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(Received for publication, May 26, 1995; and in revised form, October 4, 1995) From the
We have previously identified a mitochondrial DNA polymorphism
(a C
Mutations in the mtDNA have been implicated in the pathogenesis
of different clinical syndromes(1, 2) . In the past 7
years, pathogenic large-scale rearrangements as well as point mutations
in the human mtDNA have been described, most of them heteroplasmic (i.e. mutated mtDNA co-existed with the wild-type mtDNA).
Point mutations in mitochondrial tRNA genes seem to be particularly
frequent in neuromuscular disorders, possibly because of their
generalized effect on mitochondrial protein synthesis, and consequent
impairment of multiple oxidative phosphorylation enzyme
complexes(3) . Several pathogenic mutations in the
mitochondrial tRNA We have
described a patient with a multisystem mitochondrial disorder
including: progressive external ophthalmoplegia, seizures, diabetes,
cardiomyopathy, and retinopathy, harboring yet another mutation in the
mitochondrial tRNA
Figure 1:
Characterization of transmitochondrial
cybrid lines. Three cell lines containing essentially 100% wild-type
mtDNA and three cell lines containing >99% mutated mtDNA (at
position 3256) were characterized in detail and used in the subsequent
studies. Panel A illustrates the PCR-RFLP analysis of the 3256
mutation. PCR products originated from wild-type genomes have an
additional CfoI site, creating a diagnostic size difference in
a nondenaturing polyacrylamide gel after digestion of PCR products (Panel B). Mitochondrial DNA levels were estimated by Southern
blot analysis using mtDNA-specific and nuclear (18 S rRNA gene)
DNA-specific probes (Panel C). The patient-derived identity of
mtDNA in transmitochondrial cybrids was confirmed by PCR-RFLP analysis
of an unrelated polymorphism at mtDNA position 14766. A PCR fragment
encompassing the polymorphic site was digested with Tru91,
electrophoresed through a 12% polyacrylamide gel and stained with
ethidium bromide (Panel D).
To
estimate the ratio of mtDNA to nuclear DNA, approximately 5 µg of
total DNA extracted from exponentially growing cells were PvuII-digested, electrophoresed through a 0.8% agarose gel,
transferred to a Zeta-Probe GT membranes (Bio-Rad), and hybridized with
two The patient-type identity of mtDNA present in
transmitochondrial cybrids was confirmed by RFLP analysis of a
non-disease-related Tru91 polymorphism (T
Respiratory complexes activity was measured in mitochondrial
fractions isolated by standard methods (21) without digitonin.
Complexes I + III, II, II + III, IV, and citrate synthase
were measured as described elsewhere(22) . Lactate released to
the cultured medium was measured with a commercial testing kit (Sigma)
and normalized by the number of cells present at the end of the
experiment.
Figure 6:
Northern blot hybridization analyses.
Autoradiograms of total RNA, extracted from the cell lines listed on top of each lane, and hybridized to different probes are
shown. Probes are specific for the following mitochondrial transcripts: A, ND1; B, tRNA
High
resolution Northern blotting was performed essentially as previously
described(30) . For detection of the tRNA
Figure 2:
Mitochondrial functional assays in
transmitochondrial cybrid clones. A, rates of oxygen
consumption per cell of 143B, 143B/206, and the indicated
transmitochondrial cybrid lines are shown, with error bars representing ± S.D. of three determinations. B,
the graph shows a time course release of lactate to the culture medium
normalized to the number of cells. C, the histogram shows the
spectrophotometrically determined activity of different respiratory
complexes in isolated mitochondria normalized to the activity of the
matrix enzyme citrate synthase. Individual wild-type and mutant clones
are displayed in the same order as in Panel
A.
