Sister-chromatid exchanges in higher plant cells: Past and perspectives

Mutation Research, 181 (1987) 127-145 Elsevier

127

MTR 04426

Sister-chromatid exchanges in higher plant cells: past and perspectives J o r g e B. S c h v a r t z m a n Centro de Investigaciones Biolbgicas (CSIC), Vel&zquez144, Madrid 28006 (Spain) (Received 5 January 1987) (Revision received 15 April 1987) (Accepted 21 April 1987)

Keywords: Sister-chromatid exchanges; Higher plant cells.

(I) The SCE boom In science, acquisition of new knowledge in a particular field is not a continuous and progressive process. Instead, it usually occurs as a series of discontinuous bursts caused by the sudden development of specific and often simple new techniques. This is precisely the case with sister-chromatid exchange (SCE) analysis. Whereas the total number of publications on SCEs since this phenomenon was discovered in 1938 until 1973 was by far less than a hundred, at least 2672 articles have been published in this particular field from 1974 until 1986 (Table 1). What is the cause for this SCE boom? As previously mentioned, the answer to this question is relatively simple: in 1973-74 a technique was developed allowing differentiation between the two sister chromatids of metaphase chromosomes with great accuracy (Latt, 1973; Perry and Wolff, 1974). This technique involves incorporation of 5bromodeoxyuridine (BrdUrd) into DNA and the subsequent differential staining of chromatids or chromosomal regions containing different amounts

Correspondence: Dr. Jorge B. Schvartzrnan, Centro de Investigaciones Biolrgicas (CSIC), Velfizquez 144, Madrid 28006 (Spain). Abbreviations: BrdUrd, 5-bromodeoxyuridine; dNTP, deoxyribonucleotide triphosphate; dThd, thymidine; FdUrd, 5-fluorodeoxyuridine; [3H]dThd, tritiated thymidine; SCD, sister-chromatid differentiation; SCE, sister-chromatid exchange.

of this thymidine (dThd) analogue. Sister-chromatid differentiation (SCD), which is essential to visualize SCEs, was previously achieved using [3H]dThd to label DNA and autoradiography to detect the labeled chromatids (Taylor et al., 1957). However, the resolution achieved with autoradiography is rather poor and SCEs are particularly difficult to score. The BrdUrd method, on the contrary, is relatively simple and the resolution is improved significantly (Wolff and Perry, 1974). But there is yet another reason to explain the SCE boom: SCEs are the most sensitive cytological method for detecting potential mutagenic and clastogenic agents (Perry and Evans, 1975). This observation led to the utilization of the SCE test in many of the screenings performed in the expanding field of cytogenetic toxicology today.

(II) Plant cytogenetieists and the SCE boom Even though SCEs were first discovered (McClintock, 1938) and visualized (Taylor et al., 1957) in higher plants, the number of publications on SCEs in plants only accounts for about 3% of the total number of articles published in this field during the last decade (see Table 1). Whatever else this could mean, an obvious conclusion is that currently there are many more cytogeneticists working with mammalian than with plant cells. In spite of this, plant cytogeneticists have contributed significantly to the increase of our knowledge on SCEs. As previously pointed out, the experiments leading to the 3 basic discoveries made in

0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

128 TABLE 1 DISTRIBUTION OF SCIENTIFIC REPORTS ON SCEs IN H I G H E R PLANTS PER YEAR A N D C O U N T R Y OF O R I G I N 1974 Sweden U.S.A. Great Britain F.R.G. Spain G.D.R. Canada The Netherlands Bulgaria Japan Czechoslovakia

1976

2

1977 1

1 1 1

21

1978

1979

1980

2

1981

1982

2

2

1 5 1

1 3

1983

1 1 1

1 3 4

2 3 1 1

1984

1985

1

1 1

1

8 1

3

8

1

1

Sub-total (in plants) Total number of reports on SCEs

1975

1986

Total

1 1 1 1

12 3 1 7 37 8 2 3 1 1 1

2

1 2

2

3

2

5

8

8

9

9

10

5

9

8

78

30

46

104

141

148

308

295

367

274

284

383

271

2672

this field before the development of the BrdUrd method, namely: (i) Barbara McClintock's first proposal of what she called' sister-chromatid crossovers' (McClintock, 1938); (ii) the cytological demonstration that chromatid's subunits segregate semi-conservatively (Taylor et al., 1957); and (iii) the observation that SCEs are formed by breakage and polarity-restricted rejoining of the 4 polynucleotide strands involved (Taylor, 1958), were achieved working with Zea mays, Vicia faba and Bellevalia romana, respectively.

TABLE 2 PLANT SPECIES W H E R E SCEs HAVE BEEN S T U D I E D BY MEANS O F THE BrdUrd METHOD, A N D THE N U M BER OF PUBLICATIONS W H E R E EACH OF THEM WAS USED Plant species

Number of publications

A llium cepa Vicia faba Secale cereale Zea mays A Ilium sativum Hordeum vulgare Tradescantia paludosa A Ilium ascalonicum Ornithogalum longibracteatum Crepis capillaris

37 23 4 3 2 1 1 1 1 1

The first scientific report on SCEs in higher plants based on the use of the BrdUrd method occurred in 1975 (Kihlman and Kronborg, 1975). Since that publication, a total of 78 articles have been published. The distribution of publications on SCEs in higher plants per year and the country of origin is presented in Table 1. For comparison, the total number of publications on SCEs per year is also reported in this table. The list of plant species where SCEs have been studied is rather brief, probably as a reflection of the few laboratories that have been working in this field until now. Table 2 presents a list of plant species were SCEs have been investigated as well as the total number TABLE 3 LIST OF SCIENTIFIC JOURNALS W H E R E PUBLICAT I O N S ON SCEs IN H I G H E R PLANTS HAVE O C C U R R E D MORE F R E Q U E N T L Y Scientific journal

