Departments of 1 Molecular Biology and 2 Biochemistry, University of Texas Health Science Center at Tyler, Tyler, Texas 75708-3154
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ABSTRACT |
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Surfactant protein B (SP-B) is
expressed tissue specifically in the lung and is developmentally
regulated. To identify genomic regions that control SP-B expression, we
analyzed SP-B promoter activity in transgenic mice containing rabbit
SP-B 5'-flanking DNA fragments linked to the chloramphenicol
acetyltransferase (CAT) reporter gene. Results showed that
whereas the 2,176/+39-bp fragment failed to express CAT, shorter
fragments of
730/+39 and
236/+39 bp expressed CAT tissue
specifically in the lung. Further deletion of 5'-flanking DNA to
136
bp resulted in no expression of CAT. Immunostaining demonstrated that
both
730/+39- and
236/+39-bp regions expressed CAT specifically in
alveolar type II and Clara cells. The
236/+39-bp region expressed CAT at a significantly lower level than the
730/+39-bp region. CAT expression in mice containing the
730/+39-bp region was detected in
embryonic day 14 lung and attained maximum levels in
day 18 lung, indicating that the developmental expression of
CAT was similar to that of SP-B. These data show that the DNA elements necessary for cell type-specific expression are located within
236/+39 bp of the SP-B gene. Additionally, these data suggest that
the
2,176/
730- and
730/
236-bp regions contain the DNA elements
that repress and enhance SP-B gene transcription, respectively.
gene regulation; transcription; type II cell; Clara cell; respiratory distress syndrome
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INTRODUCTION |
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SURFACTANT, A LIPOPROTEIN COMPLEX, is synthesized and secreted by the alveolar type II epithelial cells of the lung. Surfactant maintains the integrity of the alveoli during respiration by reducing surface tension at the alveolar air-tissue interface (11) and serves important roles in host defense in the lung (26). Deficiency of surfactant is associated with the occurrence of respiratory distress syndrome in preterm infants (2), the leading cause of neonatal morbidity and mortality in developed countries. Surfactant protein B (SP-B), a 9-kDa hydrophobic protein, is essential for the maintenance of the biophysical properties and physiological functioning of surfactant. SP-B promotes the adsorption and spreading of surfactant phospholipids (22) and stabilizes the phospholipid monolayer formed on the alveolar surface (8). Deficiency of SP-B due to a frame shift mutation in the coding region is associated with fatal respiratory failure in infants with congenital alveolar proteinosis (18). Targeted disruption of the SP-B gene causes respiratory failure in newborn mice, further supporting the important role of SP-B in lung function (7).
SP-B mRNA is expressed in a cell type-specific manner by the alveolar
type II and bronchiolar epithelial cells of the lung (20,
30) and is under developmental and multifactorial regulation (28). Glucocorticoids and cAMP increase, whereas tumor
necrosis factor- decreases SP-B mRNA expression (3).
Our laboratory (15) previously found that a
minimal promoter containing 236/+39 bp of rabbit SP-B gene is
necessary and sufficient for high-level expression of the
chloramphenicol acetyltransferase (CAT) reporter gene in NCI-H441
cells, a cell line with the characteristics of bronchiolar epithelial
cells (Clara cells). The minimal SP-B promoter supported high-level CAT
expression in a lung cell type-specific manner in cell cultures,
suggesting that it contained a cell- or tissue-specific enhancer
(15). Our studies also identified binding sites for Sp1,
Sp3, thyroid transcription factor-1 and hepatocyte nuclear
factor-3 transcription factors in the minimal SP-B promoter
that acted in a cooperative or combinatorial manner to maintain SP-B
promoter activity (16).
Genomic regions and gene regulatory elements that direct cell-specific
and developmental expression of the SP-B gene have not been identified.
In this study, we analyzed SP-B promoter activity in mice containing
SP-B 5'-flanking DNA fragments linked to the CAT gene. Analysis of CAT
expression in transgenic mice showed that, whereas the 2,176/+39-bp
region failed to support CAT expression, shorter fragments consisting
of
730/+39 and
236/+39 bp expressed CAT in a cell- or
tissue-specific manner in the lung. Further deletion of 5'-flanking DNA
to
136 bp resulted in no expression of CAT in lung and other tissues.
CAT expression was restricted to alveolar type II and bronchiolar
(Clara) epithelial cells. The developmental expression of CAT in mice
containing the
730/+39-bp region of the SP-B gene was similar or
identical to endogenous SP-B gene expression.
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EXPERIMENTAL PROCEDURES |
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Plasmid construction and generation of transgenic mice.
