- Email: [email protected]
THE STRUCTURE OF THE CENTROMERIC REGION OF CHO CHROMOSOMES
Cell Biology International, 1998, Vol. 22, No. 2, 127–130 Article No. cb980233
THE STRUCTURE OF THE CENTROMERIC REGION OF CHO CHROMOSOMES A. T. SUMNER* MRC Human Genetics Unit, Edinburgh, U.K. Received 17 September 1997; accepted 9 February 1998
CHO chromosomes, prepared for fluorescence microscopy, or for scanning electron microscopy, sometimes show a splitting of the centromere proper into two sister centromeres, with a space between them, while the sister chromatids are joined in the most proximal regions of the chromosome arms. It is suggested that this might represent the final stage of chromatid splitting 1998 Academic Press before the anaphase separation of chromatids. K: anaphase; centromeres; chromosomes; satellite DNA; scanning electron microscopy; uninemy of chromosomes
INTRODUCTION
MATERIALS AND METHODS
The centromere is where the sister chromatids are joined to each other until the onset of anaphase, and where the chromosomes are attached, through the kinetochore, to the spindle. The centromere normally appears as a simple primary constriction of the chromosome. However, in the large chromosomes of certain plants and insects, the structure of the centromeric region appears more complicated, the sister chromatids being joined on either side of the centromere proper, while the sister centromeres themselves are separated by a gap (Lima-de-Faria, 1955, 1956, 1958; Bajer, 1958, 1968), and may contain two or more chromomeres (Lima-de-Faria, 1958). Such a structural differentiation does not appear to have been reported from mammalian chromosomes, which are generally much smaller than those studied by the authors referred to above. In this short note, evidence from fluorescence microscopy and from scanning electron microscopy is presented, to show that the centromeric region of CHO chromosomes is sometimes differentiated into a region where the sister chromatids are separated, and regions on either side where they remain joined throughout metaphase.
CHO cells were grown in RPMI 1640 medium, supplemented with fetal calf serum. Colcemid was added 3 h before harvesting, to produce a final concentration of 0.25 ng/ml, to accumulate the cells in metaphase. For fluorescence microscopy, cells were resuspended in Stenman’s hypotonic (Stenman et al., 1975) at 4C for 10 min, and then centrifuged on to slides using a Shandon Cytospin (1500 r.p.m. for 15 min). Chromosomes were prepared for scanning electron microscopy by a modification of the method of Martin et al. (1994). The cells were treated with 0.075 (hypotonic) potassium chloride, and fixed in methanol–acetic acid (3:1). The fixed cells were spread on 22-mm square glass coverslips, but immediately before they dried, they were flooded with 45% aqueous acetic acid, and after a few seconds were plunged into glutaraldehyde solution (2.5% glutaraldehyde in cacodylate buffer, pH 7.4, containing 1% sucrose). The coverslips were left in glutaraldehyde overnight (approximately 18 h) before dehydration through graded alcohols and critical point drying. The chromosome preparations were given a thin coating of platinum and examined in a Hitachi S800 field emission SEM at 5 kV. RESULTS
*Present address: 7 Smileyknowes Court, North Berwick, East Lothian EH39 4RG, U.K. (e-mail: [email protected]). 1065–6995/98/020127+04 $30.00/0
A proportion of CHO chromosomes stained with ethidium bromide, about 10–20%, show a darker 1998 Academic Press
128
Cell Biology International, Vol. 22, No. 2, 1998
Fig. 1. Cytospin preparation of metaphase CHO chromosomes, stained with ethidium bromide, showing holes at the centromeres (arrows).