Number of publications

Chromosoma Mutation Research Experimental Cell Research Caryologia Experientia Hereditas Microscopica Acta Theoretical and Applied Genetics

22 17 8 5 4 4 3 3

129 TABLE 4

G2

SOME OF T H E MOST PROLIFIC A U T H O R S ON SCEs IN H I G H E R PLANTS Author's name

Number of publications

Cortrs, F. (Spain) Gutirrrez, C. (Spain) Schvartzman, J.B. (Spain) Schubert, I. (G.D.R.) Kihlman, B.A. (Sweden)

18 15 12 10 9

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06)

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Fat[free monocentricrlng~

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of publications where each of these species was used. Note that Allium cepa and Vicia faba are by far the most preferred materials. Indeed, 80% (63 out of 78) of the publications on SCEs in higher plants use either of these two species. Concerning the scientific journals where most of these articles were published, a list is presented in Table 3 including those journals where publications on SCEs in higher plants have occurred more frequently. Finally, Table 4 presents a list of the most prolific authors in this field.

(III) The SCE basics As mentioned earlier, the occurrence of SCEs was originally deduced by Barbara McClintock (1938) after studying the aberrant mitotic behavior of ring chromosomes in maize. Fig. 1 illustrates this behavior and its mitotic consequence. After McClintock, other cytogeneticists analyzed this phenomenon in Zea mays (Schwartz, 1953a,b), Crepis capillaris (Dubinin and Nemtseva, 1964), and Viciafaba (Peacock, 1963; Geard, 1976, 1984). All of these investigators confirmed and extended McClintock's observations. The works of Geard (1976, 1984) are particularly noteworthy as they suggest the existence of reversals of polarity along the chromosomal DNA, a possibility that has been additionally analyzed only in X-rayinduced ring chromosomes of the Chinese hamster (Peacock et al., 1973; Wolff et al., 1976). Taylor and co-workers (1957) were the first to visualize SCEs using the light microscope. The experiment they performed was aimed to determine the mode of segregation of nascent DNA. The results obtained are the first experimental

Fig. 1. Diagrammatic representation of the formation of a dicentric ring due to the occurrenceof an SCE,and its mitotic consequence.

evidence demonstrating the semi-conservative segregation of chromatid subunits. Following this pioneer work of Taylor and co-workers (1957), other investigators used [3H]dThd and autoradiography to study DNA segregation and SCEs in higher plant chromosomes. However, due probably to the poor resolution of the method, the results achieved were quite controversial (LaCour and Pelc, 1959; Wood and Schairer, 1959; Peacock, 1963; Zweidler, 1964; Geard and Peacock, 1969; Sparvoli and Gay, 1973). Actually, Taylor and co-workers (1957) were not the first to study DNA segregation in plant chromosomes. A year before them Plaut and Mazia (1956) labeled meristematic cells of Crepis capillaris with carbon-labeled []4C]dThd and found in the autoradiographs that the label was not necessarily equally distributed by the mitotic process. This was probably due to SCEs. In any event, most of the controversy generated during those years on chromosome strandedness (Peacock, 1963; Zweidler, 1964; Gim~nez-Martin and L6pez-Sfiez, 1965; Trosko and Wolff, 1965; Peacock, 1979) and isolabeling (Peacock, 1963; Wolff and Perry, 1974; Luchnik and Porjadkova, 1977; Schubert et al., 1979a) was definitively solved using the BrdUrd method. Another significant advantage of the BrdUrd method is that it allows two alternative experimental protocols: cells can be grown for two consecutive cell generations in the presence of

130 3'5'

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Fig. 2. Diagrammatic representation of the BrdUrd-labeling patterns and differential staining of sister chromatids throughout 2 consecutive cell generations. At the left, when both cell cycles occur in the presence of BrdUrd. At the right, when the first cell cycle occurs in the presence of BrdUrd and the second one in the presence of dThd. Bars represent polynucleotide strands (continuous, unsubstituted; discontinuous, BrdUrd-substituted). Note that in either case SCD is achieved at the second division after the beginning of DNA labeling.

B r d U r d or only during the first one, the second cell cycle takes place in the presence of unlabeled d T h d (Fig. 2). In either case, S C D is achieved at the second division after D N A labeling. SCEs m a y occur throughout the two cell cycles, and those visualized in second-division chromosomes are the sum of all the exchanges that have occurred during those two consecutive cell generations (Fig. 3).

(IV) Some of the problems approached by plant cytogeneticists using the BrdUrd method Since the B r d U r d method was developed and applied to the study of plant chromosomes for the first time by Kihlman and K r o n b o r g (1975),

several other investigators have employed this technique to answer a wide variety of questions related to the SCE phenomenology. I will briefly discuss some of the problems that have been approached as well as the main conclusions achieved in each particular field.

