Complete Repopulation of Mouse Mitochondrial DNA-less Cells With Rat Mitochondrial DNA Restores Mitochondrial Translation but Not Mitochondrial Respiratory Function

Corresponding author: Jun-Ichi Hayashi, Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan., jih45{at}sakura.cc.tsukuba.ac.jp (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

By the fusion of mtDNA-less (⁰) cells of Mus musculus domesticus with platelets from different species, mtDNA repopulated cybrids were obtained for finding the mtDNA species that could induce mitochondrial abnormalities. Expression of mitochondrial dysfunction might be expected in these cybrids due to incompatibility between nuclear and mitochondrial genomes from different species. The results showed that mouse ⁰ cells could receive mtDNA from a different mouse species, M. spretus, or even mtDNA from the rat, Rattus norvegicus, and that the introduced rat mtDNA, but not M. spretus mtDNA, caused mitochondrial dysfunction, even though rat mtDNA could restore normal mitochondrial translation in the cybrids. Considering that mitochondrial respiratory complexes consist of nuclear DNA- and mtDNA-coded polypeptides, these observations suggest that the nuclear and mitochondrial interactions required for replication, transcription, and translation of introduced rat mtDNA must be less stringently controlled than those required for formation of normal respiratory complexes. As no procedure for introduction of mutagenized mouse mtDNA into living cells has yet been established, these findings provide important insights into generating mtDNA-knockout mice.

MITOCHONDRIAL DNAs (mtDNAs) with point mutations in the mitochondrial tRNA genes and with large-scale deletions including several mitochondrial tRNA genes have been shown to be closely associated with mitochondrial encephalomyopathies (for reviews, see LARSSON and CLAYTON 1995 ; WALLACE 1999 ). Besides the clinically distinct syndromes of mitochondrial diseases, many kinds of mtDNA mutations have been identified in association with human aging and with various age-associated disorders including diabetes and neurodegenerative diseases (LARSSON and CLAYTON 1995 ; WALLACE 1999 ). Although the pathogenicities of these mtDNA mutations were proved by cotransmission of the mutant mtDNAs and respiration deficiency to mtDNA-less (⁰) human cells (CHOMYN et al. 1991 ; HAYASHI et al. 1991 , HAYASHI et al. 1994 ; KING et al. 1992 ; TROUNCE et al. 1994 ), there is as yet no convincing evidence to explain whether accumulation of these pathogenic mutant mtDNAs in tissues is responsible for the expressions of various clinical phenotypes of mitochondrial diseases.

Establishment of mtDNA-knockout mice could provide a model system for studying exactly how pathogenic mutant mtDNA is transmitted and distributed in tissues, resulting in the pathogenesis of mitochondrial diseases that show various clinical phenotypes. However, no procedures are available for introduction of mutagenized whole mouse mtDNA into mitochondria in living cells or even into isolated mitochondria. On the other hand, introduction of mtDNA from different species could be attained by the use of cell fusion techniques. Considering that the nuclear and mitochondrial genomes in eukaryote species have evolved harmoniously and that various nuclear DNA-encoded factors are required for the expression of mtDNA-encoded polypeptides and the subsequent assembly into respiratory complexes (for review, see ATTARDI and SCHATZ 1988 ), introduction of mtDNA from different species into mouse somatic cells or fertilized eggs by cell fusion techniques could reduce mitochondrial respiratory function, probably due to the incompatibilities of the nuclear and mitochondrial genomes of different species (HAYASHI et al. 1983 ). Therefore, this seems to be the only way at present to generate mtDNA knock-out mice with mitochondrial diseases.

