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1 Kinesiology and Health Science, Faculty of Pure and Applied Science, York University, Toronto, Ontario, Canada M3J 1P3; and 2 Department of Chemistry, National Industrial Research Institute of Nagoya, Nagoya 462, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Mitochondrial transcription factor A (Tfam) is a nuclear-encoded gene product that is imported into mitochondria and is required for the transcription of mitochondrial DNA (mtDNA). We hypothesized that conditions known to produce mitochondrial biogenesis in skeletal muscle would be preceded by an increase in Tfam expression. Therefore, rat muscle was stimulated (10 Hz, 3 h/day). Tfam mRNA levels were significantly elevated (by 55%) at 4 days and returned to control levels at 14 days. Tfam import into intermyofibrillar (IMF) mitochondria was increased by 52 and 61% (P < 0.05) at 5 and 7 days, respectively. This corresponded to an increase in the level of import machinery components. Immunoblotting data indicated that IMF Tfam protein content was increased by 63% (P < 0.05) at 7 days of stimulation. This was associated with a 49% (P < 0.05) increase in complex formation at the mtDNA promoter and a 65% (P < 0.05) increase in the levels of a mitochondrial transcript, cytochrome-c oxidase (COX) subunit III. Similarly, COX enzyme activity was elevated by 71% (P < 0.05) after 7 days of contractile activity. These results indicate that early events in mitochondrial biogenesis include increases in Tfam mRNA, followed by accelerations in mitochondrial import and increased Tfam content, which correspond with increased binding to the mtDNA promoter region. This was accompanied by increased mitochondrial transcript levels and elevated COX activity. These data support the role of Tfam as a regulatory protein involved in contractile activity-induced mitochondrial biogenesis.
mitochondrial biogenesis; mitochondrial protein import
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE PROCESS OF MITOCHONDRIAL biogenesis in response to contractile activity in skeletal muscle requires the coordination of both the nuclear and mitochondrial genomes (11). Although the mitochondrion contains its own genetic material, the majority of its protein is nuclear encoded, including the proteins involved in mitochondrial DNA (mtDNA) transcription and replication. A vital component of the mitochondrial transcription machinery is the protein mitochondrial transcription factor A (Tfam; for review, see Ref. 25), which binds upstream of both heavy-strand promoter and light-strand promoter (LSP) and activates mtDNA transcription (8, 20). Tfam is also involved in mtDNA replication. During this process, transcription acts to prime mtDNA heavy-strand replication at the LSP (3), resulting in the formation of a RNA-DNA hybrid, known as the R-loop. This stable formation is processed to form a primer for the subsequent leading strand replication (34).
A recent study found that a homozygous knockout of Tfam was lethal during embryonic development in mice, whereas heterozygous knockout mice exhibited a reduced amount of mtDNA, mitochondrial transcripts, and oxidative enzyme activities in heart tissue (15). Muscle-specific disruption in the Tfam gene confirmed its importance in regulating mitochondrial mRNA and mtDNA content in striated muscle tissue and indicated that proper mitochondrial gene expression is necessary during myocardial development (30). In addition, the transfection of antisense plasmids in culture, designed to reduce the expression of Tfam, effectively decreased the levels of mitochondrially encoded transcripts (13). In contrast, the forced overexpression of Tfam produced the opposite effect (18).
These studies indicate a functional role for Tfam in regulating mtDNA and RNA content in striated muscle. However, the importance of Tfam in regulating mitochondrial gene expression in skeletal muscle undergoing mitochondrial biogenesis requires further investigation. A rapid increase in mitochondrial biogenesis can be induced in skeletal muscle by a period of chronic contractile activity. Results compiled from several studies indicate divergent responses between Tfam mRNA and protein levels in mtDNA-depleted muscle cells (1, 14, 22), and it has been suggested that a posttranscriptional mechanism may be important in regulating Tfam abundance (23, 25). We have recently shown that posttranslational import into mitochondria is an inducible process in striated muscle undergoing rapid mitochondrial biogenesis (26), and we wished to determine whether Tfam expression could be regulated in this manner.
Therefore, the purpose of this work was to identify potential mechanisms involved in regulating Tfam expression in skeletal muscle undergoing mitochondrial biogenesis. Here, we report a sequential increase in Tfam mRNA expression that precedes the elevation of its in vitro import into mitochondria, as well as the subsequent increases in Tfam protein levels, mtDNA binding, mitochondrial transcripts, and oxidative enzyme activity.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In vivo stimulation protocol. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium (60 mg/kg body wt), and two electrodes were sutured to either side of the common peroneal nerve. Electrode wires were passed subcutaneously from the thigh to the base of the neck, where they were attached to an external stimulator unit secured to the back of the animal (26). After a 1-wk recovery period, the tibialis anterior (TA) and the extensor digitorum longus (EDL) muscles were stimulated (10 Hz, 0.1-ms duration) for 3 h/day for 1, 2, 3, 4, 5, 7, and 14 days. The contralateral limb was used as a control for all animals (n = 3-6 experiments).