(1) The mechanism of sister-chromatid differentiation The mechanism leading to the differential staining of sister chromatids after B r d U r d incorporation has been studied by several investigators. As m a n y of these investigators used different protocols, a unified interpretation is rather difficult. Nevertheless, at least in the case of the so-called F P G (fluorescent-plus-Giemsa) tech-

131

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5

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FIRST

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PHASE

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THE

5'3'

S

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SECOND

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SSCOND

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Fig. 3. Diagrammatic representation of labeling patterns, diftrerc.ntial staining of sister chromatids, and SCEs throughout 2 consecutive cell generations. For explanations see Fig. 2. Double-head arrows indicate the occurrence of SCEs. Note that the SCEs visualized at the second division are the sum of all the exchanges that have occurred throughout the last 2 consecutive cell generations.

nique (Perry and Wolff, 1974), the mechanism is now clearly understood. In this method, air-dried preparations are first treated with a fluorochrome, usually Hoechst 33258, although other agents can be used as well (Hazen et al., 1985). Fluorochromes act as photosensitizers (Scheid, 1976). Subsequently, slides are irradiated with ultraviolet (UV) light or long-wave UV light. This treatment causes debromination and eventually single-strand breaks in BrdUrd-substituted chains (Lyon, 1970). The next step in the protocol is to incubate the slides in a buffer at relatively high temperatures. This treatment leads to a preferential extraction of nucleoproteins from those chromatids that have been damaged more intensely (Ockey, 1980). Gonz~dez-Gil and Navarrete (1982) reported that this differential extraction can be improved significantly if the slides are subsequently incubated

with 5 N HC1 at 20 °C for 15 rain. In any case, the slides are finally stained with Giemsa, resulting in a preferential staining of the less disorganized chromatid, that one containing more dThd (Fig.

4). In contrast with this method, some authors found, after introducing several modifications in the original protocol, that the more BrdUrd-substituted chromatid was preferentially stained. This was the case, among others, of Tempelaar et al. (1982) who developed a Feulgen-staining procedure for plant chromosomes. In this case, after fixation root tips are immediately hydrolyzed at 2 8 ° C in 5 N HC1. BrdUrd-substituted DNA is known to have a stronger affinity for proteins as compared with dThd-containing DNA (Gordon et al., 1976). Therefore, as irradiation with UV light is specifically avoided prior to hydrolysis in this

132

Fig. 4. 'Harlequin-like'chromosomesof Allium cepa L. stained by means of the FPG technique at the second division after DNA substitution with BrdUrd throughout the last 2 consecutive cell generations.

particular protocol, it is expected that the chromatid containing more dThd and less BrdUrd would be preferentially degraded. Consequently, the more BrdUrd-substituted chromatid would be preferentially stained. Basically, this is the rationale for the so-called 'reverse SCD' (Schvartzman and Tice, 1982).

(2) BrdUrd incorporation into plant DNA and its effects on cell cycle kinetics Haut and Taylor (1967) were the first to report that BrdUrd incorporation into plant DNA is not as easy as it is in the case of cultured mammalian cells. This could be due to the larger size of deoxyribonucleotide triphosphate (dNTP) pools that cells of higher plants appear to have. However, they also noted that when the endogenous synthesis of thymidilic acid is inhibited, BrdUrd incorporation is greatly enhanced. To illustrate this observation further, it was found that treating Chinese hamster ovary (CHO) cells with 1 . 1 0 -5 M BrdUrd in the absence of FdUrd leads to about 91% substitution of BrdUrd for dThd in the nascent strands (Zwanenburg et al., 1984). On the contrary, a 1 • 10 -4 M BrdUrd treatment leads to less than 20% substitution in Allium cepa root-tip cells. However, when this treatment occurs simultaneously with 5 - 1 0 - 6 M 5-fluorodeoxyuridine

(FdUrd), a specific inhibitor of thymidilate synthase (Cohen et al., 1958), substitution of BrdUrd for dThd becomes greater than 50% (Jordao, personal communication). Therefore, in most of the experiments requiring BrdUrd incorporation into the DNA of plant cells, FdUrd is used. Schvartzman and Cortts (1977) and Cortts and Gonz~lez-Gil (1982) investigated the effects of BrdUrd incorporation on cell cycle kinetics in onion root tips. They found that treatment with 1 • 10 -7 M FdUrd and 1 • 10 -4 M BrdUrd causes a significant delay in cell cycle progression. This is particularly evident when cells are treated in earlyand mid-S. In contrast, there is no apparent delay when BrdUrd-substituted DNA is used as template during the following cell generation in the absence of BrdUrd and FdUrd. In general, substitution of BrdUrd for dThd does not appear to affect adversely cell proliferation kinetics. Indeed, Evans and Filion (1980) employed the BrdUrd method to measure cell cycle time in Viola faba and Zebrina pendula. They took advantage of the strategy developed by Tice and co-workers (1976) which is based on the capacity offered by the BrdUrd method to differentiate between cells that had developed 1, 2 or 3 consecutive cell cycles in the presence of BrdUrd (see Fig. 5). They determined that at 20 o C, the cell cycle time is 18.5 h for Vicia faba and 17.0 h in the case of Zebrina pendula. These figures are in good agreement with measurements performed using other methods.

