Relationship between sister-chromatid exchanges and heterochromatin or DNA replication in chromosomes of Crepis capillaris

Mutation Research, 190 (1987) 271-276

271

Elsevier

MTRL 0967

Relationship between sister-chromatid exchanges and heterochromatin or D N A replication in chromosomes of Crepis capillaris B. Dimitrov Institute of Genetics, Sofia 1113 (Bulgaria) (Accepted 7 November 1986)

Keywords: Sister-chromatid exchanges; Heterochromatin; DNA replication; Crepis capillaris.

Summary The C-band patterns, DNA late replication patterns and distribution patterns of spontaneous and 7-rayinduced SCEs in Crepis capillaris chromosomes were studied. The fluorescence plus Giemsa (FPG) technique was used for detection of SCEs and late-replicating chromosome regions after unifilar incorporation of BrdU into DNA. An asynchronous replication of both euchromatic and heterochromatic chromosome regions was established. The frequency of SCEs is increased about 2-fold by 1.5 Gy 7-rays. The localization of the sites of SCEs was analyzed with special reference to eu- and heterochromatin and early- and latereplicating regions. The data obtained showed that SCEs were distributed nonrandomly along the chromosomes. Preferential occurrence of SCEs was observed in the following chromosome regions: at the junction between eu- and heterochromatic regions, the latter being rich in late-replicating DNA; at the junction between early- and late-replicating regions, the latter not being C-band positive. Certain heterochromatic regions were more rarely involved in SCEs than expected on the basis of their length. The lowest incidence of SCEs was found in the centromeric regions. Very similar distribution patterns of spontaneous and 7-ray-induced SCEs were observed. The possible role of the differences in the time of replication of the different chromosome regions in the formation of SCEs is discussed.

Many studies have been carried out to clarify the molecular nature of sister-chromatid exchanges (SCEs) since Taylor et al. (1957) first described this phenomenon by means of autoradiographic methods. A better resolution of the various aspects of SCE, however, became possible by the use of methods, based on incorporation of BrdU in Correspondence: Dr. B. Dimitrov, Institute of Genetics, Sofia 1113 (Bulgaria).

DNA. The most sensitive one is the fluorescence plus Giemsa technique (Perry and Wolff, 1974). Studies of the frequency of SCEs in euchromatin and heterochromatin, which differ in some of their properties, have considerable significance for the clarification of SCE nature. Investigations of the distribution of SCEs between the euchromatin and heterochromatin have been made in different species and by different approaches. The data from most of these studies, however, are con-

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272 tradictory. Most studies on human and animal chromosomes report higher frequencies of SCEs in the euchromatin and only in a few cases SCEs are more frequently found in the heterochromatin (see ref. Schubert and Rieger, 1981). Analyses of SCEs in plants are scarce. The simultaneous study of the frequency of SCEs in eu- and heterochromatin and in early- and late-replicating regions of chromosomes could give information useful for the clarification of the mechanisms of formation of SCEs, because some authors (Painter, 1980) proposed that the difference in the time of replication of individual replicon clusters seemed to play a role in the formation of SCEs. Having in mind scanty and contradictory results, especially with regard to plants, we decided to examine the intrachromosomal localization of spontaneous and 7-ray-induced SCEs in cells of Crepis capillaris. This plant is ideally suited for this kind of examination, because its karyotype consists of 3 pairs of easily identifiable chromosomes, signified as A, D and C. The localization of the sites of SCEs has been made with particular care for the euchromatin and heterochromatin and the chromosomal regions with early and late replication of DNA.

Material and methods

The experiments were carried out with primary roots of Crepis capillaris (2n = 6). A visualization of SCEs was performed by a FPG technique adapted for Crepis capillaris after certain modification of the method developed for Vicia faba by Kihlman and Kronborg (1975). A pulsechase technique based on a substitution of the thymidine with a 5-bromouracil into DNA was employed to obtain harlequin staining. For this purpose the roots, 1-2 mm long, were incubated for 12 h in a solution of 10 -4 M bromodeoxyuridine (BrdU), 10 -8 M fluorodeoxyuridine (FdU), and 10- 6 M uridine (Urd). Thereafter they were treated for 12 h with a second solution, containing 10 - 4 M thymidine (dThd) and 10 -6 M

