Heterochromatin (C bands) in bovine chromosomes

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1 Hereditas 73: (1973) Heterochromatin (C bands) in bovine chromosomes K. M. HANSEN Anatomy Department B, University o f Copenhagen The John F. Kennedy Institute, Glostrup Department of Obstetrics and Gynaecology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark (Received August 22, 1972) Metaphase chromosomes from cattle leukocytes were stained for constitutive heterochromatin. It was possible to demonstrate heterochromatin in the centromere regions of the autosomes but not of the X chromosome. The short arm and the centromere region of the Y chromosome were more faintly stained than the centromere regions of the autosomes. The staining pattern of the centromere regions of the autosomes together with the segregation of the centromere block in anaphase indicate that bovine autosomes may be telocentric and not arcrocentric. The heterochromatin patterns of man, mouse and cattle are compared. In all mammals so far examined constitutive heterochromatin is present in chromosomes as centromeric, interstitial, and telomeric (terminal) heterochromatin (ARRIGHI and Hsu 1971; CHEN and RUDDLE 1971; Hsu and ARRIGHI 1971). It is regarded as a state of a chromosome or a part of a chromosome rather than a substance (LIMA-DE- FARIA and JAWORSKA 1968). Heterochromatin and euchromatin often show different stain affinity. Certain base pairs have greater affinity to the fluorochrome quinacrine mustard (QM) than other base pairs. Depending on the sequence of base pairs, varying fluorescence intensity of entire chromosomes or parts of chromo- (Ciba) per 5 ml of blood suspension for 3 hours. somes occurs (CASPERSSON et al. 1969; WEISBLUM Heterochromatin was stained according to the and HASET 1972). method described by ARRIGHI and Hsu (1971). Identification of the karyotype of cattle by Subbing of slides was omitted. Staining for 15 means of QM and fluorescence microscopy has minutes with Giemsa solution (Merck) diluted shown that the centromere regions of the auto with phosphate buffer, ph 7.0. somes fluoresce faintly or not at all (HANSEN 1971, 1972). Several of the telomeres of the large autosomes also show faint fluorescence. The centromeric region of the X chromosome seems to be without fluorescence, but it is difficult to identify. The centromere region of the Y chromosome cannot be identified. As the faint or lacking fluorescence could be caused by heterochromatic regions, the chromosomes of cattle were stained for constitutive heterochromatin. Material and methods Nine clinically normal animals, 4 heifers and 5 bulls, of Red Danish Dairy Breed were examined. Blood samples were collected and incubated as previously described (HANSEN 1972). The cells were treated with 1 drop of 0.02 Yo Colcemid Results All autosomes showed distinct staining of the chromatin in the centromere region, centromeric heterochromatin (ARRIGHI and Hsu 1971). In 5

2 66 K. M. HANSEN Fig. 1. Karyotype of cattle stained for constitutive heterochromatin. The chromosomes are placed arbitrarily according to decreasing length. Fig. 3. X and Y chromosomes from different metaphase plates. Most of the X chromosomes have no centromeric heterochromatin. In some cases a shadow is visible in the region. The Y chromosomes show a tendency to terminal heterochromatin. Fig. 2. Metaphase plate. The long arrow indicates the X, the short arrow the Y chromosome. Note that the X chromosome has no centromeric heterochromatin and that the staining of the Y chromosome differs from the centromere regions of the autosomes. some autosomes the centromeric heterochromatin appears as large black patches, in others as small black areas (Fig. 1). The euchromatin is light grey. The telomeres of the large autosomes are faintly euchromatin stained. NO centromeric heterochromatin is visible in the X chromosome (Fig. 1, 3, 4). Nor does the Y chromosome show proper centromeric heterochromatin. The long arm of the Y chromosome is stained as euchromatin (Fig. 1, 2). The short arm and the centromere region are definitely dark but intermediate between heterochromatin and euchromatin. In a few slides the staining gave rise to a faint band formation on the chromosomes (Giemsa banding?). Fig. 4 shows a cell in