Figure 3:
Mitochondrial protein synthesis in
transmitochondrial cybrid clones. Fluorograms of mitochondrial
translation products labeled with [
Figure 4:
Densitometric analysis of high molecular
weight mitochondrial translation products. Fluorograms of mitochondrial
translation products labeled with [
Steady-state levels of two
subunits of COX were measured by Western blot. Both the mtDNA-encoded
subunit COX II, and in a lesser extend, the nuclear-encoded subunit COX
IV were reduced in the mutant clones (Fig. 5). In mutant clones,
COX II mean value was only 25% of the wild-type, while COX IV mean
value was 42% of wild-type values. The ratio of COX IV/COX II was 1.6
for 143B, 1.9 ± 0.4 (mean ± S.D.) for wild-type clones,
4.0 ± 2.5 for mutant clones. The mtDNA-less cell line 143B/206
had normal levels of COX IV but lacked COX II (Fig. 5).
Figure 5:
Steady-state levels of two COX subunits.
The figure shows a Western blot of isolated mitochondria from different
cell lines incubated simultaneously with two antibodies specific to COX
subunits II and IV. Color development was stopped before previously
determined half-maximum band intensities.
The relative levels of tRNA
Figure 7:
High resolution Northern hybridization.
Total RNA extracted from different cell lines was electrophoresed
through a 20% polyacrylamide gel, electrotransferred to a nylon
membrane, and hybridized to tRNA-specific probes. Panel A shows hybridization to a tRNA
The increasing number of reports on mtDNA mutations
associated with human diseases has created the need for functional
studies to corroborate the genetic data. Although features such as:
heteroplasmy, evolutionary conservation, and clinical-genetic
correlations are strong indicators of etiologic mtDNA mutations, they
cannot replace functional studies in providing prove for pathogenicity.
Several potentially pathogenic mutations in the mitochondrial
tRNA The present
study tried to establish a correlation between a C Homoplasmic
mutant clones (>99% mutated mtDNA) had a severe deficiency in
respiratory chain function, as shown by oxygen consumption, lactate
production and enzymes activity. They also showed a 75% reduction in
the steady-state levels of a mitochondrially synthesized polypeptide
(COX II), and a 15-80% deficiency in synthesizing different mtDNA
encoded polypeptides when compared to wild-type clones. The partial
reduction in the nuclear-encoded subunit IV of COX could be explained
by the primary deficiency of COX II, which would limit the number of
properly assembled holoenzymes. However, this explanation may not be
satisfactory because COX IV was present at normal levels in
mitochondria isolated from the mtDNA-less 143B/206 line, even though
COX II was completely absent. The observations described above
suggest that the mitochondrial dysfunction associated with the C Besides the role of the 3256
mutation in tRNA function and RNA processing, some of our results are
also compatible with alternative pathogenetic mechanisms. The 3256
mutation is located within the last base pair footprinted by a
mitochondrial transcription termination factor(33) ,
potentially altering the binding of this trans-acting factor, that
could lead to an unbalance in the levels of transcripts located
upstream and downstream of the termination site(34) . In
vitro, the 3243 mutation (located in the middle of the termination
factor binding site) reduces transcription termination, leading to an
increase in the levels of downstream transcripts(34) . It is
possible that the 3256 mutation has an opposite effect (i.e. it strengthens transcription termination), therefore reducing
transcription of downstream genes. This hypothesis would be compatible
with the observed reduction in ND1 and tRNA As in previous
reports(12, 18) , we found some phenotypic
heterogeneity among mutant clones. Part of this variation may be
explained by the aneuploid character of these transformed cell lines.
Although we found partial quantitative abnormalities in transcripts and
polypeptides produced by mutant cell lines, we do not know if alone,
they can account for the severe oxygen consumption impairment observed.
Therefore, we cannot exclude that the 3256, as well as other
tRNA
Volume 271,
Number 4,
Issue of January 26, 1996 pp. 2347-2352
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
T
Transition at Position 3256 of the Human Mitochondrial Genome
THE EFFECTS OF A PATHOGENIC MITOCHONDRIAL tRNA POINT MUTATION IN
ORGANELLE TRANSLATION AND RNA PROCESSING (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
T transition at position 3256, within the mitochondrial
tRNA
gene) in a patient with a multisystem disorder.
