Copyright 0 1982 by the Society of America

SEQUENCE OF SEPARATION: ROLE OF CENTROMERIC

BALDEV K. VIG

Department of Biology, University of Nevada at Reno, Reno, Nevada 89557

Manuscript received April 7, 1982 Revised copy accepted August 20,1982

ABSTRACT The late -early anaphase cells from various tissues of male Mus musculus, M. poschiavinus, M. spretus, M. castaneus, female and male Bos taurus (cattle) and female Myopus schisticolor (wood lemming) were analyzed for that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y is the first one to separate, as is the X or Y in the cattle. These are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late repli- cating appears to separate later than the active X. In the wood lemming the three pairs of autosomes with the least amount of centromeric constitutive heterochromatin separate first. These are followed by the separa- tion of seven pairs of autosomes carrying medium amounts of constitutive heterochromatin. Five pairs of autosomes with the largest amounts of consti- tutive heterochromatin are the last in the sequence of separation. The sex chromosomes with medium amounts of constitutive heterochromatin around the centromere, and a very large amount of distal heterochromatin, separate among the very late ones but are not the last. These observations assign a specific role to centromeric constitutive heterochromatin and also indicate that nonproximal heterochromatin does not exert control over the sequence in which the centromeres in the separate. It appears that qualitative differences among various types of constitutive heterochromatin are as important as quan- titative differences in controlling the separation of centromeres.

T is now known that at the junction of meta-anaphase of , the I chromosomes in a given genome separate at their centromeres in a nonran- dom, genetically determined sequence to release the two daughter . Following earlier studies by VIG and WODNICKI(1974), such sequences have been established for man (VIG1981a; MEHES1978) besides for Chinese hamster (VIC and MILTENBURGER1976; SINCHand MILTENBURGER1977), Vicia faba (MURATAand VIG 1980), Haplopappus gracilis and Crepis capillaris (FAROOK and VIG1980), and Potorous tridactylus (VIG1981b). In man, chromosomes 28, 2 and some others separate earliest, whereas D group chromosomes are the last, and G group is usually next to last to separate. In P. tridactylus, the smallest, acrocentric Y2 is the last, whereas Y1 and X are among the last few. There appears to exist a straight forward relationship between the position of a centromere in sequence of separation and the amount of centromeric hetero- in the Potorous genome. The last separating Y2 has the largest

Genetics 102: 795-806 December, 1982 796 B. K. VIG quantity of C chromatin, whereas the X and Y1 are next in order. In this organism chromosomes 4 and 5, which are the earliest to separate at their centromeres, have the least amount of C chromatin. The situation with man, however, is not so simple. The last separating chromosomes have rDNA close to the centromere but not the largest amount of C chromatin. Nonetheless chromosomes 2 and Y, which also have large quantities of C chromatin, are among the last few to separate. Even though only casual, this correlation suggests a possible role of centro- meric heterochromatin in controlling the separation of daughter centromeres. We have tried to answer this question by studying such correlations using chromosomes with no, or a little, C chromatin and those with large quantities of C chromatin in the same genome. The data show that at least one function of the so-called junk DNA (genetically inactive C chromatin) is the control of centromere separation.

