Kostadin Gecheff

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.

Production of Recombinant Human Lactoferin in Transgenic Barley

ABSTRACT Introduction of human lactoferrin (hLF) gene, a cationic iron-binding glycoprotein, in the genome of commercial Bulgarian barley (Hordeum vurgare L.) cultivar was performed via particle bombardment. A rapid, efficient, and reproducible system was used to generate green, fertile, transgenic plants. Mature zygotic embryos from dry seeds of malting cultivar Ruen were bombarded with two plasmids (pAFT105 and pUBLF) containing human lactoferrin gene. The embryos were co-transformed either with pDM302 containing selectable marker gene (bar) for herbicide resistance and plasmid pAct1-F carrying uidA gene forβ-glucuronidase activity, or with plasmid pAHC25 including bar and uidA genes both under the control of the ubiquitin (Ubi) promoter. Forty eight individual regenerants were selected on Bialaphos (glufosinate-ammonium) and subjected to PCR analysis using primers corresponding to the sequence of hLF gene. The introgression of hLF cDNA was confirmed in 27 plants from 16 lines. The expression of recombinant human lactoferrin was proved by Southern and Western blot analyses. Transmission of the hLF gene to T1 progeny was studied. In this study we present an efficient system for introduction of gene encoding valuable recombinant protein. These data suggest that commercial barley can be used as a bioreactor for molecular farming ofpharmaceutical proteins.

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

Summary.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 6H5H 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.

Karyotype reconstruction modulates the sensitivity of barley genome to radiation-induced DNA and chromosomal damage

The potential of cytologically reconstructed barley line D-2946 to cope with the major lesions that hamper genome integrity, namely DNA single- and double-strand breaks was investigated. Strand breaks induced by γ-rays and Li ions were assessed by neutral and alkaline comet assay. Repair capacity after bleomycin treatment was evaluated by agarose gel electrophoresis under neutral and alkaline conditions. Frequencies of radiation-induced chromosome aberrations were also determined. Results indicate that radiation-mediated constitutive rearrangement of the chromosome complement has led to a substantial modulation of the sensitivity of barley genome towards DNA strand breaks, produced by ionising radiation, Li ion implantation and bleomycin in an agent-specific manner, as well as of the clastogenic response to γ-rays. Based on these findings, reconstructed barley karyotype D-2946 can be considered a candidate radio-sensitive line with reduced ability to maintain genome integrity with respect to both DNA and chromosomal damage.

DNA methylation pattern in a barley reconstructed karyotype with deleted ribosomal gene cluster of chromosome 6H

A reconstructed barley karyotype (T-35) was utilised to study the influence of chromosomal rearrangements on the DNA methylation pattern at chromosome level. Data obtained were also compared with the distribution of Giemsa N-bands and high gene density regions along the individual chromosomes that have been previously described. In comparison to the control karyotype (T-1586), the DNA methylation pattern was found to vary not only in the reconstructed chromosomes but also in the other chromosomes of the complement. Significant remodelling process of methylation pattern was found also in the residual nucleolus organiser regions (NOR) on chromosome 5H as a consequence of deletion comprising the whole NOR of chromosome 6H in T-35. Moreover, differences between corresponding segments of the homologues with respect to some other chromosome locations were also observed. Repositioning of genomic DNA methylation along the metaphase chromosomes following chromosomal reconstruction in barley seems to be essential to ensure correct chromatin organisation and function.

Effects of caffeine on the frequencies and location of chemically induced chromatid aberrations in barley

Abstract We have studied the influence of caffeine on the frequencies and location of MNNG and MH-induced aberrations in a reconstructed barley karyotype. We found a mutagen-dependent mechanism of caffeine action that was affected by the nature of the clastogen applied. Both mutagens induced chromatid aberrations of the delayed type. Post-treatment with caffeine significantly increased the frequencies of aberrations induced by these agents; the effect was more clearly pronounced with MNNG. There was no delay in the appearance of aberrations induced by MNNG (as opposed to MH) after caffeine post-treatment, an observation that is difficult to explain with the well-known mechanism of caffeine inhibition of post-replication repair. After treatment of barley with MNNG or MH, some intrachromosomal regions were preferentially involved in induced aberrations. Combined action of either of the mutagens with caffeine in general revealed a rather random distribution pattern of induced aberrations. Nevertheless, certain chromosomal segments were more sensitive to caffeine potentiation. Because it has been suggested that caffeine potentiation of the frequency of chemically induced chromosomal aberrations is connected with an inhibition of post-replication repair of primary lesions, it seems likely that the effect of caffeine on aberration distribution is coupled with more or less pronounced differential effectivity of repair processes in individual chromosomal regions.

