The functional organization of mitochondrial genomes in human cells
© Iborra et al; licensee BioMed Central Ltd. 2004
Received: 11 December 2003
Accepted: 24 May 2004
Published: 24 May 2004
We analyzed the organization and function of mitochondrial DNA in a stable human cell line (ECV304, which is also known as T-24) containing mitochondria tagged with the yellow fluorescent protein.
Mitochondrial DNA is organized in ~475 discrete foci containing 6–10 genomes. These foci (nucleoids) are tethered directly or indirectly through mitochondrial membranes to kinesin, marked by KIF5B, and microtubules in the surrounding cytoplasm. In living cells, foci have an apparent diffusion constant of 1.1 × 10-3 μm2/s, and mitochondria always split next to a focus to distribute all DNA to one daughter. The kinetics of replication and transcription (monitored by immunolabelling after incorporating bromodeoxyuridine or bromouridine) reveal that each genome replicates independently of others in a focus, and that newly-made RNA remains in a focus (residence half-time ~43 min) long after it has been made. This mitochondrial RNA colocalizes with components of the cytoplasmic machinery that makes and imports nuclear-encoded proteins – that is, a ribosomal protein (S6), a nascent peptide associated protein (NAC), and the translocase in the outer membrane (Tom22).
The results suggest that clusters of mitochondrial genomes organize the translation machineries on both sides of the mitochondrial membranes. Then, proteins encoded by the nuclear genome and destined for the mitochondria will be made close to mitochondrial-encoded proteins so that they can be assembled efficiently into mitochondrial complexes.
Mitochondria are the centre of energy production in the cell. They are usually numerous and polymorphic, and their overall shape depends on a balance between the fusion and fission of individual mitochondria . These processes are controlled by several proteins , including dynamin-related protein 1 (Drp1) [3, 4], and the mitofusins (for example, Mfn 1 and 2) . Mitochondria also move in a complex way through living cells , probably carried by actin/myosin- and dynein/kinesin-based motors along the microfilament and microtubule networks [7, 8].
In most human cells, mitochondria contain 103–104 copies of a circular genome of 16,569 base-pairs that encodes two ribosomal RNAs, 22 tRNAs, and 13 polypeptides that form parts of the respiratory-chain located in the inner mitochondrial membrane [9, 10]. A noncoding regulatory region harbours an origin of replication plus two promoters, one on each of the two strands. Transcription from these promoters generates polycistronic transcripts that are processed to produce mature rRNAs, tRNAs, and mRNAs; a transcript generated from one of the promoters also primes mitochondrial DNA (mtDNA) replication. Protein-coding regions of the resulting RNAs are translated by ribosomes within the mitochondrion. However, most mitochondrial proteins are encoded by the nucleus; they are made by cytoplasmic ribosomes and imported through specialized pores into mitochondria where they combine with those encoded by the mitochondrial genome [11, 12].
As might be expected, mutations affecting mitochondria lead to a complex set of pathologies and the resulting disorders are the most frequent of the inborn errors of metabolism, with an estimated incidence of at least one in 10,000 live births . Some causative mutations arise in the nuclear genome (for example, in NDUFS4, 7, and 8 in Leigh and Leigh-like syndromes), others in the mitochondrial one. For example, A to G transitions in nucleotides 3243 and 8344 in mtDNA underlie mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) and myoclonic epilepsy with ragged red fibres (MERFF), respectively. Unlike normal individuals in whom most mtDNA copies are genotypically similar (homoplasmic), most patients suffering from mtDNA disorders have dissimilar copies (heteroplasmic). Therefore, it is of interest how the number of copies of mtDNA is regulated, how homoplasmy is maintained in normal individuals, and how quickly mutations spread through the mtDNA population to give rise to the mutant phenotype [13–15]. Unfortunately, we still lack precise answers to such questions in man as not enough is known about the organization of individual genomes; fortunately, more is known about the organization in yeast [1, 2].
We now examine the organization of mitochondrial genomes in a human cell line that contains mitochondria tagged with the yellow fluorescent protein (YFP). This line is derived from the bladder carcinoma line, ECV304 – which is also known as T-24  – and individual mitochondria can easily be seen in the thin cytoplasm of these epithelial-like cells.
A stable cell line with YFP-tagged mitochondria
mtDNA is concentrated in foci in fixed cells
Properties of mitochondria and their genomes.
