Department of Health Sciences, Boston University, Boston, Massachusetts 02215
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ABSTRACT |
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Previous work showed that protein and mRNA levels of the "fast" isoform of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) are markedly increased in unloaded slow-twitch soleus muscles, suggesting pretranslational control of gene expression [L. M. Schulte, J. Navarro, and S. C. Kandarian. Am. J. Physiol. 264 (Cell Physiol. 33): C1308-C1315, 1993]. However, because of the difficulty of measuring transcription rates from whole muscle, transcriptional activation of the SERCA1 gene with unloading has not been confirmed. Because SERCA1 pre-mRNA levels can reflect transcriptional activity, in the present study SERCA1 introns were sequenced to allow intron-directed RT-PCR measurement of SERCA1 pre-mRNA. These data were then compared with changes in SERCA1 mRNA expression in control and unloaded soleus muscles. After 2, 4, and 10 days of unloading, SERCA1 pre-mRNA and mRNA transcript levels increased significantly by two-, three-, and sevenfold, respectively (P < 0.01). Parallel increases in SERCA1 pre-mRNA and mRNA suggest transcriptional activation of the endogenous SERCA1 gene by muscle unloading. SERCA2, the cardiac/slow-twitch skeletal muscle isoform, was not markedly increased by unloading, and RNase protection assays showed no change in alternative splicing of SERCA1 or SERCA2 primary transcripts. With use of in vivo plasmid injection, the activity of a reporter gene driven by 3.6 kb of the SERCA1 5'-flanking region increased fivefold in 7-day-unloaded soleus muscles. Comparison of the magnitude of transcriptional activation of endogenous and constructed SERCA1 genes by unloading confirms the fidelity of using intronic RT-PCR to examine muscle gene transcription rates and suggests that cis-acting elements sufficient for regulating unloading-induced transcriptional activation are contained in this promoter construct.
reverse transcriptase-polymerase chain reaction; run-on assay
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INTRODUCTION |
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PHYSICAL ACTIVITY PATTERNS have a strong modulatory effect on muscle-specific gene expression and thereby skeletal muscle phenotype. Fast-twitch muscle becomes slower with increased weight bearing, whereas slow-twitch muscle becomes faster with decreased weight bearing (reviewed in Ref. 5). These changes are due to alterations in the isoforms and expression levels of proteins involved in excitation-contraction coupling, cross-bridge cycling, and energy metabolism. Typically, the regulation is controlled at a pretranslational level (5). However, because of the difficulty in assessing transcription rates in adult muscle, very few studies have been able to demonstrate transcriptional control of endogenous muscle genes by physical activity or other physiological stimuli.
A critical problem when the standard nuclear run-on assay is used to assess transcription rates from whole tissue is that the percentage of transcriptionally active nuclei isolated from different samples is unknown. Additionally, the amount of tissue needed to isolate enough transcriptionally active nuclei for just one observation is impractical, thus precluding statistical analysis (14). An alternative approach recently presented to measure changes in transcription rate is to compare the ratio of pre-mRNA to mature mRNA levels in control and experimental tissue (12). To make this comparison, intron-directed RT-PCR is used to measure pre-mRNA levels, and Northern blotting or protection assay is used to measure mature mRNA levels from total tissue RNA. Thus, similar to the run-on assay, this in vivo analysis represents transcriptional activity of the endogenous gene. A change in the transcription rate of the endogenous gene is critical information for comparison with subsequent studies on transcriptional mechanisms using promoter fragments driving reporter genes.
