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I Schubert

Publications and source records attributed to I Schubert.

At least 19 recordsLinked to original sources
PubMed2001

DNA damage and repair in Arabidopsis thaliana as measured by the comet assay after treatment with different classes of genotoxins.

The three protocols of the comet assay A/N, A/A and N/N were for the first time applied to the plant species Arabidopsis thaliana. The purpose of the experiments was to establish conditions for genotoxic exposure causing DNA damage in Arabidopsis nuclei. This is required for comprehensive gene expression profiling with the intention to screen for genes involved in response of Arabidopsis cells to genotoxic stress. Five chemicals belonging to different classes of mutagens (the monofunctional alkylating agents N-methyl-N-nitrosourea and methyl methanesulfonate, the polyfunctional alkylating agent mitomycin C, the radiomimetic bleomycin and the herbicide maleic hydrazide) were tested. Except for maleic hydrazide, dose-dependent increases in DNA damage were found using the A/N comet assay protocol. While a rapid repair of bleomycin-mediated SSBs and DSBs was found, no significant reduction of DNA migration was observed up to 48h after treatment with the monofunctional alkylating agents.

Alkylating Agents↗
PubMed2001

The comet assay detects adaptation to MNU-induced DNA damage in barley.

We have established the comet assay for detection of DNA damage in barley. Immediately after treatment with the monofunctional alkylating agent MNU, a dose-dependent increase of DNA damage (mainly DNA breaks) was detected by the alkaline denaturation/neutral gel electrophoresis (A/N) variant of the comet assay in nuclei isolated from root tip meristems and from young leaves. A reduction of damage was observed within meristematic nuclei but not in differentiated leaf nuclei 48h after treatment. Adaptive pretreatment with a nontoxic dose of cadmium chloride prior to challenge treatment with MNU reduced the frequency of chromatid type aberrations, micronuclei and aneuploid cells as well as the amount of DNA in comet tails of meristematic nuclei.

Alkylating Agents↗
PubMed2001

Chromatin organization and its relation to replication and histone acetylation during the cell cycle in barley.

We have studied the replication time, nuclear organization and histone acetylation patterns of distinct chromatin domains [nucleolus organizers (NORs), centromeres, euchromatin and heterochromatin] of barley during the cell cycle. The Rabl orientation of chromosomes, with centromeres and telomeres located at opposite nuclear poles, was found to be maintained throughout interphase. Replication started at the rDNA loci within nucleoli and then proceeded from the euchromatic distal chromosome regions toward the heterochromatic pole. Centromere association frequently occurred in mid- and late S-phase, i.e., during and after centromere replication. Euchromatin, centromeres and heterochromatin were found to be enriched in acetylated histone H4 (except for lysine 16) during their replication; then deacetylation occurred. The level of deacetylation of H4 in heterochromatin was more pronounced than in euchromatin. Deacetylation is finished in early G2-phase (lysine 8) or may last until mitosis or even the next G1-phase (lysines 5 and 12). The NORs were found to be most strongly acetylated at lysines 5 and 12 of H4 during mitosis, independently of their potential activity in nucleolus formation and rDNA transcription. The acetylation pattern of chromosomal histone H3 was characterized by low acetylation intensity at centromeres (lysines 9/18) and pericentromeric regions (lysine 14) and more intense uniform acetylation of the remaining chromatin; it remained fairly constant throughout the cell cycle. These results have been compared with the corresponding data published for mammals and for the dicot Vicia faba. This revealed conserved features as well as plant- or species-specific peculiarities. In particular, the connection of acetylation intensity of H4 at microscopically identifiable chromatin domains with replicational but not with transcriptional activity during the cell cycle seems to be conserved among eukaryotes.

Acetylation↗
PubMed2001

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis.

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.

Flow Cytometry↗
PubMed2001

[Confidentiality within the scope of secondary data research--approaches to a solution of the problem of data concentration].

In order to implement general data protection requirements and internationally recognised ethical requirements, research with personal health and social data demands a specific framework for the secure handling of confidential data. In the process of transferring data from the health service providers to the place where they are analysed, an important role is played by a so-called trust centre, responsible for pseudonymisation of personal and institutional identifiers. An undesirable concentration of data in the trust centre can be avoided by early separation of data in the data transfer institution: the trust centre receives only the identifier to be pseudonymised, while the health provision data are sent direct to the analysing institution, where they can be matched with the pseudonyms from the trust centre, with the help of a unique case number. The possibility of reidentification, which exists mainly in large (pseudonymised) data sets, can be reduced by use of an appropriate pseudonymisation process (e.g. insuree-based pseudonymisation by health service providers for sampling of insurees). The measures described here are suitable for protecting confidentiality and for further improving data security in the handling of confidential personal and institutional data.