Plasmid pBLCAT6 containing the 2,176/+39-bp fragment of the SP-B gene
was digested with HindIII and KpnI or
SacI to release SP-B-CAT fusion DNA constructs containing
2,176/+39- and
730/+39-bp fragments of the SP-B gene. Plasmid
pSKCAT containing the
236/+39-bp fragment of the SP-B gene was
digested with XbaI and KpnI to release a
SP-B-CAT fusion DNA construct containing the
236/+39-bp fragment of the SP-B gene. Plasmid pBLCAT6 containing
136/+39 bp of SP-B 5'-flanking DNA was digested with HindIII and
KpnI to obtain a SP-B-CAT construct containing the
136/+39-bp SP-B fragment. The linear SP-B-CAT fusion DNA fragments
were purified by agarose gel electrophoresis and dissolved in 10 mM
Tris · HCl, pH 7.4, at a concentration of ~100 ng/ml.
Dot and Southern blot analysis of tail DNA.
Genomic DNA from mouse tails (0.6-1 cm) was isolated with a DNA
isolation kit from 5 Prime 3 Prime (Boulder, CO) or QIAGEN (Valencia, CA), and the DNA concentration was determined by measuring absorbance at 260 nm. Dot and Southern blot analyses of DNA were carried out according to standard methods with 5-10 µg of
genomic DNA and Hybond N+ (Amersham) as the transfer membrane. The
membranes were hybridized with 32P-labeled full-length CAT
cDNA as the probe, washed under high-stringency conditions, and exposed
to X-ray film to obtain an autoradiograph.
CAT enzymatic assay and CAT ELISA.
Mouse tissues were suspended in 5 volumes (vol/wt) of cold 0.25 M
Tris · HCl, pH 7.8, containing 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin and leupeptin, and 0.5 µg/ml of benzamidine and homogenized with a high-speed homogenizer (Tekmar Tissuemizer). Homogenates were freeze-thawed and centrifuged at 12,000 g for 5 min, and the supernatants were removed and stored at
70°C. The CAT activity of the tissue extracts was determined by the
liquid scintillation counting method (24) after the
extracts were heated to 60°C for 10 min to inactivate the endogenous
acetylase. CAT levels were also measured by ELISA with a CAT ELISA kit
(Boehringer Mannheim).
Determination of protein. The protein concentration of the tissue extracts was determined by the Bradford (5) method with the Bio-Rad protein assay reagent and bovine serum albumin as the protein standard.
RNA isolation and Northern blot analysis. Total RNA from tissues was isolated by the acid-phenol method with TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. The RNA was separated by electrophoresis on agarose gels (1%) containing 20 mM MOPS and 1.1% formaldehyde and transferred to Hybond N+ membranes by capillary action with saline-sodium citrate (20×) as the transfer solution. The membranes were hybridized with 32P-labeled full-length mouse SP-B cDNA and CAT cDNA probes, washed, and exposed to X-ray film to obtain an autoradiograph.
Immunohistochemical localization of CAT and SP-B.
Adult mice were killed, and their lungs were perfused with saline to
remove blood. The perfused lungs were instilled with formalin or ExCell
fixative for 20 min under 20 cmH2O pressure and then
removed and stored in the fixative for 24 h. The fixed lungs were
dehydrated and embedded in paraffin, and serial 6-µm sections were
cut for histology. Tissue sections were serially incubated with
polyclonal CAT (5 Prime 3 Prime), human SP-B (Chemicon
International, Temecula, CA), or nonimmune IgG antibodies at 1:200
dilution; biotinylated secondary antibody; and horseradish peroxidase-labeled streptavidin (Innovex Biosciences, Richmond, CA).
Sections were then stained with 3-amino-9-ethylcarbazole (AEC)
according to the kit instructions and counterstained with hematoxylin.
For double immunostaining, the sections were stained first with AEC for
CAT and then with TrueBlue substrate (Kirkegaard and Perry,
Gaithersburg, MD) for SP-B.
Developmental regulation of CAT. F1 heterozygous male transgenic mice were mated with wild-type female mice, and the female mice were examined daily for the copulatory plug. The gestational age of the embryos was estimated based on the day that the copulatory plug was first detected in the female (day 0). The female mice were killed at different stages of gestation, and the embryos were collected. Lungs were removed for the preparation of extracts with the aid of a dissecting microscope.