Transcriptional activation is likely to play a role in one of the most striking examples of muscle phenotype remodeling, i.e., the marked induction of the "fast" sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) isoform in slow-twitch skeletal muscle in response to biomechanical unloading. SERCA spans the longitudinal membrane of the sarcoplasmic reticulum (SR), where Ca2+ is stored between contractions. Because of its role in sequestering Ca2+ from the myoplasm, SERCA is the major rate-limiting protein for muscle relaxation. The slow-twitch soleus muscle has a nearly equal distribution of SERCA1a, the adult fast-twitch muscle isoform (6, 7, 34), and SERCA2a, the slow-twitch/cardiac muscle isoform (13, 18, 35). By comparison, the fast-twitch extensor digitorum longus expresses 99% SERCA1a protein at levels six times that of the soleus (34). After 2 wk of unloading, although there is little change in SERCA2 expression, soleus muscle SERCA1 mRNA and protein levels, SR Ca2+-ATPase activity, and relaxation rate increase to levels that are 50% of a control fast-twitch muscle (26). As early as 4 days after unloading, the soleus muscle expresses significantly greater mRNA and protein levels of SERCA1, suggesting pretranslational regulation of the SERCA1 gene.
The purpose of the present study was to determine whether increased SERCA1 expression in the soleus muscle by unloading is due to transcriptional activation. To assess the relative rate and extent of SERCA1 transcriptional activation, soleus muscles were studied after 1, 2, 4, and 10 days of muscle unloading. Similar increases in SERCA1 pre-mRNA and mature mRNA were observed at 2, 4, and 10 days of unloading, suggesting transcriptional activation of endogenous SERCA1. For comparison, the activity of an injected reporter gene driven by 3.6 kb of the SERCA1 5'-flanking region was increased fivefold in soleus muscles after 7 days of unloading. Taken together, these data demonstrate the fidelity of using pre-mRNA analysis to assess relative transcription rates, and they show that unloading activates transcription of SERCA1.
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MATERIALS AND METHODS |
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Hindlimb unloading. Female Wistar rats (3 mo old) were randomly assigned to control or hindlimb-unloading groups (n = 8 in each group at each time point). To induce muscle unloading, the rat's hindlimbs were suspended by elastic tail casts, as described previously (15). After 1, 2, 4, or 10 days, control and unloaded animals were anesthetized with pentobarbital sodium (40 mg/kg), and soleus muscles from right and left hindlimbs were removed, quickly weighed, and immediately processed for RNA isolation.
Total RNA isolation. Right and left soleus muscles were combined and homogenized in denaturing solution for RNA isolation with use of Ambion's Totally RNA kit, which is based on the guanidinium-thiocyanate RNA isolation method (9). Total RNA samples were treated with DNase I for 30 min at 37°C to degrade any genomic DNA (plus 5 mM MgCl2 and CaCl2). Mg2+ and Ca2+ were then chelated with EDTA, and the DNase I was heat denatured for 5 min at 90°C. RNA was then precipitated with lithium chloride and resuspended in 0.1 mM EDTA-water, and the RNA concentration was calculated by ultraviolet spectrophotometry at 260 nm.
Probes and mRNA analysis. Northern blotting was performed following standard procedures (2). Probes for SERCA1 (34) and SERCA2a (13) were generated from cDNA clones of the respective 3'-untranslated region (UTR) and labeled with random priming, as previously described (24). SERCA mRNA transcript levels were determined from densitometry of autoradiogram signals and normalized to levels of 18S rRNA. A 5'-end-labeled oligonucleotide was used to probe 18S rRNA (24). Blot reprobing was performed without stripping, because the transcript sizes differ sufficiently to allow adequate separation of signals.