Recently, we isolated ⁰ cells from various mouse cell lines (INOUE et al. 1997A , INOUE et al. 1997B ). This enabled us to examine whether they could receive mtDNA from different species in the absence of influence of host mouse mtDNA, or whether such chimeric cybrids with nuclear and mitochondrial genomes of different species showed reduced mitochondrial respiratory function. The results showed that cybrids with mtDNA from Mus spretus or even from the rat, Rattus norvegicus, could be isolated. Chimera cybrids with M. spretus mtDNA showed normal mitochondrial respiratory function. However, when mtDNA was introduced from the less-related species R. norvegicus, the resultant chimera cybrids showed very low mitochondrial respiratory activity, even though rat mtDNA could replicate and induce normal translation of rat mtDNA-coded polypeptides. Therefore, generation of mice with rat mtDNA could be equivalent to generation of mtDNA-knockout mice.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cells and cell culture:
Mouse ⁰ B82 cells (INOUE et al. 1997B ) resistant to BrdUrd (BrdUrd^r) and mtDNA repopulated cybrids from various species were grown in normal medium: RPMI1640 (Nissui Seiyaku, Tokyo) containing 10% fetal calf serum, 50 ng/ml uridine, and 0.1 µg/ml pyruvate.

Isolation of platelets and introduction of mtDNA into mouse ⁰ B82 cells:
Introduction of platelet mtDNA into ⁰ B82 cells was carried out by the fusion of platelets with ⁰ B82 cells in the presence of 50% (w/v) polyethylene glycol (PEG) as described previously (ITO et al. 1999 ). The fusion mixture was cultivated in selection medium RPMI1640 without pyruvate and uridine, in which even cybrids with very low COX activity have been shown to grow (ISOBE et al. 1998 ).

Southern blot analysis of mtDNA:
Total cellular DNA (1–2 µg) extracted from 2 x 10⁵ cells was digested with the restriction enzyme HindIII or BamHI (Nippon Gene, Japan), and restriction fragments were separated in a 1% agarose gel, transferred to a NYTRAN membrane, and hybridized with [-³²P]dATP-labeled mouse or rat mtDNA. Radioactivities of fragments were measured with a bioimaging analyzer, Fujix BAS 5000 (Fuji Film, Japan).

Northern blot analysis of transcripts of mtDNA:
Total cellular RNA was extracted with an ISOGEN RNA isolation kit (Nippon Gene, Toyama, Japan). Total denatured RNA (10 µg) was subjected to electrophoresis in a 1% agarose gel containing formaldehyde and then transferred to a NYTRAN membrane. The membrane was hybridized with [-³²P]dATP-labeled probes of whole rat mtDNA.

Analysis of mitochondrial translation products:
Mitochondrial translation products were labeled with [³⁵S]methionine as described previously (INOUE et al. 1997A ). Proteins in the mitochondrial fraction were separated by 0.85% SDS, 12% polyacrylamide gel electrophoresis. For quantitative estimation of [³⁵S]methionine-labeled polypeptides, the dried gel was exposed to an imaging plate for 12 hr and the radioactivities of polypeptides were measured with a bioimaging analyzer.

Measurements of oxygen consumption and mitochondrial respiratory complex activities:
The rate of oxygen consumption was measured by trypsinizing cells, incubating the suspension in phosphate-buffered saline, and recording oxygen consumption in a polarographic cell (1.0 ml) at 37° with a Clark-type oxygen electrode (Yellow Springs Instruments, OH; KANEKO et al. 1996 ). For biochemical analyses of mitochondrial respiratory chain complex activities, cells in log-phase growth were harvested, and complex I + III and complex IV activities were measured as described previously (MIYABAYASHI et al. 1984 , MIYABAYASHI et al. 1989 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Characterization of mouse ⁰ B82 cells with respect to acceptance of mouse mtDNA:
As mouse ⁰ B82 cells (INOUE et al. 1997B ) are derived from M. m. domesticus, we carried out their mtDNA repopulation using species related to M. m. domesticus as mtDNA donor to find the mtDNA species that could induce abnormalities in mitochondrial respiratory functions. mtDNA was transferred by fusing ⁰ B82 cells with platelets and isolating the resultant cybrids. Before transferring mtDNA from different species, repopulation of ⁰ B82 cells with mtDNA of M. m. domesticus was carried out to exclude the following possibilities: first, that the ⁰ B82 cells were mutant cells unable to allow replication of mtDNA as in the case of the mouse embryos disrupted gene for mitochondrial transcription factor A (Tfam; LARSSON et al. 1998 ); second, that the apparent mtDNA repopulated ⁰ B82 cells, i.e., cybrids, were revertant B82 cells containing sufficient recovered internal B82 mtDNA, which initially might have remained in such a small amount that it could not be detected by PCR and Southern blot analysis (INOUE et al. 1997B ) in the early stage of their clonal isolation; third, that drug treatment for excluding mtDNA from parental B82 cells (INOUE et al. 1997B ) might have left some heritable lesions in nuclear-coded factors involved in the expression of mitochondrial respiration function.