Urd. The roots remained in 0.05% colchicine for 2 h before fixation. The irradiation with 1.5 Gy 6°Co -r-rays was done 2 h after the incubation of the material in the second solution. Fixation was done in ethanol-acetic acid (3:1) at 4°C for 12 h. Before fixation the whole procedure was performed in the dark. After rinsing in 0.01 M sodium-citrate buffer (pH 4.7), the material was incubated in 0.5% pectinase at 30°C for 100 min. Squashing was done in 45% acetic acid and the coverslips were removed after freezing by liquid nitrogen. After hydration (100%, 90°70, 70%, 30% and distilled water) the preparations were incubated for 1 h at 30-32°C in 0.01070 ribonuclease made in 0.5xSSC. Then the preparations were washed in 0.5 x SSC and stained for 25 min in a solution of 33258 Hoechst (1 mg fluorochrome was dissolved in 1 ml ethanol; 1 ml of this solution was added to 200 ml of 0.5xSSC). After that the preparations were washed, then mounted in the same buffer, covered with coverslips and sealed with rubber cement. They were irradiated for 1 h by a mercury-quartz lamp with wavelengths of 260-380 nm from a distance of 15 cm. Thereafter they were washed in 0.5 x SSC, incubated at 60°C in the same buffer, rinsed in 0.17 M phosphate buffer (pH 6.8) and stained in 3070Giemsa solution (Gurr's R 66) in the same buffer. After being airdried, the preparations were mounted in DPX. Demonstration of the C-banding patterns in the karyotype of Crepis capillaris was performed by slight modification of the method of Marks (1975). The best differentiation of the heterochromatin blocks was obtained after staining with Giemsa Merck. The patterns of late replication were determined by means of the FPG technique: the roots, 2 mm long, were incubated in the dark in 10-8 M FdU for 2.5 h, then transferred into a solution of BrdU and FdU for 4 h which corresponds with the appearance at metaphase of cells which were in late-S phase at the time of BrdU addition and were fixed. The seedlings were placed in 0.05% colchicine solution prior to fixation. The localization of C-bands and regions of late replication and SCEs was determined on

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photographs. The exact positions were given as ratios between their distance from the centromere and the whole length of the chromosomal arm. 20 metaphases were analyzed for both the C-bands and the regions of late replication. A statistical comparison between the frequency of SCEs per segment and that expected for a random distribution was performed by calculating the upper and lower confidence limits with significance levels of 1°70 and 5070.

Results

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SCEs have been differentiated in these experiments after unifilar BrdU incorporation into DNA (one replication cycle in the presence of BrdU). As seen from Fig. 1, a very clear differentiation of the two sister chromatids occurs under these conditions, which allows the localization of SCEs. The mean frequency of SCEs was 9.4/cell. "rIrradiation of the Crepis capillaris cells containing unifilarly substituted chromosomes enhances the frequency of SCEs as shown in our previous works (Dimitrov, 1981, 1985). In the present experiments 19.2 SCEs/cell were observed after exposure of root tips to 1.5 Gy 6°Co "r-rays. Data about C-band patterns and the patterns of late replication of the chromosomes of Crepis capillaris are presented in Fig. 2. All 3 chromosomes contain intercallary constitutive heterochromatin presented as C-bands. In A chromosome C-bands are present only in the short arm, while in D and C chromosomes they are found in both arms (Fig. 2a). The differential staining of metaphase chromosomes by the FPG technique after BrdU incorporation during the late-S phase results in a pale staining of the BrdU-substituted chromosome segments. Late-replicating regions

Fig. 1. Metaphase cells of Crepis capillaris showing SCEs detected after BrdU incorporation into DNA for one replicating period and stained with the FPG technique. (a) Spontaneous SCEs. (b) SCEs induced by 1.5 Gy of ~,-rays. Bar represents 10 /~m.

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Fig. 3. Distribution of spontaneous and ~,-ray-induced SCEs in Crepis capillaris chromosomes. Each chromosome was divided A into 16, D into 10 and C into 8 equal-length segments. The centromeric regions are marked by broken lines. Ordinate, absolute number of SCEs. The continuous lines parallel to the abscissa mark the 1°70 upper and lower confidence limits; the broken lines parallel to the abscissa mark 5070confidence limits for the values expected on the basis of length-proportional distribution. The light squares under certain segments indicate the positions of late-replicating regions; the black squares show C-banded regions. (a) Distribution of 1800 spontaneous SCEs; (b) distribution of 1800 SCEs induced by "t-rays.

