- Email: [email protected]
[12] Mitochondrial DNA transcription initiation and termination using mitochondrial lysates from cultured human cells
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[12] M i t o c h o n d r i a l D N A T r a n s c r i p t i o n I n i t i a t i o n a n d Termination Using Mitochondrial Lysates from Cultured Human Cells
By
PATRICIO FERNANDEz-SILvA, VlCENTE MICOL, and GIUSEPPE ATTARDI
Introduction The complete nucleotide sequence of human and several other mammalian mitochondrial DNAs (mtDNAs) has been determined. T M In human mitochondria, the main initiation sites for transcription of the heavy (H) and light (L) strands of the D N A have been identified in vivo and in vitro, 5-9 the discrete transcription products have been mapped, I° and their structural and metabolic properties have been characterized, n-15 By contrast, little is known about the enzymatic machinery involved in transcription in mammalian cells and the regulation of this process. One mitochondrial transcription factor, mtTFA, necessary for high levels of specific initiation of transcription at the L-strand promoter and rRNA-specific H-strand promoter, has been identified and c l o n e d . 16'17 A different DNA-binding factor, 1 S. Anderson, A. T. Bankier, B. G. Barrell, M. H. de Bruijn, A. R. Coulson, J. D r o u i n , I. C. Eperon, D. P. Nierlich, B. A. Roe, F. Sanger, P. H. Schreier, A. J. Smith, R. Staden, and I. G. Young, Nature (London) 290, 457 (1981). 2 M. J. Bibb, R. A. Van Etten, C, T. Wright, M. W. Walberg, and D. A. Clayton, Cell (Cambridge, Mass.) 26, 167 (1981). 3 S. Anderson, M. H. de Bruijn, A. R. Coulson, I. C. Eperon, F. Sanger, and I. G. Young, J. MoL Biol. 156, 683 (1982). 4 M. N. Gadaleta, G. Pepe, G. De Candia, E. Quagliarello, E. SbisL and C. Saccone, J. Mol. Evol. 28, 497 (1989). 5 j. Montoya, T. Christianson, D. Levens, M. Rabinowitz, and G. Anardi, Proc. Natl. Acad. Sci. U.S.A. 79, 7195 (1982). 6 M. W. Walberg and D. A. Clayton, J. Biol. Chem. 258, 1268 (1983). 7 B. K. Yoza and D. F. Bogenhagen, J. BioL Chem. 259, 3909 (1984). 8 D. J. Shuey and G. Attardi, J. Biol. Chem. 260, 1952 (1985). 9 D. D. Chang and D. A. Clayton, Cell (Cambridge, Mass.) 36, 635 (1984). 10 D. Ojala, C. Merkel, R. Gelfand, and G. Attardi, Cell (Cambridge, Mass.) 22, 393 (1980). 11 R. Gelfand and G. Attardi, Mol. Cell Biol. 1, 497 (1981). lZ j. Montoya, G. Gaines, and G. Attardi, Cell (Cambridge, Mass.) 34, 151 (1983). 13 G. Gaines and G. Attardi, J. Mol. BioL 172, 451 (1984). 14 G. Gaines and G. Attardi, Mol. Cell Biol. 4, 1605 (1984). is G. Gaines and G. Attardi, J. Biol. Chem. 262, 1907 (1987). 16 R. P. Fischer and D. A. Clayton, J. Biol. Chem. 260, 11330 (1985). 17 M. A. Parisi and D. A. Clayton, Science 252, 965 (1991).
METHODS IN ENZYMOLOGY,VOL. 264
Copyright © 1996by Academic Press, Inc. All rights of reproduction in any form reserved.
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mTERF (mitochondrial transcription termination factor), involved in termination of transcription at the 16 S r R N A / t R N A Leu(vvR)boundary, has been extensively characterized in our laboratory. 18'19On the other hand, nothing is known about the mtRNA polymerase itself and the probable existence of other factors involved in transcription initiation at the downstream, whole H-strand specific promoter. 2° Submitochondrial transcription systems have proved to be useful tools to study mtDNA transcription. These systems are easy to manipulate, and they have already provided valuable information about some aspects of mtDNA transcription, such as the energetic requirements for initiation and the sequences necessary for an effective and accurate initiation and termination of transcription. 9'21,22Two different types of such "open" transcription systems have been developed. One of them utilizes partially purified protein components to promote transcription from mtDNA-derived templates6'9'e2; the other one uses the S-100 of a mitochondrial lysate programmed by exogenous templates. 8'18'2m3 We have used the latter system extensively because of its efficiency and reproducibility, and particularly because it carries out several of the in vivo transcription activities, being at the same time accessible to external manipulation. Using as a template a plasmid construct that allows the reproduction of the main events of in vivo transcription (Fig. 1), we have greatly improved the soluble transcription system previously developed in this laboratory. 8'21 In particular, the overall initiation and termination activities of the mitochondrial lysate have been increased about 8- and 15-fold, respectively, relative to the old protocol. Using this improved system, we have characterized in detail the effects on the transcription initiation and termination events of varying the protein and the template concentrations, the ATP level, the ionic strength, and the Ca 2+ and Mg 2+ concentrations. We have also used this system to characterize the termination-promoting activity of affinity-purified mTERF. 24 This transcription system is also expected to be very useful for the identification and purification of other factors active in mtDNA transcription. In this chapter, we describe a modification of the soluble transcription 18 B. Kruse, N. Narasimhan, and G. Attardi, Cell 58, 391 (1989). 19 A. Daga, V. Micol, D. Hess, R. Aebersold, and G. Attardi, J. Biol. Chem. 268, 8123 (1993). 20 A. Chomyn and G. Attardi, in "Molecular Mechanisms in Bioenergetics" (L. Ernster, ed.), p. 483. Elsevier, Amsterdam, 1992. 2l N. Narasimhan and G. Attardi, Proc. Natl. Acad. Sci. U.S.A. 84, 4078 (1987). 22 R. P. Fischer, J. N. Topper, and D. A. Clayton, Cell 50, 247 (1987). 23 B. Kruse, N. N. Murdter, and G. Attardi, Methods Mol. Biol. 37 (In Vitro Transcription and Translation Protocols) (1995). 24 V. Micol, P. Fern~indez-Silva, and G. Attardi, this volume [15].
