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Analysis of rRNA genes in variets Brassica napus
Dofková, Květoslava ; RNDr.Roman Matyášek, CSc. (referee) ; Kovařík,, Aleš (advisor)
Brassica napus (AACC, 2n = 38) is an allotetraploid species derived from the parentel diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). The aim of thesis was to carry out the genetic and epigenetic analysis of high-copy rRNA genes (or rDNA) in several varieties of hybrid species B. napus. The experiments involved determining the ratio of parental genes in hybrids, sequencing and methylation analysis of the promoter region of rDNA. Using Southern hybridization, it was revealed significant variability in the number of parental rDNA units between each variety. Data from sequence analysis were in good agreement with the results of Southern blot. Genetic recombination between parental rDNA units was revealed in one variety by DNA sequencing of promotor region. To study methylation, bisulfite sequencing was performed. It was found out that rDNA units of B. rapa origin have a higher value of methylation than units originated from B. oleracea.
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Monitoring the influence of stress factors on yeasts of the genus Metschnikowia using molecular techniques
Kuljovská, Tereza ; Kovalčík, Adriána (referee) ; Němcová, Andrea (advisor)
Molecular techniques are used widely nowadays, especially in the identification of various species of microorganisms, and provide reliable and accurate results in a relatively short period of time. This work deals with the characterization of Metschnikowia yeasts exposed to various stress factors and the monitoring of their genomic DNA by PCR-DGGE and PFGE methods. The work was focused on the analysis of ribosomal rDNA, specifically the ITS1 and 5,8-ITS2 regions and genes encoding the domains D1/D2, that are part of the large 26S rDNA ribosomal subunit, which are commonly used in the characterization of fungal eukaryotic communities. Three types of stress factors were selected for the experiments: osmotic stress (NaCl environment), oxidative stress (addition of H2O2 to the medium) and nutritional stress (addition of hemp flour / leaves and flowers as a carbon source). The analysis was performed for particular strains Metschnikowia andauensis, Metschnikowia pulcherrima, M. chrysoperlae, M. shanxiensis, M. sinensis and M. zizyphicola. The results showed that addition of the NaCl, H2O2 and hemp components at higher concentrations to the production media does not disrupt the ribosomal DNA when detected by PCR-DGGE. Mutations have not been observed by comparing these strains with yeast that was cultivated under optimal conditions. Despite stress factors, PFGE analysis of karyotypes showed that DNA of some yeast species does not prove any damage and remain intact. Remaining strains proved certain degree of damage, and bands were not detected on the gel for these strains. In the given circumstances, it can be stated that the high adaptability of these species to a stress environment makes them promising biotechnology producers. These yeasts have great potential for usage in agriculture as a tool for biocontrolling of fruit or vines.
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Use of DGGE to analysis and identification of selected microorganisms
Jankeje, Kristína ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
Presented diploma thesis is focused on use of DGGE to analysis and identification of selected microorganisms. PCR-DGGE is a method that allows direct characterization of the microbial community in the natural environment without necessity of cultivation. A literature review is devoted to the principle of the method, current applications and its limitations too. In experimental part microbial DNA was isolated and used as a template for PCR reaction. Microbial DNA was then amplified using the universal eukaryotic primers that target the D1/D2 domain of the 26S subunit of ribosomal DNA. To improve specificity and sensitivity of detection nested PCR was chosen using outer and inner primer pairs. Generated amplicons (250 bp) were consequently separated by DGGE. The analysis of selected microorganisms by DGGE technique was performed after optimization of electrophoresis conditions (in particular the denaturing gradient extent and separation time). Despite the optimization, mutual differentiation among individual yeast strains was not possible since each reference strain was represented by several bands in the same positions. In conclusion DGGE profile obtained from wine musts is discussed. Present bands suggest the major presence of non-Saccharomyces yeasts, yeast-like strain A. pullulans is present in the minority and Saccharomyces yeasts are probably present too. The technique remains open for further optimization, particularly as regards the conditions of polymerase chain reaction.
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Monitoring the influence of stress factors on yeasts of the genus Metschnikowia using molecular techniques
Kuljovská, Tereza ; Kovalčík, Adriána (referee) ; Němcová, Andrea (advisor)
Molecular techniques are used widely nowadays, especially in the identification of various species of microorganisms, and provide reliable and accurate results in a relatively short period of time. This work deals with the characterization of Metschnikowia yeasts exposed to various stress factors and the monitoring of their genomic DNA by PCR-DGGE and PFGE methods. The work was focused on the analysis of ribosomal rDNA, specifically the ITS1 and 5,8-ITS2 regions and genes encoding the domains D1/D2, that are part of the large 26S rDNA ribosomal subunit, which are commonly used in the characterization of fungal eukaryotic communities. Three types of stress factors were selected for the experiments: osmotic stress (NaCl environment), oxidative stress (addition of H2O2 to the medium) and nutritional stress (addition of hemp flour / leaves and flowers as a carbon source). The analysis was performed for particular strains Metschnikowia andauensis, Metschnikowia pulcherrima, M. chrysoperlae, M. shanxiensis, M. sinensis and M. zizyphicola. The results showed that addition of the NaCl, H2O2 and hemp components at higher concentrations to the production media does not disrupt the ribosomal DNA when detected by PCR-DGGE. Mutations have not been observed by comparing these strains with yeast that was cultivated under optimal conditions. Despite stress factors, PFGE analysis of karyotypes showed that DNA of some yeast species does not prove any damage and remain intact. Remaining strains proved certain degree of damage, and bands were not detected on the gel for these strains. In the given circumstances, it can be stated that the high adaptability of these species to a stress environment makes them promising biotechnology producers. These yeasts have great potential for usage in agriculture as a tool for biocontrolling of fruit or vines.
