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
DOI: 10.1126/science.1160226

Abstract

Full Text (subscription required) - HTML PDF

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.

This week's event came towards the end of a rather hot and humid spell - a strong and gusty wind blew up during the day, but had thankfully abated by the time I left work to ride over to Stony Stratford, where the evnt was being held on the F5u/10 course (actually about 11.4 miles in our configuration.  My new route over is to ride up V10 then across to Watling St on H3 and on to Stony.  This was pretty straightforward, and a good warmup.

For the event itself, I tried to keep the gears modest for the climb up to Nash, and by and large succeeded.  My HR quickly rose to respectable levels, and indeed (probably due to the warm conditions) rose over 190bpm for much of the event.

I felt reasonably good, and finished as first vet on standard, about 51 seconds behind Tony Parks.  Most of us found it rather warm!  This puts me (just) top of the vets league.

A week at residential school (seven 12 hour days indoors, with cafeteria food and too much beer) is not the best way to prepare for a 50 mile time trial.  On the morning, we were greeting with warm and humid conditions, with a little light wind (headwind northbound) and a few light showers.

I started late in the field, number 83, and found the first 4 or 5 miles pretty quick.  But then is truggled a bit to keep in a rhythm.  No traffic problems other than tractor overtaking me just before  a descent shortly before my second turn at the Buckden roundabout - this held me up until a sprinted past.  I was cheered on by Carol at the roadside, which always helps.  I finished in 1:58:58, which is I think my best '50' in the last three seasons (but is bizarrely slower than my first 50 miles in the NM&H CA '100' two weks ago, on the same section of road.  I was reasonably happy with this - I kept my HR above 180 for most of the race.  The event was won by Jason Gurney (Team MK) who improved by about 4 minutes to record a 1:46!

One sad thing was that a rider from the organising club came a cropper just after finishing, and cut his head.

Full results

Jones et al (2008) PNAS July 22, 2008 vol. 105 no. 29 10023–10027

Full text (requires subscription); Abstract (free access)

Tasmanian devils (Sarcophilus harrisii) are the largest extant marsupial carnivore - male specimens can weigh up to 14kg, while females can reach 9kg.  They have long been extinct on the Australian mainland.  Recently, an infectious tumour (Devil Facial Tumour Disease, or DFTD) has afflicted the remaining population of Tasmanian devils, resulting in considerable population decline.

DFTD is a peculiar disease: it's an infectious tumour which is spread via  injury.  Technically, and unusually, transmission follows transfer of tumour cells via injury - the tumours are said to be allografts - the tumour cells afflicting an individual are Tasmanian devil cells (albeit abnormal) but they are genetically distinct from the afflicted individual.  Tasmanian devils are a bit rough, and frequently injure each other by facial injuries - this is the route by which infection occurs.  A similar disease, Canine Transmissible Venereal Tumour (CTVT) is found in dogs - it is transmitted during copulation.

DFTD was first reported in 1996 - by 2007 it had reached about half of the range occupied by Tasmanian devils, and has resulted in declines of up to 89% in some populations.  As one might imagine, the appearance of this condition results in a strong selective pressure,  This paper investigates the consequences of this DFTD on reproductive strategies in Tasmanian devil populations. I've come to this paper as a non-specialist (I'm a Drosophila geneticist, and this paper is essentially an ecology paper).

Prior to the appearance of DFTD, Tasmanian devils would live to abut 5 or 6 years old, generally breeding from their second year (breeding in successive years - iteroparity).  It seems these marsupials are adapted for a "breed quick, die young" kind of strategy: reproduction is a tough business, with males for example losing about 25% of body mass in a breeding season.  It is in light of this that the authors have investigated the consequences of DFTD on breeding age in infected populations, and find a shift to semelparity.

The authors compare the frequency of animals aged 3+ years in populations sampled before and after DFTD invasion.  It is pretty clear that not only has the frequency of devils of age 3+ years dropped markedly (in some locations older animals are entirely absent), but that the frequency of one year old females that are breeding has increased - in some locations from 0% to over 50%.  So what are the authors' conclusions?  Principally, they conclude that this is an adaptation to a strong selective pressure - a possible mechanism being that reduced population density permits more rapid growth of individuals and therefore permitting earlier reproduction.  An alternative hypothesis, that the cause of earlier reproduction is a reduction in interference competition is given short shrift (though I don't have the expertise to understand why!).

An interesting paper, on a fascinating disease!

Terminology

semelparous - breeds once before death (see this article on reproduction)

iteroparous - breeds in several successive seasons (see this article on reproduction)

allograft