NEW STAINING METHOD FOR THE DETECTION OF SISTER-CHROMATID EXCHANGES IN BrdU-LABELLED CHROMOSOMES

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1 J. CM Set. 33, (1978) 185 Printed in Great Britain Compdny of Biologists Limited i<)j8 NEW STAINING METHOD FOR THE DETECTION OF SISTER-CHROMATID EXCHANGES IN BrdU-LABELLED CHROMOSOMES P. ALVES AND J. JONASSON Institute of Medical Genetics, University of Uppsala, V. Agatan 24, S Uppsala, Sweden SUMMARY A direct Giemsa technique is described for the selective staining of BrdU-substituted chromatin. It can advantageously be used for sister-chromatid exchange analysis and for the detailed cytological study of the mouse kinetochores. Presumptive inversions within the centromeric region of chromosomes 4 and 18 in the C S7 Black/6J mouse are described. INTRODUCTION Methods have recently been developed that allow for the rapid scoring of sisterchromatid exchanges in the metaphase chromosomes of cells that have been grown for two replication cycles in the presence of 5-bromodeoxyuridine (BrdU) (Latt, 1973; Perry & Wolff, 1974; Korenberg & Freedlender, 1974). By means of the altered Hoechst and/or Giemsa-staining properties of BrdU-substituted chromatin these methods will positively stain only chromatids containing a parental DNA strand which does not contain BrdU. We report here a simple procedure, based upon the principles of the Giemsa-11 technique (Bobrow, Madan & Pearson, 1972), by which it is possible to stain instead the BrdU-substituted chromatin. The present direct Giemsa technique produces an intense magenta staining of fully BrdU-substituted chromatin, whereas half-substituted chromatin remains pale. Non-substituted chromatin is refractory to staining. The microscopical resolution and information gained is far greater than what is generally achieved with the conventional FPG technique (Perry & Wolff, 1974). MATERIALS AND METHODS Cells C i7 Bl melanoma Clone PG19. This cell line from the mouse has been fully described by Jonasson, Povey & Harris (1977). Cell culture The cells were maintained in plasticflasksin Eagle's Minimal Essential Medium (Eagle et al. 1956) plus 10 % foetal calf serum. 5-bromodeoxyuridine was added to a final concentration of 15 /ig/ml growth medium o, 12, 24 or 36 h prior to harvesting. Thereafter the cultures were

2 18 6 P. A Ives and J. Jonasson kept in the complete dark. Details of culture techniques can be found in Klein, Bregula, Wiener & Harris (1971) and subsequent papers in that series. Preparation of cells for chromosome analysis Cells in tissue culture were treated with colchicine at a concentration of o - i /tg/ml for a period of 60 min. They were then exposed to a hypotonic solution of 0-56 % KC1 for 8 min and fixed (with 2 changes of fixative) in 1 part acetic acid and 3 parts methanol for at least 30 min. Spreads were made on dry slides and air-dried. Microscopical analysis The metaphase spreads were stained for 10 min at room temperature in a 2 % solution of Giemsa R66 (Gurr) in 0-3 M Na 2 HPO 4 adjusted to ph 10-4 with 1 N NaOH. The preparations were rinsed in distilled water, dried and examined under a 100 x oil-immersion planapochromatic lens in a Zeiss photomicroscope. Kodak SO mm film was used for photography. As with all chromosome-banding techniques slides may respond differently to the staining procedure. However, by altering the ph between 102 and 106 and the phosphate concentration between 0-2 and 04 M one can generally achieve satisfactory results. The study of the mouse kinetochores is often made easier if the slides are preincubated in the buffer at 42 C for 10 min before staining. RESULTS AND DISCUSSION The metaphase chromosomes of cells grown in the absence of BrdU are refractory to Giemsa staining with the present technique (Fig. 1). After one complete cycle of DNA replication in the presence of BrdU all chromosomes appear faintly stained, with a weak G-band differentiation along both chromatids. One of the two kinetochores on each chromosome is totally resistant to the Giemsa stain (Fig. 2 A). A cytologically detectable lateral asymmetry in the centromeric region of mouse chromosomes was first described by Lin, Latt & Davidson (1974) after studies involving the BrdU quenching of Hoechst fluorescence. They put forward the hypothesis that the phenomenon may reflect the unequal distribution of thymidine between the two complementary polynucleotide chains of mouse satellite DNA (22 vs 45 %) described by Flamm, McCallum & Walker (1967). Replication of such DNA in the presence of BrdU should result in the incorporation of more BrdU into one of the daughter helices than the other. This hypothesis can also fully explain our observations, obtained with the present direct Giemsa technique. The parental strand of DNA with a high thymidine content should in our case be represented by a nonstaining kinetochore at the 1st division. The segregation pattern of the non-staining kinetochores at the 2nd and 3rd divisions is in agreement with this notion (see further below). In the present material we also found that after one complete cycle of DNA replication in the presence of BrdU, both chromosomes 4 and both chromosomes 18 were involved in apparent 'sister-chromatid exchanges' within the centromeric region (Fig. 2B) in all cells examined. Sister-chromatid exchanges on other chromosomes were only rarely seen. The point of breakage of chromosome 18 was always located in

