DNA methylation and chromosomal rearrangements in reconstructed karyotypes of Hordeum vulgare L.

One standard and two reconstructed barley karyotypes were used to study the influence of chromosomal rearrangements on the distribution pattern of DNA methylation detectable at the chromosome level. Data obtained were also compared with Giemsa N-bands and high gene density regions that had been previously described. The effect of chromosomal reconstruction in barley seems to be decidedly prominent in the repositioning of genomic DNA methylation along metaphase chromosomes. In comparison to the standard karyotype, the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complements not subjected to structural alterations. Moreover, differences may occur between corresponding regions of homologues. Some specific chromosomal bands, including the nucleolus-organizing regions, showed a relative constancy in the methylation pattern, but this was not the case when the two satellites were combined by translocation in chromosome 6H(5H) of line T-30. Our results suggest that epigenetic changes like DNA methylation may play an important role in the overall genome reorganization following chromosome reconstruction.

Further evidence for a high position-specific effect in the action of chemical mutagens on the chromosomes of barley.

Further convincing evidence for the decisive role of chromosome constitution in the processes underlying the specific induction of structural mutations by chemical mutagens is described. The most important conclusion to be drawn from the results obtained in experiments with maleic hydrazide (MH) and ethyl methanesulphonate (EMS) is that segments 39 and 47, situated next to the secondary constrictions of standard chromosomes 6 and 7, when tandemly combined by reciprocal translocation in chromosome 7(6) of reconstructed karyotype T-21, behave in a rather similar way. This is independent of the nature of the chemical mutagens applied, as far as the distribution pattern of induced chromatid aberrations is concerned. The phenomenon may be characterized as follows: (1) the segments in question in the new position appeared to be the most pronounced aberration "hot spots", the expressivity in segment 39 always being higher; (2) the pronounced "hot spot" character of these segments proved to be due mainly to their preferential involvement in intercalary deletions and duplication-deletions; (3) the specific constitution of chromosome 7(6) resulted in the majority of cases in a marked increase of the region involved in the aberration types mentioned above in segment 39. This is one of the very few examples of true position effect in the expression of chemically induced structural mutations at the chromosome level.

Multiple reconstruction of barley karyotype resulting in complete cytological marking of the chromosome complement.

A new reconstructed barley karyotype, PK88, which is a quadruple homozygote for three unequal translocations, 1-2, 3-4, 5-7, and one pericentric inversion in chromosome 6, was studied. As a result of these chromosome rearrangements, a complete cytological marking of the complement has been achieved. Due to the specific intra or interchromosomal transfer of particular bands, Giemsa staining of somatic chromosomes provided clear-cut indications about the localization of translocation and inversion breakpoints. It was established that the long arms of chromosomes 1, 2, 4, 5 and 7 and the short arm of chromosome 3 have been involved in interchanges 1-2, 3-4, and 5-7. The breakpoints of pericentric inversion proved to be located proximally to the short (satellite) arm and distally in the long arm of chromosome 6. PK-88 offers an essential gain in resolution power and extension of the areas of application in cytogenetics over other reconstructed karyotypes produced so far in barley.

Position-specific effects in the mutagenic action of mitomycin C on the chromosomes of Hordeum vulgare L.

The mutagenic action of mitomycin C (MMC) on the chromosomes of two reconstructed karyotypes of barley was studied. MMC-induced chromatid aberrations were found to be distributed non-randomly along the chromosomes. The regions situated next to the secondary constrictions of chromosomes 6 and 7 appeared to be clearly pronounced aberration "hot spots". In these segments, intercalary deletions and duplication-deletions were the most frequently induced aberration types. The comparative analysis of the frequency and localization of MMC-induced aberrations in the chromosomes of the two karyotype variants, which differ from each other by the position of the "hot spot" segments, provided new evidence about the influence of the segment transposition on the "hot spot" expressivity. The most remarkable finding obtained in the study is that the size of the segment involved in both intercalary deletions and duplication-deletions proved to be strongly affected by the structural peculiarity of the reconstructed chromosome. The possible reasons underlying this finding are discussed.

Production and identification of new structural chromosome mutations in barley (Hordeum vulgare L.).