Molecular Cytogenetic Characterization of a New Reconstructed Barley Karyotype

ABSTRACT Multicolor fluorescence in situ hybridization (FISH) was applied to mitotic metaphase chromosomes of the standard barley variety Freya and a new reconstructed karyotype, PK 88–19, with complete cytological marking of the chromosome complement in order to map physically the rearrangement breakpoints induced by gamma-irradiation. Three repetitive DNA sequences, namely, GAA satellite sequence, Afa-family subclone pAs1, and rDNA clone pTa71, were used as FISH probes. The hybridization sites produced were found to be distributed in specific locations along the chromosomes, thus creating conditions for more detailed cytogenetic characterization of the reconstructed karyotype. The comparative analysis of the distribution patterns of FISH signals in the reconstructed chromosomes resulting from three reciprocal translocations (1H-5H, 2H-7H and 3H-4H) and two pericentric inversions (4H3H and 6H) on the one hand, and the respective standard chromosomes, on the other, allowed a precise identification of the putative regions where the rearrangement breakpoints have occurred. In addition, three new minor rDNA sites located in the long arms of chromosomes 2H, 5H and 6H were identified. Due to the clear morphological distinctions of the different chromosome types and the available detailed positions of their rearrangement breakpoints, PK 88–19 may offer an essential gain in the resolution power over other reconstructed karyotypes in various research areas of barley cytogenetics.

CHIAS-based positioning of recombination hotspots and Giemsa bands in a multireconstructed barley karyotype

Abstract The chromosome complement of reconstructed barley karyotype PK 88 was analyzed by computer-aided Chromosome Image Analysis System (CHIAS). Fine mapping of Giemsa N-bands and regions with increased meiotic recombination activity along each individual chromosome was achieved. It was also found that CHIAS-visualized condensation profiles can be utilized as a reliable criterion for subtle differentiation of hetero- and euchromatin domains within a defined chromosomal regions. Application of CHIAS on karyotypes with distinct chromosome morphology was found to be an appropriate and reliable tool for screening of changes in chromatin compactness and its functional characteristics.

Antimutagenic effects of stressors in higher plants

SummaryThe protective (antimutagenic) effect triggered by ‘conditioning’ treatment with low doses of mutagens, sublethal heat shock or compounds known as inducers of stress proteins prior to ‘challenge’ treatment with a high clastogen dose is described. Criterium of protection (‘clastogenic adaptation’) is the reduced quantity of chromatid aberrations induced by combination of ‘conditioning’ plus ‘challenge’ treatment as compared to only ‘challenge’ treatment of root tip meristems of higher plants (Vicia faba andHordeum vulgare). This anticlastogenic effect is absent in metabolically inactive cells and becomes counteracted by impairment of protein synthesis or inhibition of ‘G2 repair’. The data are indicative of at least 3 different inducible repair functions specific for certain types of DNA damage and reminiscent of those involved in the ‘adaptive response’ of bacterial and mammalian cells.ZusammenfassungDie Arbeit faßt die protektiven (antimutagenen) Effekte zusammen, die beobachtet wurden, wennVicia faba oderHordeum vulgare vor der Einwirkung mit hohen Clastogenkonzentrationen einer ‚konditionierenden‘ Vorbehandlung mit niedrigen Clastogenkonzentrationen, Hitzeschocks oder Induktoren von Hitzeschockproteinen unterworfen wurden. Kriterium der Protektion (‚clastogene Adaptation‘) war die signifikant verringerte Häufigkeit induzierter Chromatidenaberrationen, die bei kombinierter Einwirkung von ‚konditionierender‘ Vorbehandlung und nachfolgender Einwirkung mit hohen Clastogendosen in Wurzel-spitzenmeristemen beobachtet wurden. Derartige anticlastogene Effekte blieben in metabolisch inaktiven Zellen aus und wurden durch Einwirkung von G2-Reparaturinhibitoren kompensiert. Die vorliegenden Daten weisen auf protektive Funktionen (Reparaturprozesse?) hin, die durch ‚konditionierende‘ Vorbehandlung induziert werden und für bestimmte DNA-Schadenstypen spezifisch zu sein scheinen. Die beobachteten Phänomene erinnern an den bei Bakterien und Säugerzellen beschriebenen ‚adaptive response‘.Краткое содержаниеРабота обобщает защитные (антимутагенные) эффекты, которые наблюдались, когдаVicia faba илиHordeum vulgare, перед воздействием кластогенами высоких концентраций, подвергались «кондиционированно й» предварительной обработке кластогенами низких концентраций, тепловому шоку или обработке индукторами белков теплового шока. Критерием защиты («кластогенная адаптация») служила сигнификантно уменьшающаяся частота индуцированных хроматидных аберраций, которые наблются в меристемах корневых окончаний при комбинированном воздействии «конденсированной» предварительной обработке и последующего воздействия высокими дозами кластогенов. Антикластогенные эффекты подобного рода отсутствуют в метаболически неактивных клетках и компенсируются воздействием ингибиторов репарационных процессов в G2-фазе. Представленные данные указывают на защитные функции (процессы репарации?), которые индуцируются вследствие «кондиционированн ой» предварителыной обработки и, кажется, являются специфическими для определенных типов повреждений ДНК. Наблюдаемые феномены напоминают описанные у бактерий и в клетках млекопитающихся адаптивные реакции.