w o r k of H e n e e n a n d Nichols was p e r f o r m e d on a n a d a p t e d cell line of t h e m u n t j a e . W h i l e t h e y d i d find t h a t c h r o m o s o m e l o c a t i o n was d e f i n i t e l y n o n - r a n d o m in t h e s e cells, t h e o b s e r v a t i o n of a large a m o u n t of v a r i a b i l i t y of c h r o m o s o m e p o s i t i o n raised t h e question of t h e effect of culturing on t h e p h e n o m e n o n . Boss (1954) d e m o n s t r a t e d t h a t homologous p a i r i n g of c h r o m o s o m e s t e n d s to increase in c u l t u r e d cells. Since H e n e e n
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and Nichols observed an increase in homologous pairing over t h a t expected b y chance, their significant results might have been d u e to culture artifact. Alternatively, the variability which t h e y observed might have been the result o f culturing. Recently, N u r (1973) has questioned some of their statistical assumptions and, if he is correct, IIeneen and Nichols actually observed a r a n d o m chromosome arrangement. F o r these reasons, it was t h o u g h t desirable to s t u d y chromosome positioning in uncultured cells not subjected to either anti-mitotic agents or excessive hypotonic treatment.
Materials and Methods Uncultured corneal cells from tile Chinese hamster (2N~22) were obtained by the method of Fredga (1964). Uncultured testicular cells, mitotic and meiotic, were obtained by a method essentially the same as that published by Stock et al. (1972). Hypotonic treatment (0.07 % sodium citrate) for periods of up to 5 minutes was utilized to obtain cells with sufficient spreading of chromosomes to allow for recognition of chromosomes. In no case, however, were the cells which were studied distorted out of the radial configuration. Within each meiotic cell, the neighbors of each of the 11 bivalents or dyads were ranked from nearest to farthest measuring centromere-to-centromere distances. For corneal and testicular mitotic ceils, only the closest 3 neighbors were recorded and ranked for each chromosome. Results Cytological Observations Mitotic metaphase cells from the cornea had a radial distribution of chromosomes such as t h a t cited b y N u r (1973) as indicative of nondistortion of chromosome position. The six smallest chromosomes of the complement were invariably within a circle formed b y the centromeres of the remaining 16 chromosomes (Fig. 1 a). Identification of specific chromosomes was difficult due to their tight packing on the metaphase plate. This was also observed to be a problem in the m u n t j a c (Heneen and Nichols, 1972). Stronger or extended hypotonic treatment, while making chromosome identification easier, destroyed the radial chromosome arrangement (Juricek, 1973). N o indication of homologous association in the corneal cells was observed other t h a n the central location of the six smallest chromosomes. B o t h mitotic and meiotic cells from the testis had a radial distribution of chromosomes identical to t h a t observed in corneal cells (Figs. 1 and 2). The mitotic cells consisted of two populations: (1) cells with no homologous pairing (Fig. 1 b) ; and (2) cells in which homologous pairing was evident. In the latter case, the range of pairing was from very few homologously paired chromosomes per cell (Fig. I c, e) to cells in which there was complete homologous pairing of chromosomes
Fig. 11. (a) Radial metaphase cell from corneal epithelium. The chromosomes do not lie in one focal plane. Note central location of small chromosomes (b) Testicular mitotic metaphase cell with no pre-meiotie pairing. (c) Testicular mitotic metaphase cell with incomplete pre-meiotic pairing. (d) Testicular mitotic metaphase ceils. Cell to left has complete pre-meiotic pairing. Cell to right has complete pairing except for chromosomes number 2. The homologues of pair 1 separate the homologues of pair 2. (e) Interpretive drawing of e. (f ~nd g) Interpretive drawings of (d)
(Fig. l d , f). I n all cases, t h e smaller c h r o m o s o m e s were p a i r e d before t h e larger chromosomes. No cell was o b s e r v e d in which chromosomes 1 a n d 2 were p a i r e d in t h e a b s e n c e of p a i r i n g b y all o t h e r chromosomes. 1 Bars on the figures represent 5 lzm.