spot in the middle of the centromere, as if there were a hole there (Fig. 1). The rest of the chromosomes show a much narrower, undivided constriction. This is confirmed by scanning electron microscopy, where a similar proportion of chromosomes show a hole at the centromere: the two arms are connected by a pair of fibres (Fig. 2), and the sister chromatids are joined on either side of the constriction, not within the constriction itself. Measurements indicate that the fibres connecting the two arms range in thickness from about 10 nm up to about 70 nm. DISCUSSION The observations described in this paper show that in CHO chromosomes, as in certain large plant and insect chromosomes (Lima-de-Faria, 1955, 1956, 1958; Bajer, 1958, 1968), the centromere can be differentiated into three regions: a central constriction, where the sister chromatids are already separated at metaphase; and flanking regions, consisting of the most proximal parts of the chromosome arms, where the sister chromatids are still joined together. Only a proportion of CHO chromosomes show these ‘split’ centromeres, and it might be wondered if they are some sort of artefact. Although the high concentration of acetic acid used to prepare chromosomes for scanning electron microscope might cause more extensive extraction of proteins than the standard methods of preparing chromosomes, there is no evidence in the scanning electron micrographs for an artificial mechanical split, while for the observations by fluorescence microscopy, no acetic acid was used. In fact, previous studies by electron microscopy appear
to show centromeric structure similar to that described here, for example, Figures 9.12 and 9.14 in DuPraw (1970), and Figure 4 in Stubblefield and Wray (1971). Wanner and Formanek (1995) have also illustrated a chromosome prepared for scanning electron microscopy and stained with platinum blue, which binds to DNA, and this chromosome has a hole in the DNA at the centromere (their Fig. 4). The fact that a variety of different methods of preparing chromosomes produce similar results gives one confidence that the observations described in this paper of holes at the centromeres of CHO chromosomes are not an artefact. The centromeres of large plant chromosomes often appear to contain two or more chromomeres in each of the separated sister centromeres (Limade-Faria, 1958). No such subdivision of the centromeres was detected in the CHO chromosomes, however. The split centromeres probably represent the penultimate stage of the splitting of chromosomes into daughter chromosomes. The centromeres may perhaps only be split briefly before the whole chromosome divides at anaphase, and if the separation of different chromosomes is asynchronous, as occurs in other mammals (Me´hes and Bajno´czky, 1981; Vig, 1983), it would be expected that only a proportion of the chromosomes would show split centromeres. It has been shown previously (Sumner, 1991) that there is a sequence of splitting of chromosomes during mitosis, starting with the euchromatic arms, followed by the paracentromeric heterochromatin, and finally the centromeric region itself. The observations presented here suggest that the centromeres themselves split in two stages, firstly the centromeres proper, and finally,
Cell Biology International, Vol. 22, No. 2, 1998
129
Fig. 2. Scanning electron micrographs of CHO chromosomes, showing centromeric regions divided into sister centromeres. (A) and (B) show the same chromosome at different magnifications, as do (D) and (E). Scale bars represent the following lengths: (A) and (D): 2 ìm; (B), (C), (E) and (F): 1 ìm.
at the metaphase/anaphase transition, the juxtacentromeric heterochromatin that forms the proximal parts of the chromosome arms. If this interpretation is correct, then the situation in CHO cells differs from that described by Lima-de-Faria (1958) for plant chromosomes, in which the centromere is already split by mid-prophase, and from that described by Bajer (1958), in which a hole between the sister centromeres appeared to result from the initial interaction of the kinetochores with the spindle in early prometaphase. It is not possible at this stage to determine whether this represents a real difference between the behaviour of plant and animal chromosomes, or is a result of the greater difficulties of observing details in the much smaller CHO chromosomes, or is a consequence of the different preparative techniques used. The structural division of the centromeric region could parallel functional and compositional differences. It was proposed some years ago that the
function of paracentromeric heterochromatin could be to hold sister chromatids together (Lica et al., 1986; Sumner, 1991), and to provide a substrate for the action of topoisomerase II at the beginning of anaphase, to separate the chromatids (Sumner, 1991). Recent work suggests that topoisomerase II is present in the paracentromeric regions, but may be absent from a small region at the centromere itself (Sumner, 1996a). Particular satellite DNAs—alphoid satellite in man, and minor satellite in mouse—appear to be associated with the centromere proper, whereas the human ‘classical’ satellites and the mouse major satellite are located paracentromerically and form blocks of heterochromatin (Joseph et al., 1989; Mitchell et al., 1992). Unfortunately, little is known about the composition of centromeric DNA in Chinese hamster chromosomes, but if an organism could be found in which the centromeric DNAs were well characterized, and the splitting of centromeres