MATERIALS AND METHODS Three genera, including four species of Mus (mouse), Bos taurus (cattle) and Myopus schisticolor (wood lemming) were used for this study (Table 1).Cells from various tissues of male Mus and bone marrow cells from female wood lemming were prepared by routine methodology following 1- or 2-hr Colcemid injection before sacrifice. In the case of the mouse, preparations were also made without pretreatment with colcemid. Cattle lymphocytes from both male and female animals were grown in chromosome medium 1A for 72 hr, and preparations with or without Colcemid were made. The slides were stained with Giemsa. C banding was achieved by using BaOH, following the method of FREDGAet al. (1976). For comparison of separation of the active vs. inactive X chromosomes in Bos, the lymphocyte cultures were treated with 1 X M BrdUrd during 70-76 hr postculture and fixed soon after. These cells were processed as if for sister exchange analysis and stained with Giemsa. The inactive, late replicating X showed large, lightly stained segments along its length, as expected. It could be easily distinguished from the active X which showed only small light-stained, late replicating regions. Analysis of centromere separation was carried out as described earlier (VIG 1981a). In Mus and Myopus the chromosomes were classified as showing separation or not showing separation. In Bos, however, the degree of separation was further categorized as 0 (centromere appeared as single unit, no evidence of separation), 1 (centromere initiated separation but not completely separated into two identifiable units) and 2 (centromere completely separated into two daughter units). The purpose of this study was to compare the relative position of separation of centromeres with different amounts of C chromatin within a given genome and correlate the amount of C chromatin with early vs. late separation of the centromere. The of all mouse species studied and the sex chromosomes of cattle have no detectable C chromatin, whereas all of the autosomes in both of these species have easily detectable C chromatin. Therefore, studies in mouse and cattle were limited to analysis of the relative positions of chromosomes showing no C chromatin with those showing C chromatin, i.e., to find out the number of C chromatin-carrying chromosomes that separate before the Y chromosome in any laboratory mouse or before the sex chromosomes in cattle. Because of difficulty of quantitative differentiation between chromosomes with C chromatin, comparison between various chromosomes with C bands was not made. The wood lemming genome (Figure 1) was arbitrarily classified into four categories: (1)three pairs of chromosomes (nos. 2, 4 and 6) with very small amounts of centromeric C chromatin, (2) seven pairs with medium quantity of C chromatin, (3) five pairs with rather large quantities, and (4) the X with medium quantity of centromeric heterochromatin as well as a very large amount of distal constitutive heterochromatin displayed as uniformly dark-staining region (equivalent to homogeneously staining region). Studies with C-banded bone marrow preparations were carried out to record the number of chromosomes separating in various categories. No attempt was made (or thought necessary) to identify individual chromosomes in a given group. CENTROMERIC HETEROCHROMATIN 797

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2 3 FIGURE 1.-A C-banded of a female wood lemming (2n = 32) in which one of the X chromosomes has lost about ?+ distal constitutive heterochromatic segment in the long arm. The chromosomes are organized according to FREDCAet al. (1976) and marked for the quantity of constitutive centromeric heterochromatin as indicated by comparison of from six different cells. I = light band, m = medium band, and h = heavy band. FIGURE2.-A C-banded cell from bone marrow of M. musculus domesticus showing only Y chromosome (arrow) having separated at its centromere. This cell was treated with Colcemid. It should. however, be noted that Colcemid does not effect the sequence of centromere separation (M. F. FICUEROA and B. K. Vrc, unpublished reusults). FIGURE3.-A C-banded cell from bone marrow of M. musculus domesticus showing that Y chromosome (arrow) and an autosome (arrowhead) separated at their centromeres. Cells in which an autosome showed separation simultaneously with, or before, the Y separated were rare. This cell was prepared from bone marrow not treated with Colcemid.

RESULTS The studies carried out with mouse centered on the position of Y chromosome in the sequence. As Table 1shows the cells from a variety of strains were used. CENTROMERIC HETEROCHROMATIN 799

TABLE 2

Separation of Y chromosome vs. autosomes in Mus cells showing only one chro- mosome separating at the centromere

No. and type of earliest separating chromosomes

Auto- Species Strain Cells Y somes + X

M. musculus domesticus CBA 200 198 2 BALB 120 118 2 NZB 72 72 Rb (5.14) 26 26 Rb (14.14) 42 39 3 Rb (9.14) 32 30 2 Rb (6.17) 34 32 2 M.poschiavinus 250 245 4 M.castaneus 52 51 1 82 80 2 M. spretus 30 30

The data (Table 2) show that the Y chromosome almost always separated before any other chromosome initiated separation at the centromere (Figure 2). In a few cells some chromosome other than Y showed earliest separation, or sepa- rated apparently simultaneously with Y. It must also be added that in one mouse analyzed in our laboratory at the University of Nevada at Reno about 15% of the cells showed a chromosome other than Y to separate first (Figure 3). However, this incidence was not observed in any other laboratory where analyses were carried out (Table 1). Besides, one specimen from M. castaneus and two from M. spretus also showed Y chromosome separating first. M. poschiavinus differs from the parental species, M. musculus, in having accumulated seven pairs of Robertsonian translocations so that 2n = 26. The genetic material, including those of the sex chromosomes, however, appears to have been preserved in toto. It was, therefore, an expected finding when, of 200 cells analyzed from bone marrow, all but one showed the Y to separate earliest (Figure 4). In cattle, a similar situation prevailed (Table 3). In females generally one or both X chromosomes showed initiation of separation or complete separation before any autosomes separated (Figure 5). Where autosomal separation is shown for comparison (last row in case of every animal), both X chromosomes showed complete separation. There were a few isolated instances in which an occasional autosome showed separation before both Xs had completely sepa- rated. In all animals studied, the total number of cells showing such deviation was small (six of 260 cells). However, no cell showed the separation of an autosome without at least one X showing separation. A comparison between the sex chromosomes and autosomes in male cattle also showed the sex chromosomes to separate earlier than the autosomes. Table 4 shows that almost no separation of autosomal chromosomes takes place unless sex chromosomes have separated (Figure 6). As in female cattle, the 800 B. K. VIG