We isolated a respiration-competent cybrid clone CyMmd with exogenously imported mtDNA from the same species as that of the nuclear genome, i.e., M. m. domesticus (Table 1), by fusing respiration-deficient ⁰ B82 cells with platelets from an old inbred B6 strain of mice in the presence of PEG followed by nutritional selection without pyruvate and uridine to exclude unfused respiration-deficient ⁰ B82 cells (Table 1). Since no colonies were grown from the fusion mixtures in the absence of PEG, the possibility of isolation of revertant B82 cells with recovered host B82 mtDNA as the apparent cybrids could be excluded. Southern blot analyses of BamHI fragments showed that CyMmd cybrids contained mtDNA of M. m. domesticus (Fig 1), suggesting that ⁰ B82 cells retained the ability to receive exogenously introduced mouse mtDNA and allow its replication. Finally, restoration of mitochondrial translation activity indicated by [³⁵S]methionine labeling of mitochondrially synthesized polypeptides (Fig 2) and the resultant restoration of mitochondrial respiration activity (Fig 3) were also observed in CyMmd cybrids. Therefore, ⁰ B82 cells did not receive any heritable lesions in nuclear DNA-coded factors required for the expression of normal mitochondrial respiration on drug treatment for mtDNA depletion, and thus CyMmd cybrids could be used as positive controls in the following experiments.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Identification of mtDNA species in cybrid clones by Southern blot analysis of BamHI restriction fragments. mtDNA of M. m. domesticus gave an 8-kbp fragment, whereas mtDNA of M. spretus gave a 16-kbp fragment. Lanes: B82, B82 cells; Ms, mtDNA prepared from liver of M. spretus; 0, 0 B82 cells; Mmd, a cybrid clone CyMmd with mtDNA from M. m. domesticus; Ms1 and Ms2, cybrid clones CyMs1 and CyMs2 with mtDNA from M. spretus.

View larger version (135K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Analysis of mitochondrial translation in cybrid clones by SDS-polyacrylamide gel electrophoresis. Lanes: B82, B82 cells; 0, 0 B82 cells; Mmd, a cybrid clone CyMmd with mtDNA from M. m. domesticus; Ms1 and Ms2, cybrid clones CyMs1 and CyMs2 with mtDNA from M. spretus. HeLa, HeLa cells. ND5, COI, ND4, Cytb, ND2, ND1, COII, COIII, ATP6, ND6, ND3, ATP8, and ND4L are polypeptides assigned to mtDNA.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Comparison of mitochondrial respiration activities in cybrids with mtDNA from M. m. domesticus and cybrids with mtDNA from M. spretus. (A) Complex I + III activity; (B) complex IV activity; (C) O2 consumption. B82, B82 cells; 0, 0 B82 cells; Mmd, a cybrid clone CyMmd with mtDNA from M. m. domesticus; Ms1 and Ms2, cybrid clones CyMs1 and CyMs2 with mtDNA from M. spretus.

Isolation of cybrids with mtDNA from M. spretus:
We previously found that mouse cybrids with the nuclear background of M. m. domesticus but with mtDNA from the different subspecies M. m. molossinus showed completely restored mitochondrial respiratory function (INOUE et al. 1997A ). In this study, transfer of mtDNA to ⁰ B82 cells was carried out using platelets from the different species M. spretus, which belongs to the same genus Mus, and is sufficiently related to make interspecies F₁ hybrids. After ⁰ B82 cells possessing the nuclear background of M. m. domesticus were fused with platelets from M. spretus in the presence of PEG, colonies grown in selective medium without pyruvate and uridine were isolated as cybrid clones CyMs1 and CyMs2 (Table 1). No colonies were obtained from fusion mixtures in the absence of PEG.

