Mutagenesis, Vol. 15, No. 3, 277-279,
May 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
HaeIII induces position-dependent chromosomal breakage in barley (Hordeum vulgare L.)
Institute of Genetics `D.Kostoff', 1113 Sofia and 1 De Montfort University, Norman Borlaug Centre of Plant Science, Institute of Genetic Engineering, 2232 Kostinbrod-2, Bulgaria
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Abstract |
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The pattern of localized chromosomal breakage induced by the restriction endonuclease HaeIII in reconstructed barley karyotypes T-1586 and T-21 was investigated. It was found that nucleolus organizing regions (NORs) of chromosomes 6 and 7 (segments 46 and 38, respectively), containing actively transcribed ribosomal (r)DNA, as well as segments 39 and 47, both containing condensed rDNA repeats, are the most pronounced aberration hot-spots in T-1586. The number of aberrations observed in these segments was three to five times higher than theoretically expected. The intrachromosomal distribution of chromatid aberrations in karyotype T-21, where the NOR-bearing segments in chromosomes 6 and 7 change their position, revealed a substantial difference in the aberration hot-spot behaviour. A position-specific increase in aberration clustering was observed, most pronounced in segments 38 and 47. On the other hand, segment 46 retained its initial sensitivity, while segment 39 in the new position lost its previous status as a mutation hot-spot. The data are indicative of the expressivity of aberration hot-spots generated after treatment with this restriction endonuclease being influenced by their distinct chromosomal location.
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Introduction |
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There are many studies indicating that the chromatid aberrations induced by various mutagenic agents are not randomly distributed along the chromosomes. The most convincing data in plants were established with experimentally reconstructed karyotypes (Schubert et al., 1986
). Amongst the factors influencing the pattern of intrachromosomal distribution of induced aberrations, the karyotype constitution was proved to be of primary importance (Rieger et al., 1977; Schubert et al., 1985; Gecheff, 1991).
Following characterization of the restriction endonucleases as efficient clastogens in both mammalian (Bryant, 1984; Natarajan and Obe, 1984) and plant (Stoilov et al., 1996) genomes, their differential activity along the individual chromosomes was also analysed (Obe et al., 1986; Balajee et al., 1994; Folle and Obe, 1995, 1996; Gecheff et al., 1997).
Analysis of the intrachromosomal distribution of chromatid aberrations induced by restriction endonucleases recognizing different DNA sequences has shown nearly the same pattern of localized breakage (Folle and Obe, 1995, 1996). In a later study it was shown that the rDNA regions are amongst the most pronounced aberration hot-spots produced by different restriction endonucleases in barley (Gecheff et al., 1997).
The repositioning of aberration hot-spot segments was proved to play an essential role in the intrachromosomal distribution of chemically induced structural mutations (Schubert et al., 1986; Gecheff, 1991). In this respect, position-dependent induction of chromosomal damage after treatment with restriction enzymes might also be expected.
The present study is an attempt to shed some light on the problem, taking advantage of the availability of reconstructed barley karyotypes with an altered chromosomal localization of the nucleolus organizing regions (NORs).
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Materials and methods |
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Germinating seeds of reconstructed barley karyotypes T-1586 and T-21 were utilized as the experimental material. Karyotype T-1586, containing a reciprocal translocation between chromosomes 3 (3H) and 4 (4H), permits clear identification of all chromosomes and was used as a control. Karyotype T-21 is identical to T-1586 with respect to the 3–4 translocation and in addition contains a reciprocal translocation between chromosomes 6 (6H) and 7 (5H). As a result, the NOR-bearing segments in chromosomes 6 (6H) and 7 (5H) exchange their positions.
Restriction endonuclease HaeIII (12 U/µl; Sigma) was used as the inducer of chromosomal damage due to its previously established activity in the barley genome (Stoilov et al., 1996).
Treatment with HaeIII was performed essentially as previously described (Stoilov et al., 1996). Briefly, after permeabilization of the primary roots with Driselase and conditioning with the respective digestion buffer the material was exposed to 500 U/ml of the enzyme for 3 h at 37°C. Metaphase block, fixation of the material and preparation of Feulgen stained squashes were done as before (Gecheff, 1989). The recovery times used in our experiments (19 and 22 h after application of the restriction endonuclease) were chosen to coincide with the maximal frequency of aberrations produced by HaeIII, namely 25–28% damaged cells. About 700 cells per recovery time were scored. To analyse the localization of the four types of chromatid aberrations the metaphase chromosomes were subdivided into 53 segments of nearly equal sizes (Figures 1 and 2). The centromeres and secondary constrictions were designated individually. The segments were numbered with respect to their position in the standard barley karyotype. The data are pooled from at least two independently performed experiments and analysed statistically according to the formula of Rieger et al. (1975).
