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Analysis of recombinant clones of apoptotic nucleases in "leaf factory" system upon coinfiltration with modifying genes
LOMNICKÁ, Anna
TBN1 is a nuclease with antitumor activity. The main goal of this work was to estimate how TBN1 and its modificated variants are stable in the ?leaf factory? system used for its production and whether it can be enhanced or influenced by chosen potential ?modificators? i. e. silencing supressors, transcription factors, glycosyltransferases and kinases. Nicotiana benthamina plants were infiltrated with the mixture of Agrobacterium tumefaciens strains bearing the nuclease plant expression vectors and co-infiltrated with the ?modifying? vectors. The nuclease and protein analyses revealed that nuclease TBN1 wt and its modificated variants are stable in the used ?leaf factory? system as to their molecular mass, only quantitative changes were detected. Expreximents showed that activity and production of the nucleases increased upon coinfiltration with silencing supressor and decreased upon coexpression with chosen transcription factor. Glycosyltransferases and kinases influenced activity and production only insignificantly. The experiments also revealed that modificated variants of TBN1 have different molecular weight suggesting that different N-glycosylation domains have different length of sugar chain and influence on nuclear activity. Our data show that this expression in planta seems to be suitable for production for study of antitumor activity of these nucleases.
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Expression of SANT/HTH Myb mRNA, a plant morphogenesis-regulating transcription factor, changes due to viroid infection
LOMNICKÁ, Anna
Potato spindle tuber viroid (PSTVd) belongs to plant-pathogenic, circular, non-coding RNAs. Its propagation is accompanied by (mis)regulation of host genes and induction of pathogenesis symptoms including changes of leaf morphogenesis depending on the strength of viroid variant. We found strong genotype-dependent suppression of tomato morphogenesis-regulating transcription factor SANT/HTHMyb (SlMyb) due to viroid pathogenesis. Its relative mRNA level was found to be significantly decreased in PSTVd-sensitive tomato (cvs Rutgers and Heinz 1706) due to degradation processes, but increased in PSTVd-tolerant (cv. Harzfeuer). In heterologous system of Nicotiana benthamiana, we observed a SlMyb-associated necrotic effect in agroinfiltrated leaf sectors during ectopic overexpression. Leaf sector necroses were accompanied by activation of nucleolytic enzymes but were suppressed by a strongly pathogenic PSTVd variant. Contrary to that, PSTVd's effect was inhibited by the silencing suppressor p19. It was found that in both, Solanum lycopersicum leaves and N. benthamiana leaf sectors, SlMyb mRNA degradation was significantly stronger in viroid-infected tissues. Necroses induction as well as gene silencing experiments using the SANT/HTH-Myb homologues revealed involvement of this Myb in physiological changes like distortions in flower morphogenesis and growth suppression.
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Dynamika pohybu a umlčování transgenních lokusů HP1 v živých buňkách
Ondřej, Vladan ; Kozubek, Stanislav ; Lukášová, Emilie ; Matula, Pa. ; Matula, Pe. ; Kozubek, Michal
Intranuclear localization and mobility of Cy3 labelled transgene loci were studied in living cells. Observations showed occurrence of several transgene copies in each cell nucleus. The majority of copies were localized in the centromeric heterochromatin defined by HPlbeta-GFP fusion protein, the minority of copies in euchromatin. The tracking of loci showed restricted diffusive motion of these in the short-time range. During long-time observation of HPlbeta domains and transgene loci, we have found that in most cases the proximity of these objects decreased within time. The changes of positions of HPl domains were very small but transgene loci displayed directional movement towards the relevant HPl domain. Our data records support the idea that the cell nucleus consists of several separated higher-order compartments. The genes relocate within these compartments, which is finally connected with changes of their expression status.
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A comparison of SH3 domains' tyrosine phosporylation influence on their binding capacity
Tatárová, Zuzana ; Novotný, Marian (advisor) ; Kuthan, Martin (referee)
Understanding the impact of protein phosphorylation is very important for the formation of dynamic biological processes such as gene silencing, cell growth, differentiation or apoptosis. This work deals with the phosphorylation of a protein-interaction module known as SH3 domain and the influence of phosphorylation on its ligand-binding capacity. SH3 domain is a part of a large number of enzymes directly involved in signal transduction as well as adapter proteins without enzymatic activity. Many studies have shown the importance of tyrosine sites within SH3 domain in regulatory mechanisms of proteins by using either mutants that cannot be phosphorylated, mutants mimicking the negative charges created by phosphorylation or by evidence of in vivo phosphorylation. The work also includes bioinformatic analysis, which further expand our knowledge of SH3 phosphorylated proteins and confirms that phosphorylation of the tyrosine sites is conserved among proteins containing the SH3 domain.