Southern blot analysis of BamHI fragments showed that CyMs1 and CyMs2 cybrids contained M. spretus mtDNA, its amount being comparable to that in CyMmd cybrids (Fig 1). These CyMs cybrids also showed normal activities of mitochondrial translation (Fig 2), mitochondrial respiratory complex I + III, complex IV, and of O₂ consumption (Fig 3, A–C). Therefore, the CyMs cybrids isolated by interspecies mtDNA transfer from M. spretus (Table 1) are normal in mtDNA content, mitochondrial gene expression, and mitochondrial respiratory functions, reflecting the absence of extensive interspecies incompatibility between the nuclear and mitochondrial genomes from M. m. domesticus and M. spretus. Normal activities of mitochondrial respiratory complexes were also observed in tissues of congenic strain B6mt^spr mice, which are completely equivalent to CyMs cybrids in possessing the nuclear genome of M. m. domesticus and mitochondrial genome of M. spretus (data not shown).

Isolation of cybrids with mtDNA from rats:
Since the available species most closely related to M. m. domesticus outside the genus Mus is Rattus norvegicus (rats), platelets from Wistar strain rats were fused with ⁰ B82 cells (Table 1). Small colonies were grown in selection medium without pyruvate and uridine and were isolated as cybrid clones CyRn (CyRn1 and CyRn2). However, their growth was recovered in normal medium with pyruvate and uridine. Using these CyRn cybrids, we examined whether rat mtDNA causes significant reduction in mitochondrial respiratory function in mouse ⁰ B82 cells or whether it does not replicate effectively to provide a sufficient amount of rat mtDNA in the cells.

Southern blot (Fig 4A) and Northern blot (Fig 4B) analyses of CyRn cybrids unambiguously showed that they possessed sufficient rat mtDNA and its transcripts. Moreover, the translation activity in mitochondria deduced from [³⁵S]methionine incorporation was restored in CyRn cybrids with rat mtDNA to a level comparable to that in CyMmd cybrids with mouse mtDNA (Fig 5). However, their activities of complex I + III and complex IV and O₂ consumption were simultaneously reduced to only 20–50% of those in CyMmd cybrids (Fig 6). Furthermore, CyRn cybrids showed significant reduction of growth in selection medium without pyruvate and uridine.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Identification of rat mtDNA and its transcripts in cybrid clones CyRn1 and CyRn2. Lanes: L6TG, rat myoblast L6TG cells; 0, 0 B82 cells; Mmd, a cybrid clone CyMmd with mtDNA from M. m. domesticus; Rn1 and Rn2, cybrid clones CyRn1 and CyRn2 with mtDNA from R. norvegicus. (A) Southern blot analysis of HindIII restriction fragments. mtDNA of R. norvegicus gave 6.5, 4.1, 2.6, and 2.1 kbp fragments. As Southern blot analysis of HindIII restriction fragments was carried out using a probe of rat mtDNA, mouse mtDNA in CyMmd was not hybridized with rat mtDNA probe. (B) Northern blot analysis. Whole rat mtDNA was used as a probe. The low signal for CyMmd would be due to a weak cross-reaction of the probe to mouse mitochondrial RNAs. 16S and 12S indicate 16S rRNA and 12S rRNA, respectively.

View larger version (127K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Analysis of mitochondrial translation in cybrid clones with R. norvegicus mtDNA by SDS-polyacrylamide gel electrophoresis. Lanes: L6TG, rat myoblast L6TG cells; 0, 0 B82 cells; Mmd, a cybrid clone CyMmd; Rn1 and Rn2, cybrid clones CyRn1 and CyRn2. HeLa, HeLa cells. ND5, COI, ND4, Cytb, ND2, ND1, COII, COIII, ATP6, ND6, ND3, ATP8, and ND4L are polypeptides assigned to mtDNA. Note that the mobilities of the ND2 polypeptide were slightly different in the mouse and rat.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Comparison of mitochondrial respiration activities in cybrids with mtDNA from M. m. domesticus and R. norvegicus. (A) Complex I + III activity; (B) complex IV activity; (C) O2 consumption rate. B82, B82 cells; 0, 0 B82 cells; Mmd, a cybrid clone CyMmd; Rn1 and Rn2, cybrid clones CyRn1 and CyRn2.