Computer Security↗
PubMed2001

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.

Chromosome Mapping↗
PubMed2001

Alteration of chromosome numbers by generation of minichromosomes -- is there a lower limit of chromosome size for stable segregation?

Practical applications of minichromosomes, generated by de novo composition or by truncation of natural chromosomes, rely on stable transmission of these chromosomes. Functional centromeres, telomeres and replication origins are recognized as prerequisites for minichromosome stability. However, it is not yet clear whether, and if yes, to what degree the chromatin content has a qualitative or quantitative impact on stable chromosome transmission. A small translocation chromosome, which arose after X-irradiation of a reconstructed field bean karyotype, comprised approximately 5% of the haploid metaphase complement and was found to consist of three pieces of duplicated chromatin and a wild-type centromere. This chromosome was stably transmitted through all meristematic and pollen grain mitoses but was frequently lost during meiosis (66% loss in hemizygous and 33% in homozygous condition). This minichromosome was only a little smaller than stably segregating translocation chromosomes (comprising approximately 6% of the genome) of a euploid field bean karyotype. The duplications specific for this minichromosome did not influence meiotic segregation when associated with non-duplicated chromatin of other chromosomes. In comparison with minichromosomes of other species, the possibility of a lower size limit for a stable chromosome transmission must therefore be considered which might be based, for instance, on insufficient lateral support of centromeres or on insufficient bivalent stability due to the incapability of chiasma formation.

Animals↗
PubMed2000

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Base Sequence↗
PubMed2000

Opportunism knocks?

Explore the source record for details and available documents.

Academies and Institutes↗
PubMed2000

Detection of specific DNA lesions by a combination of comet assay and FISH in plants.

We have studied comet formation on Vicia faba nuclei embedded in agarose and treated with the endonucleases DNase I (to produce SSBs or DSBs at random sites), FokI (to produce DSBs preferentially within FokI repeats), or EcoRI (to produce DSBs at random sites but not within FokI elements). DNase I-induced SSBs were detected when enzyme treatment was followed by alkaline denaturation. DSBs efficiently mediated comet formation using neutral conditions. FISH with DNA probes, detecting specific chromosomal domains such as FokI element-containing heterochromatin, NORs, or telomeres, was done on comets. The distribution of FISH signals between the head and tail of comets indicated to which degree these domains were damaged and reflected the distribution of cleavage sites for the applied restriction endonucleases within these domains. The data confirmed the expectation that the observed comet formation was based on enzyme-specific DNA breakage.

DNA, Plant↗
PubMed2000

Adaptation to alkylation damage in DNA measured by the comet assay.

The alkylating mutagens N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS) were studied for their potential to induce DNA strand breaks and abasic (AP) sites in meristematic nuclei of Vicia faba root tips by the comet assay. The alkaline unwinding/neutral electrophoresis (A/N) and alkaline unwinding/alkaline electrophoresis (A/A) protocols were used for detection of DNA damage. With the A/N comet assay, less DNA damage was seen after conditioning pretreatment with a low dose prior to a high challenging dose of alkylating mutagens as compared to application of the high dose only, whereas a nearly additive effect was seen when the A/A comet assay was used. Adaptation was even more obvious when AP sites were revealed by the AP-endonuclease activity of exonuclease III. The adaptation observed with the A/N comet assay was abolished by pretreatment with the protein synthesis inhibitor cycloheximide. These data suggest that the comet assay is able to detect on molecular level a phenomenon resembling clastogenic adaptation.

Adaptation, Physiological↗
PubMed2000

Evolutionary conservation of kinetochore protein sequences in plants.

The evolutionary conservation of structural/functional kinetochore proteins has been studied on isolated nuclei and pro-/metaphase chromosomes of mono- and dicot plants. The cross-reactivities of antibodies against human CENPC, CENPE and CENPF, and against maize CENPCa with the centromeric regions of mitotic chromosomes of Vicia faba and/or Hordeum vulgare are shown. Putative homologs of the kinetochore protein SKP1 (suppressor of kinetochore protein 1p of yeast) were found in both species and of CBF5p (centromere binding factor 5 of yeast) in barley. Antibodies against synthetic peptides derived from partial sequences encoding these proteins were produced and recognized the centromeric regions on mitotic chromosomes as detected by indirect immunofluorescence.

