title: I-OmiI and I-OmiII: two intron-encoded homing endonucleases within the Ophiostoma minus rns gene.
abstract: The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3' overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; Department of Botany, Faculty of Science, Suez University, Suez, Egypt.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Hafez
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
authors.forename: Tuhin Kumar
authors.initials: TK
authors.lastname: Guha
authors.affiliation: Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Electronic address: hausnerg@cc.umanitoba.ca.
authors.forename: Georg
authors.initials: G
authors.lastname: Hausner
mesh_terms: D003001:Cloning, Molecular; D004272:DNA, Mitochondrial / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020459:Genes, rRNA; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D055156:Ophiostoma / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004272:DNA, Mitochondrial; D004720:Endonucleases
keywords: DNA cutting enzymes; In vivo endonuclease assays; Mitochondrial DNA; Mobile elements; Mobile introns