3 New method for sister-chromatid exchanges 1 18 Fig. 1. The metaphase chromosomes of a cell grown in the absence of BrdU and stained with the present technique. Fig. 2 A. The metaphase chromosomes of a cell grown in the presence of BrdU for a period of 12 h prior tofixation. There is a faint G-banding pattern on both chromatids. Note also the different staining of the two kinetochores on each chromosome. Fig. 2B. Partial karyotype of the same cell as in Fig. 2 A. Both chromosomes 4 and both chromosomes 18 show apparent 'sister-chromatid exchanges' which presumably correspond to centromeric inversions (see text). Note also the different size of the nonstaining kinetochores on the two chromosomes number 1. The non-inverted centric fusion between chromosomes 5 and 15 is a characteristic feature of the melanoma cell line.

4 188 P. Alves andj. Jonasson the middle of the kinetochore, whereas the corresponding point on chromosome 4 was always closer to the tip of the chromosome (Fig. 2 B). This consistent feature of homozygosity for 'sister-chromatid exchanges' within the centromeric region of chromosomes 4 and 18 can easily be explained on the basis of the hypothesis of Lin et al. (1974) if one assumes that an inversion of the DNA double helix has occurred within the centromeric region of these chromosomes. (Throughout this work, we will use the concept of the chromatid as a continuous DNA double helix of which the centromeric satellite DNA is an interspersed and integral part.) The thymidine-rich chain of satellite DNA of the inverted segment will then be linked to the opposite DNA chain of neighbouring segments (see e.g. Angell & Jacobs, 1975). The morphology of chromosome 18 in the present material is in fact compatible with such an inversion being present in the 'lower' segment of the centromeric region (Fig. 2B). Under favourable conditions, a second break point can be detected close to the edge of the centromeric region. On the other hand, the rearranged chromosome 4 does not show any sign of having three segments, which one should expect for an inversion. Since chromosome 4 cannot positively be identified as having a true inversion one should also, perhaps, consider the possibility that it might have arisen by a process of centric fusion followed by centric fission. This would give the right chromosomal appearance, using the present method, if the thymidine-rich chain of satellite DNA was associated with the same DNA chain in both interacting chromosomes. Alternatively, if it was associated with the opposite DNA chains in the two chromosomes, a reciprocal translocation between the centromeric regions would produce the observed phenomenon. In any event, the chromosome will undoubtedly carry a segment of satellite DNA that is inverted relative to that of the rest of the chromosome. Therefore, we think it is appropriate to talk about 'centromeric inversions' being present even if some of them may eventually turn out to represent centric fusion/fission or reciprocal translocation events rather than true inversions. The same two 'centromeric inversions' as those observed in the present material were also identified in normal lymphocytes from C 57 Black/6J mice (kindly provided by Dr H. Wigzell) from which the melanoma was originally derived. We have seen that polymorphism for such inversions exists between different inbred strains of mice. Fig. 3 A. The metaphase chromosomes of a cell grown in the presence of BrdU for a period of 12 h prior tofixation. The slide was preincubated in the buffer before staining (see Materials and methods). A 'centromeric dot' (see text) can be seen on the pale chromatid on most chromosomes. Its position generally agrees with the 'narrowest point' of the centromeric region. This can be seen on some chromosomes which have consistently got more than one 'centromeric dot', e.g. chromosome 19, which is the small one at the bottom of the photograph. This chromosome has got two dots wide apart. An unidentified chromosome in the centre has got a tandemly repeated dot. A 'centromeric dot' is also present on the small centric fragment in the centre of the photograph. Note also that the 'centromeric inversion' present on chromosome 18 is clearly visualized with its two break points in the bottom left corner. Fig. 3B. The metaphase chromosomes of a normal diploid C 67 Black/6J lymphocyte grown in the presence of BrdU for a period of 48 h prior to fixation. This cell is shown only for the comparison with Fig. 3 A.