130
could be demonstrated, it would be predicted that the centromere proper and the proximal parts of the chromosome arms might contain different DNA fractions. So far, split centromeres similar to those reported here in CHO cells have not been found in mouse chromosomes, and although occasional images of conventionally prepared (methanol–acetic acid fixed) human metaphase chromosomes appear to show images that could be interpreted as split centromeres, nothing of this sort has been seen in human chromosomes prepared using the techniques described in this paper. There appears to be no logical reason why the centromeres themselves should remain joined until the end of metaphase, and of course they are not joined in the first meiotic metaphase. Meiosis is, of course, a rather different situation, but the mechanical problems are similar. Chromosomes are generally held to be unineme, having only a single DNA molecule running throughout the length of an unreplicated chromosome. This has never been shown by electron microscopy, where even the centromeric constriction appears to be crossed by multiple fibres (e.g. DuPraw and Bahr, 1969; Squarzoni et al., 1994). It is therefore worth noting that the thinnest single fibres crossing the centromeric constriction that were observed in this study were in the region of 10 nm thick, and thus comparable with the thickness of a single nucleosomal fibre (also 10 nm). In conclusion, the centromeric region of CHO chromosomes appears to consist of three segments: the centromere proper, which is already split into two sister centromeres at late metaphase, and the most proximal regions of the two chromosome arms, which are still joined at this stage. This structural differentiation could well reflect compositional and functional differentiation. ACKNOWLEDGEMENTS I should like to thank Bill Christie for setting up cell cultures for me, and Andrew Ross for processing specimens for electron microscopy. I would also like to thank the Photographic Department, MRC Human Genetics Unit, for preparing the illustrations in this paper. REFERENCES B A, 1958. Cine´-micrographic studies on mitosis in endosperm. IV. The mitotic contraction stage. Exp Cell Res 14: 245–256.
Cell Biology International, Vol. 22, No. 2, 1998
B A, 1968. Behavior and fine structure of spindle fibers during mitosis in endosperm. Chromosoma 25: 249–281. DP EJ, 1970. DNA and Chromosomes. New York: Holt, Rinehart and Winston. DP EJ, B GF, 1969. The arrangement of DNA in human chromosomes, as investigated by quantitative electron microscopy. Acta Cytologica 13: 188–205. J A, M AR, M OJ, 1989. The organisation of the mouse satellite DNA at centromeres. Exp Cell Res 183: 494–500. L LM, N S, H BA, 1986. Mouse satellite DNA, centromere structure, and sister chromatid pairing. J Cell Biol 103: 1145–1151. L--F A, 1955. The division cycle of the kinetochore. Hereditas 41: 238–240. L--F A, 1956. The role of the kinetochore in chromosome organisation. Hereditas 42: 85–160. L--F A, 1958. Recent advances in the study of the kinetochore. Int Rev Cytol 7: 123–157. M R, B W, H RG, W G, 1994. Efficient preparation of plant chromosomes for highresolution scanning electron microscopy. Chromosome Res 2: 411–415. M´ K, B´ K, 1981. Non-random centromere division: analysis of G-banded human chromosomes. Acta Biol Acad Sci Hung 32: 55–59. M A, J P, H D, G J, 1992. The organization of repetitive DNA sequences on human chromosomes with respect to the kinetochore analyzed using a combination of oligonucleotide primers and CREST anticentromere serum. Chromosoma 101: 333–341. S S, C C, S S, V A, M NM, 1994. Preparation of chromosome spreads for electron (TEM, SEM, STEM), light and confocal microscopy. Chromosoma 103: 381–392. S S, R M, R NR, 1975. Preparation and spread of unfixed metaphase chromosomes for immunofluorescence staining of nuclear antigens. Exp Cell Res 90: 87–94. S E, W W, 1971. Architecture of the Chinese hamster metaphase chromosome. Chromosoma 32: 262–294. S AT, 1991. Scanning electron microscopy of mammalian chromosomes from prophase to telophase. Chromosoma 100: 410–418. S AT, 1996a. The distribution of topoisomerase II on mammalian chromosomes. Chromosome Res 4: 1–10. S AT, 1996b. Problems in preparation of chromosomes for scanning electron microscopy to reveal morphology and to permit immunocytochemistry of sensitive antigens. Scann Microsc Suppl. 10: 165–176. S AT, R A, 1989. Factors affecting preparation of chromosomes for scanning electron microscopy using osmium impregnation. Scann Microsc Suppl. 3: 87–99. V BK, 1983. Sequence of centromere separation: occurrence, possible significance, and control. Cancer Genet Cytogenet 8: 249–274. W G, F H, 1995. Imaging of DNA in human and plant chromosomes by high-resolution scanning electron microscopy. Chromosome Res 3: 368–374.