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Fig. 2. (a) Cell in diakinesis. Scale indicates 10 microns. (b) Cell in metaphase of meiosis II Thus pre-meiotic pairing (see review by Brown, 1972) appears to be a gradual process occurring over several cell cycles, and there seems to be a sequence observed by the chromosomes during this process.
Statistical Analysis o/Neighbor Ranking Four cell types were examined for chromosome patterning: (1) five mitotic cells from the cornea; (2) three testicular mitotic cells with complete homologous pairing; (3) ten cells in diakinesis; and (4) ten cells in metaphase of meiosis II. A total of four animals provided the cells which were examined. Table 1 shows the frequency of ranked associations of all chromosomes with respect to chromosome 1 in ten cells in diakinesis. Note that in seven of the ten cells, chromosome 11 was one of the three chromosomes closest to chromosome 1. Because of the number of tables generated by neighbor analysis, eleven per cell type, only this sample table is included in this report. The rest of the data can be found in Juricek (1973). For each chromosome of each cell type, the mean rank value was calculated for each of its neighbors. This appears as the last column of Table 1. Since only the first three neighbors of each mitotic chromosome were recorded, it was necessary to assume that the remaining 18 ranks were randomly filled for this calculation. Table 2 is a summary table of the mean ranks for cells in diakinesis. If chromosomes are randomly distributed in the nucleus, the expected value of each mean rank would be 5.50 for meiotic cells and 6.00 for mitotic cells. If one specific chromosome is invariably the closest neighbor
Non-random Metaphase Chromosome Distribution. I
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Table 1. Chinese hamster diakinesis: frequency of ranked association for reference chromosome 1 Neighbor chromo-
Ranks
somes
1
2
3
4
5
6
7
8
9
10
0 0 0 2 0 3 0 1 0 4
2 1 1 0 0 1 1 1 3 0
1 0 0 1 2 1 1 0 1 3
1 0 1 1 3 1 2 0 1 0
2 2 0 0 1 0 0 3 1 1
1 2 3 0 0 0 1 1 0 2
0 1 0 2 2 2 0 1 2 0
0 1 3 0 1 0 2 1 2 0
0 2 0 2 1 2 1 2 0 0
3 1 2 2 0 0 2 0 0 0
Mean rank
I
2 3 4 5 6 7 8 9 10 11
5.7 6.7 6.8 6.1 5.4 4.4 6.4 5.7 4.8 3.0
Table 2. Chinese hamster diakinesis : summary of mean ranks l~eler-
Neighbor chromosomes
ence
chromo-
1
2
3
4:
5
6
7
8
9
10
11
--
5.7 -8.0 6.8 6.4 7.0 5.8 6.4 7.8 6.2 7.2
6.7 7.6 -5.2 4.7 7.1 5.9 6.5 5.5 7.5 6.2
6.8 5.2 4.4
6.1 6.2 3.5 3.7 -6.0 6.0 6.4 5.7 6.2 6.8
5.4 5.7 5.6 4.9 6.2
4.4 3.9 4.6 4.4 5.4 5.0 -5.6 4.5 4.6 4.9
6.4 6.0 6.3 5.9 6.3 5.7 6.1 -5.3 3.8 5.4
5.7 5.5 3.9 4.7 4.6 3.2 4.1 4.9 -4.1 3.7
4.8 3.4 5.9 5.2 4.8 4.8 4.0 2.4 4.3 -3.3
3.0 4.6 4.9 4.8 5.7 4.4 4.3 4.2 3.6 3.3 --
somes
1
2 3 4 5 6 7 8 9 10 11
6.9 7.9 9.4 6.9 7.3 7.9 8.2 8.0 7.9 6.0
4.0 4.5 5.0 5.3 5.4 5.9 5.8
5.9 5.1 4.9 5.5 5.7
