|
|
Searching for interaction partners associated with the transcriptional apparatus of yeast linear plasmids
Ľalíková, Kristýna ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
The pGKL plasmids are a type of yeast linear double-stranded DNA plasmids found in the cytoplasm of yeast Kluyveromyces lactis. The plasmid gene transcription process involves the plasmid transcription apparatus, which shows similarity to the transcription apparatus of poxviruses. Poxviruses cause a range of serious diseases. Thus pGKL plasmids offer a safe way to study some of the mechanisms of these viruses without the risk of infection. The plasmid transcription apparatus comprises non-canonical RNA polymerase, helicase, and capping enzyme as its main components. Although RNA polymerase has been thoroughly characterized and described, little focus has been on the interactions between proteins within the transcription apparatus. Therefore, this study aims to shed light on this fundamental aspect of the gene expression of pGKL plasmids. The main goal of the thesis was to prepare a system that would facilitate the search for interaction partners of the pGKL transcription apparatus and verify the interactions between chosen proteins in yeast cells. The foundation of this system relies on the use of optimized genes and the yeast two-hybrid system method. The development of the experimental method has begun successfully, and it's expected to conclude soon, although further optimizations are still...
|
|
|
Preparation and validation of a system for the study of regulation of gene expression of yeast linear cytoplasmic plasmids
Horáčková, Kamila ; Vopálenský, Václav (advisor) ; Čáp, Michal (referee)
There is currently very few information about the transaltion of linear cytoplasmatic plasmids occured in yeast cells Kluyveromyces lactis. However, there is a relatively well developer information about their transcription apparatus. A study of transkript linear plasmids revealed an atypical organization at the 5ʼ end. Those ends contain nontemplate polyadenylation and they are missing the N7 methylguanosine hat. Because of the presence of this structure, which is localized at 5ʼend of plasmids specific mRNA, raised a question regarding the iniciation of the translation. The present thesis is focused on the preparation of reporter systém suitable for studying the influence of a number of the nontemplate adenosins, which were added at the 5ʼ ends of mRNA linear plasmids. The frist step was making a construction of dual yeast cell plasmids carring two reporters genes, which are under the controle of two different promoters. After a successfull construction, the aktivity of promoters TEF1 and PGK1 was measured, whereby the promoter TEF1 proved twice stronger. The transcription start site of both promotor was determined. The second step was the construction of a reporter system directly in yeast cell plasmid pGKL. Reporter genes were under the controle of two promoters originating from the pGKL...
|
|
|
Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal
Transcription is the control point of gene expression. This process relies on protein complex of multisubunit RNA polymerases, which are extremely conserved among all cellular organisms. Transciption of extrachromosomal hereditary elements such as organelles, viruses and plasmids is dependent on host cellular RNA polymerases or intrinsic RNA polymerase is contained within these elements. Putative non-canonical two-subunit RNA polymerase is also encoded by linear cytoplasmic plasmids of the yeast Kluyveromyces lactis and most likely transcribes genes of these plasmids. Besides the two subunits of RNA polymerase encoded by linear plasmids of Kluyveromyces lactis there are another two estimated components of the transcription apparatus, namely capping enzyme that adds the cap to 5' mRNA ends and putative DExD/H box helicase. Characterization of the unique and underexplored transcription machinery of Kluyveromyces lactis plasmids was the principal objective of this work. The main goal was to: 1) clarify evolutionary origin of the linear plasmid transcription apparatus; 2) describe architecture of the linear plasmid transcription complex in vivo focused on putative RNA polymerase binding partners; 3) reveal mechanisms of transcription initiation and termination of the yeast linear plasmids. The main...
|
|
|
Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal
Transcription is the control point of gene expression. This process relies on protein complex of multisubunit RNA polymerases, which are extremely conserved among all cellular organisms. Transciption of extrachromosomal hereditary elements such as organelles, viruses and plasmids is dependent on host cellular RNA polymerases or intrinsic RNA polymerase is contained within these elements. Putative non-canonical two-subunit RNA polymerase is also encoded by linear cytoplasmic plasmids of the yeast Kluyveromyces lactis and most likely transcribes genes of these plasmids. Besides the two subunits of RNA polymerase encoded by linear plasmids of Kluyveromyces lactis there are another two estimated components of the transcription apparatus, namely capping enzyme that adds the cap to 5' mRNA ends and putative DExD/H box helicase. Characterization of the unique and underexplored transcription machinery of Kluyveromyces lactis plasmids was the principal objective of this work. The main goal was to: 1) clarify evolutionary origin of the linear plasmid transcription apparatus; 2) describe architecture of the linear plasmid transcription complex in vivo focused on putative RNA polymerase binding partners; 3) reveal mechanisms of transcription initiation and termination of the yeast linear plasmids. The main...
|
|
|
Characterization of non-canonical RNA polymerase encoded by the yeast linear plasmids
Sýkora, Michal ; Vopálenský, Václav (advisor) ; Macíčková Cahová, Hana (referee) ; Valášek, Leoš (referee)
Transcription is the control point of gene expression. This process relies on protein complex of multisubunit RNA polymerases, which are extremely conserved among all cellular organisms. Transciption of extrachromosomal hereditary elements such as organelles, viruses and plasmids is dependent on host cellular RNA polymerases or intrinsic RNA polymerase is contained within these elements. Putative non-canonical two-subunit RNA polymerase is also encoded by linear cytoplasmic plasmids of the yeast Kluyveromyces lactis and most likely transcribes genes of these plasmids. Besides the two subunits of RNA polymerase encoded by linear plasmids of Kluyveromyces lactis there are another two estimated components of the transcription apparatus, namely capping enzyme that adds the cap to 5' mRNA ends and putative DExD/H box helicase. Characterization of the unique and underexplored transcription machinery of Kluyveromyces lactis plasmids was the principal objective of this work. The main goal was to: 1) clarify evolutionary origin of the linear plasmid transcription apparatus; 2) describe architecture of the linear plasmid transcription complex in vivo focused on putative RNA polymerase binding partners; 3) reveal mechanisms of transcription initiation and termination of the yeast linear plasmids. The main...
|
guest ::
