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RNA recombination in the turnip crinkle virus system: An analysis of sequences and structures required for RNA recombination
Abstract
In this dissertation, I report on the first in-depth study of RNA recombination in any virus system. In turnip plants infected with turnip crinkle virus (TCV) genomic RNA, satellite RNA D (sat-RNA D) and certain altered transcripts of sat-RNA C, sat-RNA D/C recombinants are found to accumulate. This exchange of RNA can be classified as targeted, aberrant homologous recombination as varying lengths of sat-RNA D are found joined to one of 5 consecutive bases within sat-RNA C. These 5 nucleotides are contained within a larger sequence, Motif I, which is one of three sequences we propose act as replicase recognition signals. We propose that recombination between these RNAs occurs utilizing a replication dependent mechanism in which the replicase, after synthesizing a full length or nearly full length sat-RNA D molecule, reinitiates polymerization at Motif I within sat-RNA C to generate a chimeric D/C species. Detailed analysis of the role of Motif I in this exchange of RNAs was carried out. The sequence of the signal is not required for infectivity of sat-RNA C transcripts but is required for RNA recombination. Thirteen different point mutations were introduced into and around Motif I; all alterations allowed infectivity of the transcripts yet several of these same base changes abolish RNA recombination. Small insertions and deletions generated at three unique restriction sites in sat-RNA C had no effect upon transcript infectivity, however, some of these alterations were found to affect recombination. The addition of residues at the predicted 5$\sp\prime$ border of Motif I (a Bam HI site) or 32 residues upstream (a Mlu I site) allowed recombination to occur; a deletion of 4 bases 13 bases downstream (an Apa I site) did not allow for RNA exchange. Secondary structure modelling of a 78 base region encompassing Motif I indicated that a dual-stem loop structure could be formed. Analysis of the sequences of the loops suggests that the two loops may interact. Two base alterations which appeared to disrupt the formation of the larger stem were selected for further analysis. Compensatory mutations were generated to repair the nucleotide mismatches introduced by these two alterations; repairing of the base pairing (replacement of the original A:U pair with a G:C pair) restored recombination activity.
Subject Area
Molecular biology
Recommended Citation
Cascone, Pamela Josephine, "RNA recombination in the turnip crinkle virus system: An analysis of sequences and structures required for RNA recombination" (1992). Doctoral Dissertations Available from Proquest. AAI9305810.
https://scholarworks.umass.edu/dissertations/AAI9305810