Although there were several indicators suggesting a pathogenetic role
for this mtDNA polymorphism, its heteroplasmic nature made functional
and molecular studies difficult to interpret. We have now fused
enucleated fibroblasts from the patient with a mtDNA-less cell line to
generate transmitochondrial cybrids harboring different proportions of
mutated and wild-type mtDNA. Individual clones harboring essentially
100% wild-type or >99% mutated mtDNAs were characterized and studied
for respiratory capacity, respiratory chain enzymes activity,
mitochondrial protein synthesis, and RNA steady-state levels and
processing. Our results showed that cell lines containing exclusively
mutated mtDNAs respire poorly, overproduce lactic acid, and have
significantly impaired activity of respiratory complexes I and IV.
Molecular studies showed that mutant clones have a decrease in
steady-state levels of mitochondrial tRNA
, and a
partial impairment of mitochondrial protein synthesis and steady-state
levels, suggesting that these molecular abnormalities are involved in
the pathogenetic mechanism of the mtDNA 3256 mutation.
gene have been
described(3, 4, 5, 6, 7, 8, 9, 10) .
One of these, an A
G transition at nucleotide -3243
(numbers according to Anderson et al.(11) ), is seen
most frequently in patients with mitochondrial encephalomyopathy,
lactic acidosis, and stroke-like episodes (MELAS). (
)The
functional consequences of the 3243 mutation have been extensively
analyzed(12, 13, 14) , though no final
conclusion could be drawn from these observations. The 3243 mutation
segregates with a respiration dysfunction and a partial impairment in
mitochondrial protein synthesis, but the molecular mechanisms
associated with these abnormalities are not understood. Because cells
with essentially 100% mutated mtDNAs have higher levels of an
intermediate transcript (termed RNA 19) encompassing the
tRNA and two adjacent RNAs, it was suggested that
this processing abnormality would either deprive the cell from adequate
levels of mature transcripts (12) or that RNA 19 would
interfere with translation by ``stalling'' mitochondrial
ribosomes(15) . Similar processing abnormalities have been
observed in association with two other mutations in the mitochondrial
tRNA
gene(7, 16) , suggesting that
abnormal processing could be a common pathogenetic mechanism associated
with mutations in this particular mitochondrial gene.
gene at position 3256. More
recently, a second family with MELAS, harboring the same mtDNA mutation
was identified in Japan(17) . In the present report we
establish the association between this mutation and an oxidative
phosphorylation dysfunction, and explore potential pathogenetic
mechanisms.
Cell Lines
The human osteosarcoma-derived cell
line 143B(TK) and its mtDNA-less derivative 143B/206
were a kind gift of Dr. Michael P. King (Columbia University, New
York). Growth conditions and the characterization of the 143B/206 line
were described elsewhere(18) . Transmitochondrial cybrids were
grown in the absence of uridine, except when functional or molecular
studies were to be performed. In these cases, uridine (50 µg/ml)
was added to the medium 48 h before the experiment. A fibroblast line
from a patient harboring an heteroplasmic (48%) mtDNA mutation at
position 3256 was obtained and characterized previously(3) .
The selected clones used in this study were frozen into several
aliquots that were thawed to perform specific experiments (including
DNA analysis). This procedure assured that no significant time in
culture elapsed between the different analyses.
Production and Genetic Characterization of
Transmitochondrial Cybrids
Enucleated fibroblasts from the
patient were fused to the mtDNA-less 143B/206 cell line as previously
described(12, 18) . Parental cell lines were killed
either by bromodeoxyuridine (fibroblasts) or by the lack of uridine in
the medium (143B/206). Individual cybrid colonies were first analyzed
by collecting a few cells (approximately 100 cells) from the initial
foci, exposing their DNA by a micro alkaline lysis
procedure(19) , and genotyping the mtDNA for the 3256 position
by restriction fragment polymorphism (RFLP) of PCR-amplified fragments (3) (Fig. 1, A and B). To determine
the sensitivity of the RFLP assay, we PCR-amplified a DNA fragment
encompassing the mtDNA 3256 position (between nucleotides 3316 and
3353) from a wild-type and a mutant transmitochondrial cybrid line.