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6 190 P. Alves andj. Jonasson One can therefore conveniently use these ' centromeric inversions' as chromosomal markers (unpublished results). In this context it should also be mentioned that Lin & Davidson (1974) observed that all metacentric chromosomes of the tobacco mouse, and also of other mouse cells, show contralateral symmetry in the centromeric region. They concluded: 'These results suggest that centric fusion involves the maintenance of DNA polarity through the centromere and that the thymidine-rich chain of satellite DNA in the centromeric region is associated with the same DNA chain in every mouse chromosome'. Holmquist & Comings (1975) confirmed these results. Obviously, one cannot take their interpretations literally, but their results might indicate that for functional reasons complete inversions of the centromeric region possibly do not occur in the mouse. To this matter, we should like to add the following observations. Two metacentric chromosomes are regularly present in the PG19 cell line. One of them is a homologous Robertsonian translocation of the two chromosomes 12, which does show the expected contralateral symmetry, whereas the other is a Robertsonian translocation between chromosomes 5 and 15, which does not show contralateral symmetry in spite of the bipartite nature of its centromeric region (Fig. 2 B). If one supposes that completely inverted kinetochores do not exist in the mouse, the interpretation of this structure in accordance with the hypothesis of Lin et al. (1974) would require a centromeric inversion and the subsequent loss of non-inverted segments, if any occurred at the microscopical level. This would in a great majority of the cases be contradictory to the model for the formation of Robertsonian translocations that Dancis & Holmquist (1977) recently have proposed, since their model predicts that no loss of chromosome material occurs at centric fusions. If, on the other hand, completely inverted kinetochores do exist in the mouse and, therefore, one should explain the centric fusion between chromosomes 5 and 15 accordingly, the basis of their model (viz. a strict conservation of' satellite polarity' within the telomere) is suspended. In our preparations, under certain conditions, it is possible to assign a morphological direction to each kinetochore in the following way. The centromeric region is provisionally divided in two main segments, viz. a telomere proximal segment, and a telomere distal segment. The borderline between these two segments is demarcated by the 'centromeric dot', a minute structure which does not show any lateral asymmetry, which we think may very well correspond to the attachment site of the spindle fibres (Fig. 3). (We propose to use this nomenclature even if this dot may not be identical to the centromeric dots in Nigella described by Marks (1977).) The 'proximal segment' of the centromeric region, which includes the end of the chromosome, Fig. 4. Detail of a cell stained under the same conditions as in Fig. 3 A, where the only 'centromeric dot' (arrow) present on the centric fusion chromosome 5 and 15 (vertical) can be clearly seen (see text). Fig. 5. The metaphase chromosomes of a cell grown in the presence of BrdU for a period of 24 h prior to fixation. There is one dark and one pale chromatid on each chromosome. Approximately half of the pale ones have a non-staining kinetochore. Sister-chromatid exchanges can be seen on some chromosomes.