t o a n o t h e r , t h e m e a n r a n k w o u l d b e 1.00. I n T a b l e 2, n e i g h b o r b i v a l e n t 5 h a s a m e a n r a n k of 3.7 w i t h r e f e r e n c e b i v a l e n t 4. T h e r e c i p r o c a l a s s o c i a t i o n h a s a m e a n r a n k of 4.0. B o t h v a l u e s i n d i c a t e t h a t t h e s e c h r o m o s o m e s are closer to each o t h e r t h a n would be e x p e c t e d u n d e r a r a n d o m h y p o t h e s i s . I n the same table, the m e a n ranks for t h e association of b i v a l e n t s 2 a n d 3 (7.6, 8.0) i n d i c a t e t h a t t h e s e t w o c h r o m o s o m e s are farther from each other than would be expected. The mean rank values within each table were in turn ranked from l o w e s t t o h i g h e s t a c r o s s t h e r o w s . T h i s a p p e a r s i n T a b l e 3 f o r cells i n
318
Diane K. Juricek Table 3. Chinese hamster meiosis II: ranking of mean ranks
ReferNeighbor chromosomes ence chromo- 1 2 3 4
5
6
7
8
9
10
11
2.0 2.0 3.0 1.0 3.0 1.0 3.0 5.0 2.0 1.0 --
somcs
1 2 3 4 5 6 7 8 9 10 11
-10.0 10.0 10.0 8.0 5.0 7.0 10.0 9.0 7.0 6.0
9.0 -9.0 8.0 9.0 9.0 9.0 9.0 8.0 10.0 7.5
7.0 4.0 -4.0 5.0 6.0 4.0 4.0 5.0 4.5 4.0
8.0 3.0 7.0 -7.0 4.0 10.0 8.0 7.0 9.0 5.0
6.0 7.0 7.0 7.0 -10.0 5.0 7.0 4.0 8.0 9.5
4.0 9.0 7.0 2.0 10.0 -8.0 6.0 3.0 3.0 3.0
5.0 8.0 5.0 9.0 4.0 7.5 -2.0 10.0 4.5 7.5
10.0 6.0 2.0 6.0 6.0 7.5 2.0 -6.0 6.0 9.5
3.0 1.0 4.0 4.0 1.0 3.0 6.0 3.0 -2.0 2.0
1.0 5.0 1.0 4.0 2.0 2.0 1.0 1.0 1.0 -1.0
Aa Bb
82.0 93.0
87.5 98.5
47.5 58.5
68.0 79.0
70.5 81.5
55.0 66.0
62.5 73.5
61.0 72.0
29.0 40.0
19.0 23.0 30.0 34.0
a Column sum of row ranks, b Corrected column sum of row ranks.
meiosis I I . L o w r a n k values i n d i c a t e close association while a high r a n k v a l u e indicates non-association. D a t a for t h e r e m a i n i n g cell t y p e s can be f o u n d in J u r i c e k (1973). T h e c o l u m n sums of t h e r o w r a n k s were c a l c u l a t e d w i t h i n each table. F o r purposes of c o m p a r i s o n b e t w e e n m e i o t i c a n d m i t o t i c cell types, 11 was a d d e d t o t h e t o t a l s of each c o l u m n sum for m ei o t i c cells (Table3). A modified Z 2 t e s t ( F r i e d m a n , 1937) of t h e c o l u m n sums was p e r f o r m e d for each cell t y p e . F o r b o t h m e i o t ic cell t y p e s t h e chi-square v a l u e (Z~0 ~ 4 4 . 3 for diakinesis; Z~0 ~ 4 4 . 1 for meiosis I I ) i n d i c a t e d a proba b i l i t y of less t h a n 1 in a t h o u s a n d t h a t t h e o b s e r v a t i o n s came f r o m a p o p u l a t i o n of cells which h a v e a r a n d o m c h r o m o s o m e distribution. T h e r a n d o m h y p o t h e s i s was n o t r e j e c t e d for either m i t o t i c cell type. H o w e v e r , such a result was e x p e c t e d since 18 of t h e 21 r an k s were assumed to be r a n d o m in order t o complete t h e r a n k i n g s to allow comparisons a m o n g all four cell types. Th e c o m p a r i s o n a m o n g t h e four cell t y p e s was accomplished b y r a n k i n g t h e c o l u m n sums of t h e r o w r a n k s w i t h i n each cell t y p e (Table 4) A Z ~ t e s t of these four rankings r e j e c t e d i n d e p e n d e n c e (g~ = 3 1 . 6 , p ~ 0.001). So, a l t h o u g h t h e results f r o m corneal an d t e s t i c u l a r m i t o t i c cells were non-significant in t e r m s of c o l u m n sums of r o w ranks, t h e r a n k i n g of t h e c o l u m n sums conforms to t h e r a n k i n g of c o l u m n sums in t w o s t at i s t i c a ll y n o n - r a n d o m populations.