These PCR fragments were subcloned into a plasmid vector (TA
cloning/pCR II kit from Invitrogen). Individual bacterial clones were
isolated, and their plasmid was tested for the presence or absence of
the 3256 mutation. We mixed known amounts of purified wild-type and
mutant plasmid corresponding to 10, 5, 2, 1, 0.5, and 0.1% wild-type
mtDNA (for a final concentration of plasmid that was similar to the
estimated amount of target mtDNA in the DNA samples extracted from
cybrid clones). These mixtures were PCR-amplified in parallel with DNA
from the three mutant transmitochondrial cybrid lines used in our
studies. By overexposing the gel for 4 days with an intensifying screen
we were able to visualize 1% wild-type mtDNA in the test mixture.
P-labeled probes. One probe was a 2.8-kb PCR fragment
encompassing the mtDNA D-loop region (nucleotide positions
13956-175). The second probe was a 5.8-kb EcoRI insert
from a construct containing the nuclear-encoded 18 S rDNA
gene(20) . The fragments were labeled with a random primer
labeling kit (Boehringer Mannheim). Filter hybridization was performed
as recommended by the manufacturer (Bio-Rad) with 5
10
cpm/ml of each probe (specific activity of approximately
0.7-1 10
cpm/µg). The mtDNA appears as a
single 16.6-kb band, while the 18 S rRNA gene sequence appears as a
13-kb band in the Southern blot (Fig. 1C). The ratio
between the two bands was determined by scanning and analyzing a
shortly exposed x-ray film with the software IMAGE 1.57 (NIMH,
freeware). C at
nucleotide position 14,766) previously identified in this
patient(3) . A PCR-amplified fragment encompassing mtDNA
positions 14682-14810 was digested with Tru91,
electrophoresed through a 12% nondenaturing polyacrylamide gel, and
stained with ethidium bromide.
Respiratory Function Assays
Oxygen consumption was
measured in a 1.5-ml reaction chamber (Gilson, Inc.), heated to 37
°C and equipped with a Clark-type platinum polarographic electrode.
Measurements were made with 5 10
exponentially
growing cells (in the presence of uridine), resuspended in 1.6 ml of
Dulbecco's modified Eagle's medium without glucose and 5%
dialyzed fetal bovine serum as previously described(18) . Analysis of Mitochondrial Translation
Products
Exponentially growing cybrids, 143B and 143B/206 cells
(1.2 10 on 60-mm Petri dishes), were labeled with
[S]methionine (1175 Ci/mmol, 250 mCi/ml) for 30
min at 37 °C in 2 ml of methionine-free Dulbecco's modified
Eagle's medium supplemented with 50 µg/ml uridine, 100
µg/ml emetine, and 5% dialyzed FBS as previously
described(12) . Equal amounts of total cellular protein
(determined by the Bio-Rad protein assay), were analyzed on
15-20% and 12.5% SDS-polyacrylamide gels, and subjected to
fluorography. The gels were dried on filter paper and exposed to X-Omat
autoradiography film overnight at -80 °C. Quantitation of
mtDNA-encoded polypeptides on fluorograms of appropriate densities was
performed by densitometry using the software IMAGE 1.57 (NIMH,
freeware).
Immunoblotting
Immunoblottings were performed
using cytochrome c oxidase (COX) II polyclonal and COX IV
monoclonal antibody (kind gifts of Drs. Anne Lombes and Armand Miranda,
respectively). Ten µg of mitochondrial proteins were separated onto
15% SDS-PAGE gels, and transferred to PVDP membrane (Immobilon,
Bio-Rad). Prestained protein standards were used as molecular weight
markers and to provide visual confirmation of transfer efficiency.