are present in all 3 chromosomes. The two latereplicating regions in the long arm of D and one in C correspond to the C-bands, while in A, latereplicating regions are located in the euchromatin. The nucleolus-organizing regions, which reside on the D chromosome also show late replication (Fig. 2b). As seen from Fig. 3a spontaneous SCEs are nonrandomly distributed along the chromosomes. A preferential involvement of some segments in SCEs is observed in all 3 chromosome pairs. The frequency of exchanges in segments 8, 11, 13 and 14 in A, 4, 5, 6 and 8 in D, 4 and 6 in C exceeds the upper confidence limits for a random distribution. The positions of the segments with high frequency of SCEs do not correlate with those of the Cbands, but at least in some segments e.g. 4 and 6 of D, 4 and 6 of C more probably correspond to the euchromatin-heterochromatin junctions. In these segments of C the observed frequencies of SCEs considerably exceed the 1070 upper confidence limit (Fig. 3a). More pronounced occurrence of SCEs in all 3 chromosomes at the junctions of early- and latereplicating regions was observed. Some of them as segments 4 and 6 of D and 4 of C correspond to the C-bands. The late-replicating regions in chromosome A have euchromatic nature, and segments 8, 13 and 14 correlating with the junction of earlyand late-replicating regions show a higher frequency of SCEs than expected, the latter two surpassing the 1070 upper confidence limit (Fig. 3a). Lower frequencies than expected for a random distribution are observed in some segments. These include the centromeric segment and its adjacent segments as well as the telomeric regions of the 3 chromosomes. Some of the regions with low frequency coincide with the heterochromatic regions as in the case of the short arms of A and C, where the heterochromatin occupies about half of the lenght of the arms. SCEs in the short arm of C chromosome are almost missing (Fig. 3a). As can be seen from Fig. 3b the distribution of SCEs, induced by ~/-rays, is similar to the distribution of spontaneous ones, nearly the same

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segments of A and D chromosomes containing high frequencies of SCEs. There is a slight difference in A, where the induced frequency of SCEs in the centromere regions is much higher than the spontaneous one. In chromosome C the spontaneous high frequency of SCEs in segment 4 is missing after 3,-rays. At the same time in the heterochromatic regions of the short arms of A and C chromosomes the frequencies of SCEs were lower than expected for random distribution.

Discussion

The present data indicate a nonrandom distribution of SCEs within the chromosomes of Crepis capillaris. Some regions possess high sensitivity towards both spontaneous SCEs and those induced by ,y-rays, while in other regions the frequency is lower than expected for random distribution. These results are partially at variance with those obtained by other investigators in plants. In a reconstructed karyotype of Viciafaba, Schubert et al. (1979) observed random distribution of SCEs along the chromosomes with the exception of a nucleolus-organizing region, where a higher frequency of exchanges than expected was found. A nonrandom distribution was reported by Schvartzman and Cort6s (1977) and Cort6s et al. (1985) in the chromosomes of Allium cepa. The frequency of SCEs was found to be lower than expected on a random basis in regions corresponding to heterochromatin (C-bands). In the satellite-bearing chromosome of Secale cereale, Friebe (1978) also found deficiency of SCEs in heterochromatic regions. Our own observations confirm that SCEs occurred with a lower frequency than expected in the heterochromatic regions of plant chromosomes. This is particularly evident in the short arms of A and C chromosomes, where heterochromatin occupies more than 5007o of their length, the latter allowing conclusive observations. The simultaneous mapping of C-bands and latereplicating chromosome regions as well as the division of the chromosomes into small-size segments (0.5 #m) enable us to estimate a high incidence of

SCEs at the junctions between heterochromatin and euchromatin, and at the junctions between early- and late-replicating regions. These results are relevant with respect to the mechanisms of formation of SCEs and will be discussed later in this paper. A preferential occurrence of SCEs at the junction between euchromatin and heterochromatin has been reported for some animal species such as Indian muntjac (Carrano and Wolff, 1975), kangaroo rat (Bostock and Cristie, 1976), Chinese hamster (Kato, 1979; Crossen, 1983), and wallaby (Kato, 1979). Nevertheless, the mechanisms of clustering of SCEs at the junction were not fully understood. Carrano and Johnston (1977) and Kato (1979) have suggested that these chromosome regions represent a 'locally uncoiled' state of the nucleoprotein complexes, which makes them less available for repair enzymes that turn the DNA damage into SCEs. Our results proposed a different explanation. The observed higher frequency of SCEs at the junctions of early- and latereplicating regions not only when the latter correspond to the heterochromatin, but also when the junctions are euchromatic suggests that the differences in the time of replication of the individual chromosome regions could play an important role in the formation of SCEs. This suggestion is based on the fact that SCEs are formed only in time of replication of DNA (Wolff et al., 1974) and it is consistent with the replication model of Painter (1980). According to this model the replicon cluster is a functional unit for the formation of SCEs. During the replication of DNA (S phase) double-strand breaks occur at the junction between two adjacent replicon clusters, one being completely replicated and the other one only partially replicated. SCEs occur in this case as a result of the possibility of recombination of a daughter strand from one replicon cluster with a nonreplicated part of the adjacent cluster. Factors which block the progress of the replication fork extend the time during which the replicon cluster remains next to the partially replicated one and in this way they increase the possibility for recombination of daughter strands of the duplicated cluster with the parent strands from the partially

