Buried within the genomes of many microorganisms are genetic elements that encode rare-cutting homing endonucleases that assist in the mobility of the elements that encode them, such as the self-splicing group I and II introns and in some cases inteins. There are several different families of homing endonucleases and their ability to initiate and target specific sequences for lateral transfers makes them attractive reagents for gene targeting. Homing endonucleases have been applied in promoting DNA modification or genome editing such as gene repair or “gene knockouts”. This review examines the categories of homing endonucleases that have been described so far and their possible applications to biotechnology. Strategies to engineer homing endonucleases to alter target site specificities will also be addressed. Alternatives to homing endonucleases such as zinc finger nucleases, transcription activator-like effector nucleases, triplex forming oligonucleotide nucleases, and targetrons are also briefly discussed.

Background: Mitochondrial porins, or voltage-dependent anion-selective channels (VDAC) allow the passage of small molecules across the mitochondrial outer membrane, and are involved in complex interactions regulating organellar and cellular metabolism. Numerous organisms possess multiple porin isoforms, and initial studies indicated an intriguing evolutionary history for these proteins and the genes that encode them. Results: In this work, the wealth of recent sequence information was used to perform a comprehensive analysis of the evolutionary history of mitochondrial porins. Fungal porin sequences were well represented, and newly-released sequences from stramenopiles, alveolates, and seed and flowering plants were analyzed. A combination of Neighbour-Joining and Bayesian methods was used to determine phylogenetic relationships among the proteins. The aligned sequences were also used to reassess the validity of previously described eukaryotic porin motifs and to search for signature sequences characteristic of VDACs from plants, animals and fungi. Secondary structure predictions were performed on the aligned VDAC primary sequences and were used to evaluate the sites of intron insertion in a representative set of the corresponding VDAC genes. Conclusion: Our phylogenetic analysis clearly shows that paralogs have appeared several times during the evolution of VDACs from the plants, metazoans, and even the fungi, suggesting that there are no "ancient" paralogs within the gene family. Sequence motifs characteristic of the members of the crown groups of organisms were identified. Secondary structure predictions suggest a common 16 β-strand framework for the transmembrane arrangement of all porin isoforms. The GLK (and homologous or analogous motifs) and the eukaryotic porin motifs in the four representative Chordates tend to be in exons that appear to have changed little during the evolution of these metazoans. In fact there is phase correlation among the introns in these genes. Finally, our preliminary data support the notion that introns usually do not interrupt structural protein motifs, namely the predicted β-strands. These observations concur with the concept of exon shuffling, wherein exons encode structural modules of proteins and the loss and gain of introns and the shuffling of exons via recombination events contribute to the complexity of modern day proteomes.

Group I introns are intervening sequences that have invaded tRNA, rRNA and protein coding genes in bacteria and their phages. The ability of group I introns to self-splice from their host transcripts, by acting as ribozymes, potentially renders their insertion into genes phenotypically neutral. Some group I introns are mobile genetic elements due to encoded homing endonuclease genes that function in DNA-based mobility pathways to promote spread to intronless alleles. Group I introns have a limited distribution among bacteria and the current assumption is that they are benign selfish elements, although some introns and homing endonucleases are a source of genetic novelty as they have been co-opted by host genomes to provide regulatory functions. Questions regarding the origin and maintenance of group I introns among the bacteria and phages are also addressed.

Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes. A variety of reagents have been developed in recent years that can generate targeted double-stranded DNA cuts which can be repaired by the error-prone, non-homologous end joining repair system or via the homologous recombination-based double-strand break repair pathway provided a suitable template is available. These genome editing reagents require components for recognizing a specific DNA target site and for DNA-cleavage that generates the double-stranded break. In order to reduce potential toxic effects of genome editing reagents, it might be desirable to control the in vitro or in vivo activity of these reagents by incorporating regulatory switches that can reduce off-target activities and/or allow for these reagents to be turned on or off. This review will outline the various genome editing tools that are currently available and describe the strategies that have so far been employed for regulating these editing reagents. In addition, this review will examine potential regulatory switches/strategies that can be employed in the future in order to provide temporal control for these reagents.

Endoconidiophora resinifera (=Ceratocystis resinifera) is a blue-stain fungus that occurs on conifers. The data showed that the Endoconidiophora resinifera mitochondrial genome is one of the largest mitochondrial genomes (>220 kb) so far reported among members of the Ascomycota. An exceptional large number of introns (81) were noted and differences among the four strains were restricted to minor variations in intron numbers and a few indels and single nucleotide polymorphisms. The major differences among the four strains examined are due to size polymorphisms generated by the absence or presence of mitochondrial introns. Also, these mitochondrial genomes encode the largest cytochrome oxidase subunit 1 gene (47.5 kb) reported so far among the fungi. The large size for this gene again can be attributed to the large number of intron insertions. This study reports the first mitochondrial genome for the genus Endoconidiophora, previously members of this genus were assigned to Ceratocystis. The latter genus has recently undergone extensive taxonomic revisions and the mitochondrial genome might provide loci that could be applied as molecular markers assisting in the identification of taxa within this group of economically important fungi. The large mitochondrial genome also may provide some insight on mechanisms that can lead to mitochondrial genome expansion.

Fungi isolated from Tomicus piniperda (L.) galleries in infected trap logs, standing trees, and directly from insects were identified using morphological features and molecular data obtained from the mitochondrial and nuclear DNA region. Identified strains represented Leptographium wingfieldii Morelet, Leptographium procerum (Kendr.) Wingf., Leptographium lundbergii Lag. & Melin sensu Jacobs & Wingfield, Ophiostoma ips (Rumb.) Nannf., Ophiostoma minus (Hedg.) H. & P. Syd., and Sphaeropsis sapinea sensu lato. Leptographium wingfieldii is believed to be a potentially pathogenic introduced fungus, but sequence data suggest a possible connection between it and the teleomorph of Ophiostoma aureum (Robinson-Jeffrey & Davids.) T.C. Harrington (reported from British Columbia and the western United States). Our data also show that the ex-type culture of Leptographium terebrantis Barras & Perry, a species very similar morphologically to L. wingfieldii, also grouped with L. wingfieldii. We also identified strains of Leptographium truncatum (Wingf. & Marasas) Wingf.; this species has been synonymized with L. lundbergii, but our data indicate that these are distinct species, and therefore, the name L. truncatum should be reinstated. We also report the extended presence of L. procerum in Ontario. Previously viewed as a “southern” species frequently associated with pine-root decline diseases, it has been infrequently reported from New York state and but once each from Ontario and Quebec.