Poly(vinylidene fluoride) blot membranes were incubated for 1 h with
10% milk in phosphate-buffered saline with 0.05% of Tween 20 as
blocking agents. Membranes were then incubate with COX II polyclonal
antibody and COX IV monoclonal antibody (both individually and
together), for 14 h at 4 °C, and subsequently incubated with
anti-mouse IgG conjugated to alkaline phosphatase, anti-rabbit IgG
conjugated to alkaline phosphatase, or both (Sigma). Bands were
developed by incubation with 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt and nitro blue tetrazolium chloride (Life
Technologies, Inc.) for 10 s.Northern Blotting
Total RNA of each cybrid line
was isolated by acid guanidinium thiocyanate-phenol-chloroform
extraction (23) . Twenty µg of total RNA was
electrophoresed on 1.2% agarose, 5% formaldehyde gels and transferred
to nylon membrane. The filters were hybridized overnight at 42 °C
with specific mtDNA probes (see below). For detection of
tRNA we used a synthetic 48-nucleotide
oligonucleotide (between mtDNA positions 3304 and 3257) end-labeled
with [
-
P]ATP. The probe for the detection
of ND1 mRNA was obtained by gel purification of a 786-bp fragment
(corresponding to mtDNA positions 3337-4121) produced by
digestion of a 1426-bp PCR fragment (primers spanning nucleotide
positions 3116-3125 and 4542-4546) with RsaI and EcoRI. The 16 S rRNA probe was a 1555-bp PCR fragment
corresponding to mtDNA positions 1690-3246, obtained by PCR
amplification. The tRNA
, tRNA
, and
tRNA
probe was a 204-bp PCR fragment obtained by
digestion of a 382-bp PCR fragment (spanning mtDNA positions
4260-4542) with RsaI. The 204-bp fragment corresponding
to positions 4260-4464 was gel-purified and used as template. A
1.9-kb
-actin mRNA probe was obtained by EcoRI digestion
of a cloned insert(32) . All double-stranded DNA templates were
P-labeled by the random primer method (Boehringer
Mannheim). Northern hybridizations were performed in 50% formamide, 5
Denhardt's solution, 0.1% SDS, 100 µg/ml denatured
salmon sperm DNA, 25 mM sodium phosphate pH 6.8, 5
SSC, and washed twice in 1 SSC, 0.1% SDS at room temperature
for 1 h and subsequently in 0.25 SSC, 0.1% SDS at 55 °C for
1 h, and finally in 0.1 SSC at 55 °C for 1 h. The blot in Fig. 6was subsequently hybridized and stripped with different
probes in the following order: tRNA, ND1,
tRNA
, 16 S RNA, and finally
-actin.
; C, 16S
rRNA; D, tRNAs
; E,
nuclear-coded
-actin. The ethidium bromide-determined positions of
28 S and 18 S rRNAs are shown on the left of each panel. Band
assignments were based on molecular weight and probe specificity. RNA
19 corresponds to an intermediary transcript composed of 16 S rRNA
+ tRNA
+ ND1. Transfer RNA-specific probes
cannot detect the small molecular weight tRNAs because they run out of
the agarose gel.
we
use the same probe described above. The mitochondrial tRNA
was detected with a
P-labeled PCR fragment
corresponding to mtDNA positions 1460-1776. The same blot was
first hybridized with a tRNA
probe, stripped, and
hybridized with a tRNA
probe.
Statistical Analyses
Comparisons between different
cell line groups were done by Student's t test using Mac
Statworks software package (Cricket Software Inc.).
Isolation and Characterization of Transmitochondrial
Cybrids
Thirty-three individual cybrid clones were isolated from
two independent fusions of enucleated fibroblasts from the patient
(containing 48% mutated mtDNA) with mtDNA-less 143B/206 cells. The vast
majority of the transmitochondrial cybrids were essentially homoplasmic
at position 3256. Only four clones were heteroplasmic (ranging from 50
to 80% mutated mtDNA). Mitochondrial genotype analysis was performed
after no longer than 10 population doubling (<10
cells)
by collecting and analyzing mtDNA from approximately 100 cells during
the transfer of initial cell foci from cloning rings to larger wells
(see ``Materials and Methods''). Three homoplasmic mutant and
three homoplasmic wild-type lines were used in the subsequent studies.
