Botanical Studies (2011) 52: 137-144.
MOLECULAR 
 BIOLOGY
In vitro mitochondrial nucleoid DNA replication and transcription are affected by the associated mitochon-drial membrane
Bao-Lin LI1,2, Yih-Shan LO1, and Hwa DAI1,*
(Received June 29, 2010; Accepted July 19, 2010)
ABSTRACT. Fractionation of lysed mitochondria of mung bean seedlings by discontinuous sucrose gradient centrifugation resulted in three major and two minor fractions of mitochondrial nucleoid-membrane complexes based on their sedimentation density. Noticeably different quantities of mitochondrial membrane were found to associate with the different mitochondrial nucleoid fractions, suggesting that mitochondrial DNA, mtDNA binding proteins and membrane may be constituted in a heterogeneous organization among those fractions and result in different sizes, shapes and densities. Moreover, mitochondrial nucleoid-membrane complexes from these five fractions displayed differential abilities of in vitro DNA replication/synthesis and transcription. This implies, for the first time, that some biological activities such as DNA replication/ synthesis or gene transcrip­tion of the mitochondrial nucleoids can be affected by the specific membranes associated with them.
Keywords: Mitochondrial membrane; Mitochondrial nucleoid-membrane complex; Mitochondrial nucleoids; mtDNA replication; mtDNA transcription; Mung bean.
INTRODUCTION
Mitochondrial nucleoids are relatively stable assemblies of mitochondrial nucleoproteins with mtDNA in situ (Nass
et al., 1965, Kuroiwa, 1982, Stevens, 1981, Spelbrink et
al, 2001). Purification of mitochondrial nucleoids has been accomplished by applying a moderate detergent treatment to purified mitochondria followed by gradient centrifuga-tion (Suzuki et al., 1982; Miyakawa et al., 1987; Newman et al., 1996; Dai et al., 2005). Mitochondrial nucleoids isolated from a defined density in a 15% to 30% sucrose gradient following routine procedures (Miyakawa et al., 1987) exhibit a relatively homogeneously-sized popula­tion in higher plants (Dai et al., 2005). These mitochon-drial nucleoids contain mitochondrial nucleoproteins and membrane components. Isolated mitochondrial nucleoids are self-sufficient in the processes of mtDNA replication and transcription (Kuroiwa, 1982; Miyakawa et al., 1996; Sakai et al., 2004; Dai et al., 2005). Recently, most stud­ies on mitochondrial nucleoids have focused on the func­tion of their binding proteins. The molecular functions of nucleoid proteins in humans and in yeast have been particularly well-studied (Newman et al., 1996; Wang and
Bogenhagen, 2006; Bogenhagen et al., 2008; Spelbrink, 2010). However, the functional role played by the mem-brane component associated with mitochondrial nucleoids or mtDNA have been largely ignored. It was previously reported that the mitochondrial membrane plays an im-portant role in mitochondrial DNA synthesis and mtDNA segregation (Echeverria et al., 1991; Boldogh et al., 2003; Meeusen and Nunnari, 2003). In bacteria, the ancestor of mitochondria, the initiation of chromosomal DNA replica-tion depends on the specific association of chromosomal DNA with the membrane at a region close to the replica-tion origin that is recognized by specific membrane pro-teins (Hoshino et al., 1987; Yung and Kornberg, 1988).
We have previously observed the membranes associ-ated with mitochondrial nucleoids of mung bean seedlings under electron microscope. Moreover, the phospholipids compositions: cardiolipin (CL), phosphoglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC) of the membranes of these mitochondrial nucleoids are similar to those of the whole mitochondria (Dai et al., 2005). Here we showed that the majority of the nucleoids derived from the detergent-treated mitochondria by the conventional processes were fractionated into five mitochondrial nucleoid-membrane complexes according to their sedimentation density. The amounts of mtDNA and membrane components of these five mitochondrial nucleoids-membrane complexes varied from one another. Moreover, the mitochondrial nucleoid-membrane complexes from each one of these five fractions displayed differential abilities of in vitro DNA replication/
2Current address: School of Chemistry and Material Science, Shaanxi Normal University, Xi'an, Shaanxi 710062, P.R. China.
