Mutagenesis, Vol. 16, No. 1, 85-89,
January 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Termini of human chromosomes display elevated rates of mitotic recombination
Department of Radiation Oncology, 344 Gail Borden Building, University of Texas Medical Branch, Galveston, TX 77555-0656, USA
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Abstract |
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The strand-specific in situ hybridization technique of CO-FISH was used to probe telomeres of human mitotic cells in order to determine the spontaneous frequency of crossover. This approach allowed the detection of recombinational crossovers occurring anywhere along the length of individual chromosomes, including reciprocal events taking place between sister chromatids. Although the process of sister chromatid exchange (SCE) is the most prominent type of recombination in somatic mammalian cells, our results show that SCEs accounted for less than a third of the recombinational events revealed by CO-FISH. It is concluded that chromosomal regions near the termini of chromosome arms undergo extraordinarily high rates of spontaneous recombination, producing terminal crossovers whose small size precludes detection by standard cytogenetic methods. That similar results were observed for transformed epithelial cells, as well as primary fibroblasts, suggests that the phenomenon is a common characteristic of human cells. These findings are noteworthy because, although telomeric and subtelomeric DNA is known to be preferentially involved in certain types of recombination, the tips of somatic mammalian chromosomes have not previously been identified as preferred sites for crossover. Implications of these results are discussed in terms of limitations imposed on CO-FISH for its proposed use in directional hybridization mapping.
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Introduction |
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By capping the otherwise reactive ends of linear chromosomes, telomeric sequences are thought to protect the genome from untoward acts of recombination. In seeming contradiction to this role, telomeric, subtelomeric and telomere-like sequences of chromosomes from a number of organisms have repeatedly been shown to behave as `hot-spots' for various types of recombination (Alvarez et al., 1993
; Bouffler et al., 1993, 1996; Murnane and Yu, 1993; McEachern and Blackburn, 1996; Preston, 1997; Riboni et al., 1997; Wintle et al., 1997; Day et al., 1998; Bailey et al., 1999).
Arguably, the most prominent source of mitotic recombination in mammalian cells is that from sister chromatid exchanges (SCEs). Because standard cytogenetic techniques used to measure SCEs are considered quite robust in their ability to detect terminal crossover events, it is significant that the distribution of SCEs along the length of chromosomes does not show a telomeric bias (Crossen et al., 1977). Based on these findings, it is reasonable to conclude that telomeric regions of mitotic chromosomes are not unusually susceptible to crossover. A plausible alternative explanation is that conventional cytogenetic methods simply lack the resolution to discern putative crossovers that cluster at the tips of chromosomes. To examine this possibility, we employed the technique of chromosome orientation–fluorescence in situ hybridization (CO-FISH) (Goodwin and Meyne, 1993) to mark the telomeres of sister chromatids. CO-FISH is a strand-specific in situ hybridization technique that can be used to determine the 5'
3' polarity of a nucleotide target sequence with respect to the pterqter orientation of the chromosome (Meyne and Goodwin, 1995; Bailey et al., 1996a,b). This allowed us to detect crossover events (including SCEs) that occurred anywhere between the p and q termini of a given chromosome (see Figure 1).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The spontaneously transformed EJ30 human male epithelial cell line was a gift from Charles Waldren (Colorado State University). AG1521 normal primary human foreskin fibroblasts were obtained from the Coriell Institute for Medical Research (Aging Cell Repository, Camden, NJ). Both cell types were cultured in
-MEM (Gibco BRL, Grand Island, NY) containing 10% fetal bovine serum and were maintained, under humidified conditions, in 5% CO2, 95% air.
The CO-FISH procedure of Goodwin and colleagues (Meyne and Goodwin, 1995; Bailey et al., 1996a,b) was used, with the following modifications. Cells were synchronized at the G1/S boundary (Tobey et al., 1988), then allowed to progress through S phase and into the first mitosis in the presence of 30 µM bromodeoxyuridine (BrdU) and 10 µM bromodeoxycytidine (BrdC) (Sigma Chemical Co., St Louis, MO). Colcemid (Gibco BRL) was added to a final concentration of 0.1 µg/ml during the last 3–4 h of culture. Cells destined for SCE analysis were cultured in precisely the same way, but were allowed to undergo the requisite two replication rounds in BrdU and BrdC to achieve sister chromatid differentiation. Cells were suspended using trypsin/EDTA solution (Gibco BRL), washed in serum-free medium, fixed in 3:1 methanol:acetic acid and spread onto glass microscope slides, all using routine cytogenetic techniques. Differential staining of sister chromatids was performed by the standard FPG technique (Perry and Wolf, 1974).
