Plant Molecular Biology 6: 417-427, 1986 © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
A DNA topoisomerase type I from wheat embryo mitochondria Manuel Echeverria, Marie Th6r6se Martin, B6r6nice Ricard I & Simon Litvak Institut de Biochemie Cellulaire et Neurochimie du CNRS, 1 rue Camille Saint-Sdens, 33077 BordeauxCedex, France 1Current Address: Laboratoire de Physiologie Vdgdtale Inra-Bordeaux Pont de la Maye
Summary In order to study DNA replication and expression in wheat mitochondria our laboratory has been seeking to develop a system that supports DNA synthesis and transcription, either in isolated mitochondria from wheat embryos or in a mitochondrial lysate from the same source deprived of endogenous DNA in vitro. We have characterized some of the enzymes involved in the DNA synthesis and transcription process. In this study we describe a DNA topoisomerase activity. Broken mitochondria from wheat embryos can actively relax negatively supercoiled DNA (pBR322, pAT153, etc...). The enzyme is intramitochondrial: the activity is detected only when intact organelles are broken by non-ionic detergent. Most of the topoisomerase activity found in the broken mitochondria is recovered in the mitochondrial lysate. It is stimulated by Mg 2+ and has an optimum salt concentration, KC1 or NaC1, between 50 mM and 100 mM. ATP has no effect on this activity. Ethidium bromide, berenil, novobiocine and nalidixic acid, compounds currently used to characterize DNA topoisomerases, do not affect the relaxation of supercoiled DNA by the wheat mitochondrial activity. On the other hand N-ethylmaleimide has a strong inhibitory effect indicating that sulfhydryl groups are essential for enzyme activity. The molecular weight of the enzyme as determined by glycerol gradient sedimentation, is about 110 kd. Another important feature of the mitochondrial lysate DNA topoisomerase is the ability to relax positively supercoiled DNA, a property of eukaryotic topoisomerases I.
Introduction Mitochondrial DNAs (mt DNA) from different organisms show considerable diversity in structure and genetic organisation even though they are assumed to carry out essentially the same genetic functions. Mitochondrial DNAs from animal cells are closed circular molecules about 15 kbp in length. Organelle genome from lower eukaryotes can be circular (yeast) or linear (Paramecium, Tetrahymena) and range in size from 15 kbp to 75 kbp. Mitochondrial genomes from higher plants are unusually large and variable in size ranging from about 200 kbp in Brassica to 2400 kbp in Cucumis melo. Electron microscopic analysis of mt DNA isolated from plant tissues shows a heter-
ogeneous array of molecules, mostly linear, with a low percentage of circles ranging from 1/xm to 30 ~m in contour length (for reviews see 1-3). Within the last few years a general structure for the mitochondrial genome of plants has emerged based on restriction enzyme mapping of DNA. The first model was proposed for Brassica campestris (4). According to this model the mitochondrial genome is composed of a master circle of 218 kbp containing a 2 kbp repeated sequence and could give rise by intramolecular recombination to two smaller circular molecules of 135 kbp and 83 kbp. A similar genomic structure, involving recombination events between circular DNA species through repeated sequences, was proposed for maize (5) and wheat(3).
418 In contrast to the advances obtained in the understanding of the structure of plant mt DNA, very little is known about the enzymes involved in its replication and expression. Among these activities an important group is constituted by DNA topoisomerases, which modify the topology of DNA (6, 7). DNA topoisomerases, from prokaryotic and eukaryotic cells, have been classified into two classes: DNA topoisomerases I and II. Type I topoisomerases catalyze different reactions by introducing a single strand break on double stranded DNA in the absence of ATE They can relax negatively supercoiled DNA (eukaryotic topoisomerases I can also relax positively supercoiled DNA), knot and unknot single stranded DNA, catenate and decatenate nicked DNA. Type II topoisomerases, act by a reversible double strand break of the DNA. They can relax supercoiled DNA, knot and unknot, catenate and decatenate intact double stranded DNA. The prokaryotic topoisomerase II, DNA gyrase of Escherichia coli is the best example, can also supercoil DNA. Most of type II topoisomerase activities require ATE We have begun to characterize different enzyme associated with DNA replication and expression in wheat mitochondria. We have already described a specific mitochondrial DNA polymerase (8, 9). Here we demonstrate the presence of a DNA topoisomerase in whole mitochondria and a mitochondrial lysate from wheat embryos. The properties of this enzyme are compared to those of a DNA topoisomerase I most probably a nuclear enzyme extracted from a wheat germ high speed supernatant (10-12).