FIGURE4.-A cell from bone marrow of Colcemid-treated M. poschiavinus showing the Y chromosome (arrow) separated at its centromere. FIGURE5.-A cell from lymphocytic culture from female B. taurus. One of the two X chromo- somes (arrow) shows initiation of separation, whereas the other X (arrowhead) and all autosomes are still held at the centromere. FIGURE6.-X chromosome (arrow) in male B. taurus showing separation in a cell from lympho- cytes in which neither Y (arrowhead) nor any autosome has separated. CENTROMERIC HETEROCHROMATIN 801

TABLE 3 B. taurus: separation of sex chromosomes in relation to autosomes in females

No. of chromosomes showing none (O), partial (1) or complete (2) separation No. of X/X homologs" Autosomesh cells ana- Animal lyzed 0/1 1/1 0/2 1/2 2/2 x 1 2 z 1 55 18* 13 38798 6 12 27 11 38 H-661 50 23 9 46288 2 4 2 3 5 4 8 6 20 26 H-3014 50 21 6 10 6 1 88 1 2 1 1 1 2 1 1 2 4 8 2 6 8 H-3020 50 12 2 13 4 8 78 1 2 1 1 1 2 1 1 9 18 4 32 36 X = Total number of chromosomes, not pairs. Number refers to a total of 18 pairs of Xchromosomes, with one homolog showing no separation (0) and the other showing partial separation (I),13 pairs with each of the two homologs showing partial separation, etc. *Total number of autosomes (not pairs) showing separation in cells analyzed to obtain data in the corresponding row for the X/X homologs. In animal 1, for example, no autosome showed separation in the cell preparation from which the corresponding 49 X/X pairs were studied, but 38 autosomes showed separation (27 partial and 11 complete) in cells in which six pairs of X/X chromosomes showed complete separation for each of the two homologs. males also show the separation of autosomes in cells that have both sex chromosomes completely separated. Exceptions, how-so-ever rare, were found (in seven of 416 cells analyzed). Even though neither of the sex chromosomes has detectable C chromatin, or has an extremely small quantity (see EVANS, BUCKLANDand SUMNER1973), the X generally initiated separation before did Y. Thus, in animal NB-79, 25 cells showed initial or complete separation of X chromosome without Y showing any separation, in contrast to only four Y chromosomes showing separation when X had not separated at all. The respec- tive figures for other animals in the sequence given in Table 3 are 17 vs. 4

FIGURE7.-A BrdUrd-labeled cell from lymphocytes of female B. tourus distinguishing the late replicating X (arrow) from the active X (arrowhead). The latter showed earlier separation in about 75% of the cells. Note an early replicating part in the long arm of the so-called inactive X. This was evident in almost all of the cells in which BrdUrd was incorporated for up to the last 8 hr of culturing. FIGURE8.-A cell from Colcemid-treated bone marrow of M. schisticolor showing centromere separation in six chromosomes (arrows). All of these were identified as having very light or light C bands and, hence, least amount of constitutive centromeric heterochromatin in the genome. FIGURE9.-A cell similar to the one shown in Figure 8 except that most of the chromosomes show clear centromere separation or initiation of separation. All six light C-banded chromosomes have separated, as have the medium C-banded ones 802 B. K. VIG

TABLE 4

B. taurus: separation of sex chromosomes in relation to autosomes in males"

No. of chromosomes showing none (O), partial (I), or complete (Z), separation No. of X/Y heterologs Autosomes cells an- Animal alyzed 0/0 1/0 1/1 2/0 2/1 2/2 0/1 0/2 1/2 2 122

NB-79 50 1867264 1 88 1 2 11 5 10 4 13 17 2 70 14 22 3 14 3 4 4 128 6 12 4 13 17 HB-90 64 15 14 10 5 5 2 1 2 108 2 4 44 1 2 22 1 2 11 6 12 4 24 28 AB-5 50 10569441 78 1 2 11 2 4 224 1 2 22 7 14 5 23 28 AB-4 50 1548562 80 1 2 11 1 2 11 8 16 26 26 6 32 4 118 25 60 2 4 44

a For explanation see footnotes in Table 3.