The homoplasmic nature of the mtDNA 3256 nucleotide was confirmed in
expanded cultures (Fig. 1, A and B). Our RFLP
assay for the detection of wild-type sequences was sensitive to 1%
wild-type mtDNA (see ``Materials and Methods''), indicating
that all three mutant clones had >99% mutated mtDNA. Mitochondrial
DNA levels were also measured by Southern analysis using a multicopy
nuclear gene (18 S rRNA) as internal reference (Fig. 1C). Mutant clones had slightly elevated mtDNA
levels (mtDNA signal/nDNA signal = 12.8 ± 4.1 (mean
± S.D.) when compared to wild-type clones (8.1 ± 1.1) and
143B (6.9) levels. The patient-derived mtDNA identity of
transmitochondrial cybrids was confirmed by RFLP analysis of an
unrelated mtDNA polymorphism previously identified in the patient (3) (Fig. 1D).Functional Mitochondrial Assays
Functional
mitochondrial assays showed a clear deficiency in the respiratory chain
activity in mutant clones (Fig. 2). Oxygen consumption was
severely impaired in these clones (Fig. 2A; mean
reduction of 82%, p = 0.003). Accordingly, lactate
production was increased in mutant clones (Fig. 2B),
also suggesting a defective oxidative phosphorylation. The activity of
different respiratory complexes and citrate synthase showed a
significant deficiency of complexes I (mean 30% of wild-type values, p = 0.01) and IV (mean 22% of wild-type values, p = 0.03) in mutant clones relative to wild-type lines (Fig. 2C). We have not noted significant differences
between the growth characteristics of wild-type and mutant clones.
Synthesis and Steady-state Levels of Mitochondrial
Polypeptides
The effect of the mtDNA 3256 mutation in
mitochondrial protein synthesis was investigated by labeling cells with
[S]methionine in the presence of emetine, a
cytoplasmic ribosome inhibitor. Thirty-minute-labeled cells were
solubilized and analyzed by 15-20% SDS-PAGE (Fig. 3, left panel). Mutant clones showed a mild reduction in
mitochondrial protein synthesis. High molecular weight polypeptides
appeared to be particularly reduced in mutant clones, a trend that was
better observed in a 12.5% SDS-PAGE (Fig. 3, right
panel). We quantitated the intensity of the
S signal
for the individual polypeptides present in the 12.5% gel (i.e. only the higher molecular weight polypeptides). Mutant clones had
mean values that ranged from 20% (ND1) to 85% (ATP6) of the of
wild-type mean values (Fig. 4).
S]methionine
after electrophoresis through a 15-20% SDS-PAGE (left
panel) and a 12.5% SDS-PAGE (right panel) are shown for
the different wild-type and mutant cybrid lines. After 30-min labeling
in the presence of emetine, equal amounts of an SDS lysate of total
cellular protein (50 µg) were loaded in each lane. Bands were
assigned according to Attardi(31) .
S]methionine
after electrophoresis through a 12.5% SDS-PAGE were scanned and
quantitated by densitometry. The histogram represents the different
band intensities. Note the different level of impairment of specific
polypeptides in the mutant cell lines.
Mitochondrial RNAs Steady-state Levels and
Processing
Because of previously reported RNA processing
abnormalities associated with mitochondrial tRNA mutations, we analyzed RNA species surrounding the
tRNA
by Northern blot hybridization (Fig. 6).