(3) Chromosome replication banding The great resolution achieved with the BrdUrd method prompted the use of this technique to study the chromosomal pattern resulting when cells are labeled with BrdUrd at different times throughout the S phase. Dobel and co-workers (1978) studied DNA late-replicated bands in a reconstructed karyotype of Vicia faba. They found that not all heterochromatic C-bands are late replicating. In particular, centromeric C-bands do not replicate late whereas all interstitial C-bands do. A different although not controversial conclusion was achieved by Cortts and co-workers (1980) after studying the late-replicating pattern of Allium cepa chromosomes. These authors found, as it has been previously shown using [3H]dThd and autoradiography (Schvartzman and Diez, 1977), that het-

133 AFTER S

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P R E S E N C E OF BrdUrd

P R E S E N C E OF BrdUrd

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Fig. 5. Diagrammatic representation of labeling patterns, differential staining of sister chromatids, and SCEs, throughout 3 consecutive cell generations. For explanations see Fig. 2. Note that first- and second-cycle SCEs are detected as non-reciprocal exchanges in third-division chromosomes. On the other hand, third-cycle SCEs involving the native (unsubstituted) DNA strand are detected as reciprocal exchanges.

erochromatic C-bands are exclusively located at the telomeres whereas both telomeric and pericentromeric DNA are late replicated. By labeling alternatively early- or late-replicating DNA with BrdUrd, they also showed the occurrence of

early-replicating small bands that are immersed within large blocks of late-replicating pericentromeric DNA. In addition, the existence of late-replicated segments of dimensions smaller than the width of a chromatid was also shown

TABLE 5 COMPARISON OF THE BASELINE FREQUENCIES OF SCEs IN D I F F E R E N T PLANT SPECIES Species

Chromosome constitution

DNA/cell (pg)

SCEs/cell

S C E s / p g DNA

Reference

Secale cereale Vicia faba Vicia faba Secale cereale Tradescantia paludosa Allium cepa Allium ascalonicum Hordeum vulgate Allium cepa Allium satioum Zea mays Zea mays

TB-BB TT-TB TB-BB TT-TB TT-TB TT-TB TB-BB T'I'-TB TB-BB TB-BB TI'-TB TI'-TB

25.0 44.0 44.0 25.0 54.0 54.0 47.0 20.0 54.0 60.0 16.8 16.8

10.3 20.6 29.1 16.9 43.5 44.8 41.6 20.6 62.4 104.0 37.0 46.0

0.41 0.48 0.66 0.67 0.80 0.83 0.88 1.03 1.15 1.73 2.20 2.73

Friebe et al. (1982) Kihlman and Kronborg (1975) Kihlman and Andersson (1982) Friebe et al. (1982) Andersson (1985) Schvartzman et al. (1979a) Cort6s et al. (1983c) Schubert et al. (1980) Schvartzman et al. (1979a) Cort6s et al. (1983c) Khuong and Schubert (1987) Chou and Weber (1980)

134 (Cortrs and Escalza, 1983). Altogether these observations confirm the great resolution that is possible to be achieved using the BrdUrd method, and clearly demonstrate the complexity of the organization of chromosomal DNA for replication. Another important finding in this context is the observation by Schubert and Rieger (1979) that in some chromosomes of Vicia faba, interstitial late-replicating DNA occurs in the form of asymmetric bands. These bands are assumed to be indicative of clusters with unequal dThd distribution between the two complementary polynucleotide chains (Lin et al., 1974).

(4) The frequency of SCEs in different plant species Thus far, SCEs have been studied in only a few plant species (see Table 2). Different species have different chromosome numbers and genome sizes. Therefore, in order to compare the SCE yields between species it has been suggested that this frequency should be expressed per D N A content, i.e. per picogram of D N A corresponding to the haploid chromosome complement (Kihlman and Kronborg, 1975). Also, it should be remembered that the SCE yield is significantly affected by the labeling conditions. In this regard several aspects should be considered: the number of replication rounds where the cells had been exposed to BrdUrd (Schvartzman and Cortrs, 1977; Schvartzman et al., 1979a); the concentration of BrdUrd (Wolff and Perry, 1974; Kato, 1974; Gutirrrez et al., 1983); and whether or not FdUrd was employed, as well as its concentration (Escalza et al., 1985). Table 5 presents the baseline frequency of S C E s / pg of DNA that has been observed for different plant species. Note that if all the different factors aforementioned are considered, the frequencies of SCEs in all the species are quite similar. The only possible exception would be Zea mays. (5) SCEs: spontaneous or induced? This long-lasting debate refers to the problem of whether some SCEs occur spontaneously or if all of them are induced by the methods used to achieve SCD. The question was originally outlined by Taylor (1958) who was concerned with the effects of DNA-incorporated [3H]dThd. Indeed, Brewen and Peacock (1969) showed that in peripheral lymphocytes from a human male hetero-

zygous for a ring chromosome, the frequency of induced dicentrics is a dose (tritium)-dependent process. This observation was interpreted as an indication that SCEs are radiation-induced events. The BrdUrd method overcomes the radiation problem. However, it was early demonstrated that increasing concentrations of BrdUrd induce a concomitant increase in the yield of SCEs, at least within certain ranges (Wolff and Perry, 1974; Kato, 1974; Gutirrrez et al., 1983). So, the problem was to determine whether or not there is a baseline frequency of SCEs which is independent of BrdUrd. Gutirrrez and co-workers (1981, 1983, 1984a) have developed several mathematical equations to analyze the results obtained using different experimental protocols. This methodology led them to postulate that in Allium cepa, 0.06 SCE occurs per picogram of DNA and cell cycle independently of BrdUrd substitution. This was confirmed by Sutou (1981) who arrived at a very similar conclusion after studying the frequency of dicentric induction in unlabeled ring chromosomes of the Chinese hamster. However, the possible spontaneity of SCEs was recently challenged by Holmquist (1983) who claims that the existence of adult human beings, with apparent normal intelligence, who carry a ring chromosome in almost every cell is not compatible with the notion that SCEs occur spontaneously 'in vivo'. To summarize, the debate continues. The occurrence of BrdUrd-independent SCEs is now generally accepted, although their spontaneity is still questioned. Interestingly enough, the word 'spontaneous' is progressively avoided. Indeed, 'spontaneous' only means that we are ignorant of the possible causes.