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replicated cluster which results in the formation of SCEs. Such a possibility exists for the clusters in the junctions of early- and late-replicating chromosome regions. Natural differences in the time of replication of the clusters in these junctions exert a beneficial action in the abnormal recombination of DNA strands leading to the formation of SCEs with higher frequency compared to the junctions and the replicon clusters where replication takes place simultaneously. In conclusion the present results seem to favor the hypothesis that the difference in replication time between the chromosomal subunits is an important factor in the formation of SCEs. Further investigations with different approaches including biochemical techniques are necessary to elucidate if this is a general mechanism responsible for the formation of SCEs as proposed by some authors (Painter, 1980).

References Bostock, C.J., and S. Christie (1976) Analysis of the frequency of sister chromatid exchange in different regions of chromosomes of the kangaroo rat (Dipodorays ordii), Chromosoma, 56, 275-287. Carrano, A.V., and G.R. Johnston (1977) The distribution of mitomycin C-induced sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac, Chromosoma, 64, 97-107. Carrano, A.V., and S. Wolff (1975) Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac, Chromosoma, 53, 361-369. Cort6s, F., P. Escalza, J.M. Rodrigues-Higueras and J. Monoz-Garcia (1985) Frequency and distribution of spontaneous and induced SCEs in BrdU-substituted satellized chromosomes of Allium cepa, Mutation Res., 109, 249-257. Crossen, P.E. (1983) DNA replication and sister chromatid exchange in 9qht, Cytogenet. Cell Genet., 35, 152-153.

Dimitrov, B. (1981) Intrachromosomal distribution and mechanisms of producing spontaneous and gamma-raysinduced sister chromatid exchanges in Crepis capillaris, in: Proc. of Genet. Cytogenet. Conf., 10-11 November 1981, Sofia, pp. 113-122 (in Bulgarian). Dimitrov, B. (1985) Intrachromosomal distribution patterns of SCEs and structural chromosome aberration in Crepis capillaris, Mutation Res., 147, 291. Friebe, B. (1978) Untersuchungen zum Schwesterchromatidenaustausch bei Secale cereale, Microscop. Acta, 81, 159-165. Kato, H. (1979) Preferential occurrence of sister chromatid exchanges at heterochromatin-euchromatin junctions in the wallaby and hamster chromosomes, Chromosoma, 74, 307-316. Kihlman, B.A., and D. Kronborg (1975) Sister chromatid exchanges in Vicia faba, I. Demonstration by a modified fluorescent plus Giemsa (FPG) technique, Chromosoma, 51, 1-10. Marks, G.E. (1975) The Giemsa-staining centromeres of Nigella damascena, J. Cell Sci., 18, 19-25. Painter, R.B. (1980) A replication model for sister chromatid exchange, Mutation Res., 70, 337-341. Perry, P., and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatid, Nature (London), 251, 156-158. Schubert, I., and R. Rieger (1981) Sister chromatid exchanges and heterochromatin, Hum. Genet., 57, 119-130. Schubert, I., S. Sturelid, P. D6bel and R. Riger (1979) Intrachromosomal distribution patterns of mutagen-induced sister chromatid exchanges and chromatid aberrations in reconstructed karyotypes of Viciafaba, Mutation Res., 59, 27-38. Schvartzman, J.B., and F. Cortes (1977) Sister chromatid exchanges in AIlium cepa, Chromosoma, 62, 119-131. Taylor, J.H., P.S. Woods and W.L. Hughes (1957) The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine, Proc. Natl. Acad. Sci. (U.S.A.), 43, 122-128. Wolff, S., J. Bodycote and R.B. Painter (1974) Sister chromatid exchanges induced in Chinese hamster cells by UV irradiation at different stages of the cell cycle: The necessity of cells to pass through S, Mutation Res., 25, 73-81. Communicated by F.H. Sobels