Molecular biology of giant viruses' DNA replication machinery
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Viruses are the most widespread and abundant entity on this planet, further constituting the largest part of the genosphere. The majority of these infectious agents are miniature, having been described as being smaller than the smallest bacteria. Even though they encode a limited number of viral proteins, they still obtain the bulk of the material they require for their replication and propagation from the infected host cell. Recently, this traditional concept of viruses has been shaken up by the breakthrough finding of a new group of viruses, the Giant Viruses. They have been assigned this definition due to their amazingly and surprisingly large genomic size. The vast majority have their own replication machinery. They have been discovered in the sea, where they prefer to infect amoebas and other marine microorganisms. For the purpose of this study, we focused on three of these giant viruses; Mimivirus, Marseillevirus, and Cafeteria roenbergensis virus (CroV). The aim of the study was to comprehend how these giant viruses replicate and propagate their genetic material through the generations, to have reached a point where their genome size is comparable to normal-sized bacteria. For this reason, an extensive biochemical analysis on the molecular biology of giant viruses' DNA replication machinery was performed, hoping to obtain new insights into the evolution and lifestyle of these unique viruses. We specifically focused on what we considered to be two of the most important DNA replication proteins; the Proliferating Cell Nuclear Antigen (PCNA) and Flap structure-specific Endonuclease 1 (FEN1). Our goal was to determine their properties. The protocols performed were a series of protein expression procedures, during which the particular synthetic genes were cloned in a selection of expression vectors and were then expressed in bacteria (i.e. E.coli host expression strains). Depending on the protein expression efficiencies, some trial protein purification procedures followed. For the first few months of the project, however, it was impossible to obtain any conclusive results concerning the expression of the proteins. The synthetic genes were proving to be extremely difficult to express in vectors containing an expression tag. Only when we switched to un-tagged expression vectors, much later on in the project, did we start getting better and more promising results. This was a particularly useful outcome in itself, as it revealed that enhanced expression of the PCNA and FEN1 proteins preferentially occurs when no expression tags are present. Towards the end of the project, some protein purification trials were performed, but unfortunately they only resulted in an incredibly low protein purity level. The discovery of these distinctive viruses has not only incited scientists to maybe rethink and change their view about the general nature of viruses, but it has also begun to alter and question the outlook regarding the history of life as a whole. As the investigation is still in its very early stages, there are many aspects concerning the giant viruses still to be discovered. This in the end could essentially teach us a great deal more than we ever hoped to expect, and therefore it is of great significance and importance to continue with this research.
Thesis, MPhil Master of Philosophy
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