[12]
mtDNA TRANSCRIPTIONIN MITOCHONDRIALLYSATES IHR
5 5 9 4 ~ 6 ~ S ~ _ ~ 30563 2 2759 9
Hinc]I
,,,oo';B,5,oo, Tt
j,
57 ~
oo,
131
IL~
//
r'~,,
pTER
Mb/ run-off transcript termination transcript
4t5nt 254 nt
S 1 probe $1 protected termination transcript
FIG. l. Map of the clone pTER used for in vitro transcription. The map positions of the H-strand run-off and terminated transcripts, of the probe used in the $1 protection assays, and of the Sl-protected terminated transcripts are shown. IHR, Upstream initiation site for H-strand transcription; IL, initiation site for L-strand transcription. Modified, with permission, from A. Daga, V. Micol, D. Hess, R. Aebersold, and G. Attardi, J. Biol. Chem. 268, 8123 (1993).
system utilizing the 13,000 g supernatant (S-13) of a mitochondrial lysate from small-scale cell culture samples. Furthermore, we report an adaptation of the mobility shift assay for the semiquantitative detection of mTERF in the S-13 fraction from small amounts of cells, which allows an easy comparison of its relative content in different cell lines and under different growth conditions. Procedures
Reagents and Solutions. All glassware should be baked at 180° at least 4 hr. Solutions should be prepared with diethyl pyrocarbonate (DEPC)treated and autoclaved distilled water. 2 M Sucrose in autoclaved distilled water: sterilize by filtration and store frozen at - 2 0 ° 0.5 M Dithiothreitol (DTT): dissolve in autoclaved distilled water and store in aliquots at - 2 0 ° (add to buffers just before use) 0.5 M Phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO): dissolve in either ethanol or 2-propanol and store in aliquots at -20 ° (add to buffers just before use) Polyoxyethylenesorbitan monolaureate (Tween 20, Sigma) NKM buffer: 1 mM Tris-HC1, pH 7.4 at 25°, 0.13 M NaC1, 5 mM KC1, 7.5 mM MgCl2 Homogenization buffer: 10 mM Tris-HCl, pH 6.7, 10 mM KCI, 0.15 mM MgCI2
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STUDYING MITOCHONDRIAL GENE EXPRESSION
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Mitochondria suspension buffer: 10 mM Tris-HCl, pH 6.7, 0.25 mM sucrose, 0.15 mM MgCl2 Mitochondria lysis buffer: 25 mM HEPES-KOH, pH 7.6, 5 mM MgC12, 0.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% glycerol v/v TD buffer: 25 mM Tris-HC1, pH 7.4, 135 mM NaC1, 5 mM KC1, 0.5 mM NazHPO4 Trypsin solution: 5 g/liter trypsin(i-300) (from porcine pancreas, ICN Biomedicals, Irvine, CA), 3.2 g/liter penicillin, 0.2 g/liter streptomycin, 0.003% phenol red w/v TBE: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA Transcription buffer (2x): 20 mM Tris-HCl, pH 8.0, 20 mM MgCI2, 2 mM DTT, 200/zg/ml bovine serum albumin (BSA), 20% (v/v) glycerol; sterilize by filtration and store at - 2 0 ° NTP mixture (25 x): 25 mM ATP, 2.5 mM GTP and CTP, 0.25 mM UTP Transcription stop buffer: 10 mM Tris base, 0.3 M sodium acetate, pH 7.0, 15 mM EDTA, 0.5% w/v SDS, final pH adjusted to 8.0 with HC1 0.5% SDS buffer: 10 mM Tris-HCl, pH 7.4, 0.2 M NaC1, 10 mM EDTA, 0.5% w/v sodium dodecyl sulfate (SDS) T3 transcription buffer (5x): 200 mM Tris-HCl, pH 7.9, 30 mM MgCI2, 10 mM spermidine, 50 mM NaCI DNase buffer (5x): 100 mM Tris-HC1, pH 7.6, 50 mM CaCI2, 50 mM MgC12 S1 hybridization buffer: 80% v/v formamide, 40 mM PIPES-HC1, pH 6.4, 380 mM NaC1, 0.5 mM EDTA; store at - 8 0 ° $1 digestion buffer (10x): 400 mM sodium acetate, 30 mM ZnCI2, 2.5 M NaC1; adjust to pH 4.6 with HC1, filter, and store at 4° $1 stop buffer: 4 M ammonium acetate, 20 mM EDTA, 200 tzg/ml yeast tRNA Buffer C: 25 mM HEPES-KOH, pH 7.5, 100 mM KC1, 12.5 mM MgCI2, 1 mM DTT, 20% (v/v) glycerol, 0.1% (v/v) Tween 20; store at 4° Urea-dye: 7 M urea, 0.01% w/v bromphenol blue, 0.01% w/v xylene cyanol in TBE; keep at - 2 0 ° and thaw at 65° before use Tris-glycine buffer (5X): 250 mM Tris-base, 1.9 M glycine, 10 mM EDTA, pH -8.5
Preparation of S-13 of Mitochondrial Lysate An important difference in the preparation of the mitochondrial lysate with respect to the previously described protocol 8,2x is the use of Tween 20 instead of NP-40 (Nonidet P-40) in the lysis step. The substitution of NP-40 with Tween 20 dramatically increases the transcription initiation and termination activities of the lysate. [Note:The change in the detergent used
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TRANSCRIPTION IN MITOCHONDRIAL LYSATES
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to prepare the mitochondrial lysate was adopted after analyzing the effect, on the transcription initiation activities, of four different detergents (Triton X-100, sodium deoxycholate, NP-40, and Tween-20), Tween-20 being the one that gave the best results. The relative overall activities were in the ratio of 100 for Tween 20, 13 for NP-40, 5 for Triton X-100, and 3 for sodium deoxycholate. A detailed comparison between the NP-40 and Tween 20 lysates showed that the overall initiation and termination activities were, respectively, 8- and 15-fold higher when Tween 20 was used.] The Tween 20 mitochondrial lysate containing the transcription machinery has been prepared from either suspension cultures or cultures grown on a solid substrate. We have used two different protocols, depending on the amount of starting material. The preparation of the S-100 of the mitochondrial lysate from a large-scale human cell suspension culture (e.g., HeLa cells grown in high-phosphate-containing Dulbecco's modified Eagle's medium supplemented with 5% v/v calf serum) is described in detail elsewhere in this volume.24 Here, we report the preparation of the S-13 of the Tween 20 mitochondrial lysate from small-scale suspension cultures or from cell cultures grown on a solid substrate, which yield a packed cell volume ranging from 0.05 to 1.0 ml. In the case of cells grown on a solid substrate, plates that are about 80-90% confluent are used. Each plate is washed once with 5 ml TD buffer and then trypsinized with 5 ml of a 1 : 10 dilution of trypsin solution in TD buffer containing 10 mM EDTA, prewarmed to 37°. The trypsin digestion is allowed to proceed for 5 min at room temperature, and then the cell suspension is collected with a Pasteur pipette and transferred to a conical tube containing 1 volume TD buffer with 5% calf serum, placed in ice. The cells (deriving from the trypsinized plates or from a small suspension culture) are pelleted at 500 gay for 5 min and washed three times with NKM. Before the last centrifugation, the cells are transferred to a graduated Eppendorf tube(s), so that the approximate volume of packed cells can be estimated (from five plates containing a total of 107 cells one can expect a volume of packed cells around 100 tzl). After removing the supernatant from the last washing step, the pelleted cells are placed at - 2 0 ° for 15 min in order to weaken the cell membrane and facilitate the breakage in the homogenization step. The samples are placed in ice, and the pellets are then suspended in a volume of homogenization buffer corresponding to 10 times the original volume of packed cells (vpc) and homogenized in a Thomas homogenizer (A. H. Thomas, Swedesboro, N J) of appropriate size (0 for up to 1 ml homogenate, AA for up to 4 ml, and A for up to 10 ml) with 10 strong strokes, using a pestle rotating at approximately 1500 rpm. The degree of cell breakage obtained in this way is usually around 80-90%.