Ethidium bromide, nalidixic acid, novobiocine and N-ethylmaleimide were from Sigma. Berenil was from Hoechst. Polynucleotides were from Sigma and Boehringer-France. pBR322 and pAT153 were prepared from Escherichia coli strain JM101 (13). Wheat seeds (variety Cesar) were obtained from the Agronomical Center, La BrosseMonceaux, France. Wheat germ topoisomerase I, purified according to Dynan, W. S. et al (10) was purchased from Genofit.
Isolation of mitochondria from wheat embryos The preparation of wheat embryos and the purification of mitochondria have been described previously (8). Essentially, the crude homogenate obtained from imbided wheat embryos is subjected to differential centrifugation. The washed mitochondrial pellet is then resuspended in the appropriate buffer and sedimented through a discontinuous sucrose gradient. The band corresponding to intact mitochondria is recovered, diluted and submitted to 18000 g centrifugation. The purified mitochondrial pellet is either resuspended (8) or stored at -20°C.
Preparation of the mitochondrial lysate The preparation of the mitochondrial lysate was as described in a previous paper (9) except for the addition of bovine serum albumin (BSA) (0.05% w/v) in the extraction buffers to increase enzyme stability. Lysates usually contained 20-25 mg/ml of protein, determined by the method of Lowry et al (14).
Glycerol gradient sedimentation The (NH4)2SO 4 precipitate obtained as described for the mitochondrial lysate preparation (9) was resuspended in 1 ml of buffer A (10 mM Tris-HCl pH 8.0, 1.5 mM Mg acetate, 15 mM KC1, 0.1 mM EDTA, 1 mM 2-mercaptoethanol) and dialyzed for 2 h against 400 ml of buffer B (10 mM Tris-HC1 pH 8, 15 mM KC1, 0.1 mM EDTA, 1 mM 2-mercaptoethanol and 15% glycerol). The dialysate contained 7 - 8 mg of protein/ml. Then 30/A of buffer C (50 mM Tris-HC1 pH 7.5, 300 mM KC1 mM EDTA, 1 mM 2-mercaptoethanol) were added to 170 #1 of the dialysate containing 1 mg of protein. The 200/zl sample was layered on a 3.6 ml 15%-30% continuous glycerol gradient prepared with buffer C. Centrifugation was carried out in a TST 60 rotor at 58000 rpm for 15 hours. The gradient was resolved into 50 fractions of 75 ~tl each and the assay for topoisomerase was carried out immediately using a 4/A aliquote per assay. Parallel gradients were run with gammaglobulin, bovine serum albumin, ovalbumin and cytochrome C as
419 molecular weight markers; their sedimentation profile was determined by measuring the optical absorbance.
Results Presence of an intramitochondrial topoisomerase activity
Unless otherwise indicated reactions were carried out in 25/A containing 50 mM Tris-HCl pH 8.0, 50 mM KCI, 15 mM magnesium acetate, 1 mM dithioerythritol and 1/zg of supercoiled plasmid (pBR322 or pAT153). Reaction was started by the addition of mitochondrial extract (10 to 50/zg of protein) or 0.25 to 2 units of wheat germ topoisomerase I. Incubation was for 20 min at 26 °C and the reaction was stopped by the addition of 5 #1 of 100 mM EDTA, 0.5o/o sodium dodecyl sulfate, 0.125% bromophenol-blue and 30% glycerol. Samples were then heated 2 min at 65 °C and loaded on to a 1% agarose gel cast in a horizontal slab apparatus. The electrophoresis buffer contained 40 mM Tris-HCl, pH 8.1, 2 mM EDTA and 20 mM sodium acetate. Electrophoresis was carried out for 15 hours at 40 mA. The gel was then stained for 20 min with ethidium bromide (1/~g/ml) and the UV fluorescent material was photographed. Other enzyme activities
Glucose 6-phosphate dehydrogenase was measured as in (15). Isocitrate dehydrogenase was assayed by the method of Ochoa (16). Succinate dehydrogenase was assayed by the method of Singer et al (17). Lipoxygenase activity was assayed as in (18). DNA polymerase A was measured as described by Castroviejo et al (19) using poly rAoligo dT12_~8 as template. Under these conditions DNA polymerase A is the only DNA polymerase from wheat germ that can use this template. DNA polymerase B and C were assayed as described in (19) using calf thymus activated DNA as template in the presence of ddTTP (ddTTP/TTP = 20). ddTTP, an analog of TTP, inhibits DNA polymerase A and mitochondrial DNA polymerase. Mitochondrial DNA polymerase was assayed as in (9), using poly dA-oligo dT12_18 as template in the presence of aphidicoline (20 tzg/ml), an inhibitor of wheat DNA polymerases B and C.