These observations suggest that exogenously introduced rat mtDNA could be replicated and could provide normal translation of rat mtDNA-encoded polypeptides in mitochondria of mouse ⁰ cells possessing only the mouse nuclear genome. However, these cybrids were not respiration competent, since the introduced rat mtDNA eventually could not restore complete mitochondrial respiratory function, probably due to incompatibility between rat mtDNA-coded polypeptides and mouse nuclear DNA-coded polypeptides or mouse mitochondrial inner membranes that are required to assemble respiratory complexes with normal activities.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Although the mitochondrial genome replicates independently from the nuclear genome, it has been presumed that cytoplasmically introduced mtDNA of a different species could not replicate in recipient cells due to interspecies incompatibility between nuclear and mitochondrial genomes (DEFRANCESCO et al. 1980 ; HAYASHI et al. 1983 ). However, there might be mtDNA species that could replicate but induce abnormalities in mitochondrial respiratory function in mouse cells, particularly when mtDNAs from closely related species were introduced. In this study, we found that CyMs chimera cybrids and tissues of congenic strain B6mt^spr mice with M. spretus mtDNA did not show any mitochondrial dysfunction. Therefore, B6mt^spr mice with normal mitochondrial respiratory function could not be used as disease models, although a slight decrease in running time until exhaustion was reported in these mice (NAGAO et al. 1998 ). On the other hand, CyRn chimera cybrids with rat mtDNA showed significant reduction of mitochondrial respiratory function, suggesting that mice with predominant rat mtDNA could be models of mitochondrial diseases.

In the CyRn cybrids, replication and transcription of the exogenously introduced rat mtDNA and translation of its transcripts occurred normally to produce sufficient amounts of polypeptides encoded by introduced rat mtDNA under the control of factors encoded by the mouse nuclear genome. Therefore, all the mouse nuclear DNA-coded factors required for these processes to produce mtDNA-encoded polypeptides could properly recognize rat mtDNA and its transcripts, resulting in the restoration of normal translation activity in mouse mitochondria (Fig 4 and Fig 5). Thus, no detectable incompatibility was observed in rat mtDNA replication, transcription, and translation. However, incompatibility was observed in the phenotypic expression of mitochondrial respiratory functions. CyRn cybrids showed progressively reduced activities of complex I + III and complex IV and resultant reduction of O₂ consumption (Fig 6). Since they have mouse-rat chimera respiratory complexes consisting of mouse nuclear DNA- and rat mtDNA-coded polypeptides, some incompatibilities between rat and mouse polypeptides inhibited their proper assembly to form complexes with normal enzyme activities, which would be responsible for the very low mitochondrial respiratory function in the CyRn chimera cybrids. Accordingly, the nuclear and mitochondrial interactions required for rat mtDNA replication, transcription, processing of transcripts, and their translation must be less stringently controlled than those required for assembly and formation of normal respiratory complexes. It should be noted that the former interactions are protein vs. nucleic acids interactions, while the latter are protein vs. protein interactions, suggesting that protein vs. nucleic acids interactions are less stringently controlled than protein vs. protein interactions.

Success of rat mtDNA transfer to mouse cells seems to be slightly inconsistent with our previous observations that cytoplasmically introduced rat mtDNA into mouse cells containing mouse mtDNA could not be transmitted to progeny cells (HAYASHI et al. 1980 ) but could be transmitted when the rat nuclear genome was cotransferred to mouse cells by fusing mouse and rat whole cells to isolate mouse x rat somatic hybrid cells (HAYASHI et al. 1983 ). We suggested that mouse mtDNA replicated preferentially under control of the nuclear genome of the same species and that the rejection of rat mtDNA from mouse cells might be due to incompatibility between nuclear and mitochondrial genomes of different species. The apparent discrepancy between our present and previous observations (HAYASHI et al. 1980 ) with respect to the acceptance of rat mtDNA by mouse cells (Table 1 and Fig 4) may be due at least partly to the difference between whether the recipient mouse cells did (HAYASHI et al. 1980 ) or did not possess mouse mtDNA (this study). Rat mtDNA may not be propagated in competition with host mouse mtDNA but may be propagated and expressed normally when no mouse mtDNA was present in the recipient mouse cells. We are investigating this possibility by introducing mouse mtDNA into CyRn cybrids to observe whether rat mtDNA is rapidly excluded by exogenously introduced mouse mtDNA. We are also investigating which species of mtDNA is limiting in propagation to mouse ⁰ B82 cells by introducing mtDNAs from more distantly related species.