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The homogenate is immediately poured into one or more Eppendorf tubes containing 2 M sucrose in an amount corresponding to one-sixth of the volume of the homogenate, mixed, and centrifuged at 1200 g to pellet nuclei, large debris, and unbroken cells. This centrifugation is repeated, and the last supernatant is transferred to a fresh Eppendorf tube(s), and centrifuged at approximately 13,000 g in a microcentrifuge for 1 min at 4°. The mitochondrial pellet(s) thus obtained is washed twice with 1 ml mitochondrial suspension buffer by centrifuging at approximately 13,000 g for 1 min, and finally suspended in 1 vpc mitochondria lysis buffer containing freshly added Tween 20 and KCI to final concentrations of 0.5% and 0.5 M, respectively. The mitochondria are lysed by pipetting up and down repeatedly until the suspension has become clear, and then by vortexing vigorously for 30 sec. The lysate is then kept on ice 5 min, and the vortexing step is repeated once in the same way. The lysate is then centrifuged for 45 min at approximately 13,000 g in the microcentrifuge at 4 °, and the clear supernatant (S-13) is collected carefully, avoiding the fluffy layer. Aliquots of the S-13 are frozen in liquid N2 and stored at - 8 0 °. The protein concentration of the S-13 preparation is determined by the method of Bradford. 25 The transcription activity of the S-13 is stable at - 8 0 ° for at least 6 months. Repeated freezing and thawing slowly reduce this activity. In Vitro Transcription Assay For the analysis of the transcription initiation and termination activities present in the S-13 of the mitochondrial lysate, we have chosen a construct (pTER) 17 that closely reproduces the known critical regions of the in vivo template, since it contains the whole promoter regions for L- and H-strand transcription and adjacent sequences, and the termination region located at the 16 S r R N A / t R N A eeu~UUR)boundary (Fig. 1). In the standard transcription experiments, for each sample to be tested, the following components are mixed in an Eppendorf tube (placed in ice): 1/zl of the template D N A (pTER) at a concentration of 1/xg//xl, 2/xl of the 25× NTP mixture, 5 tzl of the S-13 preparation to be analyzed, 25 ~1 of 2× transcription buffer, 1 ~1 of [a-32p]UTP (400 Ci/mmol, 10 mCi/ml; Amersham, Arlington Heights, IL), and DEPC-treated autoclaved, distilled water to bring the volume to 50/zl. (Note: The protein concentration has been found to be 4-6 mg/ml in the S-13 preparation of the Tween 20 mitochondrial lysate from a small-scale preparation, to be compared to a concentration of 10-12 mg/ml in the S-100 of the Tween 20 lysate from a large-scale preparation. The concentrations of lysate and D N A template 25M. M. Bradford, Anal Biochem. 72, 248 (1976).
[121
mtDNA TRANSCRIPTIONIN MITOCHONDRIALLYSATES
135
in the transcription assay may have to be adjusted in order to find the optimal ratio for each preparation. 21) The mixtures are vortexed, briefly spun in an Eppendorf microcentrifuge, and incubated at 30° for 30 min. The reaction is stopped by adding to each sample 100/zl transcription stop buffer and 20/zg yeast tRNA, and the sample is extracted with 1 volume of phenol/CHC13/isoamyl alcohol (25:24:1, v/v), T h e nucleic acids are precipitated by adding 330/zl ethanol (prechilled at - 2 0 °) and keeping the mixtures at - 8 0 ° for 30 rain; then they are pelleted by centrifuging at full speed in a microfuge at 4° for 10 min. The pellet is dissolved in 150 tzl 0.5% SDS buffer, extracted again with 1 volume of phenol/CHC13/isoamyl alcohol (25 : 24 : 1, v/v), and the nucleic acids are precipitated as described above. The final pellet is dissolved in DEPC-treated autoclaved distilled water, and the synthesized products are analyzed in a 5% polyacrylamide (acrylamide : bisacrylamide, 29 : 1)/7 M urea gel; alternatively, if so required, the transcripts are subjected to S1 protection analysis, as described below. The pellet from each transcription reaction is dissolved in 100/zl of 1 x DNase buffer, 10 units RNase-ffee DNase I is then added, and the mixture is incubated at room temperature for 20 min in order to destroy the template DNA. After phenol extraction and ethanol precipitation, the labeled R N A pellet is dissolved in DEPC-treated autoclaved distilled water, mixed with 0.2-0.4/zg of the unlabeled R N A probe (see below), and precipitated again with NaC1 and ethanol. The new pellet is carefully suspended in 20/zl of $1 hybridization buffer by pipetting up and down repeatedly. Denaturing of the sample is achieved by heating it at 80° for 10 min, and hybridization is performed at 50° for 6 to 10 hr. After the hybridization, 200 tzl of S1 digestion buffer containing 20 tzg/ml denatured salmon sperm DNA and 250 to 400 units $1 nuclease (Boehringer Mannheim, Indianapolis, IN; 400 units//zl) are added to the sample, and the mixture is incubated at 41 ° for 30 to 45 min. Finally, 55/zl of S1 stop buffer is added to the sample, and the Sl-resistant products are precipitated by adding 2.5 volumes ethanol and pelleted by centrifuging at approximately 13,000 g for 10 min at 4°C. The final pellets are suspended in the desired volume of DEPC-treated autoclaved distilled water, 1 volume of urea-dye is added, and, after denaturing at 80° for 10 min, the samples are loaded onto a 5% polyacrylamide (acrylamide : bisacrylamide, 29 : 1)/7 M urea gel in TBE and run in the same buffer for 2-3 hr at 400 V. After electrophoresis, the gel is washed twice for 10 min with autoclaved distilled water and vacuum-dried on a sheet of Bio-Rad (Richmond, CA) backing paper at 80° for 1 hr, before exposure for autoradiography. To prepare the antisense unlabeled R N A to be used as a probe for S1 analysis of the transcripts, a 50-/zl reaction mixture is set up containing the
136
STUDYING MITOCHONDRIAL GENE EXPRESSION
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following: 1.5/xg plasmid pBSAND [containing the MaeI-MaeI fragment of pTER filled in and cloned into the Sinai site of plasmid pBS KS(+) (Promega, Madison, WI)] linearized with BamHI, 10/xl of 5 X T3 transcription buffer, 0.5 mM each of ATP, CTP, GTP, and UTP, 8 mM DTT, 40 units RNase inhibitor (RNasin, Promega; 40 units//zl), and 20 units T3 RNA polymerase (Promega; 20 units//zl). The mixture is incubated at 37 ° for 90 min, and then 20 units RNase-free DNase I (Boehringer Mannheim, 10 units//zl) is added and the mixture incubated for an additional 15 min at 37 ° in order to destroy the template DNA. After phenol extraction, the synthesized RNA is precipitated by adding ammonium acetate to a final concentration of 2 M and 2.5 volumes ethanol, then recovered by centrifugation at approximately 13,000 g for 10 min. The amount of probe obtained is determined by reading the A260. A yield of 2 to 4/zg RNA per reaction is usually obtained. The results of a typical transcription experiment are shown in Fig. 2. In lane 1 (Fig. 2), containing the labeled products, two main bands are visible, one corresponding to the H-strand runoff transcripts (H) and the other to the L-strand runoff transcripts (L). Lane 2 (Fig. 2), containing the Sl-resistant products, shows two major bands corresponding to the
M
1
2
--H
710 ~
M
71o 489
489
--L
404
404
367
367
242
• 242
,90 _
ii:i:i
ii
,90
FIG. 2. Products of in vitro transcription assay using the S-13 of a Tween 20 mitochondrial lysate and EcoRI- and H1NdIII-digested pTER clone template. Lane 1 shows transcription products (H, H-strand runoff transcripts; L, L-strand runoff transcripts). Lane 2 shows S1resistant products after hybridization of the in vitro products with the MaeI-MaeI unlabeled RNA probe (Fig. 1) (R, H-strand runoff transcripts; T, H-strand terminated transcripts).