1. total length/cell (μm)
600 ± 124a
mt DNA foci
2. lineal density in mitochondria (foci/μm)
468 ± 97d, 492 ± 102e
4. diameter (range; nm)
70f, 65 (31–132)g
7.5i, 7.1j, 9.2k, 5.8l, 10m
Superficial examination of images like those in Figure 1A,1B,1C,1D also suggests that foci were concentrated in regions poor in the YFP-tagged subunit of cytochrome oxidase, which is distributed throughout the mitochondrial matrix (above). This was confirmed using a cross-correlation analysis. Twelve image pairs like those in Figure 1A and 1B were pseudo-coloured green and red respectively, the red component shifted over various distances (Δx) with respect to the green component, and the value of Pearson's correlation coefficient (r P ) plotted against Δx. If the red and green patterns coincide, mutually exclude each other, or are arranged randomly, r P respectively peaks, dips, or remains flat, around a Δx of zero . YFP proves to be excluded for up to 400 nm from the centre of mtDNA foci (Figure 1G), perhaps because mitochondria are thinner in this region . This might be advantageous to the cell, as there would be less functional cytochrome oxidase in the thinner region to generate damaging free radicals near the mtDNA.
A mtDNA focus contains approximately eight genomes
The number of mitochondrial genomes per focus was calculated after measuring the number of mitochondrial genomes per cell, the total mitochondrial length, and the number of foci per unit length of mitochondria (Table 1). While the first two can be estimated reasonably accurately, it is difficult to be sure that all foci are detected. Therefore we used various approaches, reasoning that if different methods with different thresholds of detection gave similar results, one estimate would lend credibility to another (Table 1, line 6). The data were analyzed as follows: (i) immunolabelling with both anti-DNA and anti-BrdU (after growth in BrdU to substitute DNA fully) gave similar results (that is, ~7 genomes/focus); (ii) the average intensity of foci in images like that in Figure 1D had an intensity 9.2× higher than the weakest (Figure 1F, legend); if the faintest focus then contains one genome, the average would contain ~9; (iii) foci had a diameter of ~68 nm (Table 1, line 4), sufficient to accommodate ~6 genomes packed as tightly as in the bacterial nucleoid ; and (iv) analysis of replication rates are consistent with each focus containing ~10 genomes (below). Taken together, these results suggest the average focus contains 6–10 genomes, again consistent with earlier results [18, 19].
Dynamics of mtDNA in living cells
After washing away ethidium, the fluorescent mtDNA foci can be seen moving continually within the (moving) mitochondria. This is illustrated using frames from time-lapse movies in which images were collected every 10 s for up to 500 s (Figure 4A,4B,4C,4D). Thus, the distance separating two adjacent foci labelled S1 in Figure 4B remained roughly constant at ~3 μm, although one focus travelled ~14 μm over 500 s. Similarly, the distance separating a terminal focus from the adjacent mitochondrial tip (S2 in Figure 4B) was always <1 μm, even though the tip moved 21 μm.
Movement of foci is superimposed on that of mitochondria, and it was analyzed in various ways . The mean square displacement (MSD) of one mtDNA focus relative to another is >0 (Figure 4F, 'DNA'), but less than that of a tip of one mitochondrion relative to another (Figure 4F, 'tip'). An apparent diffusion constant (D) for mtDNA foci of 1.1 × 10-3 μm2/s was calculated from the MSD; this is comparable to that of DNA in Drosophila nuclei . We next searched for directed or periodic movement using an autocorrelation function (ac) that relates changes in distance (Δd) seen between foci at increasing time intervals (Δt) . The correlogram in Figure 4G is consistent with a random walk (step length 0.26 μm over 10 s) in which the direction changes every ~16 s, and with only ~4% steps continuing in the same direction for ≥40 s. The distribution of Δd was normal and centred on zero (Figure 4H), also indicative of a random walk .