3 HETEROCHROMATIN IN BOVINE CHROMOSOMES 67 Fig. 4. Cattle leukocyte in the anaphase. The long arrow indicates the X chromosome, the short arrow the Y chromosome. early anaphase. It should be noted that the chromatids of the autosomes are in line during the divisional phase. This does not apply to the X and Y chromosomes. Discussion and conclusion Heterochromatin is generally defined as (1) heteropycnotic chromatin which stains dark in the interphase (HEITZ 1928), (2) genetically inert (MULLER and PAINTER 1932), and (3) synthetizing as centromeric heterochromatin (Hsu and AR- RIGHI 1971), but also as interstitial and telomeric (terminal) heterochromatin (OHNO et al. 1958; Hsu and ARRIGHI 1971). Heterochromatin occurs in the chromosomes of both plants and animals. HIETZ (1928) described heterochromatin in moss (Pellia), and LEVAN (1946) demonstrated heterochromatin in bulb plants (Allium). HENKING (1891) was the first investigator to demonstrate heterochromatin in animals (insects), and COOPER (1959) summarized the occurrence of heterochromatin in Drosophila melanogaster. In mammals, centromeric heterochromatin was demonstrated in horse and Bennett s Wallaby by FRED- GA (1964). The method of ARRIGHI and Hsu (1971) for demonstration of heterochromatin in human chromosomes can be used unmodified for the study of heterochromatin in other mammals (Hsu and ARRIGHI 1971). As heterochromatin is regarded as a state of a chromosome, as suggested by LIMA-DE-FARIA and JAWORSKA (1968), it will probably be necessary to use the same staining method for comparative studies and to give an accurate description of the type and stage of the cell examined. Fig. 1-3 show metaphase chromosomes from leukocytes of cattle. Fig. 4 shows an early anaphase stage. It appears that all the autosomes of cattle have distinct centromeric heterochromatin, corresponding to the dark centromere regions visible on QM fluorescence microscopy (HANSEN 1971, 1972). The same is observed in human chromosomes, in which, however, the centromere regions appear more distinct. The bovine X chromosome shows no centromeric hetero- chromatin (Fig. 1, 2), which is in accordance with DNA later than euchromatin (LIMA-DE-FARIA the fact that the centromere region of the X chro- 1959; LIMA-DE-FARIA and JAWORSKA 1968). mosome is difficult to identify by QM fluorescence Besides, heterochromatin contains two to three microscopy. Since heterochromatin has been times more DNA than euchromatin (LIMA-DE- demonstrated in the centromere region of all FARIA 1959). Further characteristics have been mammalian chromosomes examined up till now, reviewed by COMINGS (1971). e.g. in more than 20 mammalian species (Hsu Heterochromatin is of two types: constitutive and ARRIGHI 1971; BIACHI and AYRES 1971; heterochromatin, and facultative or functional COMINGS and MATTOCCIA 1972) and in birds, heterochromatin. Constitutive heterochromatin perhaps excepting macrochromosomes, (STEFOS keeps its heterochromatic properties under all and ARRIGHI 1971), it is remarkable that heteroconditions, whereas facultative heterochromatin chromatin cannot be demonstrated in the X is only heterochromatic in certain stages or tis- chromosome of cattle by means of the present sues, e.g. the one (inert) X chromosome in mam- method. mals (LYON 1961). The short arm and the centromere region of Constitutive heterochromatin is present mainly the Y chromosome of cattle show intermediate