RNase protection assays (RPAs) and RNA probe synthesis were performed with modifications as recommended by the manufacturer (Ambion Hybspeed RPA and Maxiscript kits, respectively), as described previously (24). Antisense RNA probes were synthesized by in vitro transcription from cDNA templates of SERCA1a or SERCA2a 3'-UTR. Probe incubated with yeast RNA was used to ensure complete digestion of the unprotected probe under the conditions of the assay. The maintenance of probe excess was monitored by ensuring that double the amount of RNA gave double the amount of signal. Protected fragments were separated on 6% denaturing urea-Tris borate-EDTA-polyacrylamide gels and used for autoradiography. Densitometry of autoradiogram signals allowed calculation of the corresponding SERCA mRNA transcript levels. Probe fragment size was assessed by comparison to an in vitro transcribed 100-nt-ladder-size standard (Ambion).Precursor mRNA analysis. The same total RNA samples used for Northern blots and protection assays were used for RT-PCR amplification of SERCA1 pre-mRNA. Intron-directed primers at the 5' and 3' ends of the pre-mRNA molecule were used for RT and PCR. To provide the intron sequence for designing these primers, rat genomic DNA was cloned and sequenced as follows: PCR primers were designed from rat cDNA sequence (34) to target exons 1 and 3 as well as exons 21 and 23. Amplification of rat genomic DNA with use of these primers gave a product containing introns 1 and 2 or introns 21 and 22, respectively. After positive SERCA1 identification by Southern blotting, the PCR products were subcloned into pGEM-T (Promega), and clones of the correct size by use of restriction analysis were sequenced (National Biosciences).
RT-PCR of SERCA1 pre-mRNA at the 5' end was performed using the following primers: intron 2 RT primer (5'-AGTGTCTGCAGCTCTGGATGGGCTG-3'), nested intron 2 reverse PCR primer (5'-AGCCCAGCTTGGCATGAGAG-3'), and exon 2 forward PCR primer (5'-AGCTCCCTGCTGAGGAAG-3'). RT-PCR of SERCA1 pre-mRNA at the 3' end was performed using the following primers: RT primer [oligo(dT)15-20], intron 21 reverse PCR primer (5'-GGAGCCATAGAGCTGAGCGGGTTA-3'), and exon 21 forward PCR primer (5'-CTCACTTCCAGTCATCGGGCTAGA-3'). RT was performed using Superscript II (Life Technologies) following instructions of the manufacturer. The entire 20 µl of the first-strand cDNA reaction was then subjected to PCR as follows. To prevent mispriming, an initial series of PCR was performed (40 s at 92°C and 40 s at 73°C minus 0.5°C per cycle for 15 rounds) followed by 12 rounds of 40 s at 92°C and 40 s at 64°C. Incorporation of [In vivo plasmid injection. The GenomeWalker kit (Clontech) was used to isolate 3.6 kb of the SERCA1 promoter and 5'-flanking region following instructions of the manufacturer. The 3.6-kb fragment was positively identified using Southern blotting (2). The product was ligated upstream of the luciferase gene in pGL3-basic (Promega) and amplified using Qiagen's endotoxin-free Mega isolation kit. The construct was ethanol precipitated and resuspended in 25% sucrose-1× PBS at 1 mg/ml. Female Wistar rats (8 wk old) were anesthetized with pentobarbital sodium (40 mg/kg), a lateral incision was made on the leg, and the soleus muscle was isolated. A 28-gauge needle filled with 50 µg of construct (50 µl) was inserted distally, and the tip was pushed to the most proximal portion of the muscle, similar to that previously described (17). While the needle was removed from the muscle the construct was injected, such that construct was evenly distributed along the needle tract. Animals were allowed to recover for 18 h and were then randomly divided into two groups: unloaded (n = 7 muscles) and weight bearing (n = 6 muscles). Muscles were removed 7 days later, homogenized in 1× passive lysis buffer (Promega), and centrifuged at 5,500 g for 20 min at 4°C. Luciferase activity of the supernatant was determined following instructions of the manufacturer (Promega) with use of a luminometer (model 20/20, Turner Designs). Pellets were resuspended in Buffer G2 (Qiagen), and total muscle DNA was isolated using Qiagen's Blood and Cell Culture DNA Maxi kit. DNA was digested using Hae III by cutting a 1.5-kb fragment from the pGL3-basic plasmid. Southern blotting was performed (2) using a random primed fragment of pGL3-basic as probe to quantitate the luciferase plasmid in muscle. Signals on autoradiograms were quantified using laser densitometry.
Statistics. An unpaired Student's t-test was used to determine statistical significance between control and unloaded groups with P < 0.05 or P < 0.01 as indicated.
