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Biomedical subjects

I Schubert

Publications and source records attributed to I Schubert.

At least 37 records · Page 2Linked to original sources

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↗

Opportunism knocks?

Explore the source record for details and available documents.

Academies and Institutes↗

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↗

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↗

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↗

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↗

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↗

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↗

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↗

DNA content, rDNA loci, and DAPI bands reflect the phylogenetic distance between Lathyrus species.

The nuclear DNA content, the proportion of A+T base pairs, the chromosomal positions of 5S and 25S rRNA genes, as well as of DAPI (4,6-diamidino-2-phenylindole) bands are described for seven species belonging to three different sections of the genus Lathyrus. These data and chromosome measurements allowed to establish precise idiograms and to discriminate most of the chromosome pairs of the seven species. The karyotypic features correlate well with the phylogenetic distances between these species.

Chromosome Banding↗

Markers to analyse the prescribing of non-steroidal anti-inflammatory drugs in ambulatory care. A guide to pursuing rational and safe prescribing.

OBJECTIVE: The aim of the study is to construct quality markers for rational prescribing of non-steroidal anti-inflammatory drugs (NSAIDs MO1A, MO1B) in such a manner that they meet three requirements: suitability to be assessed by prescription analysis, application in feedback strategies and contribution to the task of internal quality assurance. METHOD: Eight different markers for validating NSAID prescribing were developed according to pharmacological literature. The prescribing of 99 participants (high prescribers) and 15 coordinators of eight pharmacotherapy circles (second quarter 1996) served as the database. To test the validity of the markers in terms of rationality, the NSAID prescribing of 15 randomly selected participants of these circles, whose participants were not trained in the analysis of their prescribing, was compared with the prescribing of the 15 coordinators of these circles, who had considerable experience in pharmacotherapy and group auditing. In order to compare results according to the age and sex of the patients, the two groups treated with NSAIDs were also matched (460 patients in each group). The drugs are classified under the ATC code with the volume given in defined daily doses (DDDs). RESULTS: Marker 1 - the percentage of NSAID DDDs for recommended drugs, i.e. ibuprofen, diclofenac, indomethacin and naproxen was significantly higher for the coordinators in comparison with the high prescribers (P < 0.05). Therefore, marker 3 (drugs with questionable efficacy) and marker 5 ('me-too' drugs) show an inverse relation. Drugs with a long half-life (marker 2), high-risk drugs (marker 4) and newly marketed drugs (marker 6) were all seldomly prescribed by both groups of doctors. There was no difference between the two groups of prescribers concerning the proportion of elderly people treated with NSAIDs (marker 7). With reference to marker 8 - co-medication with anti-ulcer drugs - the coordinators treated 8.9% of NSAID patients with antacids and anti-ulcer drugs, the high prescribers, on the other hand, treated 12.2% (NS). CONCLUSION: The markers can be easily assessed using the information obtained from drug claims and shown to each doctor personally. They call for the doctors to pay special attention to their particular drug selection. The markers can be implemented into feedback strategies of prescribing habits.

Ambulatory Care↗

Short communication: the cell cycle dependent phosphorylation of histone H3 is correlated with the condensation of plant mitotic chromosomes

Mitotically dividing cells of Secale cereale, Hordeum vulgare and Vicia faba were studied by indirect immunofluorescence using an antibody recognizing phosphorylated histone H3. The study revealed the following features: (i) the H3 phosphorylation starts at prophase and ends at telophase in the pericentromeric chromatin, is associated with the condensation of mitotic chromosomes and is independent of the distribution of late replicating heterochromatin. (ii) Compared with other chromosome regions, the pericentromeric chromatin is histone H3 hyperphos- phorylated. (iii) The study of a semi-dicentric chromo- some revealed that only at intact centromeres is the chromatin hyperphosphorylated at H3.

Journal Article↗

Pharmacotherapeutic circles. Results of an 18-month peer-review prescribing-improvement programme for general practitioners.

