Mutagenesis, Vol. 16, No. 2, 145-149,
March 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Chromosomal composition of micronuclei in mouse NIH 3T3 cells treated with acrylamide, extract of Tripterygium hypoglaucum (level) hutch, mitomycin C and colchicine, detected by multicolor FISH with centromeric and telomeric DNA probes
1 Molecular Toxicology Laboratory, Third Military Medical University, Chongqing 400038, China
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The chromosomal composition of micronuclei (MN) induced by the model mutagens mitomycin (MMC) and colchicine (COL) as well as by acrylamide (AA) and the traditional Chinese medicine Tripterygium hypoglaucum (level) hutch (THH) in NIH 3T3 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using DNA probes for the centromere repeated minor satellite DNA and the telomeric hexamer repeat (TTAGGG). The majority of MN (78.6%) from treatment with MMC (0.1 µg/ml) did not show centromeric signals, reflecting the clastogenic action of MMC. Following treatment with COL (0.1 µg/ml), 74.5% of the MN showed centromeric signals and several telomeric signals, indicating that MN induced by this well-known aneugen were mainly composed of whole chromosomes. After treatment with AA (100, 200 and 400 µg/ml) both MN containing whole chromosomes and MN containing acentric fragments were found to increase in a dose-dependent manner, demonstrating that AA is not only a clastogen but also an aneugen. THH induced a high frequency of MN harboring whole chromosomes at all concentrations tested (5, 10 and 20 µl/ml) and produced a dose-dependent increase in fragment-containing MN, indicating that THH has both aneugenic and clastogenic potential.
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Introduction |
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Micronuclei (MN) can arise from acentric fragments induced by substances causing chromosomal breakage (clastogens) as well as from whole lagging chromosomes induced by those causing aneuploidy (aneugens). However, with the conventional micronucleus test it is impossible to distinguish between these two events. Various techniques have been developed to distinguish MN induced by clastogens or by aneugens, including measuring the size or DNA content of MN (Yamamoto and Kikuchi, 1980
; Grawé et al., 1994), C-banding to stain the centromeric heterochromatin (Verschaeve et al., 1988; Van Hummelen et al., 1992), staining with CREST antibody to identify the kinetochore (Miller et al., 1991; Channarayappa et al., 1992; Schriever-Schwemmer and Adler, 1994; Renzi et al., 1996), fluorescence in situ hybridization (FISH) with DNA probes that hybridize with the centromeres of chromosomes (Becker et al., 1990; Miller et al., 1991; Farooqi et al., 1993; Hayashi et al., 1994; Migliore et al., 1996) and FISH simultaneously using telomeric and centromeric DNA probes (Miller et al., 1992; Miller and Nüsse, 1993; Schriever-Schwemmer and Adler, 1994). Among these methods, FISH with a centromeric DNA probe allows detection of corresponding chromosomal regions on metaphase chromosomes as well as in interphase nuclei and, specifically, individual MN and the probe can be produced in unlimited amounts at any time and preparative alterations rarely affect centromeric DNA. A lot of research has indicated that the FISH method is an accurate technique to identify the composition of MN. FISH combining centromeric and telomeric probes can increase this accuracy, because it is intended to further characterize the content of MN (Kirsch-Volders et al., 1997).