Non-random Metaphase Chromosome Distribution. I
319
Table 4. Ranking of column sums for the four cell types in the Chinese hamster Cell type
Chromosome 1
2
3
4
5
6
7
8
9
10
11
Cornea Testicular mitosis Diakinesis
7.0 11.0 10.0 11.0 11.0 10.0
8.0 5.0 9.0
9.0 10.0 9.0 4.0 5.0 7.0
6.0 7.5 6.0
5.0 7.5 4.0
3.0 6.0 8.0
4.0 2.0 1.0 3.0 2.0 3.0
1.0 2.0 1.0
Meiosis II
10.0 11.0
4.0
8.0
5.0
7.0
6.0
3.0 1.0
2.0
Total
38.0 43.0 26.0 31.0 30.0 24.5 23.5 23.0 10.0 9.0
6.0
9.0
It must be concluded that there is definitely a non-random distribution of chromosomes in the dividing cell of the Chinese hamster and that the pattern of chromosome distribution is consistent among cell types.
Discussion While the results of neighbor analysis indicated that there is a consistent pattern of centromere position among four cell types from the Chinese hamster, a large amount of variability in chromosome association was observed. How much of the variability was due to the process of preparing cells for study, how much was due to errors in chromosome identification, and how much was due to an inherent variability of chromosome positioning, perhaps reflecting the dynamic nature of the structures responsible for maintaining chromosome position, cannot be determined precisely. However, the relative contribution of each of these factors can be approximated. I n addition, an assessment can be made of the extent of homologous association in the two mitotic cell types, the degree of acrocentric association during mitosis and meiosis, and the overall pattern of chromosome distribution in the four cell types.
Chromosome Identi]ication The use of non-distorted strict metaphase chromosome figures precluded the use of banding techniques for chromosome identification due to the highly condensed state of the chromosomes at this stage. Thus, chromosome size and shape were the only criteria which could be applied. While these characteristics are sufficient to determine group membership upon casual inspection, they are often not sufficient for the specific identification of each chromosome in a given cell. I n particular, the acrocentric chromosomes of pairs 7 and 8 are difficult to identify, as are the metacentric chromosomes of pairs 10 and 11 (Kate and Yosida, 1972; Rodman and Biedler, 1973). I t was this difficulty in distinguishing 21 Chromosoma(Berl.), Bd. 50
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Diane K. Juricek
among these chromosomes which led directly to the small sample size reported here; if there was any doubt about the specific identification of the chromosomes of a cell, t h a t cell was excluded from the study. In order to check the consistency of chromosome identification, five of the original meiotic chromosome figures were chosen at random and re-karyotyped after an interval of approximately ten months. A difference in identification was observed in one cell where a previously labelled 10 was called chromosome 11 in the second karyotype. Of the total of 55 chromosomes from these 5 cells, the identification of 53 was consistent. This suggests only that the criteria were consistently applied within a cell. Differential variations in chromosome condensation between cells could lead to mis-identification of chromosomes. However, the variation associated with the acrocentric or small metacentric chromosomes, which are difficult to identify, was no greater t h a n t h a t associated with chromosomes 1 and 2, which can be readily identified. I f error in identification was a major component of the observed variability, this would not be expected. Also, it should be pointed out t h a t Heneen and Nichols (1972) observed a large amount of variation in chromosome association in the muntjac. This animal has a low number of chromosomes (2n = 6 in females, 7 in males), each of which is rather easy to identify. Thus, although mis-identification of chromosomes probably occurred, the contribution of error in identification to the observed variability is considered to be minimal.
