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The role of CSL proteins in oxidative stress response of Schizosaccharomyces pombe
Tvarůžková, Jarmila ; Převorovský, Martin (advisor) ; Rallis, Charalampos (referee)
Oxidative stress represents a complex and intensely studied phenomenon tightly linked to a range of human diseases, and to aging in many organisms. A plethora of key cellular regulators, including the Notch signaling pathway, have been recently described to respond to the cellular redox status. We have characterized the role of CSL (CBF1/Su(H)/LAG-1) proteins, the effectors of Notch signaling pathway in metazoa, in oxidative stress response in fission yeast. Schizosaccharomyces pombe contains two CSL paralogs, Cbf11 and Cbf12, that have antagonistic functions in the regulation of cell cycle and cellular adhesion. Both proteins are able to bind the canonical CSL motif and activate transcription and, thus, function as genuine CSL transcription factors. We have determined that the strain lacking cbf11 is resistant to hydrogen peroxide but not to menadione, a source of superoxide anion radical. Using double knock-outs to assess genetic interactions we have revealed that the resistance of cbf11 knock-out is dependent on the antioxidants catalase and sulfiredoxin. Genes encoding these antioxidants are under transcriptional control of the Sty1 MAP kinase pathway and the Pap1 transcription factor which are also required for the resistance of Δcbf11 cells. Cbf12 is believed to play only a minor role in...
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Preparation and testing of polyclonal antibodies against Cbf11 and Cbf12, the fission yeast CSL transcription factors
Tvarůžková, Jarmila ; Půta, František (advisor) ; Moserová, Michaela (referee)
CSL (CBF1/RBP-Jκ, Suppressor of Hairless, Lag-1) protein family members are transcription factors critical for metazoan development as the effectors of Notch signaling pathway as well as in a Notch-independent manner. CSL homologues have been identified in fungal organisms lacking the Notch signaling pathway. Cbf11 and Cbf12 are antagonistic paralogous proteins that are important for proper coordination of cell and nuclear division, regulation of cell adhesion and chromosome integrity in the fission yeast Schizosaccharomyces pombe. The activities of Cbf11 and Cbf12 proteins need to be finely balanced for their proper biological function, however, chromosomally tagged alleles of these proteins exhibit properties different from wild type. Therefore, the availability of specific antibodies would greatly enhance the study of CSL proteins in the fission yeast. In this bachelor's thesis, design and preparation of imunogen for commercial antibody production followed by antibody testing is presented. Using bioinformatics, suitable immunogenic Cbf11 and Cbf12 protein fragments were selected and the corresponding DNA sequences were cloned into an expression vector. His-tagged expression was optimized in a bacterial expression system and the native protein was purified using immobilized metal affinity...
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