A DNA topoisomerase activity, ATPindependent, is easily detected in wheat embryo mitochondrial fractions, i.e. broken mitochondria and mitochondrial lysate, as evidenced by the relaxation of a supercoiled plasmid (Fig. 1 I, lanes A and B). Most of the mtDNA topoisomerase activity (87%) detected in total mitochondrial extracts is recovered in the mitochondrial lysate. No activity is detected in the membrane pellet obtained by high salt and detergent treatment of purified mitochondria (Fig. 1 I, lanes C). Mitochondrial activity amounts to 0.5-1% of the total ATP-independent topoisomerase activity detected in a wheat embryo homogenate. To confirm the mitochondrial origin of the topoisomerase activity detected in mitochondrial extracts, organelles were extensively washed and purified by sucrose gradient centrifugation. Table 1 shows different marker activities detected on the different fractions throughout mitochondria purification. Cytoplasmic marker enzymes are not detected on the purified mitochondria. Glucose 6-phosphate dehydrogenase, a cytosolic activity, is barely detectable in purified mitochondrial fractions (Table 1), and represents less than 0,04% of the total activity present in wheat embryo crude extracts (data not shown). This is 12,5 to 25 fold less than the amount of topoisomerase activity attributed to mitochondria (0.5%-1%). DNA polymerases A, B and C, associated with nuclear DNA replication (19) are also absent from the mitochondrial fraction (Table 1). On the other hand the purified mitochondrial fraction is enriched for a specific mitochondrial DNA polymerase activity (Table 1). The apparent decrease of the activity observed in washed mitochondria can be explained by the fact that the assay for mitochondrial DNA polymerase is not absolutely specific (DNA polymerase A also shows some activity under these conditions (19)), and also because purification on sucrose gradients causes some damage to organelles (see below). The purified mitochondrial fraction is also enriched for a succinate dehydrogenase activity, associated with the internal mitochondrial membrane, while isocitrate dehydrogenase which is as-
421 sociated with the mitochondrial matrix is enriched in washed mitochondria but diminished in sucrose gradient purified organelles (Table 1). This is explained by the fact that after surcrose gradient sedimentation approximately 50°70 of mitochondria are damaged as evaluated by electron microscopy and integrity tests (data not shown). To discard plastidial contamination, we measured different markers. Lipoxygenase, associated with immature plastids (18), is absent in pure mitochondria (Table 1). The absence of detectable plastid contamination was confirmed by analysis of carotenoid absortion spectra in the different fractions and electron microscopy studies (data not shown). From these studies we concluded that the topoisomerase activity detected in the purified mitochondria fraction was devoid of contaminating extramitochondrial activity. In order to prove unambiguously the intra-mitochondrial localization of this DNA topoisomerase, we measured this
activity on intact and broken wheat embryo mitochondria (Fig. 1, II). In intact mitochondria (washed mitochondria) no topoisomerase activity is detected (Fig. 1 II, A) even at the highest protein concentration tested, while in detergent treated organelles (Fig. 1 II, B) a strong activity is easily detected at an equivalent protein concentration. Triton-X-100 does not stimulate the mitochondrial DNA topoisomerase activity. Thus, DNA topoisomerase is detectable only when mitochondria are disrupted, indicating an intramitochondrial localization.
Characterization of the mitochondrial DNA topo&omerase activity The topoisomerase activity present in the mitochondrial lysate was characterized and compared to a topoisomerase I activity isolated from the post-mitochondrial supernatant of wheat germ (10-12).