(animal Z), 25 vs. 3 (animal HB-90), 16 vs. 5 (animal AB-5) and 23 vs. 2 (animal AB-4). Since one of the X chromosomes in eutherian females is heterochromatized and is thought to be generally genetically inactive, it was thought worthwhile to study the relationship between the two X chromosomes. In the lymphocyte cultures treated with BrdUrd during late S, the differentiation between active and late replicating, inactive X was easily made (Figure 7). In one animal out of 50 cells in which at least one X had separated at the centromere, 41 showed the active X as the only one separating or separating earlier than the heterochro- matic X. The remaining nine cells showed earlier separation for the heterochro- matic X or equivalent separation for both. The lymphocytes from a second female were studied for the cells showing only one X separated. In a sample of 48 cells 34 had the active X separating first, whereas the remaining (14 of 44) cells showed late replicating X separating earlier than its homologue. In the case of Myopus, as already mentioned, the genome was analyzed for separation of chromosomes showing light or no C bands, those showing mod- erate C bands and those showing heavy C bands. The division, admittedly, is arbitrary. The data were organized to permit comparison of chromosomes showing separation in a given group with any other group. Table 5 reflects the difficulty we had in finding cells in late stages of separation. Cells from animal A showed separation in only up to 14 chromo- CENTROMERIC HETEROCHROMATIN 803

TABLE 5 Centromere separation in M. schistocolor as function of centromeric constitutive heterochromatin (CH)

Separated Type and no. of separated chromosomes No. of separated cen- chromo- No. of tromeres not repre- Animal somes cells Light CH Medium CH Heavy CH XX sented"

A 1to6 30 1-6 (3.8)b 7 to 16 14 all 6 1-10 (3.0) 11, 12.15 to 32 B 1 to 6 103 1-6 (3.12) 7 to 10 10 All 6 1-4 (1.7) 11-16 17 1 All 6 8 (8.0) 3 (3.0) 18-29 30 1 All 6 All 14 (14.0) 10 (10.0) 31, 32 C 1 to 6 60 1-6 (3.4) 1 to 6 1 3 l(l.0) l(l.0) 7 to20 16 all 6 1-14 (4.6) 2-4 (0.4) l(0.06) 9, 11, 13, 15 >20 7 all 6 6-14 (12.9) 1-8 (5.4) 0-2 (1.0) 17, 20-20, 26, 27, 31 and 32 "No cell in the respective population was found with these many chromosomes showing separation. * Values in parentheses represent average number of chromosomes showing separation. somes. In animal B separation of only up to ten chromosomes was found except in one cell showing 17 and another showing 30 chromosomes separated. A more representative population was found in animal C. Since no specific technique is available to arrest cells at the junction of meta-anaphase, the availability of suitable cells depends upon chance. However, since the study aims at compar- ison between chromosomes carrying none or very little C chromatin and those carrying relatively large amounts, the data do permit some generalizations. In every animal studied, the six chromosomes with little or no C chromatin are the ones that show separation before other chromosomes do so. In the category of only one to six chromosomes having had separated, a total population of 193 cells (from all three animals) showed centromere separation of only light C- banded ones (Figure 8). It is only after these chromosomes have separated that those with medium C bands show separation (Figure 9). Generally, the ones with the largest amount of C chromatin do not show any separation until all the light and medium chromosomes have separated. The X chromosome appears to be one of the late separating chromosomes. In a sample of six cells in which at least one X had separated, most of the medium C-banded but only a few heavy C-banded chromosomes had also separated. Thus, the relative position of this chromosome in the sequence of separation may be close to the time of separation of the last few medium C-banded chromosomes or among the early heavy C-banded ones. In animal C, the reduction of the amount of terminally located heterochromatin in one X did not result in early separation in any of the 60 cells with up to six chromosomes showing separation.

