Karyological Features of Achyrocline (Asteraceae, Gnaphalieae): stable karyotypes, low DNA content variation and linkage of rRNA genes.

Many Achyrocline (Asteraceae, tribe Gnaphalieae) species are widely used in Argentina, Brazil and Uruguay as popular medicinal and aromatic plants. Achyrocline flaccida, A. satureioides, A. alata, and A. crassiuscula are distributed in Uruguay and popularly known as 'marcelas'. In order to characterize them, we performed chromosome counts, compared the karyotypes, mapped the 5S and 45S rDNA sites by fluorescent in situ hybridization, and estimated their DNA content. All species were diploid with 2n = 28 chromosomes, this being the first report for A. flaccida and A. crassiuscula. All species showed symmetrical karyotypes composed exclusively of biarmed chromosomes. DNA content estimated by flow cytometry revealed 2C values ranging from 5.73 to 6.03 pg, the amounts for A. alata and A. crassiuscula being higher than those for the other species. Cytogenetic mapping of 5S and 45S rDNA sequences in three species, A. flaccida, A. satureioides and A. alata, showed that in these species both sites co-localized in the pericentromeric region of chromosome 10. This site corresponds to the only DAPI(-) and CMA(+) band of their genomes. Southern blot hybridization of 5S and 45S rDNA on BamHI digested genomic DNA confirmed the tight linkage of these rDNA families into a single unit. Cytological data indicate that Achyrocline species are karyologically poorly differentiated, whereas the uncommon distribution of 5S and 45S rDNA suggests a close relationship with other genera of the Anthemidae tribe.

Close encounters: RIDGEs, hyperacetylated chromatin, radiation breakpoints and genes differentially expressed in tumors cluster at specific human chromosome regions.

Through analysis of published data we positioned along human chromosome idiograms (850 bands) hyperacetylated H4 chromatin (H4(+a)), regions of increased gene expression (RIDGEs), antiRIDGEs, ionizing radiation breakpoints, integration sites of highly expressed GFP reporter constructs and candidate genes differentially expressed in tumor tissues. Highly expressed regions of the human genome (especially RIDGEs) seem to be more sensitive to radiation damage. Comparatively, antiRIDGEs appear as radiation resistant. Tumor deregulated genes tend to cluster along and in the neighborhood of RIDGEs. We detected 35 clusters of genes differentially expressed in tumor tissues which colocalize with RIDGEs; 23 of these clusters also exhibit radiation damage. RIDGEs also accumulate highly expressed GFP reporter construct integration sites, evolutionary breakpoints as well as amplicons and/or deletion-prone chromosome segments in tumors. This could indicate that abnormal gene (up- or down-) regulation might require high-throughput transcription nuclear micro-environments to occur. Our results suggest that the human genome is a combination of regions with marked disparities regarding the topology of increased gene expression, ionizing radiation damage, evolutionary breakpoints, integration sites, and abnormal gene regulation.

Modulation of chromosome damage localization by DNA replication timing.

PURPOSE: Non-random occurrence of induced chromosome breakpoints (BP) has been repeatedly reported. DNA synthesis and chromatin remodeling may influence chromosome BP localization. The CHO9 X chromosome exhibits an early replicating short euchromatic arm (Xpe) and a late replicating long heterochromatic arm (Xqh). We investigated the role played by DNA replication and related chromatin remodeling processes on BP distribution in eu/heterochromatin using the CHO9 X chromosome as a model. MATERIALS AND METHODS: BP induced by etoposide, a topoisomerase II inhibitor, as well as by the S-dependent clastogens ultraviolet-C light (UV-C) and methyl methanesulfonate (MMS) were mapped to CHO9 X chromosome arms. The base analogue 5-bromo-2'-deoxyuridine (BrdUrd) was pulse-added immediately after UV-C irradiation or during etoposide and MMS treatments (40 min) to identify cells in early S-phase (Xpe labeled) or late S-phase (Xqh labeled) after indirect BrdUrd immunodetection in metaphase spreads using primary anti-BrdUrd and secondary fluorochrome-tagged antibodies. RESULTS: During early S-phase, BP induced by etoposide and MMS mapped preferentially to Xpe while BP produced by UV-C localized randomly. BP induced by all agents during late S-phase clustered in Xqh. CONCLUSIONS: Results obtained suggest that replication time of eu/heterochromatin as well as chromatin remodeling may determine BP localization on the CHO9 X chromosome.

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome.

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.

Chromosomal aberrations: formation, identification and distribution.