Tissue extraction and mitochondrial isolation. The TA and EDL muscles were excised from anesthetized animals (60 mg/kg pentobarbital sodium) and either clamp frozen in liquid nitrogen or minced and briefly homogenized. The intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial subfractions were isolated by differential centrifugation as described previously (5, 27). Freshly isolated mitochondria were used for protein import assays. Aliquots of each mitochondrial sample were also frozen for subsequent use in immunoblotting, electromobility shift assays, and cytochrome-c oxidase (COX) activity measurements.
Mitochondrial protein import. The plasmid containing the full-length cDNA encoding Tfam was cut with Xho I and transcribed with T7 RNA polymerase, whereas the plasmid containing the full-length cDNA encoding malate dehydrogenase (MDH) was cut with BamH I and transcribed with Sp6 RNA polymerase, as described previously (27). DNA and RNA were isolated by phenol extraction and ethanol precipitation. Labeled precursor proteins were translated with the use of a rabbit reticulocyte lysate system in the presence of [35S]methionine. Import reactions were initiated by mixing 20 µl (Tfam) or 12 µl (MDH) of programmed lysate with 50 µg of freshly isolated mitochondria prewarmed to 30°C. Incubation times were 5, 10, and 15 min when Tfam and MDH import rates were compared or 15 min when only mitochondria from control and stimulated muscles were compared. Mitochondria were then recovered by centrifugation through a 20% sucrose cushion at 4°C. Samples were then lysed and separated by SDS-PAGE. Gels were fixed in boiling 5% trichloroacetic acid and dried. Electronic autoradiography (Instant Imager, Packard) or film was used to quantify the radioactive signals.
Antibody production.
Recombinant Tfam protein was overexpressed in Escherichia
coli by cloning the rat Tfam cDNA (corresponding to amino acids 35-201) into the bacterial expression vector pQE30 (Qiagen,
Chatsworth, CA), which contains six additional histidine residues at
the NH2 terminus, as described previously
(12). Bacterial culture was grown in LB medium containing
ampicillin (100 µg/ml) and induced with 0.1 mM
isopropylthio-
-D-galactoside for 2 h. The
Tfam fusion protein was purified with a Ni-nitrilotriacetic
acid column as described by the manufacturer (Qiagen), and
immunizations were performed by Medical and Biological Labs (Nagoya, Japan).
Immunoblotting. Isolated IMF and SS mitochondria samples (25-35 µg) were separated by an 18% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Hybond C, Amersham-Pharmacia Biotech, Mississauga, ON). After transfer, membranes were blocked for 1 h in a 5% skim milk (wt/vol) and 1.25% (vol/vol) horse serum PBS solution and washed for 10 min in PBS. Membranes were then probed with antibodies raised against Tfam (1:2,000), the 20-kDa translocase of the outer membrane (Tom20; 1:1,000), the 60-kDa heat-shock protein (HSP60; 1:1,000; StressGen Biotechnologies, Victoria), the mitochondrial 70-kDa HSP (mtHSP70, 1:1,000; StressGen Biotechnologies), or adenine nucleotide translocase (1:1,000). Membranes were washed in a 0.05% Tween-PBS buffer and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:2,000 or 1:1,000). Signals were detected using the enhanced chemiluminescence detection system (Amersham-Pharmacia Biotech), and films were quantified using Sigma Gel, version 1.0 (Jandel Scientific, San Rafael, CA).
Electrophoretic mobility shift assay. Isolated, frozen mitochondria (25 µg) were thawed, and 20 µg/ml poly(dI-dC), 50 µM pyrophosphate, and 0.5% Triton X-100 in a buffer consisting of 0.1 M MgCl2, 50 mM dithiothreitol, 1 mM spermidine, 1 mM EDTA, and 20 mM Tris (pH 7.6) were added. Mitochondria were sonicated for 10 s at 4°C and incubated for 15 min at room temperature. Extracts were then similarly incubated with 40,000 counts/min of a 32P-labeled oliogonucleotide (5'-TTTTAACTTAAATCTTAGCATTGG-3') representing the Tfam binding site (underlined) on the rat LSP (2). Specific and nonspecific competition assays were performed by preincubating the mitochondrial extracts with a 200 M excess of cold oligonucleotides representing the Tfam light-strand binding site or the nuclear heat-shock element, respectively. After the binding reaction, the mixture was separated on a nondenaturing 4% polyacrylamide gel, fixed with a acetic acid-methanol-water (10:30:60) solution, dried, and quantified using electronic autoradiography (Instant Imager, Packard).