Table 1. Marker activities throughout mitochondria purification.* Activity
Washed mitochondria ( U n i t s / m g protein)
Glucose-6-phosphate dehydrogenase Succinate dehydrogenase lsocitrate dehydrogenase Mitochondrial D N A polymerase ~a~ D N A polymerase A a D N A polymerase B + C a Lipoxygenase
0.27 ND 0.08 150 120 180 996
0.05 0.68 0.10 70 20 10 144
0.012 1.15 0.06 70 ND ND ND
* Assay were carried out as described in Methods. Unit definition: Glucose-6-phosphate dehydrogenase (340 nm), succinate dehydrogenase (578 nm) and isocitrate dehydrogenase (334 nm): D O / m i n u t e . Lipoxygenase: u a t o m s O2/minute. D N A polymerase: pmoles d N T P incorporated/hour. a T h o u g h the assays are done under the o p t i m u m conditions for each type of D N A polymerase they are not absolutely specific for each activity (see methods and ref. 19). ND: not detected.
Fig. 1. Topoisomerase activity in mitochondrial fractions. I. The assay was carried out as described in Methods using 1 /~g of pBR322 and increasing a m o u n t s of mitochondrial extracts. F I represents supercoiled D N A and F II represents nicked circular DNA. 0) No protein added. A) Broken mitochondria: mitochondria purified on a discontinuous sucrose gradient were added to the reaction mixture containing 0,2°70 Triton-X-100 and no surcrose. Total volume fraction = 3 ml. B) Mitochondrial lysate: fraction was diluted 1/10 in buffer A just before the assay. Total volume fraction = 0.13 ml. C) Membrane pellet obtained after high salt and detergent treatment of purified mitochondria during lysate preparation (see methods). Total volume fraction = 1 ml. Equivalent activities in total mitochondrial extracts and lysate were determined by gel scanning. II. The enzyme activity was assayed as described in Methods except for the addition of 0,44 M sucrose, to maintain the osmolarity of the reaction mixture. The suspension of washed mitochondria contained 1 m g of protein/ml. Increasing a m o u n t s of protein were added: 0) No protein added. A) Intact mitochondria: suspension of washed mitochondria. B) Broken mitochondria: suspension of washed mitochondria treated with 0.2070 Triton X-100.
Fig. 2. Sedimentation of mitochondrial topoisomerase activity on a continuous 15- 30°70 glycerol gradient. A) The experimental conditions for the glycerol gradient and the activity are described in Methods. Each assay was performed using 1 #g of pAT153 and 4/zl of each fraction. Since the gradient contained 300 mM KC1, it was not necessary to add it to the reaction mixture. Arabic numbers represent gradient fractions while roman numbers represent pooled fractions.
Bovi.ne serum albumin Ovoal bumin
Molecular weight determination by glycerol gradient sedimentation Figure 2 shows the distribution profile of the mitochondrial topoisomerase activity on a 15°70-30°70 continuous glycerol gradient. The activity is found in the middle of the gradient, centered on fractions 25-27. This corresponds to a protein molecular weight of about 110 kd. To clearly distinguish topoisomerase activity from any 'nicking' activity of the mitochondrial lysate which could also relax supercoiled DNA, we analyzed the products of the reaction catalyzed by the pooled fractions of the gradient by electrophoresis on an agarose gel
Cytochrome C i
B) Parallel gradients were run with protein markers as described in Methods to estimate the molecular weight of the mt D N A topoisomerase. RF is the ratio between the gradient migration of a given protein and the total glycerol gradient volume.
Fig. 3. Analysis of the products of mitochondrial topoisomerase reaction on an agarose gel containing ethidium bromide.