(6) "Dot-like" SCEs and chromosome structure Kihlman (1975) was the first to note the occurrence of reciprocal SCEs involving material of smaller dimensions than the width (diameter) of the chromatid in Vicia faba. The occurrence of these minute or 'dot-like' SCEs was further analyzed in Allium cepa (Schvartzman and Cortrs, 1977; Schvartzman et al., 1978; Cortrs et al., 1983a,b). These investigators found that sistersubchromatid-exchanged segments can range in size from a minute dot (at the limit of resolution of the light microscope) to a very narrow line

135 (about 0.5/~m in height) running across almost the entire width of a chromatid. Later on, Kihlman's (1975) original interpretation of this phenomenon was experimentally confirmed (Schvartzman et al., 1978). In brief, dot-like exchanges are the consequence of two adjacent SCEs that have occurred in very close proximity in the same coil of Ohnuki's (1968) chromonema. Also, Cortrs and co-workers (1983a) studied 'the border of exchange' (Goyanes and Schvartzman, 1983) of hundreds of SCEs in Allium cepa and described discontinuous or 'stepwise' SCEs. They interpreted this pattern as more evidence supporting the existence of an ordered helicoidal subchromatid structure, namely: the chromonema.

(7) SCE distribution along chromosomes This question has been approached by several investigators using plant as well as animal cells, and has been extensively reviewed by Schubert and Rieger (1981). Apparently, SCEs occur more frequently in euchromatic than in heterochromatic regions (Carrano and Wolff, 1975; Schvartzman and Cortes, 1977; Friebe, 1978; Cortes and Hazen, 1981, Cortrs et al., 1983b). Also, there is a slight tendency for SCEs to occur at the junctions between euchromatic and heterochromatic blocks (Carrano and Wolff, 1975; Friebe, 1978; Cortrs, 1980; Ambros and Schweizer, 1986). In a reconstructed karyotype of Vicia faba, the secondary constriction which in this case is located in the middle of a long arm and harbors the nucleolarorganizing region (NOR), appears to be a hot-spot for SCEs (Schubert et al., 1979b). However, it is difficult to determine whether this SCE clustering occurs precisely at the N O R or as a consequence of the successive alternating occurrence of euchromatic and heterochromatic blocks flanking the NOR. In summary, SCEs can occur anywhere along the chromosomes. They occur more frequently in euchromatic than in heterochromatic regions. Finally, the junctions between euchromatic (earlyreplicating) and heterochromatic (late-replicating) blocks appear to be SCE hot-spots. (8) Factors affecting the frequency of SCEs in higher plant cells The frequency of SCEs is known to be affected

by a wide variety of internal as well as external cell factors. The frequency distribution of chromosomes or cells showing different numbers of SCEs follows Poisson expectations (Wolff and Perry, 1974; Schvartzman and Cortrs, 1977). However, in most of the publications the mean and the variance are calculated as if SCEs would follow a normal distribution. Fig. 6 shows a comparison between normal and Poisson distributions for 3 different means and their corresponding deviations. Note that as the mean diminishes, deviation between normal and Poisson increases. Based on this and other similar observations it was suggested that SCE data should be analyzed using analysis-of-variance (AOV) statistical methods which are based on the square root of SCE rather than SCE (Dufrain et al., 1979; Whorton et al., 1982).

(a) DNA content. It is generally accepted that the number of SCEs is roughly proportional to chromosome length. However, in those species where there is a significant difference in size among chromosomes, the larger ones tend to show more SCEs than would be expected according to a strict length proportionality (Ikushima and Wolff, 1974; Bloom and Hsu, 1975; Kihlman and Andersson, 1982). Correspondingly, when comparing two species with different chromosome numbers but the same cellular D N A content, the one with smaller chromosomes is expected to exhibit fewer SCEs than the species with larger chromosomes. This is the case, for example, of Crisetulus griseus and Xenopus laevis (see Table 6). (b) BrdUrd. As previously mentioned, it was early recognized that BrdUrd induces SCEs (Wolff and Perry, 1974; Kato, 1974). It was assumed that these BrdUrd-dependent SCEs occur as a consequence of the incorporation of BrdUrd into chromosomal DNA. However, Davidson and coworkers (1980) demonstrated that the SCEs induced by BrdUrd in mammalian cells under certain conditions, are largely independent of the BrdUrd content of DNA. Therefore, when considering BrdUrd-induced SCEs it should be taken into account that these SCEs are caused via at least two different mechanisms: (i) the dNTPs pool imbalance which is caused by high con-

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per c h r o m o s o m e

Fig. 6. Comparison between 'normal' and 'Poisson' distributions for 3 different means (at the left), and their corresponding deviations (at the fight). Calculations were made following a computer program written by Luis Martin-Parras.

centrations of BrdUrd independently of the BrdUrd content of DNA; and (ii) the BrdUrd content of DNA. With respect to the latter mechanism, it should be remembered that the total amount of incorporated BrdUrd depends on the efficiency of substitution of BrdUrd residues for dThd (Mazrimas and Stetka, 1978; Escalza et al., 1985), and also on the number of cell generations that the cells had been exposed to BrdUrd (Schvartzman and Cortrs, 1977; Schvartzman et al., 1979a).