Recently, KENYON and MORAES 1997 showed that human ⁰ cells could receive mtDNA from the common chimpanzee, pygmy chimpanzee, and gorilla, resulting in restoration of their mitochondrial respiratory function, whereas they could not receive mtDNA from the orangutan or other less-related primate species. The distance between humans and the common chimpanzee estimated from the sequence differences of the cytb gene in mtDNA (0.125), calculated by Kimura's two-parameter method (KIMURA 1980 ), corresponds to that between M. m. domesticus and M. spretus (0.099), which would be the limitation for keeping nuclear and mitochondrial compatibility to restore mitochondrial respiratory function, although preferential reduction in complex I activity was observed in cybrids containing imported mtDNA from closely related primates (BARRIENTOS et al. 1998 ). On the other hand, the sequence differences between humans and the orangutan (0.169) correspond to those between mice and rats (0.189), and in these combinations of nuclear and mitochondrial genomes from phylogenetically less-related species, mtDNA could not replicate (in the former case, KENYON and MORAES 1997 ), or could replicate and be translated but could not contribute to the construction of respiratory complexes with normal activities (in the latter case, Fig 6). Therefore, such mitochondrial dysfunction as induced by incompatibility between nuclear and mitochondrial genomes from less-related species might be applied in isolation of model mice with mitochondrial disorder.

As mouse mtDNA is inherited strictly maternally (KANEDA et al. 1995 ; SHITARA et al. 1998 ), different species of mtDNA could be introduced into mice by interspecies crossing using different species as females followed by repeated backcrossing of interspecies F₁ female to male mice (M. m. domesticus), leading to the isolation of congenic mice possessing mtDNA exclusively from different species. Since the most distant species that could make interspecies F₁ hybrids using male M. m. domesticus is M. spretus, we isolated CyMs cybrids, which are equivalent to the congenic B6mt^spr mice in possessing only the M. spretus mitochondrial genome and M. m. domesticus nuclear genome. However, mtDNA from M. spretus gave normal mitochondrial respiratory function to mouse ⁰ cells with the M. m. domesticus nuclear genome (Fig 3). On the other hand, mitochondrial abnormalities were observed in CyRn cybrids repopulated with rat mtDNA. Thus, introduction of rat mtDNA into mouse cells is the only way at present to isolate mtDNA-knockout mice, although model mice with mitochondrial disorders have been established by inactivation of nuclear DNA-coded genes required for the expression of mitochondrial respiratory function (for review, see WALLACE 1999 ). Since apparent failure to introduce rat mtDNA into mouse somatic cells (HAYASHI et al. 1980 ) may be due to absence of an effective procedure for selective isolation of cybrids with rat mtDNA, we are now trying to introduce rat mtDNA directly into fertilized mouse eggs to generate mice with rat mtDNA.

	ACKNOWLEDGMENTS

We are grateful to Dr. Kaoru Tsuda, Tokyo Metropolitan Institute of Medical Science, for valuable suggestions. This work was supported in part by a grant for Research Fellowship from the Japan Society for Promotion of Science for Young Scientists to K.I., by a grant for the Hayashi project of the Center for Tsukuba Advanced Research Alliance, University of Tsukuba, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by Health Sciences Research Grants for Research on Brain Science from the Ministry of Health and Welfare of Japan to J.-I.H.

Manuscript received October 18, 1999; Accepted for publication February 18, 2000.

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