The reaction was carried out as usual with 4/zl of each pooled fraction (A) or 10 t~g of protein from the mitochondrial lysate (B). 1 /~g of pAT153 was used as substrate. Once the enzymatic reaction was stopped, the products were separated by electrophoresis in a 1~/0agarose gel in the presence of 0.05 tzg/ml of ethidium bromide. In this case F I represents positively supercoiled DNA. 0 = no protein added. containing ethidium bromide (Fig. 3). In the presence of ethidium bromide the product of a topoisomerase activity, a closed relaxed circular DNA, will become positively supercoiled, while the product of a nicking enzyme, a relaxed circular nicked DNA, unable to gain positive superturns upon dye intercalation, will remain relaxed. Thus the two kinds of products can be easily distinguished in a gel containing ethidium bromide. Figure 3 confirms that the relaxation of the supercoiled plasmid catalyzed by the pooled fractions of the gradient is due to a topoisomerase activity. This is evidenced by the fastest migrating band which corresponds to positively supercoiled DNA. Most
of activity is found in pools III to V (Fig. 3, lanes A). This topoisomerase activity is clearly distinguished from some nicking activity also present in the mitochondrial lysate evidenced by the slow migrating band corresponding to F II in the gel (Fig. 3, B). The nicking activity of the mitochondrial lysate is separated from topoisomerase activity in the glycerol gradient, most of it been found in pools I and II (Fig. 3, A). The intermediate band observed in pools I and VII corresponds to positively supercoiled D N A generated by intercalation of ethidium bromide in the negatively supercoiled D N A used as substrate, not relaxed either by the topoisomerase or the nicking activity (compare
424 with the migration of the control plasmid on lane 0).
topoisomerase activity of the mitochondrial lysate (data not shown).
Substrate and salt requirement for mitochondrhTl DNA topoisomerase activity
Effect of inhibitors
The mitochondrial activity is absolutely dependent on Mg 2+ with an optimum close to 15 mM (Fig. 4, A). The same dependence is observed for the Wheat germ topoisomerase I activity (Fig. 4,
B). The mitochondrial enzyme requires KC1 between 50 mM and 100 mM. Higher concentrations are inhibitory, NaCI can replace KC1 (data not shown). A similar dependence on ionic strength is observed for the cytosolic topoisomerase activity (our own data and ref. 10). An interesting observation concerning the wheat embryo mitochondrial topoisomerase is that ATP, a compulsory substrate for many reactions of type II topoisomerases, has no effect on the DNA
We have studied the effect of some inhibitors currently used to characterize DNA topoisomerase activities from different sources (Table 2). Ethidium bromide and berenil, two trypanocidal drugs, which have been reported to inhibit mitochondrial DNA replication (20, 21), have no effect on the activity of the mitochondrial and extramitochondrial wheat germ topoisomerase I in the concentration range studied, the inhibition by ethidium bromide is detectable only at higher concentration. On the other hand N-ethylmaleimide, which blocks sulfhydryl groups of proteins, strongly inhibits both activities. Nalidixic acid and novobiocine, inhibitors of topoisomerases of type II, have no effect on both the mitochondrial and extramitochondrial activities.
Fig. 4. Effect of Mg 2+ on mitochondrial topoisomerase activity (pool IV of the glycerol gradient) and wheat germ topoisomerase I (WGTPI). The reaction was done as usual with 4/~1 of Pool IV fraction (A) or 0.25 units of WGTPI (B). These are comparable amounts of activity. One unit of activity is defined as the amount of enzyme required to convert 1 #g of F I to F II pBR322 DNA in 30 min at 37 °C.
425 Table 2. Effect of inhibitors on MT D N A topoisomerase and wheat germ topoisomerase I activity.* Inhibitor
Ethidium bromide 1 t~M 2.5 /zM 5 /~M Berenil 5 ~M 10/xM N-ethylmaleimide 0.5 m M 1 mM 2 mM Novobiocine 250 ~ g / m l Nalidixic Acid 100/zg/ml 300 p,g/ml
MT D N A topoisomerase (°7o Activity)
100 100 60 - 70
100 100 60 - 70
75 3 0 - 40 0
75 30 - 40
* Assays were carried out as described in Methods. The enzyme was preincubated for 5 minutes at 0 ° C with the inhibitor, and the reaction was started by addition of 1 #g of pBR322. The degree of inhibition was estimated by comparison with a serial dilution on the same gel.
Mitochondrial topoisomerase can relax positively supercoiled DNA A characteristic of eukaryotic DNA topoisomerases of type I that allow the distinction with the prokaryotic type I activities is their capacity to remove positive superhelical turns. To prepare a positively supercoiled plasmid we incubated pBR322 with ethidium bromide at a concentration high enough to make it positively supercoiled (22, 23). Figure 5 shows that the mitochondrial topoisomerase activity can completely relax the positively supercoiled pBR322. The same result has been previously observed with the wheat germ topoisomerase I (10).