7 New method for sister -chromatid exchanges /i* X*

8 192 P. Alves andj.jonasson differs in its staining properties from the 'distal segment' and sometimes does not show any lateral asymmetry, whereas the lateral asymmetry of the ' distal segment' seems to be constitutive. This difference is more pronounced in prometaphase cells with relatively long chromosomes than in highly contracted c-mitoses, and it may well reflect an organizational and functional difference between the two segments. Therefore, a complete inversion of the whole centromeric region should manifest itself, but we have not yet been able to obtain evidence of such an inversion of either chromosome 5 or 15 or any other chromosome in the present material. However, there is evidence for structural rearrangements on several chromosomes including the centric fusion chromosome 5 and 15. This chromosome has got one centromeric dot instead of the two that should be expected (Fig. 4). Consequently, it seems unnecessary to introduce the concept of a kinetochore having a normal morphological direction but completely inverted satellite DNA. In the melanoma material there is a variation between the individual chromosomes in the amount of centromeric heterochromatin that is present. Each segment within the kinetochore can occur small or large and is sometimes repeated (compare e.g. the two chromosomes 1 in Fig. 2B, and see also Fig. 3). Polymorphism in the mouse for the amount of centromeric heterochromatin is a well-known phenomenon (see e.g. Dev et al. 1975), which may be related to this heterogeneity. Some of the results, presented in the previous two paragraphs, are of preliminary character. Therefore, it should perhaps be pointed out that they are used only to illustrate the possible applications of the present method. However, we are currently engaged in the further characterization of the kinetochores of both the melanoma cell line and normal diploid cells. Sister-chromatid exchanges outside the centromeric region can be detected in the metaphase chromosomes of cells that have been grown for two replication cycles in the presence of BrdU (Fig. 5). The fully BrdU-substituted chromatid stains intensely magenta, whereas the half-substituted chromatid remains pale with a barely visible G-banding pattern. Approximately half of the chromosomes receive a non-staining kinetochore, which is nearly always located on the pale chromatid as expected, although occasional transpositions may occur by sister-chromatid exchange (Fig. 5). There is a difficulty with the conventional FPG technique to discriminate between cells that were in the late 5-period when the BrdU was introduced (and thus may have chromosomal regions replicated three times in the presence of BrdU when observed) Fig. 6. Detail of a metaphase cell grown in the presence of BrdU for a period of 36 h prior to fixation. There are short segments of isolabelling of the chromatids. This is presumably due to the partial completion of three replication cycles in the presence of BrdU. Fig. 7. The metaphase chromosomes of a cell grown in the presence of BrdU for a period of 36 h prior to fixation. Approximately half of the chromosomes appear with both chromatids intensely staining. A few of the pale chromatids have a non-staining kinetochore. A non-staining kinetochore is also found on a chromosome with both chromatids intensely staining present in the upper left quadrant. There are several segmented chromosomes which presumably have arisen from sister-chromatid exchanges in previous cell cycles.

9 New method for sister-chromatid exchanges 193 s

10 194 P. Alves andj. Jonasson and those cells that were actually out of the S-period. Such cells that have been actively synthesizing DNA at the beginning of the BrdU-labelling period easily reveal themselves with the present technique. An example of a cell that has partially completed three replication cycles in the presence of BrdU is given in Fig. 6. If, on the other hand, BrdU was present for less than two complete replication cycles the intensely staining chromatid appears with unstained segments. These phenomena are not easily seen with the conventional FPG technique. After three replication cycles in the presence of BrdU. on the average half of the chromosomes appear with both chromatids intensely staining. There is still a faint G-banding pattern superimposed that often allows for their identification. The other remaining chromosomes have got one pale and one intensely staining chromatid (Fig. 7). The actual number of each kind in the individual cell should be dependent on the orientation of dark and pale chromatids at the metaphase following the second replication cycle. From theoretical considerations one should expect on the average one-eighth of the chromatids to have a non-staining kinetochore and these should be pale ones. The actual number in this material is slightly higher due to the products of the 'centromeric inversions' described above. The exceptional case where a nonstaining kinetochore is found on a chromosome having two intensely staining chromatids (Fig. 7) probably represents a sister-chromatid exchange event in a previous cell cycle. Such events also produce hybrid chromosomes which in one segment of the chromosome have got both chromatids intensely staining and in another segment have got one pale and one intensely staining chromatid (Fig. 7). It is not within the aim of the present communication to give an account for the frequency of sister-chromatid exchanges in the material. This is to be presented separately. CONCLUSIONS It is concluded that the present Giemsa-staining technique has got many characteristics that in our opinion make it the method of choice for sister-chromatid exchange analysis: (1) It is a quick and simple method, and it gives excellent results. (2) There is more information to be gained from the direct staining of BrdU-substituted chromatin with the present method than from other methods presently used which all give negative staining of the BrdU-substituted chromatin. Furthermore, we conclude from the present data that ' centromeric inversions' in the mouse (which to our knowledge have not been described before) can be easily revealed by the use of the present Giemsa-staining technique. This investigation was supported by grants from the Marcus Borgstrdm Fund and the Swedish Cancer Society (1082-B77-01X).