4 68 K. M. HANSEN staining intensity (Fig. 1, 3)just as the Y chromo- Acknowledgements. - The author wishes to express his some of mice (CHEN and RUDDLE 1971; Hsu et al. appreciation to Dr. Margareta Mikkelsen, the John F. Kennedy Institute, for discussion and reading the manu- 1971). Apparently the short arm of the bovine Y script. My thanks are also due to laboratory technicians, chromosome has terminal heterochromatin (Fig. Mrs. Else Larsen and Mrs. lnger Heinze for their skilful 3,4). The distal part of the long arm is stained as assistance. The work was supported by the Danish euchromatin (Fig. 1,4). Agricultural and Veterinary Research Council, Grant No /69 and /72. The human Y chromosome has heterochromatin on the distal part of the long arm (ARRIGHI and Hsu 1971). This heterochromatin differs from Literature cited centromeric heterochromatin by showing brilliant ARRIGHI, F. E. and Hsu, T. C Localization of fluorescence (CASPERSSON et al. 1971). The Y heterochromatin in human chromosomes. - Cytogenechromosome of cattle has no similar chromatin tics 10: region and does not show stronger fluorescence BIANCHI, N. 0. and AYRES, J Heterochromatin than the autosomes (HANSEN 1972). As stated by location on chromosomes of normal and transformed cells from African green monkey (Cercopithectu aethiops). COMINGS and MATTOCCIA (1972), the cytologic - Exp. Cell Res. 68: term heterochromatin does in fact seem to CASPERSSON, T., LOMAKKA, G. and ZECH, L The comprise several biochemically different types of 24 fluorescence patterns of the human metaphase chromosomes heterochromatin. - distinguishing characters and variability. - Hereditas 67: Fig. 4 shows the segregation of the centromere CASPERSSON, T., ZECH, L., MODEST, E. J., FOLEY, G. E., regions into two blocks during the anaphase. The WAGH, U. and SIMONSSON, E Chemical differautosomes in conventionally stained metaphases entiation with fluorescent alkylating agents in Vicia from blood, skin, liver, kidney, and other organs faba metaphase chromosomes. - Exp. Cell Res. 58: of cattle often show a small, slightly lighter CHEN, T. R. and RUDDLE, F. H Karyotype anaregion between the centromere regions of the lysis utilizing differentially stained constitutive heterochromatids (heterochromatin blocks). This is chromatin of human and murine chromosomes. - Chromosoma 34: probably because the density of fibres is less in COMINGS, D. E Heterochromatin of the indian this part of the chromosomes as found by COM- muntjac. - Exp. Cell Res. 67: INGS and OKADA (1970) in telocentric chromo- COMINGS, D. E. and MATTOCCIA, E DNA of somes of mice. Rudiments of short arms are mammalian and avian heterochromatin. - Ibid. 71: often, but inconstantly observed on the autosomes I. of cattle (HANSEN 1967). In fact the autosomes of cattle are usually described as acrocentric chromosomes (SASAKI and MAKINO 1962). The short arms do not show fluorescence (HANSEN 1972) and the centromeric heterochromatin does sometimes appear irregularly angular (Fig. 1). This, in connection with the mode of segregation of the chromatin blocks in the anaphase (Fig. 4), gives reason to assume that the autosomes of cattle are telocentric. The extent of centromeric heterochromatin in relation to euchromatin varies with the stage of division of the cell and the degree of contraction of the chromosomes, as also described by AR- RIGHI and Hsu (1971). Therefore there seems to be no point in stating the extent of the heterochromatin in percentage of the arm length. Since homologous cattle chromosomes cannot be identified by the present method, it was not possible to demonstrate heterochromatin polymorphism, as has been demonstrated in human chromosomes by CRAIG-HOLMES and SHAW (1971). COMINGS, D. E. and OKADA, T. A Whole-mount electron microscopy of the centromere region of metacentric and telocentric mammalian chromosomes. - Cytogenetics 9: COOPER, K. W Cytogenetic analysis of major heterochromatic elements (especially Xh and Y) in Drosophila melanogaster, and the theory of heterochromatin. - Chromosoma 10: CRAIG-HOLMES, A. P. and SHAW, M. W Polymorphism of human constitutive heterochromatin. - Science 174: FREDGA, K Heterochromatic regions in mitotic and meiotic chromosomes of Bennett s Wallaby (Protemnodon rufogrisea DESMAREST). - Exp. Cell Res. 36: HANSEN, K. M Sex chromosomes in a freemartin. - Arsberefn. Inst. Sterilitetsforskn. 10: Identification of bovine chromosomes by the quinacrine mustard fluorescence technique. - Hereditas 69: Bovine chromosomes identified by quinacrine mustard and fluorescence microscopy. --- Ibid 70: HEITZ, E Das Heterochromatin der Moose. I. - Jahrb. Wiss. Bot. 69: HENKING, H Untersuchungen iiber die ersten EntwicklungsvorgHnge in den Eiern der Insekten. - Z. W~SS. ZOO^. 51;

5 HETEROCHROMATIN IN BOVINE CHROMOSOMES 69 Hsu, T. C. and ARRIGHI, F. E Distribution of constitutive heterochromatin in mammalian chromosomes. - Chromosoma 34: Hsu, T. C., COOPER, J. E. K., MACE, M. L. and BRINKLEY, B. R Arrangement of centromeres in mouse cells. - Ibid. 34: LEVAN, A Heterochromaty in chromosomes during their contraction phase. - Hereditus 32: LIMA-DE-FARIA, A Differential uptake of tritiated thymidine into hetero- and euchromatin in Melanoplus and Secale. - J. Biophys. Biochem. Cytol. 6: LIMA-DE-FARIA, A. and JAWORSKA, H Late DNA synthesis in heterochromatin. - Nature 217: LYON, M. F Gene action in the X-chromosome of the mouse (Mus musculus L.).- Ibid. 190: MULLER, H. J. and PAINTER, T. S The differentiation of the sex chromosomes of Drosophila into genetically active and inert regions. - Z. Indukt. Abstumm. - Vererbungsl. 62: OHNO, S., KAPLAN, W. D. and KINOSITA, R Demonstration of bi-partite spiral structure on spermatogonial anaphase chromosomes of Mus musculus. - Exp. Cell Res. IS: SASAKI, M. S. and MAKINO, S Revised study of the chromosomes of domestic cattle and the horse. - J. Hered. 53: STEFOS, K. and ARRIGHI, F. E Heterochromatic nature of W chromosome in birds. - Exp. Cell Res. 68: WEISBLUM, B. and HASETH, P. L Quinacrine, a chromosome stain specific for deoxyadenylate-deoxythymidylate-rich regions in DNA. - Proc. Nut. Acad. Sci. 69: , K. M. Hansen Anatomy Department B University of Copenhagen Universitetsparken 1 DK-2100 Copenhagen 0, Denmark