(c) Growth temperature. The growth temperature dependence of SCEs has been confirmed in several species (Kato, 1980a; Speit, 1980; Gutirrrez et al., 1981; Abdel-Fadil et al., 1982; Hern~ndez and Guti6rrez, 1983). However, different cell types show diverse temperature-response curves. This could be at least partially attributed to the narrow range of environmental variations warm-blooded animal cells can tolerate as opposed to coldblooded ones. Higher plant cells, on the other hand, have the ability to grow over a wide temper-

TABLE 6 C O M P A R I S O N OF T H E F R E Q U E N C Y O F SCEs IN T W O SPECIES W I T H T H E SAME C E L L D N A C O N T E N T BUT DIFFERENT CHROMOSOME NUMBER a Species

D N A content/cell

Chromosome number

Average D N A content/ chromosome

SCEs/cell

SCEs/chromosome

SCEs/pg DNA

Xenopus laeois Crisetulus griseus

12.6 12.6

38 22

0.22 0.57

9.5 10.8

0.25 0.49

0.75 0.85

" Data from Uggla and Natarajan (1982).

137 ature range; e.g. onion roots can grow between 5 °C and 35 o C, with 25 °C being the optimal as determined by root growth rate (L6pez-S~ez et al., 1969). Under those conditions, cell cycle time becomes longer as the culture temperature diminishes from 25 o C to 5 o C. Guti6rrez and co-workers (1981) demonstrated that in Allium cepa root tips the frequency of SCEs increases as the growth temperature diminishes and the cell cycle time becomes longer. They suggested that either the rate of fork displacement or the intracellular oxygen concentration, both of which are known to be influenced by temperature, could be responsible for this behavior. This particular question is still to be answered. Hern~ndez and Guti6rrez (1983) further analyzed SCE formation at supra-optimal growth temperatures in onion root tips. They showed that the optimal growth temperature appears to be the experimental condition where cells exhibit the lowest frequency of SCEs.

(d) Oxygen tension. Oxygen and oxygenderived free radicals are known to play an important role in mutagenesis, carcinogenesis and aging. Production of DNA damage and the potentiation of the effects induced by other clastogens have been confirmed for oxygen in a number of different cell types (Fridovich, 1978). In higher plants, Andersson and co-workers (1981) studied t h e capacity of X-rays to produce SCEs under aerobic and anaerobic conditions. They found that X-ray-induced SCEs were only slightly potentiated by oxygen. In contrast with this finding, Guti6rrez and Schvartzman (1981) noted that visible light irradiation of BrdUrd-substituted onion root tips, when performed in a nitrogen atmosphere, does not produce the high yields of SCEs otherwise induced when the same damaging treatment occurs in air. They interpreted this result as an indication that oxidizing free radicals play an important role in SCE formation, The oxygen dependence of SCEs was further analyzed by Guti6rrez and co-workers (1982, 1984, 1985). These authors incubated root meristems of Allium cepa in the presence of 5-100% oxygen and studied SCEs at the second and third division after BrdUrd substitution. These experiments clearly showed that SCE frequency increases as a function of

oxygen tension. It was suggested that oxygen seems to provoke short-lived lesions, and that oxygendependent SCEs are formed by exchanging postreplicative DNA portions. However, these studies do not address the question of whether oxygen only induces DNA lesions which, if not properly repaired, could give rise to SCEs, or if the effect is exerted during DNA replication, at the time when SCEs are thought to be formed. This question turns out to be highly relevant when considering the mechanism(s) involved in SCE formation.

(e) DNA damage. Matin and Prescott (1964) were the first to analyze the possible correlation between DNA damage and the frequency of SCEs. However, they used X-rays to provoke DNA breakage and autoradiography to visualize SCEs. Consequently, their results were not considered conclusive. Later on, Kato (1973) showed that UV light can lead to a dramatic increase in SCE frequency. Finally, Perry and Evans (1975) using the BrdUrd method, clearly established that different damaging treatments can induce SCEs with varying efficiency. They also concluded that SCEs are the most sensitive cytological method to detect potential clastogenic agents. It was demonstrated quite early that in order to increase the frequency of SCEs, damaged cells must pass through the S phase of the cell cycle (Wolff et al., 1974). In agreement with this notion, Schvartzman and Guti6rrez (1980) demonstrated that the shorter the time interval between damage induction and DNA replication the higher the efficiency of a damaging treatment in provoking the formation of SCEs. In other words, the highest increase over baseline is obtained when damage induction coincides with the beginning of the S phase (Latt and Loveday, 1978; Schvartzman and Guti6rrez, 1980). These observations led to the notion that SCEs are formed as a consequence of the replication of damaged templates. However, as we will see later on, although this is probably the most important mechanism it is not the only one that may lead to SCEs. Kihlman (1975) and Kihlman and Sturelid (1978) analyzed the ability of several chemical mutagens to produce SCEs in Vicia faba. With only the exception of bleomycin, they confirmed the observation that most chemical mutagens in-