Fission and fusion of mitochondria next to mtDNA foci
Whole mitochondria also split, which in ~90% cases occurred by progressive thinning next to a focus to leave all DNA in one daughter (Figure 5C) . As a result, some daughters arise that lack mtDNA, albeit rarely (not shown). That fission occurs next to mtDNA was confirmed by counting the fraction of mitochondrial tips in fixed cells that contained mtDNA within 500 nm of a tip (Figure 1A,1B, insets); the value (that is, 52%) was significantly higher than the 39% expected if mtDNA were distributed randomly along mitochondria (p < 0.0001; χ2 test). As the dynamin-related protein, Drp1, is found at fission sites , we immunolocalized it relative to mtDNA, and found it often lay immediately next to mtDNA (Figure 5D,5E). This was confirmed by the peaks flanking the dip in Pearson's coefficient (r P ) around a Δx of zero (Figure 5F). These results confirm and extend those of Garrido et al. , and show that mitochondria split next to mtDNA foci.
Stochastic replication of individual mitochondrial genomes
Three extreme models for the labelling kinetics are shown in Figure 6E; only one (model b) is consistent with essentially all foci containing some Br-DNA by 3 h (Figure 6C), and with the average intensity per focus being constant between 1 and 2 h before rising (Figure 6D). Then, one genome per focus would be replicated within the first 2 h, and all genomes in a focus after a complete cycle of 22 h. As the intensity per focus increases 10-fold during this period, we estimate each focus contains ~10 genomes (Table 1, line 6).
Newly-made mitochondrial transcripts are found in discrete sites
Sites containing newly-made RNA can be visualized after growing cells in bromouridine (BrU), and then immunolabelling the resulting Br-RNA [38, 39]. (Here, cells are grown in the absence of ethidium so transcription is not inhibited.) Br-RNA accumulates in nuclei and in discrete mitochondrial foci (Figure 7A,7B,7C,7D) that initially lie immediately next to mtDNA foci (Figure 7E,7F,7G). The intensity of Br-RNA labelling per focus increases progressively (Figure 7H), and declines exponentially (t1/2 ~ 45 min) during a 'chase' (Figure 7I). Quantitative analysis shows that after an initial pulse of 20 or 30 min, most Br-RNA foci colocalize with mtDNA (defined as lying within 200 nm of mtDNA), but after longer periods (or a pulse-chase) the Br-RNA foci become distributed randomly (Figure 7J). Kinetics are consistent with Br-RNA having a residence half-life in mtDNA foci of ~43 min (see Methods). This is approximately eight-fold longer than the ~5.5 min required to make a complete transcript (assuming ~50 nucleotides are polymerized every second) . This suggests that mitochondrial messages remain at transcription sites for extended periods after they have been made.
Not all mtDNA foci contain Br-RNA (Figure 7E,7F,7G), but we cannot distinguish whether the unlabelled foci are transcriptionally inactive or contain too little Br-RNA to be detected. However, there is no correlation between the amounts of DNA and Br-RNA in a focus (Figure 7K), which is consistent with some being inactive.
Mitochondrial transcription sites lie near extra-mitochondrial ribosomes
Organization and dynamics of mtDNA
We analyzed the organization and function of mitochondrial DNA (mtDNA) in a stable cell line containing mitochondria tagged with the yellow fluorescent protein (YFP). A plasmid encoding subunit VIII of cytochrome c oxidase fused with YFP, was transfected into ECV304 cells – an epithelial-like human line – and a clone (that is, cox18) expressing YFP established. The tagged subunit is incorporated into mitochondria, so that they autofluoresce (Figure 1A).
As others have found [18, 19], mtDNA is concentrated in discrete foci in both fixed and living cells (Figures 1, 2, 4). We calculated the average number of mitochondrial genomes in a focus after measuring the genome number per cell, the total mitochondrial length, and the number of foci per unit length of the mitochondrion (Table 1). While the first two can be measured reasonably accurately, it is difficult to be sure that we detect most foci. Therefore we used various approaches, reasoning that if different methods with different thresholds of detection gave similar results, one estimate would lend credibility to another. Four independent methods (involving immunolabelling with two different antibodies, analysis of profiles of the amount of DNA per focus, packing densities, and replication time) suggest that each focus contains 6–10 genomes (Table 1, line 6), which is again consistent with earlier results .