A total of 52 reciprocal translocations and 9 pericentric inversions were induced and identified in both standard and cytologically marked barley karyotypes using gamma-rays as the clastogenic agent. An analysis based upon Giemsa N-banding patterns and arm length measurements of the reconstructed chromosomes enabled a rather precise cytological localization of intra- and interchange breakpoints. This analysis was significantly facilitated and improved, especially for the identification of pericentric inversions, when the reconstructed karyotype T-1586 was used as starting material. The majority, if not all, of the aberration breakpoints proved to be localized in interband regions or in medial and terminal parts of the chromosomes, i.e., in regions which are deficient in constitutive heterochromatin. A great number of the structural mutations produced in this study contain specific cytological markers covering nearly all of the chromosomes of barley karyotype. This material might be of considerable interest in solving various problems of barley cytogenetics and chromosome engineering and especially in constructing a physical map of barley genome.

Transcriptional activity of an inversion split NOR in barley (Hordeum vulgare L.).

The transcriptional activity of NORs in a new structural mutant of barley (PK-88-4) was studied based on the positive reaction of NORs of metaphase chromosomes with AgNO3 and the number and size of silver-stained nucleoli in somatic interphase cells and meiocytes. PK-88-4 proved to contain a pericentric inversion that split NOR 7 into two unequal parts residing in the opposite arms of the reconstructed chromosome 7(5). This chromosomal rearrangement enables testing of intrachromosomal suppression of NORs and provides insight into the mechanisms of intraspecific nucleolar dominance. Both parts of the split NOR proved to be transcriptionally active, and are not subject to intrachromosomal nucleolar dominance. Thus, translocation-induced intraspecific nucleolar dominance is probably the result of interaction of NOR6 and NOR7 or other genetic factors located on the NOR-bearing chromosomes 6(6H) and 7(5H).

Different chromosomal distribution patterns of radiation-induced interchange breakpoints in barley: first post-treatment mitosis versus viable offspring.

Translocation breakpoints (TBs) induced by ionizing radiation are nonrandomly distributed along barley chromosomes. When first post-treatment mitoses were evaluated, centromeres and the heterochromatin-containing proximal segments tended to be more than randomly involved, and terminal segments to be less than randomly involved in translocations. Contrary to this, small chromosomal regions in median and distal arm positions, characterized by high recombination rates and high gene density, were identified as preferred sites for the origination of viable translocations, probably due to deviations in chromatin organization. Apparently, the position of a TB has an influence on the rate of viability versus elimination of the carrier cells. Surprisingly, TBs within centromeres and heterochromatin-containing segments seem to be more harmful for survival than those induced in gene-rich regions.

HaeIII induces position-dependent chromosomal breakage in barley (Hordeum vulgare L.).

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.

Cytological and molecular evidence of deletion of ribosomal RNA genes in chromosome 6 of barley (Hordeum vulgare).

The barley chromosomal mutant T-35, in which only one pair of satellite chromosomes is apparent, was analyzed using a range of cytological and molecular techniques. Using conventional Feulgen staining, Giemsa and silver banding, in situ hybridization, and Southern blot analysis, unequivocal cytological and molecular evidence was obtained that T-35 is a homozygous deletion of rRNA genes residing in the nucleolus organizer region (NOR) of chromosome 6. According to the criteria of arm ratio and Giemsa-banding pattern of this chromosome, the deletion involved the whole NOR, one of the breakpoints being localized in the short arm proximally to the NOR-associated heterochromatic band, the other probably in the satellite of the chromosome. As a result of this deletion, an increased activity of the rRNA genes (as indicated by the size of the silver bands) on the other NOR-bearing chromosome (chromosome 7) was observed. The possible reasons for this phenomenon are discussed.

Restriction endonucleases induce chromosomal aberrations in barley.

The clastogenic ability of the restriction endonucleases (MspI, HpaII and HaeIII) in germinating seeds of reconstructed barley karyotype was assessed. An effective induction of chromosomal aberrations after restrictase treatment was observed. The frequency, types and cell-cycle dependence of the observed abnormalities are discussed in relation to the distinct characteristics of the enzymes and the features of the plant genome. The capacity to induce aberrations was not significantly influenced by the nature of the double-strand breaks (blunt- or cohesive-ended); however, it was dependent on the methylation status of the plant DNA. The restriction enzymes displayed an S-independent mode of action revealing the transition between G1 and S as the most sensitive stage of the cell cycle in barley for induction of chromosomal damage.