138 crease the frequency of SCEs with varying efficiency. Interestingly enough, they also showed that caffeine post-treatments have no significant effect on the induced frequency of SCEs. Similar observations were made by Friebe and co-workers (1982) who studied the effects of caffeine pre- and post-treatments on UV light-induced SCEs in Secale cereale. In contrast, Hern~ndez and Guti~rrez (1985) confirmed previous observations (Guglielmi et al., 1982) suggesting that caffeine significantly increases the baseline frequency of SCEs in the absence of damaging treatments. As this effect is limited to and occurs throughout the S phase with the same efficiency, they suggested that caffeine's induction of SCEs takes place via an alteration in the pattern of DNA replication. Induction of SCEs by visible light on BrdUrdsubstituted chromosomes has been studied in detail in Allium cepa root tips. Schvartzman and co-workers (1979b) first demonstrated that irradiation of BrdUrd-substituted cells with the visible light produced by an incandescent bulb, leads to a considerable increase in the frequency of SCEs. That the DNA lesions provoked by this treatment were readily repaired was shown by studying the capacity of the same treatment to induce SCEs when irradiation took place at different times throughout 2 and 3 consecutive cell cycles (Schvartzman and Gutirrrez, 1980). More recently, Maldonado and co-workers (1985) showed that inhibition of u r a c i l - D N A glycosylase dramatically potentiates the effect of visible light irradiation on BrdUrd-substituted chromosomes. They interpreted this result as an indication that uracil a n d / o r some product of its repair could be the DNA lesion responsible for SCE formation under those experimental conditions. This interpretation is supported by the observation that incorporation of deoxyuridine monophosphate (dUMP) into DNA also leads to a significant increase in the yield of SCEs (Pardo et al., 1987). (f) Persistence of DNA lesions. The observation that both the type of lesion induced and the cell's repair capability are crucial in determining the effectiveness of a damaging treatment in provoking the formation of SCEs, led Wolff (1981) to suggest that one of the most critical factors would be the lifetime of the induced lesions. Conse-

quently, a number of workers decided to analyze DNA damage persistence using several methods (see Tice and Schvartzman, 1982; Schvartzman et al., 1984). In higher plants persistence of DNA lesions through several cell generations has been studied only in the case of visible light-irradiated BrdUrd-substituted onion root tips (Schvartzman et al., 1979b). The results obtained strongly suggest that visible light-induced DNA lesions are rapidly repaired as they do not lead to significant SCE yields one cell cycle time after irradiation. Nevertheless, as different laboratories used different methods and experimental protocols, the results obtained concerning persistence of several other specific DNA lesions often appear to conflict (see Schvartzman et al., 1984). Moreover, another possible complication in these studies is SCE cancellation. Indeed, Stetka (1979) first pointed out that if a DNA lesion that elicited the formation of a SCE persists through several cell generations, it may elicit the formation of more than one SCE at the same chromosomal locus. This would result in the cytological cancellation of at least some of such SCEs. Schvartzman and co-workers (1985) specifically designed an experiment to test this hypothesis using the '3-waydifferentiation' method (Schvartzman, 1979; Schvartzman and Goyanes, 1980). They concluded that in human lymphocytes neither UV light nor ionizing radiation or mitomycin C leads to the formation of consecutive SCEs at the same locus in successive cell generations. The consequence of DNA lesion persistence on SCEs is yet to be defined. Probably, one of the most important questions is whether or not the occurrence of an SCE involves the repair of the lesion(s) that provoked its formation (Kato, 1977a). Until an answer to this question is found, the real importance of DNA lesion persistence will remain a speculation. (9) The SCE test in cytogenetic toxicology As already mentioned, SCEs are the most sensitive cytological method to detect potential clastogenic agents. In addition, the advantages of using plant root meristems to detect SCEs induced by mutagens and carcinogens have been repeatedly enumerated (Kihlman, 1982; Kihlman and Andersson, 1982, 1984; Grant, 1982; Ugla and

139

Natarajan, 1982). Table 7 presents a list of some of the chemical as well as physical treatments that have been shown to increase the yield of SCEs in BrdUrd-substituted higher plant cells. Treatments have been ordered according to their efficiency in provoking an increase over baseline at the second division after BrdUrd substitution. Note that according to this data, quinacrine mustard (QM) appears to be the most efficient SCE inducer in higher plants.

(10) S ister-chromatid differentiation (S CD) and sister-chromatid exchanges (SCEs) related to the meiotic process There is no publication available where plant meiosis has been studied using the BrdUrd method. However, the possible relationship between chiasma formation and SCE has been approached in a number of plant species. Friebe (1980a) showed that the presence of B-chromosomes which have a pronounced effect on meiotic pairing and crossing-over, does not influence the yield of SCEs

in somatic cells of Secale cereale. He also showed that the SCE frequency is quite similar in wild, cultivated and inbred lines of rye that are known to differ significantly in chiasma frequency (Friebe, 1980b). Finally, Linnert and co-workers (1981) further confirmed this apparent lack of a relationship between crossing-over and SCE in normal and asynaptic mutants of Vicia faba. In spite of these observations, the molecular mechanisms involved in the SCE formation and meiotic crossing-over might still be similar, since in a strict sense both processes involve the recombination between two DNA duplexes (Whitehouse, 1982).