For mouse centromere detection the major DNA probe was used primarily. This probe hybridizes to the pericentric heterochromatin of all chromosomes except Y in a one-dot pattern. Another centromere probe is the minor DNA probe, which labels all chromosomes except Y at the centromere in the kinetochore region with two dots. The minor probe is recommended for FISH detection of MN containing lagging chromosomes because it is shorter and labels closer to the centromere (Chen et al., 1994; Schriever-Schwemmer and Adler, 1994; Grawé et al., 1997; Hayashi et al., 2000). Telomeres represent repetitive tandem arrays of a hexanucleotide sequence (TTAGGG) that are highly conserved through evolution and necessary for chromosome stability and DNA replication. Telomere probes show an exclusively telomeric labeling pattern in the mouse (Meyne et al., 1990). In the present study colchicine (COL) and mitomycin C (MMC) were used as positive controls for aneugens and clastogens (Schriever-Schwemmer and Adler, 1994; Schriever-Schwemmer et al., 1997; Renzi et al., 1996; Sgura et al., 1997). The suspected aneugen acrylamide (AA) and Tripterygium hypoglaucum (level) hutch (THH) were tested for their possible aneugenicity and clastogenicity. AA is an industrial chemical which has shown clastogenic effects in somatic and germinal cells and recently studies in mouse bone marrow and spermatocytes indicated that AA has aneugenic potential (Gassner and Adler, 1996; Schriever-Schwemmer et al., 1997). THH is a medicinal herb that has been used to effectively treat various immunological diseases in China, such as systemic lupus erythematosus, rheumatoid arthritis, etc. However, Wang et al. (1993) observed that THH showed similar genotoxic effects to COL in mouse bone marrow cells, which showed a significant increase in MN and positive C-mitotic effects, but no structural chromosome aberrations. Other studies in our laboratory have indicated that THH induces HPRT gene mutations dose-dependently in the HL-60 cell line. Molecular analysis indicated that THH induces 47.6% deletions and 52.4% point mutations.
In the present study MN were induced in mouse NIH 3T3 fibroblasts by COL, MMC, AA and THH and the chromosomal compositions of the induced MN were analyzed by FISH using mouse telomeric and minor DNA probes simultaneously.
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Materials and methods |
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Cell culture and treatment with mutagens
NIH 3T3 cells (permanent mouse fibroblasts) with a cell cycle of ~22 h were grown in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (1:1 ratio; Gibco BRL, Eggenstein, Germany) supplemented with 1% glutamine and 10% fetal calf serum (Gibco BRL). The cell line was a mixture of mostly hypertriploid cells (n = 68 chromosomes) and ~10% diploid cells (n = 40 chromosomes).
COL, MMC and AA were purchased from Sigma Chemical Co. (St Louis, MO). The dry root of THH was purchased from Yun Nan Medical Co. (Kun Ming, China) and prepared as proposed by Wang et al. (1993). Samples of 10 g THH were extracted in 100 ml of boiling distilled water and then the mixture was steeped at room temperature for 30 min. To ensure full extraction, the procedure was repeated three times. The supernatant was filtered with 0.8 and 0.2 µm filters and concentrated to 5 ml; 1 ml of concentrated extract was equal to 2 g THH herb. One concentration of MMC (0.1 µg/ml) and COL (0.1 µg/ml) and three concentrations of AA (100, 200 and 400 µg/ml) and THH (5, 10 and 20 µl/ml) were used in this study. All three chemicals were dissolved in bi-distilled water and filtered with 0.2 µm filters before treatment.
Slide preparation
Metaphase chromosomes
Untreated NIH 3T3 cells were allowed to proliferate for 24 h at 37°C (5% CO2) in DMEM/F12 medium. Metaphases were arrested with colcemid (final concentration 5x10–7 M) for 2–3 h. Cells were trypsinized and spun down at 800 g for 7 min. About 5 ml of hypotonic solution (0.56% KCl) was added. After 20 min incubation at room temperature cells were spun down again and the pellet was fixed with Carnoy's fixative (methanol:acetic acid 3:1). The fixative was changed twice. Finally, the suspension was dropped onto slides that had been cleaned with chromium sulfonic acid. The slides were air dried and stored at –17°C until used.
MN
After a 48 h culture the cells were treated with the test chemicals. At intervals of 24 h after the treatment the cells were trypsinized, washed with fresh medium and centrifuged without any additional hypotonic treatment. The pellet was fixed with methanol:acetic acid (8:1) for 15 min. The fixative was changed twice. Then the cell suspension was dropped onto slides. The slides were air dried and stored at –17°C until used.
DNA probes
The murine minor satellite DNA probe pMKB6 (Wong and Rattner, 1988), a 273 bp fragment that represents approximately two tandem repeats in plasmid pRP855, was a gift from Prof. R.Pearlman (York University, Canada). It was propagated in Escherichia coli DH5
. The plasmid DNA was biotinylated by the random primed procedure according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany).