We have described a DNA topoisomerase activity present in wheat embryo mitochondria able to relax negative and positive supercoiled DNA. It represents about 1°70 of the ATP-independent
Fig. 5. Relaxation o f positively supercoiled plasmid by the mitochondrial and wheat germ topoisomerase I activity. The assay was carried out in the reaction mixture containing the indicated a m o u n t of ethidium bromide and 0.05 ~tg of pBR322. The reaction was started by the addition of 10 #g of mitochondrial lysate (A), 2 units of W G T P I (B), or no protein (0).
426 wheat germ DNA topoisomerase activity. This is similar to the amount of mitochondrial topoisomerase I present in other systems (24, 25). The absence of extramitochondrial marker activities in our purified mitochondrial fraction and the fact that topoisomerase activity is detected only in broken organelles eliminates the possibility that the activity we detect is due to contamination by a cytosolic topoisomerase and points instead to the intramitochondrial localization of the enzyme. The wheat mitochondrial enzyme has the properties of a type I eukaryotic topoisomerase and is indistinguishable from a topoisomerase of type I isolated from the post-mitochondrial supernatant of wheat germ (10-12). The topoisomers obtained by treatment of a supercoiled plasmid with the mitochondrial activity are identical to those obtained with the wheat germ topoisomerase I, which changes the linking number by steps of one. Like the post-mitochondrial topoisomerase, the wheat mitochondrial enzyme has a molecular weight of about 110 kd, is ATP-independent and can relax positively supercoiled DNA. We have used different inhibitors to distinguish the mitochondrial from the cytosolic activity, no differences were found between the two enzymes. Moreover the mitochondrial and the extramitochondrial activity show the same dependence to ionic strength and both require Mg 2+ for optimal activity. It is interesting to point out that the cytosolic enzyme was originally described as a Mg2+-independent activity (10); our results indicate that the magnesium dependence of this enzyme is observed at low enzyme concentrations while raising the enzyme concentration masks the Mg 2÷ stimulation. The magnesium dependence of topoisomerase I activity has been used as a criteria to distinguish prokaryotic from eukaryotic enzymes. Nevertheless this criteria is far from absolute. The effect of Mg 2÷ on eukaryotic topoisomerase I is variable depending on the enzyme source, the purification procedure and the assay conditions (24, 30, 31). A more significant result that relates our plant mitochondrial topoisomerase to the eukaryotic group is its ability to relax positively supercoiled DNA. Prokaryotic type I activities can relax only negatively supercoiled DNA (6, 7). In conclusion the wheat embryo mitochondrial topoisomerase corresponds to an eukaryotic type I
activity, similar to the cytosolic enzyme from the same source. Most mitochondrial proteins are coded in the nuclear genome, synthesized in the cytosol and then transferred into the mitochondria, similar mitochondrial and nuclear type I topoisomerase have been isolated from Xenopus laevis oocytes (24). Also the results obtained with a topoisomerase I isolated from human acute leukemia cell mitochondria show no difference to nuclear activities (25). However in the case of rat liver the mitochondrial type I topoisomerase been distinguished from the nuclear enzyme by ethidium bromide and berenil inhibition (21, 26). This difference can be explained by a partial proteolysis of the enzyme, as suggested by the low molecular weight (44 kd) of the rat liver mitochondrial enzyme characterized (30, 31). An interesting point is that a DNA topoisomerase I, isolated from spinach chloroplasts by Siedlecki et al (27), is unable to relax positively supercoiled DNA. This is a typical property of a prokaryotic topoisomerase I. The involvement of DNA topoisomerase in animal mitochondrial DNA replication has been implied mainly from topological changes observed during its replication cycle (28, 29). The presence of the topoisomerase I activity in wheat embryo mitochondria can be particularly relevant to the general mechanism of mtDNA replication regarding the proposed structure of higher plant mitochondrial genome: a master circle able to give origin to multiple circular molecules by intramolecular recombination through repeated sequences.
Acknowledgements This work was supported by the CNRS (Action Th6matique Programm6e 'Biologie Mol6culaire V6g6tale' M S / D N 207) and by the Universit6 de Bordeaux II. We are very grateful to Drs. Michel Duguet and Gilbert Brun (Paris) for helpful discussions and to Dr. Laura Tarrago-Litvak for comments on the manuscript.
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Received 10 December 1985; in revised form 26 February 1986; accepted 18 March 1986.