Copyright 0 1982 by the Genetics Society of America SEQUENCE OF CENTROMERE SEPARATION: ROLE OF CENTROMERIC HETEROCHROMATIN 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 metaphase-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 centromeres that showed separation into two daughter centromeres and those that did not show such separation. In all strains and species of mouse the Y chromosome is the first one to separate, as is the X or Y in the cattle. These sex chromosomes are devoid of constitutive heterochromatin, whereas all autosomes in these species carry detectable quantities. In cattle, the late repli- cating X chromosome 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 genome 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 mitosis, the I chromosomes in a given genome separate at their centromeres in a nonran- dom, genetically determined sequence to release the two daughter chromatids. 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- chromatin 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 chromatid 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 Y chromosome 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 0Y # f$ B 5: &5 * B sa 2% 2 g 34 ~ ; ;g Q, ---_. --_- ~ - _-__.__- g 9 ;em2 35 2 ..L? 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K. VIG tr h I h xv ass m m m m (Ir h h m 1 2 3 FIGURE 1.-A C-banded karyotype 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 karyotypes from six different cells.