Hexamers of animal telomeric repeats (5'-TTAGGG-3') were synthesized as 30mer oligonucleotides in both the sense and antisense orientations, amplified using PCR (Ijdo et al., 1991) and labelled with digoxigenin-11-dUTP (Boehringer Mannheim). The use of biotin and digoxigenin as haptens allowed the use of two DNA probes simultaneously due to the availability of different detection systems for the haptens.
FISH
In situ hybridization was performed as described by Schriever-Schwemmer and Adler (1994) with slight modifications. Frozen slides were brought to room temperature and rehydrated in 2x SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0) for 5 min prior to treatment with 0.1% Triton X-100 in 2x SSC for 3 min. After washing the slides in 2x SSC for 2 min they were fixed in 4% paraformaldehyde for 10 min at room temperature and washed in 2x SSC for 5 min. Then the slides were denatured for 10 min in 70% formamide, 2x SSC, pH 7.0, at 72–74°C and dehydrated in an ice-cold 70, 90 and 100% ethanol series. After air drying the slides were brought to 37°C for <3 min on a warming plate. The hybridization mix contained seven parts master mix 1.0 (50% formamide, 10% dextran sulfate), one part centromeric probe (~20 ng/slide), one part telomeric probe (~12 ng/slide) and one part yeast tRNA (500 µg/ml). The hybridization mix was denatured for 5 min at 74°C and chilled on ice. Aliquots of 20 µl of the hybridization mix were added to the slides, covered with a 24x50 mm coverslip and sealed with rubber cement. Simultaneous hybridization was carried out overnight in a moist chamber at 37°C.
Signal detection
The slides were washed twice for 20 min in 30% formamide, 2x SSC, pH 7.0, at 42°C, followed by two washes for 15 min in PN buffer (0.1 M sodium phosphate, pH 8.0, 0.1% Nonidet P40) at 37°C and incubated with 40 µl PNBR (PN buffer plus 5% blocking reagent and 0.02% sodium azide; Boehringer Mannheim) for 10 min. The biotin-labeled centromeric probe was detected with 40 µl streptavidin–Cy3 conjugate (1:400, end concentration 5 µg/ml; Sigma) and the digoxigenin-labeled telomeric probe was visualized with 40 µl FITC-conjugated sheep anti-digoxigenin antibody (5 µg/ml; Boehringer Mannheim), all in PNBR buffer for 30 min at 37°C in a moist chamber. If necessary, the centromeric signals were amplified by one or two rounds of biotinylated anti-streptavidin (5 µg/ml in PNBR; Vector Laboratories, USA) and streptavidin–Cy3 conjugate. The telomeric signals were enhanced by FITC-conjugated rabbit anti-sheep antibody (5 µg/ml in PNBR; Vector Laboratories) and FITC-conjugated sheep anti-rabbit antibody (5 µg/ml in PNBR; Boehringer Mannheim). The cells were counterstained with Hoechst 33258 (0.5 µg/ml; Sigma). The slides were finally embedded in antifade solution (Johnson and Araujo, 1981) and were scored immediately or following storage for a few days at 4°C in the dark.
Scoring of the slides
Fluorescence microscopy was performed on a BX60 (Olympus) fluorescence microscope with filters for Cy3 (WIG, excitation at 510–550 nm), FITC (WIB, excitation at 470–490 nm) and Hoechst 33258 (WU, excitation at 330–385 nm).
On metaphase slides chromosomes of five different metaphase cells with 40 chromosomes were analyzed and the number of telomeric signals per chromosome was counted. To determine the fraction of MN per nucleus, all slides were randomized and coded prior to scoring. A minimum of 3000 cells/dose for each experiment was scored. MN were classified according to the following scheme: group C0T0, MN without any signal; group C0Tn, MN with telomeric signal only; group CnTn, MN with centromeric and telomeric signals. The percentages of MN belonging to groups C0T0, C0Tn and CnTn were determined as well as the number of telomeric and centromeric signals per MN counted in groups C0Tn and CnTn. For more precise information about the different types of MN induced by the mutagens the fractions of MN per 1000 nuclei belonging to groups C0T0, C0Tn and CnTn were calculated. The scoring of MN was performed by the same microscopist.