THE STRUCTURE OF THE CENTROMERIC REGION OF CHO CHROMOSOMES A. T. SUMNER* MRC Human Genetics Unit, Edinburgh, U.K. Received 17 September 1997; accepted 9 February 1998
CHO chromosomes, prepared for fluorescence microscopy, or for scanning electron microscopy, sometimes show a splitting of the centromere proper into two sister centromeres, with a space between them, while the sister chromatids are joined in the most proximal regions of the chromosome arms. It is suggested that this might represent the final stage of chromatid splitting 1998 Academic Press before the anaphase separation of chromatids. K: anaphase; centromeres; chromosomes; satellite DNA; scanning electron microscopy; uninemy of chromosomes
INTRODUCTION
MATERIALS AND METHODS
The centromere is where the sister chromatids are joined to each other until the onset of anaphase, and where the chromosomes are attached, through the kinetochore, to the spindle. The centromere normally appears as a simple primary constriction of the chromosome. However, in the large chromosomes of certain plants and insects, the structure of the centromeric region appears more complicated, the sister chromatids being joined on either side of the centromere proper, while the sister centromeres themselves are separated by a gap (Lima-de-Faria, 1955, 1956, 1958; Bajer, 1958, 1968), and may contain two or more chromomeres (Lima-de-Faria, 1958). Such a structural differentiation does not appear to have been reported from mammalian chromosomes, which are generally much smaller than those studied by the authors referred to above. In this short note, evidence from fluorescence microscopy and from scanning electron microscopy is presented, to show that the centromeric region of CHO chromosomes is sometimes differentiated into a region where the sister chromatids are separated, and regions on either side where they remain joined throughout metaphase.
CHO cells were grown in RPMI 1640 medium, supplemented with fetal calf serum. Colcemid was added 3 h before harvesting, to produce a final concentration of 0.25 ng/ml, to accumulate the cells in metaphase. For fluorescence microscopy, cells were resuspended in Stenman’s hypotonic (Stenman et al., 1975) at 4C for 10 min, and then centrifuged on to slides using a Shandon Cytospin (1500 r.p.m. for 15 min). Chromosomes were prepared for scanning electron microscopy by a modification of the method of Martin et al. (1994). The cells were treated with 0.075 (hypotonic) potassium chloride, and fixed in methanol–acetic acid (3:1). The fixed cells were spread on 22-mm square glass coverslips, but immediately before they dried, they were flooded with 45% aqueous acetic acid, and after a few seconds were plunged into glutaraldehyde solution (2.5% glutaraldehyde in cacodylate buffer, pH 7.4, containing 1% sucrose). The coverslips were left in glutaraldehyde overnight (approximately 18 h) before dehydration through graded alcohols and critical point drying. The chromosome preparations were given a thin coating of platinum and examined in a Hitachi S800 field emission SEM at 5 kV. RESULTS
*Present address: 7 Smileyknowes Court, North Berwick, East Lothian EH39 4RG, U.K. (e-mail: [email protected]). 1065–6995/98/020127+04 $30.00/0
A proportion of CHO chromosomes stained with ethidium bromide, about 10–20%, show a darker 1998 Academic Press
128
Cell Biology International, Vol. 22, No. 2, 1998
Fig. 1. Cytospin preparation of metaphase CHO chromosomes, stained with ethidium bromide, showing holes at the centromeres (arrows).