(11) The mechanism of SCE formation Despite the huge amount of information on SCEs that has been generated throughout the last decade (see Table 1), the mechanisms involved in the formation of SCEs are still a matter of speculation. The observation that DNA damage increases the frequency of SCEs led many cytogeneticists to

TABLE 7 CHEMICAL A N D PHYSICAL TREATMENTS THAT HAVE BEEN SHOWN TO INCREASE THE YIELD OF SCEs IN H I G H E R PLANT CELLS Treatment

Species

Increment factor over baseline

Reference

QM MH MMS EMS MNNG MH UV VL

Vicia faba Vicia faba Vicia faba Vicia faba Vicia faba Allium cepa Secale cereale Allium cepa Vicia faba Vicia faba Alliurn cepa Allium cepa Alliurn cepa Allium cepa Alliurn cepa Allium cepa Allium cepa Vicia faba Allium cepa

5.2 ×

Kihlman and Sturelid (1978) Kihlman and Sturelid (1978) Kihlman and Sturelid (1978) Kihlman and Sturelid (1978) Kihlman and Sturelid (1978) Cort6s et al. (1985) Friebe et al. (1982) Schvartzman et al. (1979b) Kihlman and Sturelid (1978) Kihlman and Sturelid (1978) Maldonado et al. (1985) Escalza et al. (1985) Armas-Portela et al. (1985) Cort6s and Hazen (1984) Schvartzman and Hernhndez (1980) Guti6rrez et al. (1981) Cort6s and Hazen (1984) Andersson (1981) Hernhndez and Guti6rrez (1983)

TT

MMC V L + 6-AU FdUrd Pyronin Y + green light Caffeine 5-AU 5oC

Pyronin Y Streptonigrin 35 o C

4.8 × 4.3 × 4.2 × 4.2 × 3.8 X 3.6 x 3.5 × 3.4 ×

3.3 × 2.8 × 2.5 × 1.9 × 1.8 ×

1.7 × 1.7 × 1.6 × 1.4 x 1.3 x

QM, quinacrine mustard; MH, maleic hydrazide; MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; UV, ultraviolet light; VL, visible light; "IT, thiotepa; MMC, mitomycin C; 6-AU, 6aminouracil; 5-AU, 5-aminouracil.

140 believe that the mechanisms involved in SCE formation should be related to those operating in the generation of chromosomal aberrations. In this regard, Revell's (1959) hypothesis to explain the induction of chromosomal aberrations by clastogens played an important role in prompting several groups to test whether or not aberrations and SCEs arise via the same molecular process. It was repeatedly shown that whereas a caffeine posttreatment dramatically potentiates the frequency of clastogen-induced aberrations, it has little or no effect on clastogen-induced SCEs (see Kihlman et al., 1977, 1978; Schvartzman and Hern~ndez, 1980; Kihlman and Andersson, 1982). This observation led to the notion that aberrations and SCEs are produced by mechanisms that are at least partly different. On the other hand, Lindenhahn and Schubert (1983) found that 50% of the chromatid breaks induced by hydroxyurea during the G 2 phase of the cell cycle are associated with an SCE. This and other previous observations (Schubert and Meister, 1979) led them to conclude that under certain conditions, aberrations may originate in close connection with SCEs. There is no doubt that SCEs arise during D N A replication (Wolff et al., 1974; Latt and Loveday, 1978; Schvartzman and Gutirrrez, 1980). However, whether they are formed at the replication site (Kato, 1980b) or far away, at the site where nascent replicons or replicon clusters join (Painter, 1980) is pure speculation. Nevertheless, the observation that inhibition of DNA synthesis during G 2 induces SCEs (Andersson, 1983) supports the latter hypothesis. Nascent replicon maturation has been shown to occur precisely during late-S and the G 2 phase of the cell cycle (Funderud et al., 1978; Schvartzman et al., 1981). In short, SCEs appear to be formed as a consequence of an alteration in the pattern of DNA replication and maturation. These alterations may be due to the presence of DNA lesions, dNTPs pool imbalance, D N A synthesis inhibition, etc. In any case, it seems to be clear that the molecular mechanisms involved in the formation of aberrations and SCEs, although they may be partly common in some cases, are completely independent in others.

(V) What is next in SCE research? There is no doubt that many questions on SCEs still need to be answered. It seems to me though, that most of the information on SCEs that could possibly be obtained using the BrdUrd method has already been obtained. Therefore, the only way to break this situation appears to be the development of new techniques that would allow scientists to approach new or even the same problems but from a different perspective. However, this is not an easy task. From a molecular point of view SCEs are extremely rare events and DNA damage appears to be highly inefficient in inducing SCEs. The situation is completely different concerning the utilization of the SCE test in cytogenetic toxicology. In this field there is certainly a lot to be done. However, it is not completely clear to me how this kind of work can lead to a better understanding of SCEs. It appears as if the current SCE boom is coming to an end. However, science progresses as a series of discontinuous bursts. Therefore, it can be assured that we are now closer than ever before to the SCE burst that can be expected in the near future.

Acknowledgements SCE studies have been reviewed many times during the last decade (Wolff, 1977; Kato, 1977b; Evans, 1977; Schneider et al., 1978; Latt, 1979, 1981; Latt et al., 1979). In addition there are 3 books that are entirely devoted to SCEs (Wolff, 1982; Sandberg, 1982; Tice and Hollaender, 1984). Two of these books include specific chapters on SCEs in higher plants (Kihlman and Andersson, 1982; Uggla and Natarajan, 1982). The present article was also grounded on those studies where SCEs have been analyzed in higher plant cells. However, while discussing some of the problems that have been approached by plant cytogeneticists using the BrdUrd method, a number of pioneer reports that have been carried out using animal cells were also discussed. Nevertheless, because of space limitations it was impossible to comment on every subject in depth as well as to quote every reference on each given subject. For

141 all t h e o m i s s i o n s I a d d r e s s t h e r e a d e r t o a n y o f t h e aforementioned reviews. To field whose names are not a p o l o g i e s . I w o u l d like t o and Crisanto Gutirrrez for

those scientists in the mentioned I offer my thank Raymond Tice critical reading of the

manuscript. Finally, this work was partially supported by the 'Comisibn Asesora para la Investigaci6n Cientlfica y Tecnol6gica' (CAICYT).

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