Chromosomal aberrations (CA) are the microscopically visible part of a wide spectrum of DNA changes generated by different repair mechanisms of DNA double strand breaks (DSB). The method of fluorescence in situ hybridisation (FISH) has uncovered unexpected complexities of CA and this will lead to changes in our thinking about the origin of CA. The inter- and intrachromosomal distribution of breakpoints is generally not random. CA breakpoints occur preferentially in active chromatin. Deviations from expected interchromosomal distributions of breakpoints may result from the arrangement of chromosomes in the interphase nucleus and/or from different sensitivities of chromosomes with respect to the formation of CA. Telomeres and interstitial telomere repeat like sequences play an important role in the formation of CA. Subtelomeric regions are hot spots for the formation of symmetrical exchanges between homologous chromatids and cryptic aberrations in these regions are associated with human congenital abnormalities.

Chromosome regions enriched in hyperacetylated histone H4 are preferred sites for endonuclease- and radiation-induced breakpoints.

Previously, we have shown that breakpoints induced by the endonucleases AluI, BamHI and DNase I in CHO chromosomes are localized mainly in G-light bands. Neutrons and gamma rays produced similar breakpoint clusters to endonucleases in most CHO chromosomes. Here we compare endonuclease- and radiation-induced breakpoint maps with hyperacetylation patterns of histone H4. The H4 acetylation pattern in chromosomes is similar to the pattern of G-light, or R-bands, and breakpoints are clustered in highly acetylated chromosome regions. These findings indicate that chromosomal aberrations occur more frequently in active than in inactive chromatin.

Intrachromosomal localization of aberration breakpoints induced by neutrons and gamma rays in Chinese hamster ovary cells.

Chromosome breakpoints induced by neutrons or gamma rays in Chinese hamster ovary cells were mapped to Giemsa-light or Giemsa-dark bands or to band junctions. Radiation-induced breakpoints were found to be distributed nonrandomly according to chromosome or band length. More than 60% of the breakpoints were localized in G-light bands. A group of 13 bands which corresponded to only 7% of the total chromosome length contained 22% of the breakpoints produced by neutrons and 14% of those induced by gamma rays. Seven of these 13 bands are also preferentially damaged by AluI, BamHI and DNase I as reported previously. The results indicate that chromatin and nuclear structure may play a role in the distribution of breakpoints produced by ionizing radiation and endonucleases.

Localization of chromosome breakpoints: implication of the chromatin structure and nuclear architecture.

Restriction endonucleases and ionizing radiations have been extensively used to study the origin of chromosomal aberrations. Although a non-random distribution of chromosome breakpoints induced by these agents has been claimed by several authors, the significance of the chromatin structure and nuclear architecture in the localization of breakpoints is still not well understood. Breakpoint patterns produced by endonucleases targeted to specific genome sequences or by ionizing radiations could provide additional evidence to clarify this point. Results obtained from the localization of breakpoints induced by AluI, BamHI or DNase I as well as by neutrons or gamma-rays in G-banded Chinese hamster ovary (CHO) chromosomes are presented. AluI and BamHI were electroporated into CHO cells either during the G1 or S-phase of the cell cycle. A co-localization of breakpoints was found with a preferential occurrence in G-light bands independent of the cell cycle stage in which aberration production took place. Since AluI and BamHI recognition sequences are partitioned in the housekeeping and tissue-specific subgenomes respectively, we postulated that nuclease sensitive sites in active chromatin could be the main targets for the induction of breakpoints by these endonucleases. This assumption is supported by the finding that DNase I-induced breakpoint patterns in CHO cells are similar to those produced by AluI and BamHI. Digestion of fixed CHO chromosomes with these endonucleases induced G-banding suggesting a higher sensitivity of G-light chromatin. For comparison purposes, CHO cells were irradiated with neutrons or gamma-rays and breakpoints localized in G-banded chromosome aberrations. A higher occurrence of breakpoints in G-light bands was also observed. We detected seven breakage-prone G-light bands that were preferentially damaged by the three endonucleases and by both types of radiation. These results emphasize the possible implication of the chromatin structure and the nuclear architecture in the localization of chromosome breakpoints induced by endonucleases, neutrons and gamma-rays.

Localization of chromosome breakpoints induced by DNase I in Chinese hamster ovary (CHO) cells.

DNase I was electroporated into S-phase CHO cells and induced chromosome breakpoints were localized in G-banded metaphases. More than 75% of breakpoints mapped to Giemsa-light bands, 18% to Giemsa-dark bands and about 7% to band junctions. Chromosome breakpoint clusters produced by DNase I colocalized with chromosome breakpoints induced by the restriction endonucleases AluI and BamHI in the G1- and S-phases of the cell cycle in CHO cells. Digestion of metaphase spreads with AluI, BamHI and DNase I produced G-bands, indicating that G-light bands are more sensitive to endonuclease action. The possible role of nuclease-sensitive sites in active chromatin as selective targets for the induction of chromosome breakpoints by these endonucleases is discussed.