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mtDNA TRANSCRIPTIONIN MITOCHONDRIALLYSATES
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protected fragments of the H-strand run-off (R) and terminated (T) transcripts.
Detection of Mitochondrial Transcription Termination Factor by Mobility Shift Assay The protocol for the mobility shift assay of mTERF described elsewhere in this v o l u m e 24 w a s designed for the purpose of detecting and measuring the binding activity of mTERF purified by affinity chromatography. 2° In this chapter, we describe a modification of this procedure suitable for detecting the presence and estimating the relative abundance of mTERF in mitochondrial lysates derived from small-scale cell culture samples. The amount of probe per reaction has been increased in order to saturate the binding capacity of the factor; furthermore, in this modified protocol, the gel is run in the cold and is loaded with the current on to keep the dissociation of the DNA-protein complex to the minimum possible and to give sharp bands suitable for quantitation. By performing band-shift experiments with a constant amount of probe and increasing amounts of S-13 lysate, we were able to determine the conditions for a semiquantitative assay (Fig. 3). The probe used for this assay is the double-stranded 44-mer fragment described elsewhere, 24 3'-end-labeled by filling in with [a-32p]dTTP and the Klenow fragment of Escherichia coli D N A polymerase I. 26 To set up the DNA-binding reactions, for each sample to be tested, a mixture is prepared, in an Eppendorf tube placed in ice, containing the following components: 0.1 pmol probe [labeled to a specific activity of -200,000 counts/rain (cpm)/pmol], 5/zg BSA, 5/zg poly(dI-dC) • (dI-dC), 10 tzl buffer C containing 50 instead of 100 mM KC1, and the desired amount of the S-13 of the mitochondrial lysate to be tested (usually between 1 and 4 tzl). The volume is then brought to 20 to 50/zl by adding buffer C lacking KC1, with the final KCI concentration being adjusted to 100 mM by the addition of 3 M KC1, if needed. The samples are vortexed, spun down briefly, and incubated at 25 ° for 20 min. After incubation, the samples are placed in ice and then loaded onto a 5% polyacrylamide:bisacrylamide (80:1) gel in Tris-glycine buffer containing 2.5% glycerol, which had been prerun for at least 2 hr at 200 V. After loading, the gel is run in the cold at 200-250 V for 4 hr, heat-dried at 80° for 2 hr, and exposed for autoradiography. Figure 3 shows a calibration experiment carried out by incubating a constant amount of probe (0.1 pmol per reaction) with different amounts of the S-13 preparation of a mitochondrial lysate, corresponding to a range 26 F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., "Current Protocols in Molecular Biology." Wiley(lnterscience), New York, 1987.
138
STUDYING MITOCHONDRIAL GENE EXPRESSION
A
S-13 Fraction o 0.5 1.25 2.5 5 10 15 10 25
0
[12]
Prot. (Pg)
~ F r e e Probe
B 4 3 tr
2 1 0
..
0.0
,
5.0
10.0 15.0 20.0 Protein (IJg)
25.0
FxG. 3. Mobility shift assay calibration curve using the S-13 of a Tween 20 mitochondrial lysate. (A) A constant amount of the probe was incubated with different amounts of a sample of S-13, as described in the text. The specific retarded band that has been quantified in (B) is indicated by an arrow. (B) Quantification of the specific retarded band in the gel shown in (A) performed by Phosphor-Image detection and Image-Quant analysis (Molecular Dynamics).
of cell equivalents between 1 x 104 and 5 x 105. As the quantitation of the autoradiogram shows (Fig. 3b), there is a range of amounts of S-13/ pmol probe in which the signal of the specific retarded band is proportional to the amount of lysate. (Note: For each sample to be analyzed by the semiquantitative band shift assay, we usually test two different amounts of the S-13 preparation of a Tween 20 mitochondrial lysate included in the linear portion of the calibration curve. Repeated experiments using different S-13 preparations from the same cell source have given consistent results in the relative abundance of the retarded band. The minimum amount of protein of an S-13 preparation that gives quantifiable results under our experimental conditions corresponds to - 5 x 104 cells.)
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MAPPING VERTEBRATE D - L O O P PROMOTERS
139
Acknowledgments This work was supported by National Institutes of Health Grant GM-11726 (to G. A.) and scholarships from the Fulbright/Spanish Ministry of Education and Science Visiting Scholar Program (to V, M.) and from Caltech (Gosney Fellowship, to P. F.-S.).
[13] M a p p i n g By
Promoters in Displacement-Loop Vertebrate Mitochondrial DNA
Region of
G E R A L D S. S H A D E L a n d D A V I D A . C L A Y T O N
Introduction In all vertebrates examined to date, mitochondrial (mt) D N A exists and can be isolated as a small covalently closed-circular molecule that has a promoter-containing regulatory locus called the displacement-loop (Dloop) region (Fig. 1). It is in the D-loop region where leading-strand D N A replication and the majority of mitochondrial gene transcription initiates. This chapter outlines a general strategy for locating the major promoters in the D-loop region of the mitochondrial genome of vertebrate organisms. We propose two approaches for the isolation of a D-loop region-containing D N A fragment that utilize conserved nucleotide sequences that occur within or near the D-loop region of vertebrates. Once in hand, promoters can be precisely located on this template using a crude mtRNA polymerase preparation to assay promoter activity in an in vitro transcription reaction. Rather than presenting the conventional methods for mapping the 5' ends of primary transcripts and defining boundaries of promoters in detail, those aspects unique to analyzing mitochondrial promoters are emphasized. We refer to the published methods that were used to locate D-loop region promoters of human 1 and mouse 2,3 mtDNAs as models for this type of analysis. Isolation of Mitochondria and Mitochondrial DNA from Vertebrate Cells Many of the procedures outlined in this chapter require the isolation of mitochondria and mtDNA from vertebrate cells. Methods for the purifi1 D. D. C h a n g and D. A. Clayton, Cell {Cambridge, Mass.) 36, 635 (1984). 2 D. D. C h a n g and D. A. Clayton, Mot Cell. Biol. 6, 3253 {1986). 3 D. D. C h a n g and D. A. Clayton, MoL Ceil Biol. 6, 3262 (1986).
METHODS IN ENZYMOLOGY,VOL. 264
Copyright © 1996by AcademicPress, Inc. All rights of reproduction in any form reserved.
