Manipulating the Metazoan Mitochondrial Genome with Targeted Restriction Enzymes
Hong Xu, Steven Z. DeLuca, Patrick H. O’Farrell
Science 25 July 2008:
Vol. 321. no. 5888, pp. 575 – 577
Mitochondria are the cellular organelles responsible for energy production. In humans, defects in mitochondrial function can lead to a variety of disorders, many of which are progressive. Mitochondria are also the source of much of the reactive oxygen species that cause oxidative stress – mitochondrial dysfunction is also implicated in ageing.
Mitochondria have their own genomes – in animals, the mitochondrial genome is circular, multicopy, and contains only a few tightly packed genes. Unfortunately, classical genetics has not been useful for the genetic analysis of mitochondrial genes. In this paper. O’Farrell and his group employ a novel technique to generate mutations in mitochondrial genes, and demonstrate its application to two of those genes.
The principle of the technique appears simple: a restriction endonuclease is expressed in Drosophila cells, and is targeted to the mitochondrial matrix. the restriction endonuclease is chosen such that it has a unique cleavage site in the mitochondrial genome, within one of the mitochondrial genes. Cleavage linearises the circular genome, preventing replication. Only those molecules lacking the restriction site evade cleavage and remain active: the system therefore provides an effective selection for mutations mapping within the enzymes 6 base pair recognition sequence.
Most of the work consists of using mitochondrially targetted XhoI, which cuts one in the mitochondrial genome, with a six base recongnition sequence spanning three codons (300-302) of Cytochrome oxidase subunit 1 (CoI). To test both the efficacy of mitochondrial targetting, and of cutting, the system was tried out by transfecting Drosophila cultured S2 cell. Then transgenic flies were made, bearing the UAS-mitoXhoI construct, making use of the Gal4-UAS transgene expression system (for more about Gal4-UAS, see here).
ey-Gal4>UASmitoXhoI flies (which express the transgene only in the developing eye) have defective eyes,suggesting that loss of mitochondria in those cells is occurring.
nano-Gal4>UASmitoXhoI flies (expressing in germline cells) resulted in sterility – though about 1% of these females gave occasional progeny, presumably by selecting for rare mitochondrial variants that lacl the XhoI cleavage site.
The exciting stuff follows the analysis of three lines selected in this way, each of which contains an altered base pair in the CoI gene. Interestingly, these mutants each have distinct phenotypes, one in particular leading to a progressive degenerative phenotype (and shortened lifespan). What’s cool here is that these recovered mutant strains are homoplasmic (all the mitochondrial genomes are the same), and have a single sequence change, affecting a single mitochondrial gene.
Is this approach generally applicable? Apparently there are 31 different restriction enzymes with unique cleavage sites in the Drosophila mitochondrial genome, within eight protein coding genes, and two ribosonal and three transfer RNA genes, and indeed the paper briefly reports successful usae of this approach to recover mutants in the ND2 gene.
Can this be used to investigate mitochondrial genetics in mammals? A brief discussion at the end of the paper suggests this might be possible in mice – (for which an mtDNA mutator strainis available).
I think this is a terrific and novel approach to obtaining mitochondrial mutants, and for those of us with interests in biological processes such as ageing, in which mitochondrial function is implicated, it’s an exciting development. Furthermore, the recovery of three CoI alleles with distinctive phenotypes demonstrates that for each gene that can be targetted, several alleles with distinct phenotypes may be obtained. While it remains to be seen what impact this work will have more widely, I expect it to make a big impact on mitochondrial work in Drosophila, and quite possibly contribute to understanding of the mitochondrial dysfunctions that underly number ofhuman diseases.
H. Xu, S. Z. DeLuca, P. H. O’Farrell (2008). Manipulating the Metazoan Mitochondrial Genome with Targeted Restriction Enzymes Science, 321 (5888), 575-577 DOI: 10.1126/science.1160226