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RESULTS |
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Mature SERCA1 and SERCA2a mRNA expression.
Ten days of unloading led to a sixfold increase in SERCA1 mRNA levels
relative to weight-bearing controls (Fig.
1,
top). As a result, the
relative expression of SERCA1 mRNA in unloaded soleus muscles was
~75% of that expressed in control fast-twitch tibialis anterior
muscle. In contrast, SERCA2a mRNA increased only moderately (40%) in
unloaded muscles (Fig. 1, middle).
The sixfold increase in the relative expression of SERCA1 mRNA occurs
in the face of significant reductions in total muscle RNA. At 10 days
of unloading, soleus RNA content was 56% lower (265 ± 7 and 116 ± 4 µg/muscle in control and unloaded muscle, respectively,
P < 0.01), whereas muscle mass was
36% lower than in control muscles (251 ± 3 and 161 ± 3 mg in
control and unloaded muscles, respectively,
P < 0.01). Thus RNA concentration
(µg RNA/mg wet mass) was 32% lower (P < 0.01). However, the SERCA1 mRNA
per whole muscle was still increased 2.6-fold. This level of expression
is much greater than what could be accounted for if SERCA1 mRNA just
maintained its expression during atrophy while all other cellular RNA
decreased.
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SERCA1 and SERCA2 alternative transcript expression.
SERCA1a and SERCA1b are products of alternative splicing of the SERCA1
primary transcript. SERCA1a is the adult, skeletal isoform, and SERCA1b
is the neonatal isoform expressed primarily in neonatal fast-twitch
muscle (6, 7, 34). To examine whether unloading alters the expression
of SERCA1a relative to SERCA1b, RPAs were performed using a probe that
distinguishes between the two transcripts by size. A 42-nt exon is
removed from the SERCA1b transcript while being retained in the SERCA1a
transcript (shown schematically in Fig. 2).
In agreement with results from Northern blots, the protection assay
shows a sixfold increase in SERCA1a mRNA levels (Fig. 2). SERCA1b was
also increased approximately sixfold (confirmed by a longer film
exposure; not shown). Thus unloading increases SERCA1a and SERCA1b
transcript levels to a similar extent and does not appear to
differentially affect splicing of the two isoforms compared with
control.
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SERCA1 pre-mRNA levels. The most likely method by which SERCA1a mRNA would increase in unloaded soleus muscles is through increased transcription of the SERCA1 gene. To investigate this, RT-PCR was used to measure relative SERCA1 pre-mRNA levels as a reflection of transcriptional activation. If the magnitude of increase in SERCA1 pre-mRNA of 10-day-unloaded soleus muscles equals the sixfold increase in SERCA1a mature mRNA, this would be consistent with an increased rate of SERCA1 transcription. To test the ability of RT-PCR quantitation to detect changes in SERCA1 primary transcript levels, SERCA1 mature mRNA levels were also measured by RT-PCR with use of exon-directed primers [designed from rat cDNA sequence (34)]. Results of SERCA1 mRNA measurement with RT-PCR were similar to those from Northern blotting (not shown). For SERCA1 pre-mRNA measurement, introns at the 5' and 3' ends of the SERCA1 gene were sequenced to allow for primer design (see MATERIALS AND METHODS). The sensitivity of RT-PCR amplification by use of intron-directed primers allowed detection of pre-mRNA levels within a total RNA sample.