DISCUSSION The foregoing data support the concept that the so-called "junk" or genetically inactive DNA centered around the centromeric region has a function in con- 804 B. K. VIG trolling the separation of centromere (or its replication into two daughter centromeres) at the junction of metaphase-anaphase in mitosis. A simple cor- relation appears to emerge between the quantity of centromeric constitutive heterochromatin and delay in separation of that centromere. A clear correlation can be established between a lack of detectable quantities of C chromatin and the earliest separation of that chromosome in the genome in several strains and species of mice, as well as both male and female cattle. Unfortunately, the lack of easy identification of individual autosomes and large number of chromosomes in these species precludes analysis of the whole genome. However, it is of interest that facultative heterochromatic X in cattle showed separation later than the active X. One wonders whether it has anything to do with the replication of the centromere itself, the replication of facultative heterochromatin around the centromere or activity responsible for sepa- rating the two centromeric units. The reason for a few cells observed in a mouse showing separation of some autosomes earlier than Y chromosome is not clear. However, in view of the findings that, bleomycin can disturb the sequence of centromere separation in certain Chinese hamster chromosomes (B. K. VIG, unpublished results), one may entertain the idea of some mutagens being present in the environment in which this mouse was raised. The data from wood lemming also permit a straight forward interpretation. The three pairs of autosomes with light C band or undetectable C bands are unequivocally earlier separating than those with medium quantity of C chro- matin. The later, in turn, show separation earlier than those with heavy C bands. The Xs fall in between the last two categories. This suggests that the presence (or ) of distal constitutive heterochromatin may have no bearing on the sequence of separation. This is not to say that in all species it is only the total quantity of constitutive centromeric heterochromatin that controls the separation of centromere. It is logical, considering the various types of constitutive heterochromatin available among eukaryotic , to expect qualitative differences with regard to such control. The larger the intragenomic differences, the greater the disparity expected. genome is a case in point. When we compared the two 1 chromosomes with varying amounts of C chromatin, the one with the larger amount showed late separation. However, when 1,9or 16 were compared with D group chromosomes, it is the D group that showed late separation in spite of quantitatively smaller C heterochromatin (VIG 1981a). In my opinion, it is a reflection of qualitative, rather than quantitative, aspects of constitutive heter- ochromatin. Thus, a straight forward correlation as observed in Myopus, for example, may reflect uniformity of the structure of constitutive heterochromatin in this organism. A similar situation appears to exist in P. tridactylus (VIG 1981b). One may extend the argument to chromosomes 13, 14, 15, 22, 22 and Y in man which are among the last separating chromosomes and have SAT IV as a common denominator (MIKLOSand JOHN 1979). Preliminary data with female cattle show late separation of the inactive X chromosome. One wonders whether the inactivation process also involves the centromeric region. If so, then further studies using organisms like mealy bugs, CENTROMERIC HETEROCHROMATIN 805 in which in males paternally transmitted genome is expressed as facultatively heterochromatized, could shed light on the mechanism of centromere separa- tion. It is not clear as to how centromeric constitutive heterochromatin exerts control over separation of centromeres. The delay of separation of a centromere with a large amount of paracentromeric heterochromatin is not related to late replication of heterochromatin per se. First, replication takes place in the S phase, whereas the centromeres do not separate till quite late in metaphase. Second, studies with tetraphocomelia patients (Robert’s syndrome) show a disturbance in the sequence of centromere separation (TOMKINS,HUNTER and ROBERTS1979; GERMAN1979) in that normally late-separating chromosomes, e.g., D group, show early separation. It appears unlikely that this correlation in the sequence of separation of centromeres is a reflection of change in the timings of replication of heterochromatin. Nonetheless one may also consider some alternative hypothesis. It is possible that ordered sequence of centromere separation reflects their attachment posi- tion at the nuclear envelope in interphase. One must also distinguish between a passive control vs. an active control of centromere separation. The former would be a consequence of repeated DNA with high affinity between chroma- tids, and the latter would suggest some sort of genetic control. Also, several of the photographs (e.g., Figures 1and 9) show differential separation of chromo- some arms; the C-band-positive terminal regions are generally not as well separated as the chromatids on the opposite arm. This observation involves the possibility of “differential maturation” of the paracentromeric regions of sister chromatid arms so that the maturation process parallels the replication within the chromosome. Different regions of chromosomes undergo replication at different defined times during S phase. On completion of replication different regions may need to reach maturation prior to chromatid separation, thus resulting in certain regions separating later than others. Consequently, the regions close to and involving heterochromatin would separate last. However, observations with Robert’s syndrome cells do not fit in well with such an idea, besides the fact that maturation of centromeric regions must continue up until late metaphase. Presence of Colcemid does not seem to alter the sequence of centromere separation. The data from experiments with Chinese hamster bone marrow show similar sequences in Colcemid-treated or nontreated cells (M. F. FIGUEROA and B. K. VIG, unpublished results; also see VIG 1981b). A similar conclusion has been arrived at for Rana ridibunda (BELCHEVA,KONSTANTINOV and ILIEVA 1980) and prematurely separating X in (FITZGERALDet al. 1975).