Statistical analysis
The 
2 test was used to determine differences between controls and treated samples. Trends in dose–response effects were analyzed using simple regression. Critical values were determined using a 0.05 probability of type I error.
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Results |
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Before hybridization of MN, the specificity of the probes and the hybridization were evaluated using metaphase cells. The centromeric minor DNA probe gave reproducible signals for every chromosome except Y and the signals were located at the centromere only. Various numbers of telomeric signals per chromosome were observed. Percentages were estimated taking into account five metaphases with 40 chromosomes. The distribution of the telomeric signal frequencies on chromosomes is shown in Table I
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Clear telomeric signals were obtained on nearly all chromosomes (~98.5%). However, various numbers of telomeric signals (one to four, with an average of three) per chromosome were observed depending on the focal plane and due to different copy numbers of the telomeric repeat on mouse chromosomes. About 72% of telomeric signals (574 telomeric signals) on 800 telomeres of 200 chromosomes (there are two telomeres on every chromatid and every chromosome has two chromatids) were found. Although mouse chromosomes are telocentric, the telomeric signals on the short arm telomeres were frequently seen as two separate dots and rarely mixed together. The signals on the short arm were frequently clearer and brighter compared with those on the long arm; their sizes were similar.
All MN with centromeric signals also had telomeric signals. The distributions of signal frequencies in the MN with telomeric and centromeric probes are shown in Table II.
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After treatment with MMC the percentage of centromere-negative MN (group C0T0 + group C0Tn) was higher compared with the control (1.4-fold increase), especially the percentage of MN with telomeric signals only (1.7-fold increase). With COL the percentage of centromere-positive MN (group CnTn) was higher (1.7-fold increase) than found in the controls. A statistically significant increase in the frequencies of both MN with and without centromeric signals was observed; the former increased 9.5-fold and the latter increased 2.6-fold. There were significant differences in the distributions of centromere-negative and centromere-positive MN between the two controls, MMC and COL (
2 test, P < 0.01). Following AA treatment a clear dose–response relationship was observed. The frequencies of total MN, MN with both centromeric and telomeric signals and MN without a centromeric signal were found to increase significantly in a linear dose-dependent manner (total MN, r2 = 0.9859, P < 0.01; MN with both centromeric and telomeric signals, r2 = 0.9939, P < 0.01; MN without centromeric signal, r2 = 0.8728, P < 0.05). At the highest dose (400 µg/ml) 65.9% of MN showed centromeric signals.
In THH-treated cultures the three doses tested (5, 10 and 20 µl/ml) resulted in a significant increase in the frequencies of total MN and MN with centromeric signals (group CnTn) and a very clear dose-dependent increase in MN without centromeric signal (group C0T0 + group C0Tn, r2 = 0.9966, P < 0.01) was observed. The majority of MN contained centromeric signals following treatment with concentrations of 5 and 10 µl/ml (70.7 and 69.3%, respectively). However, when treated with the highest dose of 20 µl/ml a similar frequency of total MN was found as at the lower dose of 10 µl/ml, while the percentage of MN belonging to group CnTn was found to decrease to 49.1%.
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Discussion |
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The aim of the present study was to analyze the chromosomal composition of MN induced by two suspected mutagens, AA and THH. In order to determine the efficiency of the method two model mutagens, MMC and COL, known to have clastogenic and aneugenic actions, respectively, were also used. In control cells nearly half (44.8%) of the MN revealed centromeric signals and several telomeric signals, 34.5% of MN showed only telomeric signals and 20.7% did not show any signal. These results were in the same range as observed in the control cells of other studies (Miller et al., 1992
; Miller and Nüsse, 1993; Schriever-Schwemmer and Adler, 1994). However, the proportion of MN with two centromeric signals was higher than that reported by Miller and co-workers (Miller et al., 1992; Miller and Nüsse, 1993). This may be due to the different centromeric probe used. Miller and co-workers (Miller et al., 1992; Miller and Nüsse, 1993) used the major probe, while in our studies the minor probe was used. Usually the major probe labels chromosomal centromeres with a one-dot pattern, whereas the minor probe labels them with two dots. Thus, when an entire chromosome is enclosed in a MN it shows only one centromeric signal when the major probe is used, but two signals when the minor probe is used (Chen et al., 1994; Schriever-Schwemmer and Adler, 1994).