Figure 4 shows the results of a representative RT-PCR assay: SERCA1 pre-mRNA levels in 10-day-unloaded soleus muscles were increased eightfold compared with controls. Measurement was made at the 5' and 3' ends of the SERCA1 pre-mRNA molecule by using two different sets of RT and PCR primers (location of primers shown in Fig. 4). This was done to ensure that the measurements were an accurate reflection of the total SERCA1 pre-mRNA population and did not reflect only a change specific to the region of the long molecule targeted by our primers. For example, a delayed rate of intron removal during splicing of the primer-targeted region would result in increased RT-PCR product, even if the actual number of transcripts was the same. Additionally, an oligo(dT) primer was used for cDNA first-strand synthesis during RT at the 3' end rather than an intron-directed RT primer, as was used at the 5' end. In this way, results at the 5' end include amplification of SERCA1 pre-mRNA with and without poly(A) tails, whereas results at the 3' end represent only amplification of transcripts that had been polyadenylated. Results from the 5' and 3' ends were similar, with both increasing approximately eightfold. The conclusion from these results was that the eightfold increase in SERCA1 pre-mRNA level and the sixfold increase in SERCA1 mature mRNA are consistent with transcriptional activation of the SERCA1 gene.
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Time course analysis of SERCA1 transcriptional activation by
unloading.
Once transcriptional activation of SERCA1 was demonstrated in the
10-day-unloaded muscles, expression of SERCA1 pre-mRNA and mature mRNA
was measured over a time course of unloading to assess the relative
rate and extent of SERCA1 transcriptional activation. The additional
time points were 1, 2, and 4 days of unloading. For these earlier time
points, pre-mRNA measurement by RT-PCR was made only at the 5'
end, since 5' and 3' results were the same in the 10-day
analysis. By 2 days of unloading, SERCA1 pre-mRNA and mRNA were
significantly increased. This is before significant atrophy has
occurred in the unloaded soleus muscle. At each time point the SERCA1
pre-mRNA and SERCA1 mRNA were increased to a similar extent over
controls (Fig. 5). Thus transcriptional
activation of the SERCA1 gene during unloading occurs early and is a
major mechanism regulating expression.
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Plasmid injection of a SERCA1 reporter construct.
The parallel increase in SERCA1 pre-mRNA and mRNA with unloading
indicated transcriptional activation, so steps were taken to initiate
in vivo promoter analysis. A reporter construct containing 3.6 kb of
the SERCA1 5'-flanking and promoter region was ligated upstream
from the luciferase gene. The plasmid construct was injected into
soleus muscles, allowing measurement of the activity of a SERCA1
promoter-driven reporter gene, as previously described for contractile
protein genes (17, 29). The fidelity and advantages of using direct
plasmid injection for promoter analysis in muscle have been recently
reviewed (10, 17). Luciferase reporter activity was fivefold higher in
7-day-unloaded soleus muscles than in weight-bearing controls (Fig.
6).
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DISCUSSION |
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SERCA1 is the predominant SERCA gene expressed in skeletal muscle, and its role as the rate-limiting enzyme of SR Ca2+ sequestration is essential for normal muscle function (22). However, remarkably little is known about the mechanisms regulating expression of this gene. In adult animals, SERCA1 expression is strongly regulated by muscle loading associated with weight-bearing status. SERCA1 functional activity and expression levels are increased when loading is removed from the slow-twitch soleus muscle (26) and decreased when loading is increased in the fast-twitch plantaris muscle (16). These changes in SERCA1 expression occur earlier and to a greater extent than the changes in expression of other muscle-specific genes in response to altered activity levels (5). As with other muscle-specific genes, the regulation of SERCA1 by weight bearing appears to be pretranslational (5), but the difficulty of measuring transcription rates of endogenous genes in whole muscle has precluded detailed study of the transcriptional mechanisms. To examine the transcriptional status of the endogenous SERCA1 gene in response to unloading, SERCA1 pre-mRNA and mature mRNA transcript levels were compared in control and unloaded soleus muscles. Parallel increases in SERCA1 pre-mRNA and mRNA levels suggest that transcriptional activation is a major mechanism of the unloading-induced increase in SERCA1 expression. Increased rates of primary transcript processing and nucleocytoplasmic transport are not indicated, because either of these would necessarily decrease the relative amount of pre-mRNA over time unless transcription was increased to a similar extent. An increase in the stability of SERCA1 pre-mRNA with unloading is also an unlikely mechanism, because it would indicate that, in the control condition, SERCA1 pre-mRNA is extremely unstable. That is, if this mechanism were operative in the 10-day control soleus muscles, seven of every eight SERCA1 primary transcripts would be degraded before exiting the nucleus, before having any effect on cell function.
