Bujarski, Jozef J.
Ph.D. (Doctor of Philosophy)
Department of Biological Sciences
The phenomenon of homologous RNA recombination allows RNA viruses to repair their genomes. Among plant RNA viruses, homologous recombination has been most thoroughly studied in brome mosaic bromovirus (BMV), a positive-strand, tripartite member of the Bromoviridae family. Significantly, all the studies have focused on the 3′-termini of BMV genome. In this work, we investigate the determinants and mechanisms of homologous recombination within the previously uncharted region of BMV genome; the intergenic region (INT) located on the RNA3 component. The crossovers were tested in planta by co-inoculations with pairs of BMV RNA3 constructs carrying mutagenized duplications of various parts of INT. First, we addressed recombination at the subgenomic promoter (sgp) domain of INT. The removal or the extension of the poly U tract reduced or increased recombination, respectively. The deletion of the core hairpin or its replacement by a foreign hairpin inhibited the crossovers. The nucleotide substitutions at +1 or +2 transcription initiation positions reduced recombination. The sgp core alone supported only basal recombination activity. All the crossovers mapped to the poly U tract and to the core hairpin. The observed effects on recombination did not parallel those on transcription. We postulate that recombination is primed at the poly U tract by the predetached nascent (+) strand, whereas transcription initiates de novo at the sgp core. Second, the recombination was studied within the intergenic replication enhancer (IRE) domain of INT. Including IRE in the tested region tripled the recombination frequency observed for the sgp. IRE alone supported high frequency crossovers that mapped to the conserved box B consensus sequence. The deletion of the box B greatly reduced recombination activity. We propose that the IRE domain acts as a sgp-independent recombination hot spot during a different stage of the virus life-cycle. Our findings suggest that the crossovers at IRE may occur via cleavage-religation mechanism as opposed to the template-switch mechanism predicted for sgp. The novel function of INT reported in this work reveals its multifunctionality and significance for the viral genome, therefore, rendering this region an excellent subject of recombination studies and a possible target for the remedy.
Wierzchoslawski, Rafal Henryk, "Molecular studies on homologous recombination at the intergenic region of brome mosaic bromovirus (BMV) : determinants, mechanisms, and relationship with transcription" (2004). Graduate Research Theses & Dissertations. 4021.
xi, 190 pages
Northern Illinois University
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