I am thankful to DRS.A. T. NATARAIAN(University of Lciden, the Netherlands), THOMASRODERICK and MURIELDAVISSON (Jackson Laboratories, Bar Harbor, MA), ALFREDGROW and HEINZWINKING (Medizinishcher Hochschule, Lubeck, West Germany) and D. R. HANKS(University of Nevada, Reno) for providing various types of material used in this study. All of these colleagues generously supplied live specimens and/or prepared slides when I visited their laboratories. I am also thankful to DR. F. FAROOK,Ms. FAROOKand my graduate students for their help. This study was supported by a grant from National Institutes of Health (GM 24591). 806 B. K. VIG LITERATURE CITED BELCHEVA,R. G., G. H. KONSTANTINOVand H. L. ILIEVA,1980 Sequence of centromere separation in the mitotic chromosomes in Rana Ridibunda Pall. (Amphibia, Anura). C. R. Acad. Bulg. Sci. 33: 1689-1692. EVANS,H. J.. R. A. BUCKLANDand A. T. SUMNER,1973 Chromosome homology and heterochro- matin in goat, sheep and ox studied by banding techniques. Chromosoma (Berl.) 42 382-402. FAROOK,S. A. F and B. K. VIG, 1980 Sequence of centromerr: separation: analysis of mitotic chromosomes of Crepis capillaris and Haplopappus gracilis. Bid. Zentralbl. 99: 675-682. FITZGERALD,P. H., A. F. PICKERING,J. M. MERCERand P. M. MIETHKE,1975 Premature centromere division: a mechanism of non-disjunction causing X chromosome in somatic cells of man. Ann. Hum. Genet. 38 417-428. FREDGA,K., A. GROPP,H. WINKINGand F. FRANK,1976 Fertile XX- and XY-type females in the wood lemming Myopus schisticolor. Nature 261: 225-227. GERMAN,J., 1979 Roberts' syndrome. I. Cytological evidence for a disturbance in chromatid pairing. Clin. Genet. 16 441-447. MEHES, K., 1978 Non-random centromere division: a mechanism of non-disjunction causing aneuploidy. Hum. Hered. 28 255-260. MIKLOS,G. L. G. and B. JOHN,1979 Heterochromatin and satcllitc DNA in man: properties and prospects. Am. J. Hum. Genet. 31: 264-280. MURATA,M. and B. K. VIG, 1980 Sequence of centromere separation: analysis of mitotic chrnmo- somes in a reconstructed karyotype of Vicia faba L. Biol. Zentralbl. 99 683-693. SINGH,J. R. and H. G. MILTENBURGER,1977 The effect of cyclophosphamide on the centromere separation sequence in Chinese hamster spermatogonia. Hum. Genet. 39: 359-362. TOMKINS,D., A. HUNTERand M. ROBERTS,1979 Cytogenetic findings in Roberts-SC phocomelia syndrome(s). Am. J. Med. Genet. 4: 17-26. VLG,B. K., 1981a Sequence of centromere separation: analysis of mitotic chromosomes in man. Hum. Genet. 57: 247-252. VIG,B. K., 1981b Sequence of centromere separation: an analysis of mitotic chromosomes from long-term culture of Potorus. Cytogenet. Cell Genet. 31: 129-136. VIG,B. K. and H. G. MILTENBURGER,1976 Sequence of centromere separation of mitotic chromo- somes in Chinese hamster. Chromosoma (Berl.) 5: 75-80. VIG, B. K. and J. WODNICKI,1974 Separation of sister centromeres in some chromosomes from cultured human leukocytes: a preliminary survey. J. Hered. 65: 149-152. Corresponding editor: S. WOLFF