For CO-FISH, newly synthesized DNA strands were degraded by treating fixed cells on glass microscope slides with 0.5 µg/ml Hoechst 33258 (Sigma Chemical Co.), 313 nm light for 30 min, and 3 U/µl Exo III (Gibco) at room temperature for 5 min. Tel(+) probes were made by end-labeling (TTAGGG)8 synthetic oligomers with Cy3-dUTP (Amersham, Arlington Heights, IL) using terminal transferase (Promega, Madison, WI). Complementary (CCCTAA)8 tel(–) probes were similarly tailed with digoxigenin-dUTP (Boehringer-Mannheim, Indianapolis, IN); hybridization was detected with fluorescein-conjugated anti-digoxigenin (Boehringer-Mannheim). Microscope images were captured via a CFG framegrabber (Imaging Technology, Bedford, MA), using a TEC-470 color CCD camera (Optronix, Goleta, CA) interfaced to a PC running Optimus v.4.10 software (Optimus Corp., Bothell, WA). Digital enhancement was confined to adjustments in contrast and/or color balance.
The vast majority of chromosomes within a given metaphase spread showed characteristic CO-FISH hybridization patterns, in which telomere signals at opposite ends of the chromosome were either trans or cis with respect to sister chromatids (see Results and Figure 1
). The trans/cis status of each chromosome was scored individually. Metaphase spreads often contained a few random chromosomes that failed to show full CO-FISH hybridization, either because one of the two signals was obscured by overlaps with other chromosomes or because of a weak probe signal. Such chromosomes were excluded from analysis.
Unlike CO-FISH, the visualization of SCEs requires two rounds of DNA synthesis following the introduction of deoxynucleoside analogs (Taylor, 1958; Latt, 1973). For this reason, the `first round' SCE frequencies reported in Table I are exactly half those actually observed. It should be noted that the contribution of SCEs from the first S phase is equal to (Wolff and Perry, 1975; Ponzanelli, 1997) or less than (Steka and Spahn, 1984) that from the second S phase. Consequently, values reported in Table I are, if anything, somewhat conservative regarding discrepancies that arise between cis patterns and first round SCEs.
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For the purposes of correcting cis frequencies for multiple crossovers, SCE formation can be adequately described as a random process (Tucker et al., 1986; data not shown). In this case, the frequency of SCEs that would be necessary to account for the number of cis events represents the mean (µ) of a Poisson distribution, whose sum of odd numbered terms equals the observed cis frequency (
). This is represented by the by the following expression:
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which is equivalent to:
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The latter equation was used to convert observed cis frequencies into estimates of the true crossover frequency, also referred to as the SCE equivalent frequency (Figure 3).
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Results |
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The (TTAGGG)n telomeric repeat that caps vertebrate chromosomes (Meyne et al., 1989
) has the unique property of being orientated 5'3' towards the termini of all chromosome arms (Blackburn and Szostak, 1984). Probing these sequences by CO-FISH produced a characteristic `trans' hybridization pattern in the majority of chromosomes, in which labeling was confined to the p terminus of one chromatid and the q terminus of its sister (Figures 1 and 2a and c). Crossover events occurring anywhere along the length of a chromosome, including those deriving from spontaneous SCEs (Figures 1 and 2b and f), are expected to cause the affected chromosome to exhibit the reverse `cis' pattern of hybridization, in which fluorescent signals appear at both termini of the same chromatid (Figure 2a and e).
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The key result of this investigation, summarized in Table I
, is that the frequency of SCEs is far too low to explain the number of transcis pattern switches that were observed by CO-FISH. For example, in the transformed diploid epithelial cell line EJ30 nearly one in five chromosomes (0.19) exhibited the cis CO-FISH configuration, whereas the same cell line yielded only 0.065 SCEs/chromosome. This 3-fold discrepancy is apparently not limited to cell type or to the transformed state, since similar results were observed for AG1521 primary human fibroblasts.