The mtDNA foci are attached to the cytoskeleton as they remain after most soluble proteins are extracted (Figure 2B) [22–24]. Three regions roughly equally spaced around the genome – the origin of replication of the heavy strand plus the heavy- and light-strand promoters, the light strand origin, and another region – are likely candidates for the DNA sequences responsible for attachment, as these sequences remain attached after exhaustive nucleolytic detachment . Then we can imagine the mtDNA is tethered (directly or indirectly) through the mitochondrial membranes to the kinesin motor implicated in moving mitochondria along microtubules, as a component of that motor – KIF5B  – often colocalizes with foci (Figure 3E,3F,3G,3H). Such a transmembrane linkage implies a specialized structure. This may be related to the transmembrane structures seen in trypanosomes connecting mitochondrial nucleoids to the cytoskeleton , and in yeast where they seem to play a role in replication .
The limited movement of mtDNA in living cells is also consistent with such a tethering. Cells were grown briefly in ethidium, washed, and regrown in its absence; then fluorescent mtDNA foci can be seen moving in a constrained random walk within the (moving) mitochondria . The foci have a low apparent diffusion constant of 1.1 × 10-3 μm2/s (Figure 4), which is comparable to that of the nuclear DNA of flies  but 10-fold higher than that of humans . As the foci move, some split, others fuse (Figure 5A,5B), and still others fuse to remain together for several minutes before splitting; in 49 out of 50 of such fusion-fission events the emerging foci retain the intensity of fusing foci implying that their contents do not mix. Whole mitochondria also split after progressive thinning next to mtDNA foci (Figure 5C) , and this can generate daughters without any mtDNA, albeit rarely. Fission next to foci was confirmed in fixed cells; the tips of mitochondria contain mtDNA more often than expected by chance (Figure 1A,1B, insets; see Results). Moreover, the dynamin-related cytoplasmic protein Drp1 is found at fission sites , and it often lay immediately next to mtDNA foci (Figure 5D,5E,5F). These results confirm and extend those of Garrido et al. .
While nuclear DNA is replicated only during S phase, mtDNA molecules can be replicated at any stage in the cell cycle . Some of the models that can be envisaged for the replication of molecules within a focus are illustrated in Figure 6E. For example, a focus might constitute a unit of replication in which all genomes in that focus initiate coordinately (model a). Alternatively, genomes in one focus might replicate successively, before replication switched to another focus (model c). At the other extreme, all genomes might replicate independently of all others (model b). The labelling kinetics during growth in BrdU are consistent with the stochastic model b; thus, the number of foci containing Br-DNA increases for 3 h and then remains constant (Figure 6C), and the average intensity per focus initially remains constant for 2 h before increasing (Figure 6D). Inevitably, then, some molecules will be re-replicated during one cell cycle, as has been shown previously .
Sites containing newly-made nuclear RNA can be visualized after growing cells in bromouridine (BrU), and then immunolabelling the resulting Br-RNA [38, 39]. We show here the same approach can be used to mark newly-made mitochondrial RNA; moreover, the resulting Br-RNA accumulates in discrete foci (Figure 7A,7B,7C,7D) that initially colocalize with mtDNA foci (Figure 7E,7F,7G). Pulse-chase experiments reveal this newly-made Br-RNA has a half-life of ~45 min (Figure 7I), and a residence half-time in the mtDNA foci of ~43 min (Figure 7J). This residence half-time is approximately eight-fold longer than the ~5.5 min required to make a complete transcript (calculated assuming ~50 nucleotides are polymerized every second) . This suggests that newly-made mitochondrial messages remain at transcription sites for extended periods, before they are degraded at those sites.
Mitochondria are formed by proteins encoded by both mitochondrial and nuclear genomes. We reasoned the cytoplasmic machinery generating nuclear-encoded proteins might lie immediately next to its counterpart generating mitochondrial-encoded proteins so both sets of products could combine together efficiently. Therefore, we localized newly-made mtRNA relative to two components of the extra-mitochondrial translation machinery – a ribosomal protein (S6) and a chaperone (NAC) responsible for directing nascent cytoplasmic polypeptides to mitochondria  – and to a component of the machinery (Tom22) that imports nuclear-encoded proteins into mitochondria [11, 12]; it localized with all three (Figure 8).
Plasmid pEYFP-Mito encoding subunit VIII of cytochrome c oxidase tagged with YFP (BD Clontech UK, Basingstoke, Hampshire, UK) was transfected into ECV304 (T-24) cells – an epithelial-like human female line derived from a urinary bladder carcinoma  – and a stable line with fluorescent mitochondria selected. This clone (cox18) was grown in DMEM supplemented with 10% FCS. For Figures 1C,1D and 6, medium was supplemented with 50 μM bromodeoxyuridine (BrdU; Sigma-Aldrich, Poole, Dorset, UK).