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[12] M i t o c h o n d r i a l D N A T r a n s c r i p t i o n I n i t i a t i o n a n d Termination Using Mitochondrial Lysates from Cultured Human Cells
By
PATRICIO FERNANDEz-SILvA, VlCENTE MICOL, and GIUSEPPE ATTARDI
Introduction The complete nucleotide sequence of human and several other mammalian mitochondrial DNAs (mtDNAs) has been determined. T M In human mitochondria, the main initiation sites for transcription of the heavy (H) and light (L) strands of the D N A have been identified in vivo and in vitro, 5-9 the discrete transcription products have been mapped, I° and their structural and metabolic properties have been characterized, n-15 By contrast, little is known about the enzymatic machinery involved in transcription in mammalian cells and the regulation of this process. One mitochondrial transcription factor, mtTFA, necessary for high levels of specific initiation of transcription at the L-strand promoter and rRNA-specific H-strand promoter, has been identified and c l o n e d . 16'17 A different DNA-binding factor, 1 S. Anderson, A. T. Bankier, B. G. Barrell, M. H. de Bruijn, A. R. Coulson, J. D r o u i n , I. C. Eperon, D. P. Nierlich, B. A. Roe, F. Sanger, P. H. Schreier, A. J. Smith, R. Staden, and I. G. Young, Nature (London) 290, 457 (1981). 2 M. J. Bibb, R. A. Van Etten, C, T. Wright, M. W. Walberg, and D. A. Clayton, Cell (Cambridge, Mass.) 26, 167 (1981). 3 S. Anderson, M. H. de Bruijn, A. R. Coulson, I. C. Eperon, F. Sanger, and I. G. Young, J. MoL Biol. 156, 683 (1982). 4 M. N. Gadaleta, G. Pepe, G. De Candia, E. Quagliarello, E. SbisL and C. Saccone, J. Mol. Evol. 28, 497 (1989). 5 j. Montoya, T. Christianson, D. Levens, M. Rabinowitz, and G. Anardi, Proc. Natl. Acad. Sci. U.S.A. 79, 7195 (1982). 6 M. W. Walberg and D. A. Clayton, J. Biol. Chem. 258, 1268 (1983). 7 B. K. Yoza and D. F. Bogenhagen, J. BioL Chem. 259, 3909 (1984). 8 D. J. Shuey and G. Attardi, J. Biol. Chem. 260, 1952 (1985). 9 D. D. Chang and D. A. Clayton, Cell (Cambridge, Mass.) 36, 635 (1984). 10 D. Ojala, C. Merkel, R. Gelfand, and G. Attardi, Cell (Cambridge, Mass.) 22, 393 (1980). 11 R. Gelfand and G. Attardi, Mol. Cell Biol. 1, 497 (1981). lZ j. Montoya, G. Gaines, and G. Attardi, Cell (Cambridge, Mass.) 34, 151 (1983). 13 G. Gaines and G. Attardi, J. Mol. BioL 172, 451 (1984). 14 G. Gaines and G. Attardi, Mol. Cell Biol. 4, 1605 (1984). is G. Gaines and G. Attardi, J. Biol. Chem. 262, 1907 (1987). 16 R. P. Fischer and D. A. Clayton, J. Biol. Chem. 260, 11330 (1985). 17 M. A. Parisi and D. A. Clayton, Science 252, 965 (1991).
METHODS IN ENZYMOLOGY,VOL. 264
Copyright © 1996by Academic Press, Inc. All rights of reproduction in any form reserved.
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mTERF (mitochondrial transcription termination factor), involved in termination of transcription at the 16 S r R N A / t R N A Leu(vvR)boundary, has been extensively characterized in our laboratory. 18'19On the other hand, nothing is known about the mtRNA polymerase itself and the probable existence of other factors involved in transcription initiation at the downstream, whole H-strand specific promoter. 2° Submitochondrial transcription systems have proved to be useful tools to study mtDNA transcription. These systems are easy to manipulate, and they have already provided valuable information about some aspects of mtDNA transcription, such as the energetic requirements for initiation and the sequences necessary for an effective and accurate initiation and termination of transcription. 9'21,22Two different types of such "open" transcription systems have been developed. One of them utilizes partially purified protein components to promote transcription from mtDNA-derived templates6'9'e2; the other one uses the S-100 of a mitochondrial lysate programmed by exogenous templates. 8'18'2m3 We have used the latter system extensively because of its efficiency and reproducibility, and particularly because it carries out several of the in vivo transcription activities, being at the same time accessible to external manipulation. Using as a template a plasmid construct that allows the reproduction of the main events of in vivo transcription (Fig. 1), we have greatly improved the soluble transcription system previously developed in this laboratory. 8'21 In particular, the overall initiation and termination activities of the mitochondrial lysate have been increased about 8- and 15-fold, respectively, relative to the old protocol. Using this improved system, we have characterized in detail the effects on the transcription initiation and termination events of varying the protein and the template concentrations, the ATP level, the ionic strength, and the Ca 2+ and Mg 2+ concentrations. We have also used this system to characterize the termination-promoting activity of affinity-purified mTERF. 24 This transcription system is also expected to be very useful for the identification and purification of other factors active in mtDNA transcription. In this chapter, we describe a modification of the soluble transcription 18 B. Kruse, N. Narasimhan, and G. Attardi, Cell 58, 391 (1989). 19 A. Daga, V. Micol, D. Hess, R. Aebersold, and G. Attardi, J. Biol. Chem. 268, 8123 (1993). 20 A. Chomyn and G. Attardi, in "Molecular Mechanisms in Bioenergetics" (L. Ernster, ed.), p. 483. Elsevier, Amsterdam, 1992. 2l N. Narasimhan and G. Attardi, Proc. Natl. Acad. Sci. U.S.A. 84, 4078 (1987). 22 R. P. Fischer, J. N. Topper, and D. A. Clayton, Cell 50, 247 (1987). 23 B. Kruse, N. N. Murdter, and G. Attardi, Methods Mol. Biol. 37 (In Vitro Transcription and Translation Protocols) (1995). 24 V. Micol, P. Fern~indez-Silva, and G. Attardi, this volume [15].