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Results and discussion |
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Figure 1
shows the intrachromosomal distribution of HaeIII-induced chromatid aberrations in karyotype T-1586. The most frequently produced type of rearrangements were isolocus breaks and reciprocal chromatid translocations distributed non-randomly along the individual chromosomes. Segments 46 and 38, representing the NOR of chromosomes 6 and 7, respectively, as well as segments 39 and 47 [both containing condensed ribosomal (r)DNA], appeared to be the most pronounced aberration hot-spots. The number of aberrations observed in these segments surpassed by more than three times the theoretically expected frequency for a random distribution (for segments 46 and 39 the enhancement was more than five times). It is remarkable that the increased sensitivity of segment 39 in this case is due to its preferential involvement in intercalary deletions. Another distinct feature of the data obtained is that reciprocal translocations are predominantly located in the NORs of chromosomes 6 and 7 (segments 38 and 46).
It should be noted that a similar response of segments 38 and 46 to the action of HaeIII was found in our previous studies with multi-reconstructed karyotype PK-88. Obviously, the actively transcribed rDNA repeats localized in these segments are preferential targets for the action of restriction endonucleases since the NORs also appeared as aberration hot-spots after the application of HpaII and MspI (Gecheff et al., 1997).
The data on the intrachromosomal distribution of HaeIII-induced chromatid aberrations in karyotype T-21 are presented in Figure 2. The main reason karyotype T-21 was included in this study was that the chromosome reconstruction concerns the positions of nearly all hot-spot segments. Owing to reciprocal translocation between chromosomes 6 and 7, segment 39, together with some other segments residing normally in the short arm of chromosome 6, are transposed to the short arm of chromosome 7. As a result, segments 39 and 47 become tandemly arranged. Such a repositioning leads to a substantial alteration in the hot-spot segment sensitivity to HaeIII-induced damage. The position-specific increase in aberration induction was most pronounced in segments 38 and 47, where involvement in aberrations was more than twice as high as that observed in karyotype T-1586. Thus, ~30% of all aberrations induced in karyotype T-21 were localized in these segments (the percentage involvement in aberrations of the same segments in T-1586 was ~14%).
The other NOR residing on chromosome 67 (segment 46) does not alter its sensitivity to the action of HaeIII, again being a hot-spot segment. However, segment 39 in the new position does not retain its hot-spot expressivity.
An interesting finding in this study is that chromosome segments with identical base composition show distinct differences in their position-specific response to HaeIII clastogenic activity. Both segments 38 and 46 are known to be entirely composed of transcriptionally active rDNA repeats (Subrahmanyam et al., 1994) but only one of them (segment 38) shows increased sensitivity after its repositioning in the newly reconstructed chromosome 76. Obviously, the transcriptional activity and availability of potential recognition sites alone are not sufficient to generate hot-spot segments, as their position in the karyotype also seems to be very important. This trend is even more pronounced for segments 39 and 47, which, however, contain condensed rDNA sequences (Gecheff et al., 1994) which presumably are not transcribed. Although definite conclusions concerning the role of transcription and/or chromatin organization in the observed phenomenon cannot be drawn, a local change in chromatin condensation of the transposed segments cannot be excluded. Moreover, reconstruction of the respective chromosomal regions bearing rDNA in karyotype T-21 is not expected to affect its transcriptional activity.
It should be noted that the position-specific distribution of aberrations induced by HaeIII differs significantly from that observed after treatment of the same karyotypes with various chemical mutagens (Gecheff, 1991). The nature of the primary lesion (DNA double-strand breaks in our treatment) is probably responsible for such a discrepancy.
The locus-specific induction of damage after treatment with restriction endonucleases was first established in mammalian cells. It was shown that AluI preferentially induces chromosomal aberrations and mutations in the HPRT locus in V79 hamster cells (Obe et al., 1986). Later a preferential clastogenic activity of restriction endonucleses was also observed in the interstitial telomeric repeat sequences of CHO cells (Balajee et al., 1994; Natarajan et al., 1994). It was hypothesized that the nuclease-hypersensitive regions of the genome associated with actively transcribed genes are clusters of restriction endonuclease-induced breakpoints (Folle and Obe, 1996). Our results are generally consistent with this assumption. Moreover, the data obtained in this study are indicative of the expressivity of the potential aberration hot-spots induced by restriction enzymes being modulated to some extent by altering their position in the genome.
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Acknowledgments |
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This study was partially supported by the Bulgarian National Science Fund, grant K-804.
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Notes |
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2 To whom correspondence should be addressed. Tel: +359 2 75 90 42; Fax: +359 2 75 70 87; Email: molgen{at}bas.bg
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References |
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Received on November 17, 1999; accepted on February 3, 2000.
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