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Intragenic regions (introns) are found in all classes of organism. Transcription of such genes must undergo a splicing reaction to produce the mature, functional form of RNAs. Introns can be divided into four categories by their splicing mechanisms, namely Group I, Group II, spliceosomal, and nuclear tRNA introns. The former two are self-splicing introns. Group I introns are ubiquitous, however, most metazoan mitochondrial genomes lack introns. A novel group I intron in the mitochondrial cytochrome oxidase I gene (cox1) of Cinachyrella auctraliensis, which belongs to the IB2 subgroup, encodes a putative homing endonuclease with two amino acid motifs of the LAGLIDADG family. The homing endonuclease may perform intron translocation. Splicing in the cox1 of the sponge was demonstrated by comparing the length of DNA and RNA sequences. The intron was spliced in vivo or in vitro as revealed by RT-PCR and sequencing. Group I introns are classified as ribozymes. The pre-mRNAs fold into specific configurations that facilitate attacks of free guanosine followed by two consecutive trans-esterification steps to remove the introns. The excised cox1 intron was found to form a circle with the 5¡¦-end linked to the 3¡¦-end. Two other forms of lariats were also found with the 5¡¦-end linked to the inside sequence of the intron. Mutagenesis of a key nucleotide, which participates base pairing of RNA secondary structure, in P7 region decreased the splicing activity of the intron.

Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti. / Master of Science in Life Sciences

Homing endonucleases (HEs) typically have one of four types of catalytic domains (LAGLIDADG, GIY-YIG, H-N-H, His-Cys), and a DNA-binding region(s) that provides specificity. I-CreII, which is encoded by the psbA4 intron from Chlamydomonas reinhardtii, is unusual in containing two catalytic motifs: H-N-H and GIY-YIG. A previous study showed that I-CreII cleavage leaves 2-nt 3' OH overhangs similar to GIYYIG endonucleases, but that it also has a flexible metal requirement like H-N-H enzymes. Also, alanine substitution of several conserved residues in the GIY-YIG motif and two in the H-N-H motif did not produce a clear catalytic mutant, although some variants had strongly reduced DNA binding. Thus, in order to identify the catalytic motif, I substituted additional amino acids in both domains with alanine, and identified three histidines in the H-N-H motif that are likely to be involved in catalysis. To gain insight into how I-CreII interacts with its ~30-bp homing-site DNA, three types of DNA protection analysis were performed. Hydroxyl-radical footprinting, which reveals regions of tight DNA binding, indicated that I-CreII binds strongly to a region downstream of the cleavage and intron-insertion sites, corresponding to bp 2-10 of exon 5. There was also partial protection around the cleavage site, but only on the top strand, which is consistent with the enzyme's tendency to cleave this strand first. DNase I protection, which can reveal less closely-bound regions of target DNA, gave a larger footprint than hydroxyl-radical protection, with the additional region lying upstream of the cleavage site. These results also suggest that DNA backbone-binding downstream of the cleavage site involves sugars and phosphates, whereas upstream it is mainly with phosphates. DMS protection, which probes guanines on the N-7 position in the major groove, did not provide any evidence of major groove binding (at least not through guanines). DNase I protection could also be performed on the I-CreII variants that had reduced DNA affinity. The N161A variant was instructive in that it showed reduced protection of a T-A bp very close to the cleavage site, providing support for a catalytic role for the H-N-H motif and a possible constraint for modeling. Of the GIY-YIG motif variants, the footprint of the G231E/K245A variant was distinctly useful in that it was preferentially effected downstream of the cleavage site. This result suggested the H-N-H and GIY-YIG motifs are co-linear with their targets in the homing site. Structural modeling of the GIY-YIG domain of I-CreII using the solved I-TevI domain as template provided evidence for a unique insertion in the I-CreII structure that disrupted a catalytic α-helix; the insertion is predicted to be a positively charged, hairpinlike loop anchored by two antiparallel β-strands. I propose that this insertion can explain the evolutionary conversion of this catalytic endonuclease domain into a DNA-binding domain. These findings should also help to understand other dual-motif H-N-H/GIY-YIG endonucleases, such as I-CmoeI.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.

This work presents a survey of blue-stain fungi found in Northwestern Ontario, characterization of a homing endonuclease gene within Grosmannia piceiperda and finally an examination of the introns and homing endonuclease genes found in the large ribosomal subunit gene in species of Ceratocystis; using molecular techniques and phylogenetic analysis, we studied the molecular evolution of these mobile genetic elements. The blue-stain fungi of Northwestern Ontario were identified based on phylogenic analysis of rDNA internal transcribed spacer region sequences. This data was supplemented with morphological characteristics of the fungal cultures. The second project was an examination of a LAGLIDADG homing endonuclease and its IC2 group I intron. This intron is uniquely positioned within the group I intron-encoded rps3 gene of the large subunit ribosomal RNA gene. The final chapter is an investigation of the large subunit ribosomal RNA gene in species of Ceratocystis. The 3’ segment of this gene contains several novel introns and homing endonuclease genes. There is also much diversity between strains despite their close relation on the rDNA internal transcribed spacer region phylogenetic tree. Further, our data also suggest that the single motif LAGLIDADG homing endonuclease of the rDNA mL1923 intron is likely to be an ancestor to other homing endonucleases in the area. The results of these studies demonstrate the role that these elements play in the genetic diversity observed in the blue-stain fungi.

The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016

Ophiostoma novo-ulmi is the causal agent of Dutch elm disease (DED) which has had a severe impact on the urban landscape in Canada. This research program focused on developing molecular genetic strategies to control this pathogenic fungus. The first strategy involved the development of RNA interference (RNAi) for the down-regulation of genes involved in pathogenicity. An efficient RNAi cassette was developed to suppress the expression of the endopolygalacturonase (epg1) locus which encodes a cell-wall degrading enzyme. This epg1-RNAi cassette significantly reduced the amount of polygalacturonase activity in the fungus and resulted in almost complete degradation of epg1 mRNA. The need for a native promoter to selectively down-regulate specific gene loci was addressed by developing a carbon-catabolite regulated promoter (alcA) to drive the expression of the epg1-RNAi cassette. The expression of an alcA-driven epg1-RNAi cassette resulted in the down-regulation of epg expression under glucose starvation but normal levels of expression in high glucose. The expression could therefore be controlled by culture conditions. The second strategy explored the potential of using dsRNA viruses to vector disruptive RNAi cassettes. An isolate of O. novo-ulmi strain 93-1224 collected in the city of Winnipeg, was infected by two dsRNA mitoviruses which upon sequence characterization were named OnuMV1c and OnuMV7. To assess the transmissibility of this dsRNA virus the infected isolate 93-1224 was paired with three naive isolates of the related fungi O. ulmi and O. himal-ulmi. Through the use of nuclear and mitochondrial markers it was determined that the virus OnuMV1c may not rely on mitochondrial fusion for transmission but may have a cytoplasmic transmission route. This investigation of gene expression and manipulation has provided tools to help understand gene regulation in O. novo-ulmi. It has also added to our knowledge of mitoviruses, their transmission and potential use as a biological control. By enhancing our understanding of transmissible hypovirulence this work contributes to efforts to develop a new approach to target DED as well as a potential model for the control of other fungal diseases. / Graduate / 0307 / 0306 / 0369 / jscarneiro@hotmail.com