After treatment with the typical clastogen MMC the majority of MN (78.6%) did not show centromeric signals. This result agrees well with those of other studies (Channarayappa et al., 1992; Hayashi et al., 1994; Schriever-Schwemmer and Adler, 1994). Due to the strong clastogenic effect, MN were predominantly composed of acentric fragments after treatment with MMC. In the present study the more precise results indicate that ~30% of MMC-induced MN may be composed of two or more acentric fragments.
Typically for an aneugen, 74.5% of MN contained centromeric signals following treatment with COL. The result is similar to other reports (Farooqi et al., 1993; Schriever-Schwemmer and Adler, 1994). Our results, which show more detail, indicate that >40% of COL-induced MN were composed of two or more whole chromatids. Additionally, COL also produced a significant increase in MN without a centromeric signal; similar results have been reported by others (Becker et al., 1990; Farooqi et al., 1993; Schriever-Schwemmer and Adler, 1994). This might be due to COL-induced MN containing the Y chromosome, which cannot be detected, but might also be due to a small potential clastogenic effect of COL. It has been reported that COL has clastogenic potential besides its aneugenic action (Arni and Hertner, 1997).
The percentage of MN induced by the typical aneugen COL belonging to group CnTn was substantially higher (74.5%) than that induced by the known clastogen MMC (21.4%); the distribution of centromere-negative and centromere-positive MN among the controls MMC and COL were significantly different. Based on these two model clastogen and aneugen results, it is clear that the relative aneugenic potential as well as the clastogenic activity of a particular agent can be identified by the combination of centromeric and telomeric probes used in these studies.
It has been shown previously that AA is a clastogen (Adler et al., 1988, 1994; Gutierrez-Espeleta et al., 1992) and a suspected aneugen (Adler et al., 1993; Gassner and Adler, 1996; Schriever-Schwemmer et al., 1997). AA can damage spermiogenic cells, resulting in dominant lethals and heritable translocations (Backer et al., 1989; Adler et al., 1996). It can also induce chromosomal aberrations (chromatid gaps, breaks and exchanges) in mammalian cell cultures in the presence or absence of exogenous metabolic systems as well as in mouse bone marrow cells and in mouse spermatogonia (Adler et al., 1988, 1993). Recently, studies in mouse bone marrow and spermatocytes indicated that AA has aneugenic potential (Gassner and Adler, 1996; Schriever-Schwemmer et al., 1997). Our results showed that both the frequencies of MN with centromeric signals and MN without centromeric signals increased significantly in a dose-dependent manner following treatment with AA. At a dose of 100 µg/ml the frequency of MN containing acentric fragments was slightly but not significantly higher than that of MN containing whole chromosomes (9.3 and 8.7%, respectively). At high concentrations, however, many more MN containing whole chromosomes were produced. Therefore, our results clearly indicated that AA is not only a clastogen but also an aneugen; the aneugenic action appears more prominent at higher AA concentrations.