Total RNA isolation and hybridization. Frozen TA and EDL muscle tissues were pulverized, and 200 mg of frozen powder were homogenized in 2 ml of TRIzol (Life Technologies, Grand Island, NY). Total RNA was extracted in chloroform and precipitated with isopropanol. After centrifugation at 12,000 g, RNA pellets were washed in 70% ethanol and resuspended in diethyl pyrocarbonate-treated water. RNA samples (30-50 µg) were loaded into a slot-blot apparatus or were run on a 1% agarose gel containing 0.02% formaldehyde, transferred, and fixed to a nylon membrane (Hybond-N; Amersham-Pharmacia Biotech). Membranes were hybridized overnight at 42°C with a 32P-labeled Tfam or COX III cDNA probe, as described previously (26). Stringent washes were performed with a solution containing 0.1× sodium chloride-sodium citrate and 0.1% SDS at 55 and 60°C for 15 min each. Radioactive signals were quantified as described above.
COX enzyme activity. COX activity was evaluated as described previously (5). Briefly, mitochondrial samples were diluted in a buffer containing 0.1 M KH2PO4 and 2 mM EDTA (pH 7.2) and sonicated for 10 s on ice. The enzyme activity was determined by the maximal rate of oxidation of fully reduced cytochrome c, measured by the change in absorbance at 550 nm (Beckman DU-64 spectrophotometer).
Statistics. A one-way or two-way analysis of variance and Tukey's post hoc test were used for all time-course data. Student's paired t-test was used in the analysis of all other data. All data are expressed as means ± SE, and significance was set at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Tfam mRNA.
Slot-blot analysis revealed that Tfam transcripts were significantly
elevated by 55% after 4 days of contractile activity but not before
this time point (Fig. 1A). By
14 days of stimulation, Tfam mRNA levels had returned to those found in
control tissue (Fig. 1B).
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Tfam import.
The import of Tfam was evaluated in isolated IMF mitochondria. Import
progressively increased throughout 15 min of incubation (Fig.
2A). However, compared with
the import of the matrix enzyme MDH (Fig. 2B), Tfam import
was approximately fourfold slower when expressed as a percentage of the
total available precursor protein (Fig. 2C). Analysis of
Tfam import into IMF mitochondria isolated from chronically stimulated
muscle revealed 52 and 61% increases at 5 and 7 days, respectively
(P < 0.05; Fig. 3,
A and B). No effect of stimulation on Tfam import
was observed before 5 days. Corresponding to this acceleration in Tfam
import was an increase in the proteins mtHSP70 and Tom20, important
components of the mitochondrial protein import machinery (Fig.
3C). mtHSP70 was elevated by 32 and 58%, and Tom20 was
elevated by 58 and 112% after 5 and 7 days of stimulation,
respectively (P < 0.05, n = 3-4).
In contrast, HSP60 protein levels were not significantly altered by
this protocol of contractile activity at any time point.
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Tfam protein.
Immunoblot analyses of IMF and SS mitochondria revealed the presence of
the 25-kDa Tfam protein, which was 170% greater in the IMF compared
with the SS mitochondrial subfraction (Fig.
4A). Chronic stimulation did
not alter the level of Tfam in IMF mitochondria until 7 days, when a
63% increase (P < 0.05) was evident (Fig. 4,
B and C). In addition, Tfam protein was also
elevated after 7 days of contractile activity in SS mitochondria by
40% (not shown). This increase in Tfam was not accompanied by a
uniform increase of all mitochondrial proteins because no change in the level of the adenine nucleotide translocase was evident in IMF (Fig.
4B) or SS mitochondria (not shown).
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Protein-DNA interactions at a putative
Tfam binding site.
To evaluate the effect of contractile activity on protein-DNA
interactions at the putative Tfam binding site of the LSP,
electrophoretic mobility shift assays were performed using isolated IMF
mitochondrial extracts containing endogenous Tfam, incubated with a
synthetic oligonucleotide based on the rat mtDNA LSP. Labeled probe
incubated in the absence of mitochondrial protein migrated to the
bottom of the gel (not shown in Fig.