Homologous Association I n the mitoses from the corneal epithelium, there was only slight indication of homologous pairing. Chromosomes pairs 3 (X and Y) and 10 seem to have strong homologous pairing (taken as row ranks of 2.5 or less), and chromosomes 4, 5 and 11 have somewhat weaker homologous association (taken as row ranks of 4 or less). The other six chromosome pairs are definitely non-associated. I n contrast, homologous association in mitotic cells from the testis was found for every chromosome pair (row rank of 1.0 for chromosomes 3 through 11, 2.0 for chromosome 1, and 3.0 for chromosome 2). Indeed, such association was obvious simply from looking at the cells. I t was expected that the central location of the small metacentric chromosomes, 9-11, would result in the observation of homologous association for them, as defined b y neighbor analysis. While this was true for testicular mitotic cells selected because of their homologous pairing, in corneal cells these six chromosomes were found to associate more closely with other chromosomes t h a n with their respective homolo-
Non-random Metaphase Chromosome Distribution. I
321
gues. I t appears that size of the chromosomes is very important in determining their general location in the dividing cell. The smallest chromosomes are found in the center of chromosome figures in the Chinese hamster whether or not homologous pairing exists in the cell. Acrocentric Association There was good evidence of non-homologous acrocentric association (chromosomes 6, 7 and 8) in all four cell types. Neighbor analysis indicated association (row ranks less than 4) of 6 with 7, 6 with 8, and 7 with 8 in the corneal cells. In the testicular mitotic cells, a close association was observed only between chromosomes 7 and 8. This was also the only association for cells in meiosis I I (Table 3). In diakinesis, acrocentric association was observed between chromosomes 6 and 8. It is commonly thought that the association of acrocentric chromosomes is due to the nucleolar organizing regions located on the small arms of these chromosomes (Cooke, 1972; Ohno et al., 1961). In a number of organisms the region responsible for organizing the nucleolus is, in fact, located near the satellites of acroccntric chromosomes (see Brown, 1972, for review), and Henderson et al. (1973) have recently demonstrated that the aerocentric chromosomes in cultured human lymphocytes are frequently connected by rDNA. However, Phillips and Phillips (1969) have clearly demonstrated that all the chromosomes in the Chinese hamster can, and do, form functional nucleoli. The typical localized nucleolar organizing region does not appear to exist in this organism except, possibly, on the X and u chromosomes (pair 3) (Yerganian et al., 1960; also observed in this study). If acrocentric chromosomes associate in the absence of localized nucleolar organizing regions, then that association must be due to factors other than the presence of nucleo]ar organizing regions. Furthermore, if all the chromosomes contain regions responsible for the organization of nucleoli, then acrocentric association cannot be explained on the basis of nucleolar organizing regions. The obvious similarity in size and shape between the two acrocentrie chromosomes (7 and 8) which were observed to associate closely in three cell types in the Chinese hamster suggests that size and shape of chromosomes may be the most important factors in determining chromosome distribution in the Chinese hamster. The Relative Location o] Chromosomes in the Chinese Hamster Cell The ranking of chromosome associations in neighbor analysis can be used to construct an l 1-point mode] of chromosome relations in the Chinese hamster cell. The model is suggested in part by the statistical 21.*