*Corresponding author: E-mail: bodaihwa@gate.sinica.edu.
tw; Tel: 886-2-27871176; Fax: 886-2-27838609.
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synthesis and transcription. Taken together, we conclude that different organizations among mtDNA, mtDNA bind­ing proteins and membrane components not only change the size, shapes or densities of mitochondrial nucleoid-membrane complexes, but also affect biological activities, such as DNA replication/synthesis and gene expression patterns, of these macromolecules. This is the first report demonstrating that the specific membrane components as­sociated with mitochondrial nucleoids play an important role in mtDNA gene expression.
MATERIALS AND METHODS
Isolation of mitochondrial nucleoid-membrane complexes from mitochondria
Mitochondria were isolated from 3-day old etiolated mung bean seedlings (Vigna radiate, Tainan No. 5) as pre­viously described (Dai et al., 1991).
Mitochondrial nucleoid-membrane complex fractions were then purified from mitochondria as described previ­ously with some modifications (Dai et al., 2005). In brief, the purified mitochondrial pellet was suspended in NE2 buffer (7.5% sucrose, 20 mM Tris-HCl, pH 7.6, 2 mM EDTA, 0.8 mM spermidine, 7 mM 2-mercaptoethanol, 0.4 mM PMSF) to a final concentration of 5 mg protein/ml. Mitochondria were lysed by slowly adding 20% (w/v) NP-40 to a final concentration 0.5%. After remaining on ice for 5 min with gentle mixing, the lysate was centrifuged at 14,000 x g for 20 min to remove insoluble materials.
The clear supernatant (5 ml) was layered over five dis­continuous sucrose gradients and prepared as follows: 5 ml steps containing 15% (w/v), 20% (w/v), 30% (w/v), 40% (w/v) and 50% (w/v) sucrose in buffer (20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM spermidine, 7 mM 2-mercap-toethanol, 0.4 mM PMSF) and centrifuged at 46,000 x g for 1 h.
Mitochondrial nucleoid-membrane complexes were re­covered from the supernatant lysate, bands at the 10-15%, 15-20%, 20-30%, 30-40%, 40-50% boundaries and pellet,followed by adding 2X volume of ice-cold gradient buffer and pelleting by 46,000 x g centrifugation for 1 h.
Thin-layer chromatographic phospholipid anal­ysis of mitochondrial nucleoid-membrane com­plexes
Equivalent amounts of seven mitochondrial nucleoid-membrane fractions derived from 8 mg mitochondria were extracted with chloroform/methanol. Thin layer chroma-tography (Kieselgel 60 F254, Merck) with the use of mo­lybdenum blue (1.3% molybdenum oxide in 4.2 M sulfuric acid) spray reagent was performed exactly as described above (Dai et al., 2005).
Pulsed-field gel electrophoresis
Different mitochondrial nucleoid-membrane complex fractions were resuspended in equal volumes of the gradi-
ent buffer as described. An equal volume of 1% low-melt­ing-point agarose (LGT) was added and the samples were kept at 42°C. The samples were loaded into molds and al­lowed to set on ice for 10 min. The plugs were treated with ESP (1 mg/ml proteinase K, 1% Sarkosyl, 0.5 M EDTA, pH 9) at 50°C overnight. Fresh ESP was replaced twice during incubation. The plugs were washed with ES buffer (1% Sarkosyl, 0. 5 M EDTA, pH 9) at 50°C for 2 h. After replacement with fresh ES buffer, pulse-field gel electro­phoresis (PFGE) was performed at a 30-60 s pulse time (at the ratio A:B=1), 150 V (11.8 V/cm) on a 1.2% agarose gel for 24 h in 0.5X TBE buffer (45 mM Tris-borate, 1 mM EDTA). Gels were then stained with ethidium bromide and de-stained by washing, followed by conventional Southern blotting analysis. In mitochondrial nucleoid-membrane complex in vitro DNA synthesis analysis, an autoradio-gram exposure was performed after the gels were dry. To count a-[32p]-dCTP incorporation into newly synthesized wb- and fm-DNA, after PFGE the dried gel was sliced into well-bound and fast-moving fractions according to the autoradiography and then counted in a scintillation counter (Hewlett Packard). The amount of mtDNA in each mitochondrial nucleoid-membrane complex was measured by OD after purification of the DNA in each fraction. The ratio between wb- and fm-DNA was determined by count-ing the wb-DNA and fm-DNA fractions cut from the mem-brane after the Southern blot analysis.