5A). In addition, excess
unlabeled probe was always found at the bottom of the gel in each
reaction lane (not shown). A single binding complex that was shifted to the middle of the gel was observed in the presence of the mitochondrial extract (Fig. 5A). It was selectively competed away with the
addition of a 200 M excess of unlabeled LSP oligonucleotide and was
unaffected by the addition of a nonspecific oligonucleotide
representing the nuclear heat-shock element. Assays performed with IMF
mitochondria isolated from stimulated muscle showed that Tfam binding
increased by ~50% (P < 0.05) at 7 days of
stimulation but not before this time (Fig. 5, B and
C).
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COX III mRNA and
COX activity.
Northern blot analyses of the mitochondrially encoded transcript COX
III revealed that the cellular levels of this mRNA were not elevated
until 7 days of contractile activity (Fig.
6, A and B), when a
65% (P < 0.05) increase was evident. A similar
increase was also observed with the mitochondrially encoded transcript COX II (not shown). These results corresponded closely with the 71 and
76% (P < 0.05) increases in COX holoenzyme activity
in IMF (Fig. 6C) and SS mitochondria (not shown),
respectively, observed after 7 days of stimulation.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was conducted to identify potential regulatory processes in the expression of Tfam and the relationship of Tfam to mtDNA expression during contractile activity-induced skeletal muscle mitochondrial biogenesis. Tfam was chosen because it is the only mammalian mitochondrial transcription factor known to date (25). It is a 25-kDa nuclear gene product that binds upstream of both mtDNA promoters (8), is required for mitochondrial transcription initiation (7), and is necessary for R-loop formation during mtDNA replication (34). Thus it is important for mtDNA transcription and replication.
Consistent with the rapid turnover of transcription factor mRNAs (19, 33), elevations in Tfam mRNA were the earliest detectable changes noted in the pathway leading to the increased expression of this protein (Fig. 1). Inspection of the human Tfam promoter (28) indicates that it contains putative binding sites for Sp1 and nuclear respiratory factors-1 and -2. These have been shown to be important in Tfam transcriptional activation (29, 35), and it is likely that the increase in nuclear respiratory factor-1 mRNA noted during contractile activity (33) has a role in the increase in Tfam mRNA observed in this study.
For the protein to be effective physiologically, Tfam must first be imported from the cytosol into the mitochondria. An examination of Tfam import into IMF mitochondria revealed that the uptake of Tfam was approximately fourfold slower than that of the Krebs cycle enzyme MDH (Fig. 1). MDH appears to be imported at a rate comparable to other typical matrix enzymes (27). We speculated that this dramatically slower import could be due to differences in the amino acid composition of their presequences. However, both MDH and Tfam contain ~12% positively charged residues within their cleavable NH2-terminal domain (4, 20). This suggests that both precursors are acted on by the same electrophoretic force during the import process (16). Interestingly, Matouschek et al. (17) found that precursors with longer presequences could have higher import rates because of better interactions with the mtHSP70 import motor. This does not appear to be the case here because Tfam contains a longer presequence (42 amino acids) compared with MDH (24 amino acids). Alternatively, these preproteins could differ in their affinity for the import receptor complexes. However, we have previously shown that Tfam import and MDH import are equally sensitive to inhibition of the import receptor Tom20, indicating that their binding affinity cannot account for the dramatically slower import of Tfam (10). Because precursor proteins must first be unfolded to become import competent, it is possible that the primary structure of Tfam is more resistant to chaperone-mediated unfolding and therefore possesses a slower import rate than other matrix proteins like MDH. This will require further investigation. However, the data do suggest that the slow import rate of Tfam may represent a potential rate-limiting step in the posttranscriptional regulation of the protein.
Chronic contractile activity has been shown to be highly effective at inducing mitochondrial proliferation (for review, see Refs. 11 and 21). Previous work from our laboratory has demonstrated that this protocol is capable of increasing tissue COX activity by 120% after 14 days of contractile activity (6). This is consistent with the present study, in which 7 days of contractile activity produced a 71 and 76% increase in COX activity in IMF and SS mitochondria, respectively. We have also previously shown that chronic contractile activity (7 and 14 days) accelerates the import of matrix enzymes into mitochondria, concomitant with increases in the expression of proteins of the import machinery (26). In the present study, we evaluated the earliest adaptation of protein import in response to contractile activity, and we found that Tfam import was accelerated as early as by 5 days of treatment. This result correlated with phenotypic alterations in the import machinery, as demonstrated by the elevated mtHSP70 and Tom20, but not HSP60, protein content in IMF mitochondria (Fig. 3C). These data suggest that increases in mtHSP70 and Tom20 are important for the increase in import rate to occur. This notion is supported by previous work, in which the mitochondrial import rate of the matrix protein MDH was altered by forced overexpression or underexpression of Tom20 in cultured muscle cells (10). This adaptation of the import process appears to take place before detectable changes in IMF Tfam protein levels (Fig. 4, B and C). The data lend further support to the idea that protein import is an important posttranscriptional mechanism in regulating the mitochondrial phenotype.
