Lubomir Stoilov

DNA repair precedes replicative synthesis during early germination in maize.

DNA synthesis was studied during germination by following the rate of incorporation of radioactive thymidine into high molecular weight DNA. A peak of DNA synthesis was observed between the 8th and the 12th hour, i.e. before the beginning of the semi-conservative replication of genomic DNA, accompanied by an increase in the DNA content of the embryo. By the use of nucleoid sedimentation and nick-translation it was shown that, during the first hours of germination, extensive repair occurs of the DNA single-strand breaks present in the dry embryo. As a result, the DNA of the 16-h-germinated embryo acquires the conformation typical of that of the root meristemic cells active in transcription and replication.In addition we have shown that cytoplasmic organelle (most probably mitochondrial) DNA synthesis is very active during the prereplicative state which confirms earlier microscopic data on mitochondrial biogenesis during early germination.

Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay

Comet assay was applied to study induction and repair of DNA damage produced by bleomycin in barley genome. Experimental conditions were adapted to achieve efficient detection of both DNA single‐ and double‐strand breaks. Substantial increase of the parameter “% of DNA in tail” was observed coupled with almost linear dependence from bleomycin concentration, more pronounced for the induction of DNA double‐strand breaks. Data obtained at different recovery periods displayed rapid restoration of breakage, revealing that efficient mechanisms for repair of strand discontinuities induced by bleomycin are functional in barley DNA loop domains. Environ. Mol. Mutagen., 2008.

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.

Induction and recovery of double-strand breaks in barley ribosomal DNA.

Barley nucleolus organizing regions (NORs) were previously found to behave as prominent aberration hot-spots after treatment with some restriction endonucleases. The ability of MspI for directed induction of double-strand breaks in barley ribosomal DNA was further analyzed. Ionizing radiation-produced strand breakage within the ribosomal gene clusters was also a subject of investigation. Reconstructed barley karyotypes T1586 and T35 with normal and increased expression of rRNA genes were utilized to evaluate the relationship between transcriptional activity and damage induction. Scanning densitometry of the hybridization profiles revealed that MspI is generating double-strand breaks in barley rDNA with efficiency being independent from the NOR activity. Damage induction observed after treatment with gamma-rays was also not influenced by the transcriptional status of the ribosomal genes. A tendency towards restoration of rDNA integrity after irradiation of both germinating and dry seeds was observed which is indicative for the efficient recovery of double-strand breaks in barley ribosomal DNA.

Nuclear matrices from transcriptionally active and inactive plant cells

Abstract The morphology of DNA-rich and DNA-depleted nuclear matrices isolated from transcriptionally inactive (dry embryo) and transcriptionally active (root tip meristem) plant cells has been investigated by electron microscopy. While the transcriptionally active nuclei possess a well-defined lamina and a prominent internal matrix, the inactive nuclei show a considerable difference in that they lack an apparent internal matrix. The absence of a defined internal matrix structure can be correlated with the lack of topological constraint in DNA in the nuclei of the dry embryo cells. An electrophoretic characterization of the proteins of the nuclear matrix from the two sources is also presented.

Characterization of some nuclear matrix proteins in maize

Abstract We have prepared a nuclear matrix fraction from maize seedlings and studied its protein composition. Proteins were characterized by immunoblotting analysis, two-dimensional gel fractionation and two-dimensional peptide mapping. Most of them were found to belong to the group of IF-lamina proteins, as has been shown for the matrix proteins from animals. One of these proteins was further classified as the maize version of lamin C since it possessed an isoelectric point in the range characteristic for lamin C from animals, cross-reacted with anti-lamin c antiserum and its two-dimensional peptide map contained most of the spots characteristic of the map of mouse Earlich ascite tumor lamin C. Unlike animal lamins (proteins of molecular masses in the range of 60–75 kDa), the maize lamin C possessed a molecular weight of 42 kDa. A group of proteins possessed molecular masses in the 65- to 75-kDa range and isoelectric points similar to that of animal lamin B. In addition, their 2D peptide maps revealed considerable similarity with the map of lamin B from avian erythrocytes. However, these proteins do not resemble lamin B from EAT cells neither immunologically nor by tryptic peptide mapping. Two proteins of our preparation are not lamin-like. The tryptic peptide maps of the proteins reveal similarity with some animal proteins that bind DNA tightly.

Transcriptional activity and DNA supercoiling during early germination in maize

Abstract The possible link between the ability of DNA to be transcribed and its three-dimensional organization in the eukaryotic nucleus has been studied during germination of the plant embryo. In this model system there is a gradual increase in the transcriptional activity of chromatin and no nuclear DNA replication takes place for at least the first 20–24 h following water imbibition. Maize embryo nucleoids derived by nonionic detergent and high salt treatment from nuclei isolated at different time points of germination were analysed by sedimentation in a series of sucrose gradients containing increasing amounts of the intercalating agent ethidium bromide. The sedimentation of the dry embryo nucleoids shows no dependence on the concentration of ethidium bromide while the 16-h germinated embryo possesses the titration curve characteristic of the active plant genomes. On the other hand, DNA isolated from both the dry and the germinated material behaved upon EthBr intercalation as expected on theoretical grounds, i.e. its sedimentation rate decreased with increase of the EthBr concentration. The lack of a biphasic response to EthBr titration we interpret as an indication of a lack of supercoiled DNA loops in the dry embryo nuclei.

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.

Comparative Analysis of Data Distribution Patterns in Plant Comet Assay

ABSTRACT Screening capabilities of the comet assay have a real potential to study the impact of radiation and different mutagenic sources on induction of damage in DNA in plant nuclei. Heterogeneity of DNA damage data obtained by the application of different agents leads to an inconsistency and variation of the experimental outcomes, obtained after assessment of the comet populations. Although the potential of the comet assay technology has been clearly demonstrated, many important and interesting statistical questions remain. In this respect, different types of data need different statistical designs and this usually makes statistical analysis problematic. We advocate here a greater attention to different classical statistical distributions which best fit to plant comet data. We also demonstrate that all distribution patterns of the % of DNA in tail can be fitted by a Johnson SB distribution.