spot in the middle of the centromere, as if there were a hole there (Fig. 1). The rest of the chromosomes show a much narrower, undivided constriction. This is confirmed by scanning electron microscopy, where a similar proportion of chromosomes show a hole at the centromere: the two arms are connected by a pair of fibres (Fig. 2), and the sister chromatids are joined on either side of the constriction, not within the constriction itself. Measurements indicate that the fibres connecting the two arms range in thickness from about 10 nm up to about 70 nm. DISCUSSION The observations described in this paper show that in CHO chromosomes, as in certain large plant and insect chromosomes (Lima-de-Faria, 1955, 1956, 1958; Bajer, 1958, 1968), the centromere can be differentiated into three regions: a central constriction, where the sister chromatids are already separated at metaphase; and flanking regions, consisting of the most proximal parts of the chromosome arms, where the sister chromatids are still joined together. Only a proportion of CHO chromosomes show these ‘split’ centromeres, and it might be wondered if they are some sort of artefact. Although the high concentration of acetic acid used to prepare chromosomes for scanning electron microscope might cause more extensive extraction of proteins than the standard methods of preparing chromosomes, there is no evidence in the scanning electron micrographs for an artificial mechanical split, while for the observations by fluorescence microscopy, no acetic acid was used. In fact, previous studies by electron microscopy appear
to show centromeric structure similar to that described here, for example, Figures 9.12 and 9.14 in DuPraw (1970), and Figure 4 in Stubblefield and Wray (1971). Wanner and Formanek (1995) have also illustrated a chromosome prepared for scanning electron microscopy and stained with platinum blue, which binds to DNA, and this chromosome has a hole in the DNA at the centromere (their Fig. 4). The fact that a variety of different methods of preparing chromosomes produce similar results gives one confidence that the observations described in this paper of holes at the centromeres of CHO chromosomes are not an artefact. The centromeres of large plant chromosomes often appear to contain two or more chromomeres in each of the separated sister centromeres (Limade-Faria, 1958). No such subdivision of the centromeres was detected in the CHO chromosomes, however. The split centromeres probably represent the penultimate stage of the splitting of chromosomes into daughter chromosomes. The centromeres may perhaps only be split briefly before the whole chromosome divides at anaphase, and if the separation of different chromosomes is asynchronous, as occurs in other mammals (Me´hes and Bajno´czky, 1981; Vig, 1983), it would be expected that only a proportion of the chromosomes would show split centromeres. It has been shown previously (Sumner, 1991) that there is a sequence of splitting of chromosomes during mitosis, starting with the euchromatic arms, followed by the paracentromeric heterochromatin, and finally the centromeric region itself. The observations presented here suggest that the centromeres themselves split in two stages, firstly the centromeres proper, and finally,
Cell Biology International, Vol. 22, No. 2, 1998
129
Fig. 2. Scanning electron micrographs of CHO chromosomes, showing centromeric regions divided into sister centromeres. (A) and (B) show the same chromosome at different magnifications, as do (D) and (E). Scale bars represent the following lengths: (A) and (D): 2 ìm; (B), (C), (E) and (F): 1 ìm.
at the metaphase/anaphase transition, the juxtacentromeric heterochromatin that forms the proximal parts of the chromosome arms. If this interpretation is correct, then the situation in CHO cells differs from that described by Lima-de-Faria (1958) for plant chromosomes, in which the centromere is already split by mid-prophase, and from that described by Bajer (1958), in which a hole between the sister centromeres appeared to result from the initial interaction of the kinetochores with the spindle in early prometaphase. It is not possible at this stage to determine whether this represents a real difference between the behaviour of plant and animal chromosomes, or is a result of the greater difficulties of observing details in the much smaller CHO chromosomes, or is a consequence of the different preparative techniques used. The structural division of the centromeric region could parallel functional and compositional differences. It was proposed some years ago that the
function of paracentromeric heterochromatin could be to hold sister chromatids together (Lica et al., 1986; Sumner, 1991), and to provide a substrate for the action of topoisomerase II at the beginning of anaphase, to separate the chromatids (Sumner, 1991). Recent work suggests that topoisomerase II is present in the paracentromeric regions, but may be absent from a small region at the centromere itself (Sumner, 1996a). Particular satellite DNAs—alphoid satellite in man, and minor satellite in mouse—appear to be associated with the centromere proper, whereas the human ‘classical’ satellites and the mouse major satellite are located paracentromerically and form blocks of heterochromatin (Joseph et al., 1989; Mitchell et al., 1992). Unfortunately, little is known about the composition of centromeric DNA in Chinese hamster chromosomes, but if an organism could be found in which the centromeric DNAs were well characterized, and the splitting of centromeres