We examined three potential sources of artifact to determine whether they might explain the observed excess of cis signals relative to SCEs. First, while all chromosome groups (A–G) clearly displayed the characteristic trans or cis CO-FISH hybridization patterns, we considered the possibility that certain chromosomes might be more consistent in that regard, causing them to be inadvertently over-sampled. If these same chromosomes also tended to undergo SCE at higher than average rates, then inflated estimates of transcis switching could result. Such a situation could conceivably arise, for example, if a subset of larger chromosomes had a tendency to produce more robust hybridization signals, since SCE frequency is a function of chromosome length (Morgan and Crossen, 1977). To investigate this possibility, analysis was performed on two specific chromosomes: the X chromosome, a rather average sized chromosome, and chromosome 1, the largest. These make up 2.5 and 4.1% of the AG1521 genome, respectively (Bartholdi, 1985). The observed first round SCE frequency of 0.064 per chromosome corresponds to that of an `average sized' chromosome, representing 1/46th (2.2%) of the genome. Assuming that SCE formation is roughly proportional to chromosome size (Morgan and Crossen, 1977), ~0.075 SCEs per X chromosome would be predicted, which is similar to the frequency of 0.071 actually observed. Although we did not directly measure SCEs in chromosome 1, by similar argument the predicted frequency per homolog is 0.123 (Table I). [This estimate is also in remarkable agreement with the data of Morgan and Crossen (1977), who reported that 8.4% of spontaneous SCEs in human male lymphocytes were found amongst chromosome 1 homologs. Applying this figure to our own measurements implies a SCE frequency of 0.124 per homolog per first round division.] Thus, as shown in Table I, the SCE frequency directly observed in the X chromosome, as well as that predicted to occur in chromosomes X and 1, accounted for less than half the corresponding cis patterns. These results strongly suggest that most, if not all, chromosomes express a large excess of cis signals relative to visible SCEs.
As no in situ hybridization technique is 100% efficient, it was not unusual for a metaphase spread to contain a few chromosomes that showed only one of the expected two telomeric CO-FISH signals. Lack of visible probe hybridization to a targeted telomere sequence, coupled with non-specific hybridization to the corresponding (untargeted) site on the sister chromatid, could produce a spurious cis pattern. To determine whether this second type of artifact may have produced the observed excess of cis hybridizations, digital images were first made of 973 individual chromosomes showing either the cis or trans patterns. After they were stripped of their probe signals, chromosomes were rehybridized to a single-stranded sequence that was complementary to the original telomere probe [i.e. (CCCTAA)n]. Because the two probes were labeled using different fluorochromes, the first and second hybridization patterns could be unambiguously distinguished from one another (Figure 2c and e). We then compared the original CO-FISH pattern for each individual chromosome with that produced by the second complementary hybridization. Results showed that the location of CO-FISH signals from the two hybridizations were in perfect `mirror image' opposition to one another 98% of the time and that none of the cis patterns observed after the first hybridization became trans following the second probing. This demonstrates that cis patterns do not arise from non-specific hybridization.
The third potential source of artifact that we considered involves the spreading of mitotic cells onto microscope slides during specimen preparation, which can sometimes cause sister chromatids to twist and overlap one another (Figure 2f). It has been suggested that overlaps near the centromere can occasionally be mistaken for SCEs (Becher and Sandberg, 1983). In the context of the present investigation, mistakes of this sort can be considered `self-correcting', in the sense that any twist causing a `pseudo SCE' should also produce a corresponding `pseudo cis' event. However, because centromeric twists are somewhat harder to confirm in fluorescent preparations compared with bright field staining, it is conceivable that a disproportionate number of pseudo cis patterns could result. The maximum effect that this sort of artifact could introduce was estimated by rescoring SCEs in both cell lines; this time all chromatid color switches were scored as SCEs, including those clearly associated with chromatid twisting near the centromere. The intentionally biased analysis caused estimates of SCE frequencies to increase by <10% compared with the original values, which is insufficient to account for the large excess of in cis frequencies.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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After considering various sources of potential artifact, we conclude that the large majority of cis patterns do not derive from microscopically visible SCEs. Instead, we propose that the `unexplained excess' of cis patterns represents mitotic recombination that occurs near the tips of chromosomes, producing terminal crossovers too small to be seen by standard cytogenetic techniques, such as those used to measure SCEs. We imagine that such recombination involves the symmetrical exchange of material between sister chromatids, via a mechanism similar to that of SCE formation; the excess cis signals may, in fact, derive from cryptic SCEs. Presently, however, we cannot rule out other possibilities, including that the process is occurring between the telomeric/subtelomeric regions of different chromosomes. Of possible interest in this regard is the observation that the non- homologous end joining protein DNA-dependent protein kinase seems to protect the telomeres of mouse chromosomes from illegitimate recombination (Bailey et al., 1999
). The very smallest (i.e. most terminal) crossovers that were directly observable as SCEs involved stretches of chromatin representing 2.5% of the length of chromosome 1 (data not shown), suggesting that crossovers responsible for the cis excess are probably confined to subtelomeric regions that extend some 7 Mb from the telomere proper. This implies a recombination rate within the regions in question of the order of 10–2/Mb/cell generation. Other than perhaps the specialized site-specific processes governing antibody diversification, we believe this to be highest rate of spontaneous mitotic recombination reported thus far for mammalian cells.