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Genotyping of \kur{Giardia intestinalis} isolates
ŠRÁMOVÁ, Eliška
The aim of this work was assemble isolates of Giardia intestinalis from humans and other mammals. Stools samples were examined for presence of cysts by concentration settling method. Consequently sequencing of 532 bp parts of the TPI gene after previous amplification by the nested PCR was performed. In vitro cultures of selected isolates were established using experimental model hosts, gerbils.
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Inaktivace ICAM-1 genu metodou oligonukleotidů modifikovaných lysinem
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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Introducing targeted epigenetic changes in Solanum tuberosum and Nicotiana benthamiana by plant viruses
Dušek, Jakub ; Ryšánek, Pavel (advisor) ; Barbora, Barbora (referee)
In this thesis we have focused on the induction of targeted epigenetic changes using modified Potato Virus X vector (PVX, Potato virus X). The gene coding sequence phytoene desaturase (PDS) was isloated from Solanum tuberosum and Nicotiana benthamiana plants using PCR. Primers were specifically designed as universal for both plant species and their Tm temperatures was in the range of 5 °C. The primers also added flanking restriction sites XhoI and ClaI to simplify cloning of PDS PCR products into full length PVX viral vector in AS orientation. The PVX viral vector pGR106 was later introduced into the plants using Agrobacterium tumefaciens. Infection led to the degradation of the target transcript and to efficient gene silencing (PTGS posttranscriptional gene silencing). The process has been manifested by complete or partial fading of a green plant tissue phenotype..
Some of the plants were aslso inoculated in in vitro conditions, for which a novel technique has been implemented.
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The study of epigenetic regulation of gene HLA II. Clas within family relationships
Chmel, Martin ; Černá, Marie (advisor) ; Urbanová, Jana (referee)
Introduction: At our post-genomic era the studies of epigenetic regulation constitutes one of the tools for understanding the function of genes. Epigenetic regulation can directly control the temporal and spatial gene activity or silencing. The molecular basis of these regulations are DNA bases modifications, chromatin remodeling and RNA interference. At the same time, these mechanisms have a special way of transferring genetic information to subsequent generations called epigenetic inheritance. It has been proven epigenetic deregulation of certain genes as cause for many disease. For this reason, the study of epigenome HLA genes seems particularly important because these genes play a fundamental role in regulating the immune system. Aims: The aim of this work is to create a description of epigenetic modifications within families. It is an analysis of histone modifications and DNA methylation in the promoter region of the gene HLA DQA1. The aim was also to compare the differences in epigenetic modifications between alleles and compared the differences in these modifications between generations. The results will be compared with the analysis of the level of expression of the gene HLA DQA1. Methods: From collected peripheral blood of donors were isolated DNA, RNA, and leukocytes. DNA was used for...
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Proteomic changes of malignant cells induced by short-term treatment with valproic acid.
Kostrouchová, Markéta ; Mandys, Václav (advisor)
Cancers are clonal disorders of multicellular organisms that arise by accumulation of genetic mutations and step-wise induction of epigenetic events that silence transcription of tumor suppressing genes and facilitate expression of tumor promoting genes. This allows cancer cells in combination with altered response of host organism to overcome the regulatory cascades that orchestrate proper cooperation of cells within the multicellular organism. Epigenetic regulation is executed on the level of DNA by methylation of CpG islands and on the level of post-translational modification of chromatin proteins, histones in the first place. Post-translational modifications of histones include histone phosphorylation, acetylation, methylation, biotinylation, poly(ADP ribosylation), ubiquitination and sumoylation. Histone acetylation is connected with transcription activation or "openness" of chromatin to regulation by transcriptional factors. Histones are acetylated by histone acetyltranferases (HATs). Histone acetylation is a dynamic process that is reversed by histone deacetylases (HDACs), enzymes that are able to remove the acetyl residue from the acetylated histones. A decrease in gene expression brought about by low acetylation of histones is part of the cancer specific transcription profile that is characterized...
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