[12]
mtDNA TRANSCRIPTIONIN MITOCHONDRIALLYSATES IHR
5 5 9 4 ~ 6 ~ S ~ _ ~ 30563 2 2759 9
Hinc]I
,,,oo';B,5,oo, Tt
j,
57 ~
oo,
131
IL~
//
r'~,,
pTER
Mb/ run-off transcript termination transcript
4t5nt 254 nt
S 1 probe $1 protected termination transcript
FIG. l. Map of the clone pTER used for in vitro transcription. The map positions of the H-strand run-off and terminated transcripts, of the probe used in the $1 protection assays, and of the Sl-protected terminated transcripts are shown. IHR, Upstream initiation site for H-strand transcription; IL, initiation site for L-strand transcription. Modified, with permission, from A. Daga, V. Micol, D. Hess, R. Aebersold, and G. Attardi, J. Biol. Chem. 268, 8123 (1993).
system utilizing the 13,000 g supernatant (S-13) of a mitochondrial lysate from small-scale cell culture samples. Furthermore, we report an adaptation of the mobility shift assay for the semiquantitative detection of mTERF in the S-13 fraction from small amounts of cells, which allows an easy comparison of its relative content in different cell lines and under different growth conditions. Procedures
Reagents and Solutions. All glassware should be baked at 180° at least 4 hr. Solutions should be prepared with diethyl pyrocarbonate (DEPC)treated and autoclaved distilled water. 2 M Sucrose in autoclaved distilled water: sterilize by filtration and store frozen at - 2 0 ° 0.5 M Dithiothreitol (DTT): dissolve in autoclaved distilled water and store in aliquots at - 2 0 ° (add to buffers just before use) 0.5 M Phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO): dissolve in either ethanol or 2-propanol and store in aliquots at -20 ° (add to buffers just before use) Polyoxyethylenesorbitan monolaureate (Tween 20, Sigma) NKM buffer: 1 mM Tris-HC1, pH 7.4 at 25°, 0.13 M NaC1, 5 mM KC1, 7.5 mM MgCl2 Homogenization buffer: 10 mM Tris-HCl, pH 6.7, 10 mM KCI, 0.15 mM MgCI2
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STUDYING MITOCHONDRIAL GENE EXPRESSION
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Mitochondria suspension buffer: 10 mM Tris-HCl, pH 6.7, 0.25 mM sucrose, 0.15 mM MgCl2 Mitochondria lysis buffer: 25 mM HEPES-KOH, pH 7.6, 5 mM MgC12, 0.5 mM EDTA, 1 mM DTT, 1 mM PMSF, 10% glycerol v/v TD buffer: 25 mM Tris-HC1, pH 7.4, 135 mM NaC1, 5 mM KC1, 0.5 mM NazHPO4 Trypsin solution: 5 g/liter trypsin(i-300) (from porcine pancreas, ICN Biomedicals, Irvine, CA), 3.2 g/liter penicillin, 0.2 g/liter streptomycin, 0.003% phenol red w/v TBE: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA Transcription buffer (2x): 20 mM Tris-HCl, pH 8.0, 20 mM MgCI2, 2 mM DTT, 200/zg/ml bovine serum albumin (BSA), 20% (v/v) glycerol; sterilize by filtration and store at - 2 0 ° NTP mixture (25 x): 25 mM ATP, 2.5 mM GTP and CTP, 0.25 mM UTP Transcription stop buffer: 10 mM Tris base, 0.3 M sodium acetate, pH 7.0, 15 mM EDTA, 0.5% w/v SDS, final pH adjusted to 8.0 with HC1 0.5% SDS buffer: 10 mM Tris-HCl, pH 7.4, 0.2 M NaC1, 10 mM EDTA, 0.5% w/v sodium dodecyl sulfate (SDS) T3 transcription buffer (5x): 200 mM Tris-HCl, pH 7.9, 30 mM MgCI2, 10 mM spermidine, 50 mM NaCI DNase buffer (5x): 100 mM Tris-HC1, pH 7.6, 50 mM CaCI2, 50 mM MgC12 S1 hybridization buffer: 80% v/v formamide, 40 mM PIPES-HC1, pH 6.4, 380 mM NaC1, 0.5 mM EDTA; store at - 8 0 ° $1 digestion buffer (10x): 400 mM sodium acetate, 30 mM ZnCI2, 2.5 M NaC1; adjust to pH 4.6 with HC1, filter, and store at 4° $1 stop buffer: 4 M ammonium acetate, 20 mM EDTA, 200 tzg/ml yeast tRNA Buffer C: 25 mM HEPES-KOH, pH 7.5, 100 mM KC1, 12.5 mM MgCI2, 1 mM DTT, 20% (v/v) glycerol, 0.1% (v/v) Tween 20; store at 4° Urea-dye: 7 M urea, 0.01% w/v bromphenol blue, 0.01% w/v xylene cyanol in TBE; keep at - 2 0 ° and thaw at 65° before use Tris-glycine buffer (5X): 250 mM Tris-base, 1.9 M glycine, 10 mM EDTA, pH -8.5
Preparation of S-13 of Mitochondrial Lysate An important difference in the preparation of the mitochondrial lysate with respect to the previously described protocol 8,2x is the use of Tween 20 instead of NP-40 (Nonidet P-40) in the lysis step. The substitution of NP-40 with Tween 20 dramatically increases the transcription initiation and termination activities of the lysate. [Note:The change in the detergent used
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mtDNA
TRANSCRIPTION IN MITOCHONDRIAL LYSATES
133
to prepare the mitochondrial lysate was adopted after analyzing the effect, on the transcription initiation activities, of four different detergents (Triton X-100, sodium deoxycholate, NP-40, and Tween-20), Tween-20 being the one that gave the best results. The relative overall activities were in the ratio of 100 for Tween 20, 13 for NP-40, 5 for Triton X-100, and 3 for sodium deoxycholate. A detailed comparison between the NP-40 and Tween 20 lysates showed that the overall initiation and termination activities were, respectively, 8- and 15-fold higher when Tween 20 was used.] The Tween 20 mitochondrial lysate containing the transcription machinery has been prepared from either suspension cultures or cultures grown on a solid substrate. We have used two different protocols, depending on the amount of starting material. The preparation of the S-100 of the mitochondrial lysate from a large-scale human cell suspension culture (e.g., HeLa cells grown in high-phosphate-containing Dulbecco's modified Eagle's medium supplemented with 5% v/v calf serum) is described in detail elsewhere in this volume.24 Here, we report the preparation of the S-13 of the Tween 20 mitochondrial lysate from small-scale suspension cultures or from cell cultures grown on a solid substrate, which yield a packed cell volume ranging from 0.05 to 1.0 ml. In the case of cells grown on a solid substrate, plates that are about 80-90% confluent are used. Each plate is washed once with 5 ml TD buffer and then trypsinized with 5 ml of a 1 : 10 dilution of trypsin solution in TD buffer containing 10 mM EDTA, prewarmed to 37°. The trypsin digestion is allowed to proceed for 5 min at room temperature, and then the cell suspension is collected with a Pasteur pipette and transferred to a conical tube containing 1 volume TD buffer with 5% calf serum, placed in ice. The cells (deriving from the trypsinized plates or from a small suspension culture) are pelleted at 500 gay for 5 min and washed three times with NKM. Before the last centrifugation, the cells are transferred to a graduated Eppendorf tube(s), so that the approximate volume of packed cells can be estimated (from five plates containing a total of 107 cells one can expect a volume of packed cells around 100 tzl). After removing the supernatant from the last washing step, the pelleted cells are placed at - 2 0 ° for 15 min in order to weaken the cell membrane and facilitate the breakage in the homogenization step. The samples are placed in ice, and the pellets are then suspended in a volume of homogenization buffer corresponding to 10 times the original volume of packed cells (vpc) and homogenized in a Thomas homogenizer (A. H. Thomas, Swedesboro, N J) of appropriate size (0 for up to 1 ml homogenate, AA for up to 4 ml, and A for up to 10 ml) with 10 strong strokes, using a pestle rotating at approximately 1500 rpm. The degree of cell breakage obtained in this way is usually around 80-90%.