Amino Acid Sequence↗
PubMed2000

An efficient screen for reproductive pathways using mature seeds of monocots and dicots.

Seed samples of 32 species (obligate and facultative sexuals and apomicts of monocots and dicots) were investigated by flow cytometry to reveal the pathway of reproduction. Ten different pathways of seed formation could be reconstructed considering whether the female and/or male gametes were reduced or unreduced, the embryos arose via the zygotic or parthenogenetic route and the endosperm via the pseudogamous or autonomous route. The screen is suited to select sporophytic or gametophytic mutants in sexual species, to identify pure sexual or obligate apomictic genotypes from facultative apomictic species, and to analyze the inheritance of the individual reproductive processes. Corresponding unique results are presented for Arabidopsis, Arabis, Hypericum and Poa. The screen of mature seeds by flow cytometry yielded more information about the reproductive behavior of individual plants than any other available test, and is very useful both in basic research and plant breeding.

Plant Physiological Phenomena↗
PubMed2000

Maleic hydrazide induces genotoxic effects but no DNA damage detectable by the comet assay in tobacco and field beans.

The plant growth regulator and herbicide maleic hydrazide (MH) induced a high frequency of somatic mutations in leaves of tobacco (Nicotiana tabacum var. xanthi) and a high yield of chromosome aberrations in roots of field beans (Vicia faba, karyotype ACB). In contrast, no significant increase in MH-induced DNA damage, as measured by the Comet assay, could be demonstrated in either plant species. The absence of DNA migration induced by MH was not effected in tobacco by either pH of the MH solution, the sampling time after MH treatment or continuous MH treatment for 14 days. To our knowledge, MH represents the first agent which has proved to be highly mutagenic and clastogenic but does not cause DNA damage as measured by the Comet assay in the same experimental system.

Chromatids↗
PubMed2000

N-Methyl-N-nitrosourea-induced DNA damage detected by the comet assay in Vicia faba nuclei during all interphase stages is not restricted to chromatid aberration hot spots.

The genotoxic effect of the monofunctional alkylating agent N:-methyl-N:-nitrosourea (MNU) on root-tip nuclei of the field bean, Vicia faba, has been tested by comparative application of three protocols of the comet assay. While the alkaline denaturation/alkaline electrophoresis (A/A) procedure proved to be most sensitive at low doses, the alkaline denaturation/neutral electrophoresis (A/N) procedure yielded an optimal dose-response curve within a wider dose range. With the neutral electrophoresis without alkaline denaturation (N/N) procedure only minimal response was found. MNU-mediated single-strand breaks occurred in nuclei of all interphase stages. Detection of tandemly repeated FOK:I elements on comets by fluorescence in situ hybridization showed an average involvement of these heterochromatin-specific sequences in MNU-mediated single-strand breaks. This, together with previous results, suggests that the pronounced clustering of chromosomal aberrations in heterochromatic regions after treatment with S phase-dependent mutagens is mainly due to an error-prone interference of recombinative repair and replication in damaged basic repeats of large tandem repeat arrays.

Alkylating Agents↗
PubMed2000

Histone H4 acetylation of euchromatin and heterochromatin is cell cycle dependent and correlated with replication rather than with transcription.

Reversible acetylation of nucleosomal histones H3 and H4 generally is believed to be correlated with potential transcriptional activity of eukaryotic chromatin domains. Here, we report that the extent of H4 acetylation within euchromatin and heterochromatic domains is linked with DNA replication rather than with transcriptional activity, whereas H3 acetylation remains fairly constant throughout the cell cycle. Compared with euchromatin, plant nucleolus organizers were more strongly acetylated at H4 during mitosis but less acetylated during S phase, when the nucleolus appeared to be (at least transiently) devoid of nucleosomes. Deposition-related acetylation of lysines 5 and 12 of H4 seems to be conserved in animals and plants and extended to K16 in plants. A possibly species-specific above-average acetylation at lysines 9/18 and 14 of H3 appeared in 4',6-diamidino-2-phenylindole (DAPI)-stained heterochromatin fractions. These results were obtained by combining immunodetection of all acetylatable isoforms of H3 and H4 on mitotic chromosomes and nuclei in G1, early S, mid-S, late S, and G2 phases of the field bean with identification of specific chromatin domains by fluorescence in situ hybridization or DAPI staining. In addition, the histone acetylation patterns of distinct domains were compared with their replication and transcription patterns.

Acetylation↗