ND1 levels were significantly reduced in mutant clones (mean value was
44% of values observed for wild-type clones; p = 0.005,
and 47% when normalized to
-actin; p = 0.003). An
intermediate unprocessed transcript encompassing the 16 S rRNA,
tRNA
, and ND1 (termed RNA 19) (12) was
slightly increased in mutant clones (mean value was 109% of wild-type
and 116% when normalized to
-actin), but these values were not
significantly different from the wild-type values (p =
0.68, and p = 0.24, respectively). RNA 19 could also be
detected with the tRNA
and 16 S rRNA (Fig. 6, B and C). A downstream probe, specific to the three
tRNAs cluster (Ile, Gln, and Met) also showed intermediate transcripts,
but no major differences between wild-type and mutant clones (Fig. 6D). The integrity and amount of RNA samples was
determined by hybridizing the membrane with a
-actin probe (Fig. 6E).
were also measured by high resolution Northern blots (Fig. 7). Mutant clones had a tRNA
:
tRNA
ratio that was approximately 30% lower than the
ratio observed for wild-type or 143B clones.
probe, while Panel B shows a hybridization to a tRNA
-specific
probe.
gene have been described, many of which may
share similar pathogenetic mechanisms. These include mutations at mtDNA
positions: 3243 (MELAS, ocular myopathy)(4) ; 3251
(myopathy)(8) ; 3252 (encephalopathy)(9) ; 3256
(myoclonus epilepsy with ragged-red fibers/ocular myopathy and
MELAS)(3, 17) ; 3260 (myopathy and
cardiomyopathy)(6) ; 3271 (MELAS) (5) ; 3291
(MELAS)(25) ; 3302 (myopathy)(7) ; 3303 (myopathy and
cardiomyopathy)(10) ; and a single base pair deletion between
positions 3271 and 3273 (encephalopathy)(24) .
T transition
at mtDNA position 3256 and a mitochondrial dysfunction, and to
correlate these findings with results obtained with other
tRNA
mutations. We used a transmitochondrial cybrid
system (12, 14, 26, 27) to segregate
wild-type mtDNAs from 3256 mutated mtDNAs in different cell lines. Most
transmitochondrial cybrid lines were homoplasmic soon after fusion
(after approximately 10 population doublings). Other investigators have
also observed this tendency to generate homoplasmic transmitochondrial
clones(28) . The fast segregation of these mtDNA molecules, and
the paucity of heteroplasmic clones obtained, suggest that either: 1)
The fibroblast culture was a mixture of essentially homoplasmic
wild-type or homoplasmic mutant cell lines, or 2) mtDNA heteroplasmy is
unstable in the 143B/206 transmitochondrial system.
T transition at mtDNA position 3256 is caused by an impairment in
mitochondrial protein synthesis and steady-state levels. Similar
observations have been made for other pathogenic mitochondrial tRNA
mutations(12, 14, 27) . Although different
mechanisms could account for the translation impairment, our results
suggest that, at least partially, it is caused by a reduction in the
steady-state levels of tRNA
and possibly of other
transcripts, such as ND1. It is not clear why ND1 levels were reduced
beyond what could be accounted by the increase in RNA 19 levels, but it
may be related to the location of the mutation within a transcriptional
regulatory site (see below). Bindoff et al.(7) described a different pathogenic mutation in the
mitochondrial tRNA
gene (an A
G transition at
position 3302) associated with reduced steady-state levels of
tRNA
. In their patient, however, the decrease in
free tRNA
was accompanied by a marked accumulation
of RNA 19. Although we found only a mild increase in RNA 19 levels, it
is possible (as in the case of Bindoff et al.(7) )
that patient tissues such as muscle or CNS would accumulate higher
levels of RNA 19. RNA 19 was also increased in transmitochondrial
cybrids harboring an A
G transition at mtDNA position 3243 and a
T
C transition at position 3271, both within the same tRNA
gene(12, 30) .
transcripts. However, these two transcripts seem to be
preferentially decreased in comparison to other transcripts downstream
of ND1 (see Fig. 6D).
mutations affect cellular respiration by an yet
unidentified mechanism.
)
We thank Dr. Michael King (Columbia University) for
instructions on transmitochondrial cybrid production and Dr. Salvatore
DiMauro (Columbia University) for providing us with the patient's
fibroblast line. We are also in debt with Drs. Zi-Cai Feng, Myron
Rosenthal, Thomas Sick, and Eugene Roberts (University of Miami) for
the use of their oxygen electrode.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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