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Fig. 3. (a) Eleven-point model of chromosome relationships in metaphase II from the Chinese hamster. (b) Eleven-point composite model of chromosome relationships in the Chinese hamster. (a, b) Chromosomes drawn to scale
analysis. I n all cell types, chromosomes 9, 10 and 11 were very close to each other and to the remaining eight chromosomes. This suggests a circle of eight chromosomes with 9, 10 and 11 in the center. I t is also what was observed in each cell. In Table 3, certain strong associations (taken as row ranks of 2.5 or less) can be observed: 7-8, 3-8, 4-6, 5-9, etc. Other weaker but also important associations (row ranks of 4 or less) can also be observed: 5-7, 2-4, 8-9 etc. By considering these associations and the 8 -k 3 model suggested by both statistical analysis and biological observation, a model (Fig. 3 a), which represents the most likely position of the eleven dyads, can be constructed. I n a similar way, models were constructed for the other cell types. I n order to compare these models, the sequence of the peripheral chromosomes was recorded, starting with the 7-8 complex and moving away from the 1-2 complex. The center chromosomes were recorded by listing the one closest to the first member of the peripheral sequence and moving around the models in the same direction followed for the peripheral sequence. These results appear in Table 5. By assuming that the validity of the four models is equal, a composite model (Fig. 3 h) was constructed from Table 5. If a given chromosome appeared in a specific position two or more times in Table 5, it occupied that position in the composite model. Only chromosomes 3 and 4 could not be definitely located by this :_.process. Chromosomes 9-11 were arranged as an inner circle which can revolve with respect to the outer circle. What relationship does the composite model have with reality ? I t was constructed by democratic vote from four models, each of which was
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Table 5. Chromosome sequence lor models of chromosome relationships in the Chinese hamster MeiosisII Cornea Testicular mitotic Diakinesis
8 7 8 8
7 8 7 7
Composite
8 7
5 6 3 4
2 3 4 6
4 5 5 5
6 4 6 3
4 3 5 6
1 2 1 1
3 1 2 2
1 2
10 9 11 11 10 9 11 10 9 10 9 11 (11 10
9) a
a Circularly permutated.
constructed by ignoring the high degree of variability observed and concentrating instead on an underlying pattern to the variability. As such, it represents a picture of the average position of each chromosome, which may have no correspondence with any particular meiotic cell and which definitely cannot correspond to a mitotic cell unless complete homologous pairing is present. Despite this objection to the model, it is worth considering for it highlights certain possibly fundamental relationships in the cell. The chromosomes appear to be grouped by size and shape in the composite model. Only chromosome 6, the largest acrocentrie, violates this correspondence since it is closest to chromosomes 7 and 8 in terms of size and centromere position. Furthermore, there appears to be balancing of chromosomal mass about the periphery. Both a balanced metaphase plate and the size, shape, position correlation have been noted before. The size dependency for regular segregation of the fourth chromosomes in Drosophila melanogaster led the Grells to propose the theory of distributive pairing in meiosis (E. H. Grell, 1963; R. F. Grell, 1964; R. F. Grell and Day, 1970). Also, Heneen and Nichols (1972) observed associations based on proximity in size along with a balancing of the chromosomal mass on the metaphase plate in the muntjac. Lawrence (1931) reported secondary associations in over 100 plant species. I n all cases, the associations were between chromosomes of similar size and shape. The results, then, suggest strongly that chromosome position in the cell is determined by size and configuration.