130
could be demonstrated, it would be predicted that the centromere proper and the proximal parts of the chromosome arms might contain different DNA fractions. So far, split centromeres similar to those reported here in CHO cells have not been found in mouse chromosomes, and although occasional images of conventionally prepared (methanol–acetic acid fixed) human metaphase chromosomes appear to show images that could be interpreted as split centromeres, nothing of this sort has been seen in human chromosomes prepared using the techniques described in this paper. There appears to be no logical reason why the centromeres themselves should remain joined until the end of metaphase, and of course they are not joined in the first meiotic metaphase. Meiosis is, of course, a rather different situation, but the mechanical problems are similar. Chromosomes are generally held to be unineme, having only a single DNA molecule running throughout the length of an unreplicated chromosome. This has never been shown by electron microscopy, where even the centromeric constriction appears to be crossed by multiple fibres (e.g. DuPraw and Bahr, 1969; Squarzoni et al., 1994). It is therefore worth noting that the thinnest single fibres crossing the centromeric constriction that were observed in this study were in the region of 10 nm thick, and thus comparable with the thickness of a single nucleosomal fibre (also 10 nm). In conclusion, the centromeric region of CHO chromosomes appears to consist of three segments: the centromere proper, which is already split into two sister centromeres at late metaphase, and the most proximal regions of the two chromosome arms, which are still joined at this stage. This structural differentiation could well reflect compositional and functional differentiation. ACKNOWLEDGEMENTS I should like to thank Bill Christie for setting up cell cultures for me, and Andrew Ross for processing specimens for electron microscopy. I would also like to thank the Photographic Department, MRC Human Genetics Unit, for preparing the illustrations in this paper. REFERENCES B A, 1958. Cine´-micrographic studies on mitosis in endosperm. IV. The mitotic contraction stage. Exp Cell Res 14: 245–256.
Cell Biology International, Vol. 22, No. 2, 1998
B A, 1968. Behavior and fine structure of spindle fibers during mitosis in endosperm. Chromosoma 25: 249–281. DP EJ, 1970. DNA and Chromosomes. New York: Holt, Rinehart and Winston. DP EJ, B GF, 1969. The arrangement of DNA in human chromosomes, as investigated by quantitative electron microscopy. Acta Cytologica 13: 188–205. J A, M AR, M OJ, 1989. The organisation of the mouse satellite DNA at centromeres. Exp Cell Res 183: 494–500. L LM, N S, H BA, 1986. Mouse satellite DNA, centromere structure, and sister chromatid pairing. J Cell Biol 103: 1145–1151. L--F A, 1955. The division cycle of the kinetochore. Hereditas 41: 238–240. L--F A, 1956. The role of the kinetochore in chromosome organisation. Hereditas 42: 85–160. L--F A, 1958. Recent advances in the study of the kinetochore. Int Rev Cytol 7: 123–157. M R, B W, H RG, W G, 1994. Efficient preparation of plant chromosomes for highresolution scanning electron microscopy. Chromosome Res 2: 411–415. M´ K, B´ K, 1981. Non-random centromere division: analysis of G-banded human chromosomes. Acta Biol Acad Sci Hung 32: 55–59. M A, J P, H D, G J, 1992. The organization of repetitive DNA sequences on human chromosomes with respect to the kinetochore analyzed using a combination of oligonucleotide primers and CREST anticentromere serum. Chromosoma 101: 333–341. S S, C C, S S, V A, M NM, 1994. Preparation of chromosome spreads for electron (TEM, SEM, STEM), light and confocal microscopy. Chromosoma 103: 381–392. S S, R M, R NR, 1975. Preparation and spread of unfixed metaphase chromosomes for immunofluorescence staining of nuclear antigens. Exp Cell Res 90: 87–94. S E, W W, 1971. Architecture of the Chinese hamster metaphase chromosome. Chromosoma 32: 262–294. S AT, 1991. Scanning electron microscopy of mammalian chromosomes from prophase to telophase. Chromosoma 100: 410–418. S AT, 1996a. The distribution of topoisomerase II on mammalian chromosomes. Chromosome Res 4: 1–10. S AT, 1996b. Problems in preparation of chromosomes for scanning electron microscopy to reveal morphology and to permit immunocytochemistry of sensitive antigens. Scann Microsc Suppl. 10: 165–176. S AT, R A, 1989. Factors affecting preparation of chromosomes for scanning electron microscopy using osmium impregnation. Scann Microsc Suppl. 3: 87–99. V BK, 1983. Sequence of centromere separation: occurrence, possible significance, and control. Cancer Genet Cytogenet 8: 249–274. W G, F H, 1995. Imaging of DNA in human and plant chromosomes by high-resolution scanning electron microscopy. Chromosome Res 3: 368–374.