Intrachromosomal localization of breakpoints induced by the restriction endonucleases AluI and BamHI in Chinese hamster ovary cells treated in S phase of the cell cycle.

The restriction endonucleases (REs) AluI and BamHI were electroporated into Chinese hamster ovary (CHO) cells during S phase of the cell cycle and breakpoints in G-banded metaphases were mapped to Giemsa-light or -dark bands or to band junctions. The majority of AluI- and BamHI-induced breakpoints were located in Giemsa-light bands. Both REs induced similar distributions of breakpoint clusters. The localization pattern of S phase-induced breakpoints in CHO cells is similar to the pattern of G1-induced breakpoints reported earlier. These data show that breakpoint localization for both REs is independent of the cell cycle stage (G1 or S) in which aberrations are induced and give further support to the hypothesis that nuclease hypersensitive regions (NHRs) associated with active genes play an important role in the distribution of breakpoints.

Localization of chromosome breakpoints induced by AluI and BamHI in Chinese hamster ovary cells treated in the G1 phase of the cell cycle.

Intact Chinese hamster ovary cells were exposed to the restriction endonucleases (REs) AluI or BamHI. In metaphase spreads from these cells, 300 breakpoints per RE were localized in G-banded chromosome type aberrations (dicentrics, translocations, rings, terminal and interstitial deletions). The majority of breakpoints induced by both REs were localized in G-light bands and showed a similar distribution of breakpoint clusters. RE digestion of metaphase spreads with AluI induced C-banding, and with BamHI G-banding. The data indicate that nuclease sensitive sites associated with active genes are mainly responsible for the distribution of breakpoints.

Chromatid-type aberrations induced by AluI in Chinese hamster ovary cells.

Treatment of CHO cells with AluI in the S-phase leads to chromatid-type aberrations whose frequencies are linearly correlated with dose. Treatment in the S-phase leads to fewer aberrations than treatment in the G1-phase, which is comparable to chromosomal aberration induction following X-irradiation in the G1- and S-phases. Treatment in the G1-phase leads to few chromatid-type interchanges, some of these may originate from DNA single-strand gaps induced by AluI in canonical structures of DNA and in DNA.RNA hybrids.

The restriction endonuclease AluI induces sister chromatid exchange in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were exposed to the restriction endonuclease AluI in the presence of 1.1 M glycerol. Chromosomal aberrations and sister chromatid exchange (SCE) were scored in first post-treatment metaphases following recovery times of 6-18 h. At all recovery times chromosomal aberrations were induced by the enzyme. In AluI-treated damaged cells significant elevations of SCE frequencies were found after recovery times of 12-18 h. These results indicate that SCE, unlike chromosomal aberrations, are induced only in late G1 and early S phase of the cell cycle. The lesions producing SCE are postulated as DNA single-strand breaks and gaps induced by AluI in canonical structures of DNA and in DNA-RNA hybrids.

Induction of chromosomal aberrations by DNase I.

Chinese hamster ovary (CHO) cells were treated with bovine pancreatic DNase I using the method of electroporation. The enzyme induced chromosomal aberrations in a S-phase independent manner. The frequencies of polycentric chromosomes induced in the G1 phase of the cell cycle are positively correlated with the dose of DNase I. The distributions of DNase I-induced polycentric chromosomes were overdispersed.

[Reflection microscopy of interchromosomal filaments]. / Les filaments interchromosomiques en microscopie par réflexion.

After Giemsa staining of metaphases, reflection analysis reveals interchromosomal filaments. Either simple or in bundles, these filaments connect two chromosomes or sometimes several of them in a chain-like fashion. The anchoring points are most often localized in telomeric regions (64%). The form and the location of the chromosomes can be modified by the traction forces at the level of the anchoring sites. For those located outside the telomeric regions, the distribution of the sites shows a good correlation with the size of the chromosomal arms, except for the group D which is overrepresented.

Clastogenic action of a dimethyl p-benzoquinone of animal origin.

The cytogenetic action of a volatile dimethyl p-benzoquinone found in the natural secretion of an arachnid (Acanthopachylus aculeatus Kirby) was studied in cultured human peripheral leukocytes and in mouse bone-marrow cells. Continuous and pulse treatments carried out in vitro, as well as experiments performed in vivo, induced different chromatid and chromosome aberrations suggesting that this chemical has clastogenic properties. The biology of the animal and the possible role of one of the components of its secretion as a natural mutagenic agent are discussed.