Maintenance o/Chromosome Patter~ : Interaction o] Genetic In/ormation and Physical Forces In four cell types of the Chinese hamster, a consistent pattern of chromosome associations was observed to underlie a great deal of variability in position. The general chromosome distribution, which was observed repeatedly, was that of a circle of large chromosomes formed about the smaller chromosomes. I t was suggested that the
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association of acrocentric chromosomes in this organism was due to the size and configuration of the chromosomes rather than to homologous function on non-homologous chromosomes. The composite model of chromosome relationships constructed from the four cell types also appeared to reflect a correspondence between position and size and shape. Theoretically, there are two extremes which might exist with regard to chromosome distribution in the cell: (1) the chromosomes are completely autonomous units possessing total freedom of movement in response to physical forces acting upon them; and (2) the chromosomes are completely subject to position specification, the information for which resides in the DNA. Apparently, neither extreme is realized in actuality. If chromosomes are autonomous units, then a cyclic stochastic process, in which all probabilities closely approach unity, would have to be postulated in order to account for chromosome localization in the nucleus, centromere restriction to one portion of the nucleus in sperm (Macgregor and Walker, 1973), the observations in this report, and the m a n y other indications of non-randomness of chromosome position which have been cited. I n addition, this process would have to account for the changes which occur during pre-meiotic pairing, the unique meiotic divisions, and the continuance of the chromosomal distribution pattern between generations. While physical components such as chromosome mass and the "pulling force" generated b y the division apparatus are most likely constant (Mazia, 1961), the other characteristics of the physical environment of chromosomes tend to respond to internal and external changes in a homeostatic manner. Clearly, the tendency of the cell to maintain homeostasis over a short period of time contradicts the possibility of long-term cyclic changes being achieved solely b y interacting components of the physical environment. Thus, it is highly unlikely t h a t chromosome position is maintained b y the response of autonomous chromosomes to a cyclically changing summation of physical forces acting upon them. While chromosomes are not purely autonomous, neither are they totally without autonomy. If chromosomes were distributed in the cell according to some highly structured plan contained in the DNA, then no variation of chromosome associations would be observed. The fact t h a t a good deal of variation was observed in indicative of a lack of total genetic control or, stated in another way, proof of some freedom of movement on the part of the chromosomes in response to varying physical conditions. The observations reported here can only be regarded as a beginning study of chromosome positioning in the cell. With further work, the observed variability m a y come to have deeper significance than was suggested. Individual organisms or cell types m a y have subtle differences in chromosome patterning which were not detected in this study.
Non-random Metaphase Chromosome Distribution. I
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I n four cell types from the Chinese hamster, there was a n o n - r a n d o m d i s t r i b u t i o n of chromosomes which was consistent for the four cells types. This n o n - r a n d o m n e s s was n o t e v i d e n t from casual inspection due to the large a m o u n t of v a r i a b i l i t y associated with it. The n o n - r a n d o m n e s s , the variability, a n d the size-shape-position correlation were also f o u n d b y H e n e e n a n d Nichols (1972) in their s t u d y of m u n t j a c cells. I t is quite likely t h a t a detailed analysis of chromosome position will reveal the same characteristics i n other organisms.