SERCA1 pre-mRNA and mRNA transcript measurements were made from whole
muscle RNA, and thus they reflect transcription of the endogenous
SERCA1 gene. The in vivo studies that have shown transcriptional activation of muscle-specific genes by changes in activity levels have
involved measuring a reporter gene driven by 5' fragments of
-actin (8),
-myosin heavy chain (MHC) (19), and MHCIIb (29)
promoters. Although they are informative, these reporter constructs are
not a direct assessment of the endogenous gene. Rather, they are
limited to the regions of the gene included in the construct, typically
the promoter and 5'-flanking region. Thus the reporter construct
may not include all the important cis
elements involved in the endogenous gene regulation (23). Reporter
constructs also lose any regulatory information contained in the
chromatin conformation (10). For these reasons, we considered it
important to compare results from the SERCA1 reporter construct with
those of the endogenous SERCA1 gene. In the present study, results from
the 3.6-kb SERCA1 reporter construct injected into soleus muscles were
similar to data from the endogenous SERCA1 gene: SERCA1 reporter
activity increased fivefold at 7 days of unloading, whereas
transcriptional activity of the endogenous gene increased threefold at
4 days and sixfold at 10 days of unloading.
Because the transcriptional activation of endogenous SERCA1 and the SERCA1 reporter construct were similar in unloaded soleus muscles, the 3.6 kb of the SERCA1 5'-flanking region likely contain the necessary cis elements to confer unloading-sensitive SERCA1 transcriptional regulation. The rat SERCA1 gene has been shown to have three thyroid-responsive regions and several E-boxes within the first 962 bp of the 5'-flanking region (27) and a total of 14 putative E-boxes in 2,658 bp of the 5'-flanking region (31). Although thyroid hormone [triiodothyronine (T3)] clearly regulates SERCA1 expression in vitro (27, 30) in hyperthyroid animals (32) and during development (1, 33), the role of T3 in the regulation of SERCA1 expression by weight bearing under euthyroid conditions has not been determined. Because T3 levels are not significantly altered by unloading (11), a cofactor to the T3-T3 receptor complex (21) or a novel transcription factor may be activated or upregulated with unloading to increase SERCA1 transcriptional activation. Myogenic regulatory factors that transactivate muscle-specific genes via interactions with E-boxes (4, 20) might be involved. Unloading might inactivate a transcriptional silencer or inhibitor to increase SERCA1 transcription. Transcriptional inhibitors such as Id (3) have been identified that inhibit the activity of the myogenic factors myogenin, MyoD, and MEF2, whereas expression of silencers such as ZEB (25) decrease transcription of muscle genes via direct binding to DNA promoter regions.
In conclusion, the results of this study provide evidence that muscle unloading transcriptionally activates the SERCA1 gene. This activation occurs early and leads to marked increases in SERCA1 expression. Data on the magnitude of transcriptional activation of the endogenous gene provide critically important information for comparative purposes to subsequent SERCA1 promoter analysis. Subsequent studies to identify the important cis-acting regulatory regions and trans factors directing this activation will increase our understanding of how weight bearing regulates SERCA1 and perhaps other muscle-specific genes.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Research Grant AR-41705 and by a grant-in-aid award from the American Heart Association. S. C. Kandarian is an Established Investigator of the American Heart Association.
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FOOTNOTES |
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Sequence data in this paper can be found under GenBank accession numbers AF091852 and AF091853.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. C. Kandarian, Dept. of Health Sciences, Boston University, 635 Commonwealth Ave., 4th Fl., Boston, MA 02215 (E-mail: skandar{at}bu.edu).
Received 16 October 1998; accepted in final form 12 February 1999.
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