Consistent with the elevation in Tfam protein within IMF mitochondria by the seventh day of contractile activity is the increased complex formation at the Tfam binding site on the LSP at this time (Fig. 5). Previous studies using in vitro assays showed that initiation of mtDNA transcription is highly dependent on Tfam binding to this site (7). Our results suggest that mtDNA transcription is likely increased by contractile activity because increased Tfam binding corresponds well with the elevation in the mitochondrially encoded transcript COX III after 7 days of stimulation (Fig. 6). In addition, the activity of the oxidative holoenzyme COX is not significantly elevated until this point. These data support the idea that Tfam expression is important in regulating the mitochondrial oxidative phosphorylation system, since mtDNA encodes the catalytic subunits (I, II, and III) of the COX complex. Consistent with this is the fact that Tfam protein levels are higher in IMF, compared with SS mitochondria (Fig. 3A), as is COX activity (5).
It is likely that other conditions that produce mitochondrial biogenesis do so by the sequential pattern of Tfam expression described here. For example, it has been shown that, after 5 days of thyroid hormone treatment in rats, liver Tfam mRNA levels were elevated before an increase in mitochondrial transcripts (9). This suggests that Tfam expression is well correlated with alterations in mtDNA-encoded transcripts. Furthermore, artificial elevation of Tfam in liver mitochondria led to an increased mitochondrial transcription rate, as determined by an in organello run-on assay (18). These data are consistent with the results of the present study in skeletal muscle.
It is likely that the effect of elevated Tfam within the mitochondria
is exerted at both the level of transcriptional activation as well as
mtDNA replication. Previous studies using rabbit muscle undergoing
mitochondrial biogenesis showed that the mtDNA copy number increases
following chronic contractile activity, that mtDNA content is directly
proportional to the oxidative capacity of the tissue, and that it is
well correlated with the expression of mitochondrially encoded
transcripts (31, 32). Tfam exerts its effects on mtDNA
replication by binding to the LSP (34), for which it has a
greater affinity than for the heavy-strand promoter (8).
Although the catalytic subunit of mtDNA polymerase-
appears to be
unresponsive to contractile activity, the replication factor,
mitochondrial single-stranded DNA-binding protein, is inducible during
muscle mitochondrial biogenesis (24). Additional work will
be required to determine whether the most important consequence of the
contractile activity-induced increase in Tfam expression is exerted at
the level of mtDNA replication or transcription.
In summary, we have shown that a sequential relationship exists between Tfam mRNA expression, mitochondrial import, protein content, mtDNA binding, and the expression of mitochondrial transcripts. In addition, the protein import step may be an important posttranscriptional event regulating Tfam content. Tfam appears to be a key factor responsible for the increased mitochondrial gene expression and oxidative capacity observed in contractile activity-induced mitochondrial biogenesis.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. R. Wiesner (University of Cologne, Germany) for providing the human Tfam cDNA, to Dr. A. Strauss (Washington University, St. Louis, MO) for providing the MDH cDNA, to Dr. M. Mori (Kumamoto University School of Medicine) for providing the Tom20 antibody, and to Dr. K. B. Freeman (McMaster University, Hamilton, ON, Canada) for providing the ANT antibody. We also thank P. Peticca for technical assistance with the COX activity.
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FOOTNOTES |
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This work was supported by the Natural Science and Engineering Research Council of Canada and by the Heart and Stroke Foundation of Canada.
Address for reprint requests and other correspondence: D. A. Hood, Kinesiology and Health Science, Farquharson Health Sciences Bldg., York Univ., 4700 Keele St. Toronto, Ontario, Canada M3J 1P3 (E-mail: dhood{at}yorku.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 August 2000; accepted in final form 13 October 2000.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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) represents the binding between endogenous
IMF Tfam and the oligonucleotide. Lane 2 (C) is a
competition reaction, in which a 200-fold molar excess of unlabeled
oligonucleotide representing the Tfam binding site is preincubated with
the isolated mitochondria. Lane 3 (NS) is a nonspecific
competition, in which a 200-fold molar excess of unlabeled
oligonucleotide representing the nuclear heat-shock element is
preincubated with the isolated mitochondrial sample. B: EMSA
from IMF mitochondria isolated from 5- and 7-day control and stimulated
muscle. C: graphic representation of binding complex
formation at the Tfam binding site on the light-strand promoter (fold
above control) throughout 7 days of stimulation. *P < 0.05 compared with 1 day.
