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Physical interactions of the splicing factor Prp45
Kratochvílová, Eliška ; Folk, Petr (advisor) ; Doubravská, Lenka (referee)
It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.
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Recombinant expression of RhoA protein with affinity tag.
Filipová, Barbora ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
The rate of intestinal infections associated with antibiotics has been rising steeply. Virulent toxins of C. difficile, Yersinia and many other bacteria target the RhoA protein. The protein is a GTPase involved in signalling pathways, its major function being the regulation of the actin cytoskeleton. The above mentioned toxins render this protein non- functional, which may lead up to a fatal destruction of the cell. The present diagnostic methods of these infections are insufficient. The use of protein chips and mass spectrometry to detect any RhoA protein modifications seems to provide a feasible method of determining these intestinal bacterial diseases. Therefore the purpose of this study is to use recombinant methods to prepare the desired RhoA protein with a bound affinity probe which will serve to its immobilisation on a biochip. The thus prepared protein will be subsequently used to diagnose the previously described diseases. In Czech.
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Recombinant expression of RhoA protein with affinity tag.
Filipová, Barbora ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
The rate of intestinal infections associated with antibiotics has been rising steeply. Virulent toxins of C. difficile, Yersinia and many other bacteria target the RhoA protein. The protein is a GTPase involved in signalling pathways, its major function being the regulation of the actin cytoskeleton. The above mentioned toxins render this protein non- functional, which may lead up to a fatal destruction of the cell. The present diagnostic methods of these infections are insufficient. The use of protein chips and mass spectrometry to detect any RhoA protein modifications seems to provide a feasible method of determining these intestinal bacterial diseases. Therefore the purpose of this study is to use recombinant methods to prepare the desired RhoA protein with a bound affinity probe which will serve to its immobilisation on a biochip. The thus prepared protein will be subsequently used to diagnose the previously described diseases. In Czech.
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Physical interactions of the splicing factor Prp45
Kratochvílová, Eliška ; Folk, Petr (advisor) ; Doubravská, Lenka (referee)
It is well known that chromatin posttranslational state, transcription and splicing influence each other. Nevertheless, the details of this coupling are not fully understood. In S. cerevisiae, it is possible to induce conditions, in which splicing is uncoupled from transcription. Such situation occurred in cells expressing a mutated splicing factor Prp45, whose human homolog has been proved to participate in transcription regulation and also in splicing reactions. Based on previously indicated interactions in high throughput two-hybrid screens, we have been looking for physical links between Prp45 and proteins involved in chromatin posttranslational modifications. Finding of such a link would provide insight into the relationships of gene expression processes. Using coimmunoprecipitation and affinity purification, we were unable to detect physical interactions between Prp45 and our candidate chromatin regulators. Alternative approaches are discussed. Using the precipitation techniques, we mapped the interaction of Prp46 with truncated variants of Prp45. This observation contributes to our knowledge of protein-protein interactions within the spliceosome.
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