5 research outputs found
Optimization of Neutral Comet Assay for studying DNA double-strand breaks in pea and wheat
This study describes an adaptation of the Comet assay under neutral conditions for mono- and dicotyledonous plants pea (Pisum sativum L.) and wheat (Triticum aestivum L.). Modifications concern lysis and electrophoresis steps, respectively. Electrophoresis was carried out varying the intensity of the electric field. A linear relationship between the percentages of DNA in the tail from control background with alteration of intensity was found. Trypan blue dye exclusion test was used in order to determine the intactness of nuclear membrane of the isolated nuclei from both plant model systems. Assessment was conducted on non-irradiated and irradiated nuclei on a monolayer with three doses of UVC. It was found that the share of intact nuclei (trypan blue negative ones) is about 95% in controls. Gradual dose-related increase of damaged nuclei was observed in both species, reaching statistical significance only at the higher dose applied
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
SCE formation after exposure of CHO cells prelabelled with BrdU or biotin-dUTP to various DNA-damaging agents
Formation of sister chromatid exchanges (SCE) is a mechanism of repair or bypass of DNA damage during S phase. Although SCE have been studied for a long time, the types of DNA lesions involved and the role of 5-bromodeoxyuridine (BrdU) in SCE formation are a matter of debate. We have developed a novel method of differential labelling of sister chromatids with biotin-16-2'-deoxyuridine-5'-triphosphate (biotin-dUTP) and could show that a substantial proportion of radiation-induced SCE arise via damage to BrdU-moieties. The present investigations were performed to examine the role of BrdU in the formation of SCE by the endonucleases AluI and DNase I, as well as the alkylating agent mitomycin C (MMC). CHO cells unifilarily prelabelled with biotin-dUTP or BrdU were treated and the frequencies of SCE analysed in the first post-treatment mitoses. AluI induced similar frequencies of SCE in cells prelabelled with BrdU or biotin-dUTP. DNase I induced significantly more SCE in cells prelabelled with BrdU than with biotin-dUTP. MMC induced slightly more SCE in cells labelled with biotin-dUTP than BrdU, but the difference was not significant. The possible mechanisms responsible for the enhanced SCE frequency following DNase I treatment of cells prelabelled with BrdU are discussed
STK11 gene mutations among patients with sporadic breast cancer
Germline mutations affecting STK11 (LRG_319) are profoundly studied in
relation to Peutz-Jeghers syndrome, predisposing to the development of
various cancers at multiple sites. Though somatic mutations in STK11 are
found to be present in several cancers, limited data on its involvement in
sporadic breast cancer are available. The present study aims to evaluate the
frequency and spectrum of genetic alterations in STK11 in a group of
Bulgarian patients with sporadic breast cancer. A total of 73 tumor and 22
corresponding blood specimens derived from the patients, and 10 blood
samples from clinically healthy controls were analyzed. High Resolution
Melting analysis followed by Sanger sequencing and bioinformatic prediction
tools were utilized. Seven patients (9.58%) harbored STK11 alterations, only
two (2.74%) of which are exonic: one nonsense c.322A>T; p.K108X
(deleterious) and one missense c.440G>A; p.Arg147His (of unknown
significance). Two intronic variants were also observed: c.290+36G>T and
c. *16+18C>A (novel). To our knowledge the results represent the first data
indicating presence of STK11 alterations in patients with sporadic breast
cancer. The limited number of the detected deleterious mutations indicates
that mutational inactivation of the gene is a rare event and probably plays
a minor role in sporadic breast carcinogenesis