Y2H Study

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In order to analyse physical interactions occurring between MDV proteins, MDV bait and prey clones (see Chapter 3) were transformed into yeast. Two different yeast strains were used to generate Y2H prey and bait arrays; AH109 for the preys and Y187 for the baits. The MDV clones collection was composed of 151 DNA constructs from MDV genes cloned as both pGADT7 (prey) and pGBKT7 (bait). This number represents MDV genes, which were cloned as full length, fragments and extra & intracellular domains for membrane-associated proteins. The transformed prey clones were grown on plates of single drop out media lacking leucine and the transformed bait clones were grown on plates of single drop out media lacking tryptophan. As both vectors have the appropriate cassettes of selectivity which make the transformed yeast grow on the selective plate lacking suitable amino acid. The transformed yeast clones have been sent to the Institute of Toxicology and Genetics, Karsruhe, Germany, in 96-well plate format, where the robot assisted Y2H assay. Y2H arrays were performed by testing all the proteins against each other. Since Y2H assays can generate a number of false positive interactions, each pair wise was tested in duplicates. Interactions in the Y2H screen were scored positive, if two of the two mating were positive. If only one mating grew, the interaction was scored negative and non-reproducible. The positive results from the Y2H array were visualized and analysed using the bioinformatics software Cytoscape. The indicated software was used for the assembly of the MDV protein-protein interaction network (the MDV interactome). From the list of positive, the interactions with 5 preys were removed due to their high prey count (above 30 interactio... ... middle of paper ... ... Moreover, it is mainly expressed during latency. vIL8 is a chemokine homologue, and it may be responsible for switching of the MDV infection from B lympohocytes, where the lytic replication is established, to T lymphocytes, where the latent infection and transformation occur. PP38 is mainly expressed during lytic infection, and it is suggested that it is responsible for virus reactivation from latency. In our Y2H study, it has been found that these genes, which are responsible for different stage of virus pathogenicity, interacted with each other forming a dense network (Figure 4.2.3c). Meq interacted with itself, PP38, PP24 and vIL8 protein. In addition, vIL8 protein interacted with Meq and PP24. PP38 interacted with itself and its homologues PP24 and Meq. Interestingly, there was an interaction between Meq and the small polymerase subunit of the MDV.

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