OBJECTIVE: To assess the effectiveness of the pharmacotherapeutic circle (PTC), a general practitioner (GP) prescribing-improvement programme to enhance prescribing quality and reduce drug costs. DESIGN: Combined pre- and post-intervention time-series design using an internal comparison of subgroups and an external comparative control. SETTING: Small discussion groups meeting 8 times over 18 months. PARTICIPANTS: 79 GPs exceeding the mean drug costs/patient of all Hessian physicians by > or = 40%; 10 moderators. INTERVENTIONS: Peer-review feedback of prescription patterns based on guidelines targeting 3 suboptimal prescribing areas: drug prescriptions lacking evidence-based efficacy (target A); presumptive prescribing habits (target B); and underprescribing of new, effective therapies (target C). MAIN OUTCOME MEASURES AND RESULTS: Significant decreases in prescription rates for target A drugs were recorded for varicose vein medications (p = 0.006), peripheral vasodilators (p = 0.0001) and topical antirheumatics (p = 0.0145), but not for prokinetics/enzymes/digestives. Prescribing of target B drugs such as benzodiazepines and nonsteroidal anti-inflammatory drugs declined markedly (p = 0.0019 and 0.0014, respectively). Target C drug prescriptions such as for opioids and proton pump inhibitors were not significantly increased. Highly significant reductions in prescription costs were observed for target A and B drugs, irrespective of whether GPs were stratified into high, medium or low prescribers. When mean prescribing costs for PTC participants were compared with those of a control group comprising 8000 GPs over a 21-month period, PTC GPs decreased their costs by 2%, whereas drug costs for all Hessian physicians rose by 10%. CONCLUSIONS: PTCs appear to be an effective method to optimise the quality of drug prescribing and reduce drug costs.

Cost Control↗

Assignment of linkage groups to pea chromosomes after karyotyping and gene mapping by fluorescent in situ hybridization.

Chromosomes of the pea (Pisum sativum L.) were submitted to fluorescent in situ hybridization (FISH) with probes specific for the oligonucleotides (AG)12, (AC)12, (GAA)10, and (GATA)7 and for the genes encoding 25S rRNA, 5S rRNA and the storage proteins legumin A, K and vicilin. A fourth 5S rRNA gene locus, apparently specific for an accession of the cultivar Grüne Victoria, was newly detected. This allowed all seven chromosome pairs to be distinguished by FISH signals of rRNA genes. The same was possible using a combination of oligonucleotide probes or of oligonucleotides and rRNA gene-specific probes in multicolour FISH. Rehybridization with the 5S rRNA gene-specific probe allowed us to assign vicilin genes to the short arm of chromosome 5, the single legumin A locus to the long arm of chromosome 3 and the legumin B-type genes (exemplified by legumin K) to one locus on the short arm of chromosome 6. Correlation of these data with an updated version of the pea genetic map allowed the assignment of most linkage groups to defined chromosomes. It only remains to be established which of linkage groups IV and VII corresponds to the satellited chromosomes 4 or 7, respectively.

Chromosome Mapping↗

Molecular-cytogenetic characterization of the Vicia faba genome--heterochromatin differentiation, replication patterns and sequence localization.

A comprehensive survey of the molecular-cytogenetic features of the Vicia faba chromosome complement (2n = 12) is given. It includes previous as well as new original data. Various Giemsa, restriction endonuclease and fluorochrome banding patterns, azacytidine-mediated segment extension, replication patterns, lateral A/T asymmetry and sequence localization data for tandemly arranged simple sequence repeats, dispersed repeats and coding sequences as well as histone acetylation patterns are considered. This allows not only to distinguish and characterize telomeres, subtelomeres, centromeres and the NOR, but also the structure of the 5S rRNA gene loci and two main types of interstitial heterochromatin. Additionally, it offers physical landmarks within euchromatic areas. Thus, the field bean genome, exemplified by the reconstructed karyotype ACB, belongs to the cytogenetically best investigated plant genomes.

Chromosome Banding↗