Acknowledgments. This work was supported by the Department of Biology of Emory University and the National Science Foundation Predoctoral Fellowship Program. I thank A. Alan Humphries, Jr., William Murdy, Charles Ray, Jr., Martin Shapiro, all of Emory University, and l~obert Fetner, of the Georgia Institute of Technology, for their guidance. Re~erences Boss, J. M.N.: Mitosis in cultures of newt tissue. II. Chromosome pairing in anaphase. Exp. Cell Res. 7, 225-231 (1954) Brown, W. V. : Textbook of Cytogenetics. Saint Louis: C. V. Mosby Comp. 1972 Cohen, M. M., Enis, •., Pfeifer, C. G. : An investigation of somatic pairing in the muntjac (Muntiacus muntjak). Cytogenetics 11, 145-152 (1972) Comings, D. E. : The rationale for an ordered arrangement of chromatin in the interphase nucleus. Amer. J. hum. Genet. 29, 440-460 (1968) Comings, D. E. : The structure and function of chromatin. Advanc. human Genet. a, 237436 (1972) Cooke, P. : Patterns of secondary association between tile acrocentric autosomes of man. Chromosoma (Berl.) 36, 221-240 (1972) Fredga, K. : A simple technique for demonstration of the chromosomes and mitotic stages in a mammal: chromosomes from cornea. Hereditas (Lund.) 51, 268-273 (1964:) Friedman, M. : The use of ranks to avoid the assumption of normality. J. Amer. Statist. Ass. 32, 675-701 (1937) Grell, E. I-I.: Distributive pairing of compound chromosomes in females of Drosophila melanogaster. Genetics 48, 1217-1229 (1963) Grell, R . F . : Distributive pairing: the size-dependent mechanism for regular segregation of the fourth chromosomes in Drosophila melanogaster. Genetics 52, 226-232 (1964:) Grell, 1%.F., Day, J. W. : Chromosome pairing in the oogonial cells of Drosophila melanogaster. Chromosoma (Berl.) 31, 434:44:5 (1970) Henderson, A. S., Warburton, D., Atwood, K. C.: Ribosomal DNA connectives between human aerocentrie chromosomes. Nature (Lond.) 245, 95-97 (1973) Heneen, W. K., Nichols, W. W.: Nonrandom arrangement of metaphase chromosomes in cultured cells of the Indian deer, Muntiacus muntjak. Cytogenetics l l , 153-164: (1972) Juricek, D. K. : Non-random chromosome distribution in selected cell types from the Chinese hamster. UnpublishedPh.D. dissertation, Emory University, Atlanta, Georgia, 1973 Kato, H., u T . H . : Banding patterns of Chinese hamster chromosomes revealed by new techniques. Chromosoma (Berl.) 36, 272-280 (1972)
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Lawrence, W. J. C. : The secondary association of chromosomes. Cytologia (Tokyo) 2, 352-384 (1931) Macgregor, H. C., Walker, M. H. : The arrangement of chromosomes in nuclei of sperm from the plethodontid salamanders. Chromosoma (Berl.) 40, 243-262 (1973) Mazia, D.: Mitosis and the physiology of cell division. In: The Cell (J. Brachet and A. E. Mirsky, eds.), vol. 3, p. 77412. New York-London: Academic Press 1961 Metz, C. W.: Chromosome studies on the Diptera. II. The paired association of chromosomes in the Diptera, and its significance. J. exp. Zool. 21, 213-279 (1916) Miller, O. J., Breg, W. R., Mukherjee, B. B., Gamble, A. V. N., Christakos, A. C. : Non-random distribution of chromosomes in metaphase figures from cultured human leucocytes. II. The peripheral location of chromosomes 13, 17-18 and 21. Cytogenetics 2, 152-167 (1963b) Miller, O. J., Mukherjee, B. B., Breg, W. R., Gamble, A. V. N. : Non-random distribution of chromosomes in metaphase figures from cultured human leucocytes. I. The peripheral location of the Y chromosome. Cytogeneties 2, 1-14 (1963a) Nur, U. : Random arrangement of chromosomes in a radial metaphase configuration. Chromosoma (Berl.) 40, 263-267 (1973) Ohno, S., Trujilla, J. M. : Kaplan, W. D., Kinosita, P. : Nucleolus-organisers in the causation of chromosomal anomalies in man. Lancet 1961 II, 123-125 Phillips, S. G., Phillips, D. M.: Sites of nucleolus production in cultured Chinese hamster cells. J. Cell Biol. 40, 248-268 (1969) Rodman, T. C., Biedler, J. L. : Specificity of compaction in meiotic chromosomes of the female Chinese hamster. Chromosoma (Berl.) 42, 229-246 (1973) Stevens, N. M. : The chromosomes of Drosophila ampelophila. Proc. VIIth Int. Congr. Zool. 380-381 (1907) Stevens, N. M.: A study of the germ cells of certain Diptera, with reference to the heterochromosomes and the phenomenon of synapsis. J. exp. Zool. 5, 359-374 (1908) Stock, A. D., Burnham, D. B., Hsu, T. C.: Giemsa banding of meiotic chromosomes with description of a procedure for cytological preparations from solid tissues. Cytogeneties l l , 534-539 (1972) Yerganian, G., Kato, R., Leonard, M.L., Gagnon, H . J . , Grodzins, M.S.: Sex chromosomes in malignancy, transplantability of growths, and aberrant sex determination. In: Cell physiology of neoplasia, p. 49-93. Austin: University of Texas Press 1960 Received May 29, 1974 - - January 18, 1975 / Accepted by J. H. Taylor Ready for press January 20, 1975 Diane K. Juricek Department of Biology Emory University Atlanta, Georgia 30322 U.S.A.