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STUDYINGMITOCHONDRIALGENEEXPRESSION
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The homogenate is immediately poured into one or more Eppendorf tubes containing 2 M sucrose in an amount corresponding to one-sixth of the volume of the homogenate, mixed, and centrifuged at 1200 g to pellet nuclei, large debris, and unbroken cells. This centrifugation is repeated, and the last supernatant is transferred to a fresh Eppendorf tube(s), and centrifuged at approximately 13,000 g in a microcentrifuge for 1 min at 4°. The mitochondrial pellet(s) thus obtained is washed twice with 1 ml mitochondrial suspension buffer by centrifuging at approximately 13,000 g for 1 min, and finally suspended in 1 vpc mitochondria lysis buffer containing freshly added Tween 20 and KCI to final concentrations of 0.5% and 0.5 M, respectively. The mitochondria are lysed by pipetting up and down repeatedly until the suspension has become clear, and then by vortexing vigorously for 30 sec. The lysate is then kept on ice 5 min, and the vortexing step is repeated once in the same way. The lysate is then centrifuged for 45 min at approximately 13,000 g in the microcentrifuge at 4 °, and the clear supernatant (S-13) is collected carefully, avoiding the fluffy layer. Aliquots of the S-13 are frozen in liquid N2 and stored at - 8 0 °. The protein concentration of the S-13 preparation is determined by the method of Bradford. 25 The transcription activity of the S-13 is stable at - 8 0 ° for at least 6 months. Repeated freezing and thawing slowly reduce this activity. In Vitro Transcription Assay For the analysis of the transcription initiation and termination activities present in the S-13 of the mitochondrial lysate, we have chosen a construct (pTER) 17 that closely reproduces the known critical regions of the in vivo template, since it contains the whole promoter regions for L- and H-strand transcription and adjacent sequences, and the termination region located at the 16 S r R N A / t R N A eeu~UUR)boundary (Fig. 1). In the standard transcription experiments, for each sample to be tested, the following components are mixed in an Eppendorf tube (placed in ice): 1/zl of the template D N A (pTER) at a concentration of 1/xg//xl, 2/xl of the 25× NTP mixture, 5 tzl of the S-13 preparation to be analyzed, 25 ~1 of 2× transcription buffer, 1 ~1 of [a-32p]UTP (400 Ci/mmol, 10 mCi/ml; Amersham, Arlington Heights, IL), and DEPC-treated autoclaved, distilled water to bring the volume to 50/zl. (Note: The protein concentration has been found to be 4-6 mg/ml in the S-13 preparation of the Tween 20 mitochondrial lysate from a small-scale preparation, to be compared to a concentration of 10-12 mg/ml in the S-100 of the Tween 20 lysate from a large-scale preparation. The concentrations of lysate and D N A template 25M. M. Bradford, Anal Biochem. 72, 248 (1976).
[121
mtDNA TRANSCRIPTIONIN MITOCHONDRIALLYSATES
135
in the transcription assay may have to be adjusted in order to find the optimal ratio for each preparation. 21) The mixtures are vortexed, briefly spun in an Eppendorf microcentrifuge, and incubated at 30° for 30 min. The reaction is stopped by adding to each sample 100/zl transcription stop buffer and 20/zg yeast tRNA, and the sample is extracted with 1 volume of phenol/CHC13/isoamyl alcohol (25:24:1, v/v), T h e nucleic acids are precipitated by adding 330/zl ethanol (prechilled at - 2 0 °) and keeping the mixtures at - 8 0 ° for 30 rain; then they are pelleted by centrifuging at full speed in a microfuge at 4° for 10 min. The pellet is dissolved in 150 tzl 0.5% SDS buffer, extracted again with 1 volume of phenol/CHC13/isoamyl alcohol (25 : 24 : 1, v/v), and the nucleic acids are precipitated as described above. The final pellet is dissolved in DEPC-treated autoclaved distilled water, and the synthesized products are analyzed in a 5% polyacrylamide (acrylamide : bisacrylamide, 29 : 1)/7 M urea gel; alternatively, if so required, the transcripts are subjected to S1 protection analysis, as described below. The pellet from each transcription reaction is dissolved in 100/zl of 1 x DNase buffer, 10 units RNase-ffee DNase I is then added, and the mixture is incubated at room temperature for 20 min in order to destroy the template DNA. After phenol extraction and ethanol precipitation, the labeled R N A pellet is dissolved in DEPC-treated autoclaved distilled water, mixed with 0.2-0.4/zg of the unlabeled R N A probe (see below), and precipitated again with NaC1 and ethanol. The new pellet is carefully suspended in 20/zl of $1 hybridization buffer by pipetting up and down repeatedly. Denaturing of the sample is achieved by heating it at 80° for 10 min, and hybridization is performed at 50° for 6 to 10 hr. After the hybridization, 200 tzl of S1 digestion buffer containing 20 tzg/ml denatured salmon sperm DNA and 250 to 400 units $1 nuclease (Boehringer Mannheim, Indianapolis, IN; 400 units//zl) are added to the sample, and the mixture is incubated at 41 ° for 30 to 45 min. Finally, 55/zl of S1 stop buffer is added to the sample, and the Sl-resistant products are precipitated by adding 2.5 volumes ethanol and pelleted by centrifuging at approximately 13,000 g for 10 min at 4°C. The final pellets are suspended in the desired volume of DEPC-treated autoclaved distilled water, 1 volume of urea-dye is added, and, after denaturing at 80° for 10 min, the samples are loaded onto a 5% polyacrylamide (acrylamide : bisacrylamide, 29 : 1)/7 M urea gel in TBE and run in the same buffer for 2-3 hr at 400 V. After electrophoresis, the gel is washed twice for 10 min with autoclaved distilled water and vacuum-dried on a sheet of Bio-Rad (Richmond, CA) backing paper at 80° for 1 hr, before exposure for autoradiography. To prepare the antisense unlabeled R N A to be used as a probe for S1 analysis of the transcripts, a 50-/zl reaction mixture is set up containing the
136
STUDYING MITOCHONDRIAL GENE EXPRESSION
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following: 1.5/xg plasmid pBSAND [containing the MaeI-MaeI fragment of pTER filled in and cloned into the Sinai site of plasmid pBS KS(+) (Promega, Madison, WI)] linearized with BamHI, 10/xl of 5 X T3 transcription buffer, 0.5 mM each of ATP, CTP, GTP, and UTP, 8 mM DTT, 40 units RNase inhibitor (RNasin, Promega; 40 units//zl), and 20 units T3 RNA polymerase (Promega; 20 units//zl). The mixture is incubated at 37 ° for 90 min, and then 20 units RNase-free DNase I (Boehringer Mannheim, 10 units//zl) is added and the mixture incubated for an additional 15 min at 37 ° in order to destroy the template DNA. After phenol extraction, the synthesized RNA is precipitated by adding ammonium acetate to a final concentration of 2 M and 2.5 volumes ethanol, then recovered by centrifugation at approximately 13,000 g for 10 min. The amount of probe obtained is determined by reading the A260. A yield of 2 to 4/zg RNA per reaction is usually obtained. The results of a typical transcription experiment are shown in Fig. 2. In lane 1 (Fig. 2), containing the labeled products, two main bands are visible, one corresponding to the H-strand runoff transcripts (H) and the other to the L-strand runoff transcripts (L). Lane 2 (Fig. 2), containing the Sl-resistant products, shows two major bands corresponding to the
M
1
2
--H
710 ~
M
71o 489
489
--L
404
404
367
367
242
• 242
,90 _
ii:i:i
ii
,90
FIG. 2. Products of in vitro transcription assay using the S-13 of a Tween 20 mitochondrial lysate and EcoRI- and H1NdIII-digested pTER clone template. Lane 1 shows transcription products (H, H-strand runoff transcripts; L, L-strand runoff transcripts). Lane 2 shows S1resistant products after hybridization of the in vitro products with the MaeI-MaeI unlabeled RNA probe (Fig. 1) (R, H-strand runoff transcripts; T, H-strand terminated transcripts).