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Evolution of karyotypes and sex determination in the turtle family Geoemydidae
Clemente, Lorenzo ; Rovatsos, Michail (advisor) ; Montiel Jimenez, Eugenia Elisabet (referee) ; Castiglia, Riccardo (referee)
(IN ENGLISH) The majority of studied turtles show temperature-dependent sex determination, but genotypic sex determination (i.e. presence of sex chromosomes) was identified sporadically. This thesis aims to investigate and expand our knowledge on the evolution of the karyotype and the sex determination in turtles, particularly focusing on the family Geoemydidae, a group of turtles with previously documented variability in sex determination systems. The presence of sex chromosomes was explored by a combination of conventional and molecular cytogenetic techniques for the analysis of karyotypes, distribution of constitutive heterochromatin (C-banding) and repetitive elements and comparative genome hybridization (FISH, CGH). In total, 49 species of turtles from nine different families were cytogenetically examined in this study. In the family Geoemydidae, a remarkable similarity in karyotypes was identified, consisting of 2n=52 chromosomes (which is suggested to be the ancestral diploid number for all turtles) and a similar topology of rDNA loci and telomeric repeats. Sharma et al. (1975) previously reported ZZ/ZW sex chromosomes in Pangshura smithii. However, in the analysis presented in this thesis, it is suggested a possible misidentification of these sex chromosomes due to erroneous pairing of...
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Replication and transcription of nucleolar DNA
Flusser, Michal ; Smirnov, Evgeny (advisor) ; Staněk, David (referee)
The nucleolus is the most prominent compartment of the cell nucleus and is the place where ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. Although the nucleolus has been studied for decades its structural and functional organization is still unclear. In particular, the role of various types of DNA participating in the formation of nucleoli along with ribosomal genes is not understood. The present thesis summarizes the current knowledge and views on the nucleolus, focusing on the two synthetic processes, replication and transcription, in mammalian cells. Specific features of these processes in the context of nucleolar ultrastructure remains an unsolved problem of the modern molecular biology.
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Evolution of sex chromosomes and karyotypes in boas and pythons
Charvát, Tomáš ; Rovatsos, Michail (advisor) ; Majtánová, Zuzana (referee)
- ABSTRACT - Snakes (Serpentes) are a group of squamate reptiles (Squamata) that represents more than one third of the total reptile species diversity. Snake karyotype is generally conserved with the most common chromosome number of 36 (16 macro- and 20 microchromosomes) in diploid state. It is believed that this karyotype was also present in the common ancestor of all snakes. The majority of snake species belong to the group Caenophidia and share homologous ZW sex chromosomes. Snakes from the groups "Scolecophidia" and "Henophidia" have mostly poorly differentiated, homomorphic sex chromosomes, which made them impossible to distinguish from the autosomes in the past. These snakes were for many years assumed to have ZW sex chromosomes as well. However, recent studies demonstrated not only ZW but also two non- homologous XY sex chromosome systems in non-caenophidian snakes and thus the sex determination systems in snakes are much more variable than previously thought. In this thesis, eight species of henophidian snakes (representatives from the genera Eryx, Cylidrophis, Python and Tropidophis) and one caenophidian species (Ophiophagus hannah) were examined using conventional and molecular cytogenetic methods. However, sex chromosomes were not detected in the henophidian species, only in Ophiophagus hannah,...
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Evolution of sex chromosomes in reptiles
Mazzoleni, Sofia ; Rovatsos, Michail (advisor) ; Zrzavá, Magda (referee) ; Liehr, Thomas (referee)
- ABSTRACT - Among vertebrates, reptiles represent the ideal group for the study of sex determination. Reptiles include lineages with environmental sex determination (ESD) as seen in crocodiles and tuatara, lineages with genotypic sex determination (GSD), like e.g. iguanas, chameleons, skinks, lacertid lizards and birds, and few groups which possess variability in sex determination mechanisms, i.e. geckos, dragon lizards and turtles. This thesis is focused on the evolution of sex chromosomes and sex determination in turtles. The majority of turtle species exhibit ESD, which is considered the ancestral sex determination system of this group, while GSD either as male or female heterogamety evolved independently at least five times. We investigated the presence of sex chromosomes in representative species of turtles by cytogenetic analyses. The analyses included the reconstruction of karyotypes, distribution of constitutive heterochromatin (C-banding, methylation analysis) and repetitive elements (fluorescence in situ hybridization) and comparative genome hybridization (CGH), which often characterize the degenerated Y or W and can be helpful in the identification of "cryptic" sex chromosomes. We described XX/XY sex chromosomes in seven previously unstudied Australasian chelids (Pleurodira) from the genera...
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Study of the mechanism of gene expression regulation at the level of functional organization of chromatin domains.
Hornáček, Matúš ; Cmarko, Dušan (advisor) ; Kučera, Tomáš (referee) ; Stixová, Lenka (referee)
- 1 - ABSTRACT Nucleoli are formed on the basis of genes of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In our work we study changes of FC/DFC units in the course of cell cycle. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60 to 80 %. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis but become again active...
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Analýza karyotypu vakonošů (Psychidae, Lepidoptera) metodami klasické a molekulární cytogenetiky
HEJNÍČKOVÁ, Martina
Due to their phylogenetic position, Psychidae play an important role in the investigation of the W chromosome origin in Lepidoptera. Several species of Psychidae were tested for the presence of sex-chromatin and investigated via comparative genomic hybridization. Furthermore, odd chromosome numbers and a Z univalent were observed in females. Overall, this study brings tangible evidence for the absence of the W chromosome in Psychidae, thus contributes to complex knowledge of the W chromosome evolution. In addition, karyotypes of the given species were analyzed using 18S rDNA and histone H3 probes. The results indicate relative stability of their karyotypes.
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