Cells on coverslips were fixed (20 min; 4°C) in 4% paraformaldehyde in 250 mM HEPES (pH 7.4), and antigens indirectly immunolabelled  using the following antibodies (unless stated otherwise, Affinipure or multiple labelling grade from Jackson ImmunoResearch, Bar Harbor, ME, USA): (i) monoclonal anti-DNA produced in our laboratory (mouse IgM; 5 μg/ml) + donkey anti-mouse IgM conjugated with Cy3 or Cy5 (0.5 μg/ml); (ii) affinity-purified mouse anti-BrdU (2 μg/ml; Caltag Laboratories, Burlingame, CA, USA) + donkey anti-mouse IgG conjugated with Cy3 (0.5 μg/ml); (iii) anti-tubulin (α-subunit; 1/100; Sigma-Aldrich) + donkey anti-mouse IgG conjugated with Cy3 (0.5 μg/ml); (iv) anti-KIF5B (1/100; PCP42 antibody)  + donkey anti-rabbit conjugated with Cy3 (0.5 μg/ml); (v) anti-Drp1 (1/100)  + donkey anti-rabbit IgG conjugated with Cy3 (0.5 μg/ml); (vi) anti-Br-RNA (as for anti-BrdU) + donkey anti-mouse IgG conjugated with Cy3 (0.5 μg/ml), (vii) anti-S6 (2 μg/ml; Cell Signalling, Beverley, MA, USA; this antibody yields one band in immunoblots of whole cell extract, and so we assume it does not cross-react with a subunit of the mitochondrial ribosome) + donkey anti-rabbit IgG conjugated with Cy3 (0.5 μg/ml); (viii) anti-NAC (α subunit; 1/250)  + donkey anti-rabbit IgG conjugated with Cy3 (0.5 μg/ml); (ix) anti-Tom22 C-terminus (1/500)  + donkey anti-rabbit IgG conjugated with Cy3 (0.5 μg/ml). Coverslips were mounted in Vectashield (Vector Laboratories Inc., Burlingame, CA, USA), and images generally collected at 20°C using Lasersharp software and a Nikon TE300 (objective: 60×, numerical aperature 1.4) attached to a BioRad Radiance 2000 confocal microscope (BioRad Laboratories, Hemel Hampstead, UK) . Images were analyzed using EasiVision (Soft Imaging Systems GmbH, Münster, Germany) and Excel (Microsoft) software; single confocal images were also exported from Lasersharp into Photoshop (Adobe Systems Inc., San Jose, California, USA), contrast stretched, and presented without further modification. In Figure 1E, integrated intensities in areas of ≥4 pixels were measured using EasiVision. In Figs 1E and 7J, random distributions were generated (>1,000 runs) using Excel, and P(l) = e-ρl , where P(l) is the probability, ρ is density of foci/μm (Table 1), and l is length in μm.
After incubation in BrdU and fixation (above), cells were treated (10 min) in 1% Triton X100 in PBS, rinsed in PBS, and DNA denatured (20 min) with 0.5 M HCl; cells were then washed thoroughly in PBS and immunolabelled with the anti-BrdU (above).
Two-colour images were collected using the confocal microscope (10× zoom; 40 nm pixel size), a line profile of intensities in both channels along mitochondria collected using EasiVision, and data exported to Excel. Pixel intensities in each channel were shifted manually pixel by pixel, and Pearson's coefficient computed .
Number of mitochondrial genomes per cell
A total of 106 cells were resuspended in 100 μl agarose (1% final concentration in PBS); after gelling, the block was treated successively with 0.5% SDS (30 min), 10 mg/ml RNase A in 10 mM Tris, 1 mM EDTA (1 h), and 100 μg/ml proteinase K (1 h). Blocks and known weights of plasmid DNA were applied to an agarose gel, the gel run (4 h) to resolve mtDNA, and – after staining with ethidium and illumination with uv light – the amount of mtDNA was determined by comparing intensities with those given by plasmid DNA.