[12]
mtDNA TRANSCRIPTIONIN MITOCHONDRIALLYSATES
137
protected fragments of the H-strand run-off (R) and terminated (T) transcripts.
Detection of Mitochondrial Transcription Termination Factor by Mobility Shift Assay The protocol for the mobility shift assay of mTERF described elsewhere in this v o l u m e 24 w a s designed for the purpose of detecting and measuring the binding activity of mTERF purified by affinity chromatography. 2° In this chapter, we describe a modification of this procedure suitable for detecting the presence and estimating the relative abundance of mTERF in mitochondrial lysates derived from small-scale cell culture samples. The amount of probe per reaction has been increased in order to saturate the binding capacity of the factor; furthermore, in this modified protocol, the gel is run in the cold and is loaded with the current on to keep the dissociation of the DNA-protein complex to the minimum possible and to give sharp bands suitable for quantitation. By performing band-shift experiments with a constant amount of probe and increasing amounts of S-13 lysate, we were able to determine the conditions for a semiquantitative assay (Fig. 3). The probe used for this assay is the double-stranded 44-mer fragment described elsewhere, 24 3'-end-labeled by filling in with [a-32p]dTTP and the Klenow fragment of Escherichia coli D N A polymerase I. 26 To set up the DNA-binding reactions, for each sample to be tested, a mixture is prepared, in an Eppendorf tube placed in ice, containing the following components: 0.1 pmol probe [labeled to a specific activity of -200,000 counts/rain (cpm)/pmol], 5/zg BSA, 5/zg poly(dI-dC) • (dI-dC), 10 tzl buffer C containing 50 instead of 100 mM KC1, and the desired amount of the S-13 of the mitochondrial lysate to be tested (usually between 1 and 4 tzl). The volume is then brought to 20 to 50/zl by adding buffer C lacking KC1, with the final KCI concentration being adjusted to 100 mM by the addition of 3 M KC1, if needed. The samples are vortexed, spun down briefly, and incubated at 25 ° for 20 min. After incubation, the samples are placed in ice and then loaded onto a 5% polyacrylamide:bisacrylamide (80:1) gel in Tris-glycine buffer containing 2.5% glycerol, which had been prerun for at least 2 hr at 200 V. After loading, the gel is run in the cold at 200-250 V for 4 hr, heat-dried at 80° for 2 hr, and exposed for autoradiography. Figure 3 shows a calibration experiment carried out by incubating a constant amount of probe (0.1 pmol per reaction) with different amounts of the S-13 preparation of a mitochondrial lysate, corresponding to a range 26 F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., "Current Protocols in Molecular Biology." Wiley(lnterscience), New York, 1987.
138
STUDYING MITOCHONDRIAL GENE EXPRESSION
A
S-13 Fraction o 0.5 1.25 2.5 5 10 15 10 25
0
[12]
Prot. (Pg)
~ F r e e Probe
B 4 3 tr
2 1 0
..
0.0
,
5.0
10.0 15.0 20.0 Protein (IJg)
25.0
FxG. 3. Mobility shift assay calibration curve using the S-13 of a Tween 20 mitochondrial lysate. (A) A constant amount of the probe was incubated with different amounts of a sample of S-13, as described in the text. The specific retarded band that has been quantified in (B) is indicated by an arrow. (B) Quantification of the specific retarded band in the gel shown in (A) performed by Phosphor-Image detection and Image-Quant analysis (Molecular Dynamics).
of cell equivalents between 1 x 104 and 5 x 105. As the quantitation of the autoradiogram shows (Fig. 3b), there is a range of amounts of S-13/ pmol probe in which the signal of the specific retarded band is proportional to the amount of lysate. (Note: For each sample to be analyzed by the semiquantitative band shift assay, we usually test two different amounts of the S-13 preparation of a Tween 20 mitochondrial lysate included in the linear portion of the calibration curve. Repeated experiments using different S-13 preparations from the same cell source have given consistent results in the relative abundance of the retarded band. The minimum amount of protein of an S-13 preparation that gives quantifiable results under our experimental conditions corresponds to - 5 x 104 cells.)
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MAPPING VERTEBRATE D - L O O P PROMOTERS
139
Acknowledgments This work was supported by National Institutes of Health Grant GM-11726 (to G. A.) and scholarships from the Fulbright/Spanish Ministry of Education and Science Visiting Scholar Program (to V, M.) and from Caltech (Gosney Fellowship, to P. F.-S.).
[13] M a p p i n g By
Promoters in Displacement-Loop Vertebrate Mitochondrial DNA
Region of
G E R A L D S. S H A D E L a n d D A V I D A . C L A Y T O N
Introduction In all vertebrates examined to date, mitochondrial (mt) D N A exists and can be isolated as a small covalently closed-circular molecule that has a promoter-containing regulatory locus called the displacement-loop (Dloop) region (Fig. 1). It is in the D-loop region where leading-strand D N A replication and the majority of mitochondrial gene transcription initiates. This chapter outlines a general strategy for locating the major promoters in the D-loop region of the mitochondrial genome of vertebrate organisms. We propose two approaches for the isolation of a D-loop region-containing D N A fragment that utilize conserved nucleotide sequences that occur within or near the D-loop region of vertebrates. Once in hand, promoters can be precisely located on this template using a crude mtRNA polymerase preparation to assay promoter activity in an in vitro transcription reaction. Rather than presenting the conventional methods for mapping the 5' ends of primary transcripts and defining boundaries of promoters in detail, those aspects unique to analyzing mitochondrial promoters are emphasized. We refer to the published methods that were used to locate D-loop region promoters of human 1 and mouse 2,3 mtDNAs as models for this type of analysis. Isolation of Mitochondria and Mitochondrial DNA from Vertebrate Cells Many of the procedures outlined in this chapter require the isolation of mitochondria and mtDNA from vertebrate cells. Methods for the purifi1 D. D. C h a n g and D. A. Clayton, Cell {Cambridge, Mass.) 36, 635 (1984). 2 D. D. C h a n g and D. A. Clayton, Mot Cell. Biol. 6, 3253 {1986). 3 D. D. C h a n g and D. A. Clayton, MoL Ceil Biol. 6, 3262 (1986).
METHODS IN ENZYMOLOGY,VOL. 264
Copyright © 1996by AcademicPress, Inc. All rights of reproduction in any form reserved.