Southern blot analysis
Southern blot analysis followed the conventional method (Sambrook et al., 1989). The probe used was pure mtDNA. The purity of the mtDNA was verified by PCR using primers that generate chloroplast or nuclear gene products. Neither chloroplast nor nuclear DNA was present in the probe mtDNA. A Cox3 gene fragment was generated from mitochondrial DNA by PCR using the primers: cx3-34U: gtagatccaagtccatggcct; cx3-458L: gcat-gatgggcccaagttacggc; designed to generate a mitochon­dria-encoded cox3 DNA product that was also used as a probe. The same results were obtained when total mtDNA was used as a probe.
In vitro DNA synthesis by mitochondrial nucle-oid-membrane complexes
A method used for DNA synthesis by the membrane-associated high molecular weight complex of wheat mito­chondria was used with modifications (Echeverria et al., 1991). Briefly, mitochondrial nucleoids (isolated from 2 mg of mitochondrial protein) were suspended in 800 fil of reaction buffer containing 20 mM MgCl2, 25 mM KCl, 2 mM DTT, 0.1 mg/ml BSA, 50 mM Tris-HCl, pH 7.5, 2 mM ATP, 0.2 mM each of CTP, GTP and UTP, 50 fiM each of dATP, dGTP and dTTP and 100 of α-[32p]-dCTP (3000 Ci/mmol) in Eppendorf tubes and incubated at 30°C for 30 min. The reaction was stopped by adding 100 fM of cold dCTP and 25 mM of EDTA. A 2X vol-ume of cold gradient buffer was then added followed by
LI et al. — mtDNA replication and transcription are affected by associated mitochondrial membrane
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centrifugation at 46,000 x g for 1-2 h to pellet the mito-chondrial nucleoid-membrane complexes. PFGE analysis was carried out as described above. The X-ray film was developed after drying the gel.
Mitochondrial nucleoid-membrane complex transcription in vitro
In vitro mitochondrial nucleoid-membrane complex transcription was adapted with some modifications from a mitochondrial RNA synthesis method (Martin et al., 1987; Dai et al., 2005). Seven mitochondrial nucleoid-membrane complex fractions prepared from 4 mg protein equivalent mitochondria as described in the above section were sus­pended in 300 μl reaction buffer including 10 mM Tris-base (pH 8.5), 5 mM MgCl2, sucrose 0.25 M, 1 mM DTT, base (pH 8.5), 5 mM MgCl2, sucrose 0.25 M, 1 mM DTT,0.1% BSA, 120 mM each of CTP, GTP and ATP, 90 μCi α-32P-UTP and 100 units RNAsin, and then incubated at 25°C for 30 min before adding unlabeled UTP (final con-centration: 20 μM) continuing to incubate at 25°C for 5 min. After stopping the reaction by adding SDS and CDTA to final concentrations of 1% and 30 mM, respectively, newly synthesized RNA was isolated and purified using phenol/chloroform/isoamyl alcohol. The RNA was ana-lyzed with a standard MOPS gel containing formaldehyde (Dai et al., 2005). The newly-synthesized mitochondrial RNA was counted as described above.
RESULTS
The composition and amounts of phospholip-ids of mitochondrial nucleoid-membrane com­plexes derived from different sucrose gradient fractions
The phospholipid composition of mitochondrial nucle-oid-membrane complexes derived from each one of the sucrose gradient fractions remained similar to that of the whole mitochondria (Figure 1). However, the amounts of phospholipids of mitochondrial nucleoid-membrane com­plexes varied from one sucrose gradient fraction to another (Figure 1). In general, the amounts of phospholipids of mi-tochondrial nucleoid-membrane complexes decreased with the increase of banding density of mitochondrial nucleoid-membrane complexes (Figure 1), suggesting that the diver­sity of sizes, shapes or densities among different fractions of mitochondrial nucleoid-membrane complexes may be caused by the different amounts of membrane components association with each fraction, respectively.