For Figure 2A, cells were fixed, treated with osmium, embedded in Epon, indirectly immunolabelled on ultrathin sections, contrasted with uranyl acetate, and digital images collected on a Zeiss 912 Omega electron microscope . DNA was detected using anti-DNA (above) + goat anti-mouse IgM conjugated with 10 nm particles (1:25 dilution; British BioCell International through Agar Scientific, Stanstead, Essex, UK). The diameters of clusters of gold particles seen in images were corrected for the effects of sectioning  as follows. The major (2x) and minor (2y) orthogonal axes of each cluster were measured, and the cluster diameter, D calculated from D = 2√(xy). The mean cluster diameter, , was calculated from the number of clusters (N) and the diameter of individual clusters (D1, D2,...DN) using
For Figure 2B, cells were grown (24 h) on 200 mesh nickel grids covered with a formvar film (Agar Scientific), treated (5 min; 4°C) with 0.05% Triton X-100 in cytoskeleton (CSK) buffer , washed three times with CSK buffer, fixed (30 min) with 4% paraformaldehyde in CSK buffer, and DNA indirectly immunolabelled using anti-DNA + goat anti-mouse IgM conjugated with 10 nm particles (as above); finally, grids were dried past the critical point and digital images collected on the Zeiss 912 .
Time-lapse movies of mtDNA in living cells
Cells were grown (30 min) in 0.1 μg/ml ethidium, washed with media, regrown in its absence in a heated chamber on the confocal microscope at 37°C, and images collected every 10 seconds using a large confocal pinhole of 5 mm to optimize survival rather than resolution. Coordinates of the gravity center of each ethidium (mtDNA) focus were obtained using EasiVision; data were exported to Excel for analysis, and the mean square displacement (MSD) and autocorrelation coefficient derived . As mtDNA foci move only in one dimension, the diffusion constant was calculated using 12DΔt = <Δd2>, where D is the apparent diffusion coefficient and Δd is the changes in distance seen between foci at increasing time intervals (Δt). RNA was detected in permeabilized and fixed cells using RNase A tagged with Cy3 (prepared using a kit from Molecular Probes Inc., Leiden, The Netherlands) instead of the analogous enzyme:gold complex .
Visualizing newly-made transcripts
Cells on coverslips were grown  in 2.5 mM bromouridine (BrU; Sigma-Aldrich), fixed (20 min; 4°C) in 4% paraformaldehyde in 250 mM HEPES (pH 7.4), and Br-RNA indirectly immunolabelled using mouse anti-BrdU (Caltag) and donkey anti-mouse Fc conjugated with Cy3. For pulse-chase experiments, cells were grown in BrU, washed in medium without BrU, and regrown in medium with 5 mM U. DNA was labelled using anti-DNA plus donkey anti-IgM conjugated with Cy5 (as above) or with Hoechst 33342 (100 ng/ml; 30 min). Images were collected using a Zeiss Axioplan 2 fluorescence microscope fitted with a SPOT2 CCD camera (Optivision Yorkshire Ltd, West Yorkshire, UK). The distance between foci was calculated in images like Figure 7B,7D as follows. The area, intensity, and position (that is, gravity center) of each focus was determined using EasiVision and the distance to the nearest mtDNA foci calculated using Excel. The random distribution for Figure 7J was generated as above. The residence half-life of Br-RNA in the mtDNA foci was calculated using Berkeley Madonna software (Berkeley, CA, USA). We assume the cellular concentration of BrU (and nucleotide triphosphates (NTPs)) remains constant as the BrU is incorporated continuously into Br-RNA that colocalized with mtDNA foci, before the Br-RNA disappears exponentially from those foci during the chase either through export and/or degradation. Then, d/dt[Br-RNA] = k1[NTP] - k2[Br-RNA], and the half-life is estimated from k2.
DNA containing bromodeoxyuridine
RNA containing bromouridine
- CSK buffer:
mitochondrial encephalopathy with lactic acidosis and stroke-like episodes
myoclonic epilepsy with ragged red fibres
mean square displacement
nascent peptide associated protein
- r P :
Pearson's correlation coefficient
yellow fluorescent protein.
We thank A.M. van der Bliek, E. Jones, M. Mori, R. St. Arnaud, and R. Vale for antibodies against Drp1, DNA, Tom22, NAC, and KIF5B respectively, D. Mason and J. Vazquez for help with analyzing data, and the Spanish Ministerio de Educación y Cultura and the Wellcome Trust for support.
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