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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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Yeast isogenic system as a method for study of IFI16 protein interactions with DNA
Kratochvilová, Libuše ; Šedrlová, Zuzana (referee) ; Brázda, Václav (advisor)
This bachelor thesis deals with the binding of interferon gamma-induced protein 16 (IFI16) to the secondary local structures of the G-quadruplex (G4) and its mutations in the single-hybrid yeast system (Y1H). The IFI16 protein in the cell recognizes its own and foreign or damaged DNA, is involved in the formation of the inflammasome and induces the expression of type I interferon (IFN-I). It is also involved in the regulation of transcription and restriction of viral infection. It has been shown that the IFI16 protein binds preferentially to G-quadruplex structures and is able to stabilize them by this binding. G-quadruplexes are classified as non-canonical DNA and RNA structures formed by G-rich sequences. They are easily formed under physiological conditions and are found in a number of important regulatory structures of the genome such as telomeres or oncogene promoters. They are also part of a number of viral genomes. This makes them excellent potential targets in the treatment of cancer and viral diseases. In the first part of the work, new reporter strains of S. cerevisiae yeasts were prepared by the Delitto Perfetto method, differing only in sequence with the potential for G-quadruplex formation, which was designed and analyzed by the DNA Analyzer program. The correctness of the inserted sequences was verified by PCR and Sanger sequencing and comparison with the supplied oligonucleotide sequences by the Blast program. In the second part of the work, the newly prepared strains were transformed with vectors for the expression of p53, IFI16 proteins, and the effect of IFI16-G4 binding on the expression of the gene in connection with the tumor suppressor p53 was assessed using luciferase reporter assays. The evaluation was performed on the basis of a statistical analysis of the magnitudes of the effects obtained after normalization of the luminescence signal on the optical density of the culture at a wavelength of 600 nm. The results show that the IFI16 protein has a different effect on the trans-activation potential of the p53 tumor suppressor depending on binding to emerging structures near the reporter gene promoter, and that a G4Hunter threshold of at least 1,591 had to be reached and taken into account to successfully form a G-quadruplex the potential of the sequence to successfully form at least 2 G-tetrads.
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