The mtDNA contents varied among mitochon-drial nucleoid-membrane complexes with different densities from a sucrose gradient frac-tionation
Southern blot analysis of mitochondrial nucleoid-mem-brane complexes revealed that 36.6%, 25%, 17%, 8.9% and 4.55% of the original mtDNA was present, respec­tively, in the 15/20% sucrose boundary, lysate (7.5%)/15%
Figure 1. Phospholipid compositions of mitochondrial nucle­oid-membrane complexes in different fractions after sucrose gradient centrifugation. Equivalent portions of each of the seven mitochondrial nucleoid-membrane complex fractions indicated on the right of the figure were analyzed by thin layer chroma-tography. A phospholipid sample extracted from the purified mitochondria containing 0.5 mg proteins was loaded in line 9 as the control. The phospholipid markers in lanes 1 and 10 are car-diolipin (CL), phosphoglycerol (PG), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylcholine (PC).
sucrose boundary, the lysate layer (7.5% sucrose), 20-30% sucrose boundary and 30-40% boundary (Figure 2, Table 1). In addition, the ratios between the well-bound (wb) form and the fast-moving (fm) form of mtDNA also varied from one sucrose gradient fraction to another (Figure 2B). The highest ratio between wb form DNA to fm form DNA was found in the fraction of 15/20% sucrose boundary (Figure 2B).
In vitro mtDNA replication by the different mito-chondrial nucleoid-membrane complexes
In vitro mitochondrial nucleoid-membrane complex DNA replication was performed as described in Materials and Methods. After PFGE analysis, the complexes har­vested from the lysate/15% sucrose boundary showed the most mtDNA synthesis, even though its mtDNA content was less than that in the 15/20% fraction (Figure 3 and Table 1). In addition, the wb to fm ratio of the newly syn­thesized mtDNA was much lower than that of the original template mtDNA (compare Figure 3, Panel B to Figure 2, Panel B). Self-sufficient mtDNA replication was found in all seven fractions, and even in the pellet, which was also able to accomplish mtDNA synthesis/replication. Newly synthesized mtDNA could barely be detected in the 40/50% boundary fraction. This result indicates that the mtDNA-protein-membrane complexes in these seven frac­tions carried the essential factors for DNA synthesis.
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Figure 2. The amounts of mtDNA among different mitochondrial nucleoid-membrane complex fractions generated by sucrose gradi­ent centrifugation. Panel A: Southern blot of mitochondrial nucleoid-membrane complex from various fractions generated by sucrose gradient centrifugation. Samples from the sucrose gradient fractions indicated above each lane were analyzed by pulse field gel elec­trophoresis followed by Southern hybridization with a probe derived from mtDNA. mtDNA migrated as the well-bound (wb) and fast-moving (fm) forms in the gel after electrophoresis. Panel B: Relative amount of mtDNA among the fractions after sucrose gradient centrifugation was determined by direct counting of the radioactivities of each fraction cut from the Southern blot membrane. Similar results were obtained from OD260 absorbance of the mtDNA purified from each fraction obtained from sucrose gradient. The relative amounts of mtDNA in wb and fm forms of each fraction, respectively, were measured by counting of the radioactivities cut from the Southern blot membrane.
Figure 3. In vitro mtDNA synthesis by the different mitochondrial nucleoid-membrane complex fractions. Panel A: Autoradiograph of newly synthesized mtDNA of different mitochondrial nucleoid-membrane complexes fractionated by PFGE. Panel B: Percentage of newly synthesized mtDNA in each mitochondrial nucleoid-membrane complex. The radioactive counting was performed after cutting each lane and corresponding wb/fm from the gel.
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Table 1. Comparative analysis of the relationship between mtDNA content and mitochondrial replication activity among the differ­ent mitochondrial nucleoid-membrane complex fractions.
