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Title: An evolutionary and biochemical characterization of a self-splicing group II intron and its encoded LAGLIDADG homing endonuclease in Leptographium truncatum
Authors: Mullineux, Sahra-Taylor
Supervisor: Hausner, Georg (Microbiology)
Examining Committee: Loewen, Peter (Microbiology) Court, Deborah (Microbiology) Piercey-Normore, Michele (Biological Sciences) Bonen, Linda (Biology, University of Ottawa)
Graduation Date: October 2010
Keywords: group II
Issue Date: 6-Jul-2010
Citation: Mullineux T, Hausner G. 2009. Evolution of rDNA ITS1 and ITS2 sequences and RNA secondary structures within members of the fungal genera Grosmannia and Leptographium. Fungal Genet. Biol. 46:855-867
Mullineux ST, Costa M, Bassi GS, Michel F, Hausner G. 2010. A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions. RNA (In press, RNA/2010/021840)
Abstract: Evolutionary relationships amongst strains of the fungal genus Leptographium and related taxa were inferred using the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat. To generate robust sequence alignments for phylogenetic analysis the relationship between DNA sequence variability and RNA structural conservation of ITS segments was examined. The results demonstrate that structural conservation of helical regions is facilitated by compensatory base changes, compensating insertions/deletions, and, possibly, RNA strand slippage. A high mol % G+C bias for ITS1 and ITS2 and structural constraints at the RNA level appear to limit the types of changes observed. Fifty strains of Leptographium were screened for the presence of introns within mitochondrial genes. Superimposing intron survey data onto the ITS-derived phylogenetic tree reveals that introns are absent from the small ribosomal RNA (rns) gene of all strains of L. procerum yet are found in all strains of L. lundbergii. Amongst members of L. wingfieldii, L. terebrantis, and L. truncatum intron distribution is stochastic and is not correlated to the evolutionary relationships amongst strains. A group II intron/LAGLIDADG homing endonuclease gene (HEG) composite element from the mt rns gene of L. truncatum strain CBS929.85 was characterized. Intron-catalyzed splicing was tested using ORF-less and ORF-containing precursor transcripts, and both versions of the intron readily self-splice under moderate temperature and ionic conditions (37 °C and 6 mM MgCl2). Cleavage activity of the intron-encoded protein (I-LtrII) was tested using an N-terminal His6-tagged and near native protein. The homing endonuclease cleaves double-stranded DNA 2 nucleotides upstream of the intron insertion site within the exon, generating 4 nucleotide 3’ OH overhangs. Intron splicing is not enhanced by the addition of I-LtrII and RNA-binding assays indicate that the His6-tagged protein does not bind to the intron. Phylogenetic relationships amongst the rns gene, intron, and amino acid sequences were inferred. An evolutionary model of the composite element is proposed in which the HEG invaded a group II intron and mobilized it. The mobile genetic element may be transmitted vertically amongst L. lundbergii strains and horizontally through lateral gene transfer amongst strains of L. wingfieldii, L. terebrantis, and L. truncatum.
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