It is important to recognize that multiple SCEs that occur on the same chromosome are easily observable by conventional cytogenetic methods, but that only an odd number of crossovers can produce cis patterns. Thus, double SCEs (dark field image in Figure 2f) are undetectable by CO-FISH and (rare) triple SCEs will yield only a single visible transcis switch. For this reason, cis frequencies tend to underestimate the true frequency of crossovers. To more properly quantify the shortfall of SCEs in explaining cis patterns, it is instructive to contrast the frequency of SCEs that would be required to produce all cis patterns (which we term the `SCE equivalent') against the observed SCE frequency (see Materials and methods). Since SCE equivalent frequencies generally exceed the frequency of observable cis events (Figure 3), a consideration of multiple crossovers serves to further magnify the shortfall of SCEs in explaining cis patterns, as shown in Table I. As applied to the range of frequencies for cis patterns that were actually observed (i.e. 0.17–0.26), correction factors range from 1.2 to 1.4, respectively (Figure 3). Although these corrections are not insignificant, it should be stressed that the major conclusions of this work do not depend on their use. In EJ30 cells, for example, the estimated fraction of cis patterns that are actually attributable to SCEs is only about one fourth (1/3.7) when corrected for multiple events, whereas it is roughly one third (1/2.9) when the correction is ignored. It is worth noting, however, that multiplicity corrections become increasingly severe as the frequency of observed cis events rises. As a hypothetical example, an observed frequency of 0.4 cis events per chromosome belies a true (estimated) frequency of recombination of about 0.8. Owing to the shape of the correction function, it would become increasingly difficult to specify upper bounds for estimates of the true crossover frequency when the observed cis frequency approaches 0.5 per chromosome. Consequently, CO-FISH may be of limited value for the analysis of cells with exceedingly high levels of SCEs or other crossover events.
Unlike telomere probes, which produce the characteristic trans or cis hybridization patterns amongst sister chromatids, when a complementary single-stranded probe is directed to a unique target locus (such as a gene), CO-FISH produces a single signal on only one chromatid (Goodwin and Meyne, 1993). This forms the basis for its proposed use in a related area of investigation, directional hybridization analysis, which includes the detection of cryptic inversions (Bailey et al., 1996a,b). CO-FISH easily detects inverted target sequences, because the inversion will cause the hybridization signal to `jump' from one chromatid to its sister at the same locus. In practice, because the `face-up' versus `face-down' orientation of a chromosome cannot otherwise be known with respect to the planar surface of the microscope slide, such signal switching can only be detected by comparing the target hybridization pattern against that of a concomitant secondary CO-FISH `reference' probe. Because they produce robust hybridization signals at the termini of all vertebrate chromosomes and because their absolute 5'3' direction is always known with respect to the pq arm orientation of the chromosome, it has been suggested that telomeric sequences are ideally suited for this purpose (Bailey et al., 1996a,b).
The spontaneous incidence of crossovers impacts on the use of telomere sequences as reference probes for CO-FISH in two ways. A relatively trivial problem is the frequency of telomere-cis chromosomes themselves, which represents a sizeable minority of the number of chromosomes one must examine (Table I). These would typically be discarded during directional analysis, since it is not possible to know which of the two telomeric reference signals, against which the orientation of the primary target sequence is judged, has undergone the crossover. A somewhat more serious problem is that of double crossovers, which produce a telomere-trans configuration that is indistinguishable from the native non-recombinant state, but which can also cause erroneous results by altering the relationship between target and reference hybridization signals. Presently, there is no way of identifying and eliminating such erroneous signal patterns beforehand. It is therefore important that enough chromosomes are examined from the sample population to ensure that the modal hybridization pattern that emerges represents the true consensus pattern of the native non-recombinant chromosome. Fortunately, for any reasonable set of assumptions the frequency of double crossover telomere-trans chromosomes is low enough, ~5% under a worst case scenario, such that reasonably small sample sizes are adequate. For example, based on the data presented in Table I, our calculations indicate that a CO-FISH sample of eight chromosomes is virtually guaranteed (P 
0.99) to contain enough telomere-trans chromosomes to establish the true consensus pattern between target and telomere reference signals.
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Acknowledgments |
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We thank Rainer Sachs and Stephen Marino for help in deriving the equation used to correct for crossover multiplicity, Edwin Goodwin and Susan Bailey for valuable insights and discussions and Bradford Loucas for help in preparation of the manuscript. This work was supported by John Sealy Research and Development award 2514-98 and by grant CA76260 from the National Institutes of Health.
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Notes |
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1 To whom correspondence should be addressed. Tel: +1 409 772 4244; Fax: +1 409 772 3387; Email: mcornfor{at}utmb.edu
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References |
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Received on July 12, 2000; accepted on July 13, 2000.
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