Mitochondrial nucleoid-membrane complexes separated
Relative amount of mtDNA
Relative amount of newly synthesized
by sucrose gradient (SG) fractionation
in each fraction (%)*
mtDNA in each fraction (%)**
Lysate
17.22
27.07
Lysate-15% SG
25.28
54.49
15-20% SG
36.60
7.31
20-30% SG
8.91
7.55
30-40% SG
4.55
2.30
40-50%SG
3.27
0.50
Pellet
4.17
0.77
*Determined by optical density of purified DNA and by radioactivity intensity of the corresponding lane cut from a Southern    mem­brane. Similar results were obtained with the two techniques.
**Determined by counting the corresponding lane cut from the gel after PFGE.
For the first time in this research area, we found that it is not the conventional mitochondrial nucleoids (the band at the 15-30% boundary) that perform the best in vitro mtDNA replication. Instead, two less dense complexes containing less mtDNA but a higher amount of mitochon-drial membrane show more active DNA replication capa­bility (or completion of DNA replication).
Transcription by mitochondrial nucleoid-mem-brane complexes
The in vitro transcription activity of the mitochondrial nucleoid-membrane complexes appeared to be correlated with their associated mitochondrial membranes. The high­est activity was found in the lysate fraction (26.21%, Fig­ure 5 and Table 2), which was associated with the largest amount of mitochondrial membrane among the seven frac­tionated complexes. The transcriptional activity of each complex fraction decreased in parallel with the decline in the membrane content of the complex (Figure 5 and Table 2). Most of the RNA synthesized in each fraction showed a smear pattern and lacked the two distinguishing rRNA bands transcribed in mitochondria, as shown in Lane 10 of
Figure 5. The smearing of the mitochondrial transcripts was most likely caused by RNA degradation during the lengthy time span required for mitochondrial nucleoid-membrane complex purification followed by in vitro tran­scription.
DISCUSSION
It is known that mtDNA binds nucleoproteins and forms mitochondrial nucleoids. Mitochondrial membranes are also associated with this macromolecular complex in higher plants (Dai et al., 2005). The conventional method for isolating mitochondrial nucleoids uses a suitable range of detergent concentrations to avoid the disruption of the organellar organization of the nucleoids (Kuroiwa, 1982; Miyakawa et al., 1996; Sakai et al., 2004; Dai et al., 2005). We demonstrated in this study that besides conventional mitochondrial nucleoids, which are density banded be­tween 15% and 30% in sucrose gradients and have long been focused on, other fractions harvested from the same sucrose gradient at lower sedimentation densities showed even higher biological activity in terms of in vitro DNA
Table 2. Comparison of transcriptional activity among the different mitochondrial nucleoid-membrane complexes and its correla­tion with mtDNA content.
Mitochondrial nucleoid-membrane complexes separated by sucrose gradient (SG) fractionation
Relative amount of mtDNA in each fraction (%)
Percentage of newly transcribed mtRNA in each fraction (%)**
Lysate
17.22
26.21
Lysate/15% SG
25.28
21.95
15/20% SG
36.60
14.53
20/30% SG
8.91
12.89
30/40% SG
4.55
8.33
40/50% SG
3.27
7.39
Pellet
4.17
8.71
**Determined by incorporation counting and by counting the corresponding lane cut from the gel. Similar results were obtained with both techniques.
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replication and RNA transcription. It is intriguing to find that these supra-molecular mtDNA complexes, contain­ing less mtDNA but more mitochondrial membrane, can perform more active DNA replication and RNA transcrip­tion. The most prominent in vitro mtDNA synthesis was accomplished by the band of lysate/15% boundary (Figure
3, Panels A and B, lane 2), even though this fraction con­tained less mtDNA than the 15-20% fraction (Figure 4 and Table 1). It indicates that the activity of in vitro mtDNA replication and transcription are highly dependent on the membrane attached to mitochondrial-nucleoids. Decreas­ing of membrane content in mitochondrial nucleoid-complexes cause a decline in mitochondrial nucleoid replication and transcription (Figures 4-5 and Tables 1-2). It is most likely that the membranes associated with mtDNA-protein complexes lower the sedimentation den­sity of the mitochondrial nucleoids during sucrose gradient fractionation.