Previous studies have shown that THH is an aneuploidy inducer in mouse bone marrow cells (Wang et al., 1993). In the present study the majority (~70%) of MN had a centromere after treatment with THH at concentrations of 5 and 10 µl/ml and most of them were probably composed of single whole chromosomes because almost all of them contained only one or two centromeric signals. This pattern clearly shows the aneuploidy-inducing nature of this traditional Chinese herb and supports our earlier observations and the results of Wang et al. (1993), that give us more details about this herb. However, the three doses tested (5, 10 and 20 µl/ml) resulted in a significant dose-dependent increase in the frequency of MN without centromeric signals, suggesting that THH also has clastogenic potential, especially at high concentrations. For THH the interesting thing is that the frequency of MN after treatment with THH at a concentration of 20 µl/ml was lower than at 5 and 10 µl/ml, while the percentages of MN with and without centromeric signals were very similar. The reasons for this phenomenon are unclear. A possible explanation is that the cytotoxic effect of THH reduces cell division and MN formation at high concentrations. This agrees with the fact that THH could induce C-mitotic delay effects (Wang et al., 1993). Ames tests, HPRT locus and exon deletion analyses, chromosome aberration studies, micronucleus analyses in vitro and in mouse, sister chromatid exchange studies, C-mitotic delay and aneugenic analyses, etc. have been completed for THH (Wang et al., 1993, 1995; Cao et al., 1998; Cao and Nüsse, 1999; Liu et al., 1999). All of these mutagenesis analysis systems have given positive results, indicating that THH is a complete mutagen and can induce DNA damage, gene mutations and chromosomal numerical and structural changes. Recently, our studies showed that water extracts and total alkaloids of THH have a strong ability to induce apoptosis in NIH 3T3, CHO, HL-60, Jurkat and NB4 cells, using sub-G1 peak and Annxion-V staining, DNA ladders and morphological changes (Cao and Nüsse, 1999).
FISH with a combination of telomeric and centromeric DNA probes allows further characterization of the chromosomal composition of MN and analysis of the numerical distribution of acentric fragments and whole chromosomes inside individual MN (Miller et al., 1992; Miller and Nüsse, 1993; Schriever-Schwemmer and Adler, 1994; Nüsse et al., 1996), with some restrictions. One problem is that the Y chromosome cannot be detected with the minor satellite probe (Chen et al., 1994; Schriever-Schwemmer and Adler, 1994). However, the probability of the Y chromosome being included in a MN should be less than 1/40 of all MN induced according to random loss theory, and this problem is thus negligible. Another problem is that ~30% of the telomeres of metaphase chromosomes could not be detected with the telomeric probe under the hybridization conditions used. Miller et al. (1992) reported a similar problem. A possible reason is that the length of the telomeric sequence in different chromosomes of the mouse (Greider, 1991) varies from 20 to 200kb (Kipling and Cooke, 1990), leading to corresponding differences in signal brightness. Thus, the absolute number of fragments enclosed in a MN could not be quantified precisely and it was impossible to distinguish between MN containing exactly one or two whole chromosomes and those containing both whole chromosomes and acentric fragments. However, since ~70% of the signals were found on metaphase chromosomal telomeres and most of the chromosomes showed two to four telomeric signals, the number of fragments enclosed in a MN could be estimated. MN with both centromeric and telomeric signals must contain whole chromosomes, MN with telomeric signals only are most likely composed of acentric fragments and unlabeled MN may contain interstitial fragments. MN with one minor signal and one or two telomeric signals probably contain a chromatid after centromere separation. A MN with two minor satellite signals and three or four telomeric signals is more likely composed of a chromosome before centromere separation or two chromatids after centromere separation. Of course, for more accurate quantitative determination of the number of acentric fragments and chromosomes in MN, the efficiency of labeling and detecting the telomeric probe should be further improved, by such means as using PCR to produce and label telomeric probes and using confocal fluorescence microscopy to detect some faint telomeric signals.
In summary, we have observed that MN containing whole chromosomes as well as MN containing acentric fragments in NIH 3T3 cells increased in a dose-dependent manner following treatment with AA. An extract from the traditional Chinese herb THH clearly increased MN harboring whole chromosomes at all concentrations tested and produced a concentration-dependent increase in MN with fragments. Based on our results, multicolor FISH with centromeric and telomeric probes shows promise as a technique for analyzing the chromosomal composition of MN. However, centromeric FISH alone appears to be adequate for differentiating clastogens from aneugens.
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Acknowledgments |
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The authors thank Dr Michael Nüsse of GSF, Germany, and Dr Michael M. Yang of City University of Hong Kong for their helpful comments on this manuscript. We are also grateful to Prof. R.Pearlman of York University, Canada, for generously providing the plasmid containing the mouse minor satellite DNA probe. This research was supported by NSFC contract 39400114.
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Notes |
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1 To whom correspondence should be addressed. Tel: +86 023 68752348; Fax: +86 023 65316682. Email: caojj{at}yahoo.com
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References |
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Received on June 15, 2000; accepted on September 22, 2000.
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