Experimental evolution has been used to investigate both general and specific evolutionary processes. More recently, it has also been used to resolve protein-protein interactions. Viruses assemble through a series of protein-protein interactions which must remain more favorable than any competing off-pathway reaction. By constructing chimeric viruses with genes or segments of genes from another species, foreign elements are introduced into this system of assembly. Characterization of the resulting chimeras provides information about which proteins interact, the protein-protein interacting interface, the role of particular domains, and the importance of specific residues. Chimeric viruses often exhibit a reduction in fitness, as the foreign element is unable to interact as efficiently in the system as the native element. Through experimental evolution, mutations accumulate that affect interacting partners in the system, leading to a more optimal assembly pathway. The microviruses are well-characterized single-stranded (ss) DNA bacteriophages. They are divided into three clades, represented by φX174, G4, and α3. Incidences of horizontal gene transfer between microvirus clades are unusually rare and may be due to a complex assembly pathway with multiple stages: a foreign element has the potential to disrupt a multitude of morphogenetic steps. In this study, we exchanged major spike genes between the two microvirus species φX174 and G4, then monitored the evolutionary recovery. Results can be interpreted within this thermodynamic paradigm. Although the G4-φXG chimera could only form plaques at low temperature and exhibited reduced fitness, its evolutionary recovery was relatively straightforward. The other chimera, φX-G4G, could only form plaques when complemented with two wild-type φX174 genes. Isolating a complementation-independent chimera required the passaging of mutants through a series of different environments. The first selection yielded mutations of the largest effects. First, the truncation of a protein involved in DNA synthesis was recovered, resulting in a global decrease in gene expression. Next, a recombination event at the 3' end of the foreign gene resulted in a modification of the protein’s C-terminus. These mutations were subjected to further analysis to determine why they were so critical at this early stage of experimental evolution. Subsequent passages of the φX-G4G chimera eventually yielded viable strains, with additional mutations affecting stages of late assembly. Overall, results indicate how gene exchange can drastically affect flux through the pathway. When the system is initially perturbed, the process of experimental evolution allows the pathway to return to a normalized state. The mutations isolated during this recovery stage indicates how the flux was initially altered, and how it can be restored.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/556590 |
| Date | January 2015 |
| Creators | Doore, Sarah Marie |
| Contributors | Fane, Bentley A., Fane, Bentley A., Ferriere, Regis, Palanivelu, Ravishankar, Sullivan, Matthew B., Xiong, Zhongguo |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | en_US |
| Detected Language | English |
| Type | text, Electronic Dissertation |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
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