According to the TLC analysis shown in Figure 1, the phospholipid composition of the mitochondrial nucleoid-complexes is comprised of both outer and inner mito-chondrial membrane, which mimic that of the whole mitochondria. This finding is consistent with other find­ings that mitochondrial DNA replication and transcription are associated with both the outer and inner membranes (Meeusen and Nunnari, 2003; Iborra et al., 2004; Dai et al., 2005).
As a moving picture of mtDNA, every mitochondrial nucleoid-membrane complex showed both well-bound (wb) and fast-moving (fm) components after pulse-field gel elec-trophoresis, as reported previously (Dai et al., 2005). Struc­tural DNA that has a loose structure and greater molecular weight (〉2500 kb) does not migrate into the gel and stays in the well (well-bound, wb); more compact circular or lin­ear DNA with smaller sizes (50-200 kb) is fast-moving (fm) in the gel during pulse-field gel electrophoresis (see Figures 2A and 3A). It is clearly shown in Figure 3B that the new­ly-synthesized mtDNA contained less wb DNA than did the original mtDNA (compare Figure 3B with Figure 2B). We postulate that this phenomenon may be because fm DNA replication in vitro is much faster than that of wb DNA. Alternatively, it is also possible that conversion wb to fm DNA is more efficient in vitro than in vivo. Our previous results suggest that wb and fm mtDNA may undergo DNA replication independently and that the wb form may repre­sent rolling circular replicating mtDNA molecules (before conversion to the fm form) that are too large to move in the pulsed electrical field. Alternatively, the immobility may be the result of an unusual mtDNA structure produced by mul­tiple recombination events (Backert et al., 1996; Oldenburg and Bendich, 2001)
.
Taken together, these findings imply that there may be a defined relationship between the structural organiza­tion and the functional integrity of isolated mitochondrial nucleoids. Although the structural domains of the nucleoid membranes have yet to be characterized, more active in vitro DNA replication and RNA transcription are observed when larger amounts of membrane are attached to mito-chondrial nucleoid-membrane complexes. We believe that the organization and conformation of a supra-molecular complex containing mtDNA, nucleoprotein and mitochon-drial membranes plays an important role in mitochondrial DNA replication and RNA transcription.
Figure 4. Correlation of mtDNA synthesis activity with mtDNA content in each mitochondrial nucleoid-membrane complex.
Figure 5. In vitro mitochondrial transcription by the different mitochondrial nucleoid-membrane complex fractions. Autoradi-ography of newly synthesized mitochondrial RNA is presented, and the products of in organelle mitochondrial transcription were run as a control in lane 9.
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Acknowledgments. This research was supported by re­search grants from the National Science Council and from Academia Sinica, ROC. BLL was supported by a NSC post-doctoral fellowship.
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粒線體nucleoid DNA複製和轉錄受其相連的粒線體膜系影響
李寶林 羅意珊 戴 華
中央研究院植物暨微生物學研究所
這項研究顯示粒線體基因複製和轉錄除受粒線體核蛋白的影響外，與粒線體nucleoid關聯的特定膜
元件可能也對粒線體基因的表達發揮重要作用。粒線體nucleoids可自給自足的行粒線體基因複製和轉
錄。這研究將整個粒線體以特定方式溶解後，以常規間斷蔗糖梯度離心將其依密度的不同分為七組不同
的 mitochondrial nucleoid-membrane complexes 。這不同密度的 mitochondrial nucleoid-membrane complexes
含有明顯不同量的粒線體膜系。在行體外mtDNA複製和轉錄結果中，我們確定不同粒線體的複製和轉
錄的能力深受粒線體的膜系含量影響。我們因而確認粒線體基因的表達受粒線體DNA ，粒線體核蛋白
和粒線體膜系間的相關結構，組合關係密切。
關鍵詞：線粒體膜系；Mitochondrial nucleoid-membrane complex ；線粒體nucleoids ；線粒體基因複製；
線粒體基因轉錄；綠豆。