11 New method for sister-chromatid exchanges 195 REFERENCES ANGELL, R. R. & JACOBS, P. A. (1975). Lateral asymmetry in human constitutive heterochromatin. Chromosoma 51, BOBROW, M., MADAN, K. & PEARSON, P. L. (1972). Staining of some specific regions of human chromosomes, particularly the secondary constriction of No. 9. Nature, New Biol. 238, DANCIS, B. M. & HOLMQUIST, G. P. (1977). Fusion model of telomere replication and its implications for chromosomal rearrangements. In Chromosomes Today, vol. 6 (ed. A. de la Chapelle & M. Sorea), pp Amsterdam: Elsevier/North Holland Biomedical Press. DEV, V. G., MILLER, D. A., TANTRAVAHI, R., SCHBECK, R. R., RODERICK, T. H., ERLANGER, B. F. & MILLER, O. J. (1975). Chromosome markers in Mus musculus: differences in C- banding between the subspecies M.m. musculus and M.m. molossinus. Cltromosoina 53, EAGLE, H., OYAMA, V. I., LEVY, M., HORTON, C. L. & FLEISCHMANN, R. (1956). The growth response of mammalian cells in tissue culture to L-glutamine and L-glutamic acid. J. biol. Chem. 218, FLAMM, W. G., MCCALLUM, M. & WALKER, P. M. B. (1967). The isolation of complementary strands from a mouse DNA fraction. Proc. vatn. Acad. Set. U.S.A. 57, HOLMQUIST, G. P. & COMINGS, D. E. (1975). Sister chromatid exchange and chromosome organization based on a bromodeoxyuridine Giemsa-C-banding technique (TC'-banding). Cftromosoma 52, JONASSON, J., POVEY, S. & HARRIS, H. (1977). The analysis of malignancy by cell fusion. VII. Cytogenetic analysis of hybrids between malignant and diploid cells and of tumours derived from them.y. Cell Set. 24, KLEIN, G., BRECULA, U., WIENER, F. & HARRIS, H. (1971). The analysis of malignancy by cell fusion. I. Hybrids between tumour cells and L cell derivatives.^. Cell Set. 8, KORENBERG, J. R. & FREEDLENDER, E. F. (1974). Giemsa technique for the detection of sisterchromatid exchanges. Chromosoma 48, LATT, S. A. (1973). Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. natn. Acad. Set. U.S.A. 70, LIN, M. S. & DAVIDSON, R. L. (1974). Centric fusion, satellite DNA and DNA polarity in mouse chromosomes. Science, N.Y. 185, LIN, M. S., LATT, S. A. & DAVIDSON, R. L. (1974). Microfluorometric detection of asymmetry in the centromeric region of mouse chromosomes. Expl Cell Res. 86, MARKS, G. E. (1977). The nature of centromeric dots in Nigella chromosomes. Chromosoma 62, 36o-373- PERRY, P. & WOLFF, S. (1974). New Giemsa method for the differential staining of sister chromatids. Nature, Lond. 251, (Received 28 November 1977)

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