Cell culture and transfections. MLE-12 cells, a mouse lung epithelial cell line, were maintained in HITES (hydrocortisone, insulin, transferrin, estrogen, and selenium) medium containing penicillin (100 U/ml) and streptomycin (100 µg/ml) and supplemented with 2% fetal bovine serum. These cells express detectable levels of SP-B and SP-C and therefore may have characteristics of alveolar type II cells (29). MLE-12 cells were transfected by liposome-mediated DNA transfer with LipofectAMINE. The conditions for transfection were similar to those previously described for NCI-H441 cells by our laboratory (15).
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RESULTS |
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Generation of SP-B-CAT transgenic mice.
To identify the genomic regions that control cell type-specific and
developmental expression of the SP-B gene, we constructed SP-B-CAT
fusion genes containing 2,176/+39-,
730/+39-,
236/+39-, and
136/+39-bp fragments of rabbit SP-B 5'-flanking DNA (Fig. 1). The SP-B-CAT fusion DNA constructs
were injected into mouse oocytes to generate transgenic mice, and the
transgenic founder mice were bred with wild-type mice to establish
independent lines of transgenic mice.
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Expression of CAT in transgenic mice.
We analyzed SP-B promoter activity in transgenic mice by measuring
tissue expression of CAT activity (Table
1). Results showed that CAT activity was
not detected in the lung extracts of mice from seven independent lines
derived from the 2,176/+39-CAT construct. Analysis of CAT activity in
other tissues, such as liver, kidney, intestine, stomach, trachea,
spleen, heart, and skeletal muscle, in mice from two independent lines
indicated the absence of CAT expression. Tissue-specific expression of
CAT was also analyzed by Northern blotting. Consistent with the results
of tissue expression of CAT activity, Northern blot analysis showed no
expression of CAT mRNA in lung and other tissues (data not shown).
These data indicated that the
2,176/+39-bp region of the SP-B gene is
not capable of directing CAT expression in lung or other tissues.
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Tissue-specific expression of CAT mRNA.
Because CAT expression in mice (3-6 c) derived from the
730/+39-CAT transgene was highest compared with the other lines, we
analyzed tissue-specific expression of CAT mRNA by Northern blotting.
Results showed that similar to the endogenous SP-B mRNA, CAT mRNA
expression was detected only in the lung (Fig.
3).
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Cellular expression of the CAT transgene.
SP-B mRNA is expressed in the alveolar type II and bronchiolar
epithelial cells of adult lungs (20, 30). In the adult rabbit lung, approximately equal concentrations of SP-B mRNA were detected in alveolar type II and bronchiolar epithelial cells (30). We examined the cellular expression of CAT in
transgenic mice by immunohistochemical analysis. Serial sections of the
lung tissues of adult transgenic mice for the 730/+39-bp (line
1, 3-6 c),
236/+39-bp (line 3, 3-6 c), and
wild-type littermates were processed for immunohistochemical analysis
with polyclonal antibodies to CAT and SP-B.
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Developmental expression of CAT mRNA.
We investigated the developmental expression of CAT to determine if the
730/+39-bp region of the SP-B gene was capable of controlling the
developmental expression of the CAT gene in a manner similar to that in
the endogenous SP-B gene. CAT expression in the lungs of embryonic
day 14, 15, 16, and 18 and newborn transgenic mice was analyzed by CAT assay to investigate the developmental regulation of CAT expression (Fig. 5).
CAT expression was detected in embryonic day 14 lungs and
increased markedly as a function of development to reach maximal levels
by embryonic day 18. The expression levels of CAT in newborn
mice were not significantly different from those in embryonic day
18 mice. Investigation of the developmental expression of CAT by
ELISA produced similar results (data not shown). The levels of CAT
expression in embryonic lungs in a given family varied considerably
even though they were expected to have identical sites of integration
and copy numbers of the transgene.
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Analysis of SP-B promoter function in MLE-12 cells.
Immunostaining of lung tissues from mice containing the 236/+39-bp
region of the SP-B promoter demonstrated staining for CAT in both
alveolar type II and Clara cells, although the intensity of staining
was less than that in the mice containing the
730/+39-bp region.
These data suggested that cis-DNA elements within
236 bp
are capable of directing transgene expression in both alveolar type II
and Clara cells. We analyzed SP-B promoter function in MLE-12 cells by
deletion mapping to determine if SP-B promoter fragments confer a
pattern of regulation similar to that in NCI-H441 cells and transgenic
mice. The results showed that deletion of the 5'-flanking SP-B DNA from
2,176 to
730 and
236 bp increased promoter activity by 30%;
however, further deletion to
136 bp reduced activity by
>80% (Fig. 6). The pattern of SP-B
promoter regulation in MLE-12 cells is similar to that observed in H441 cells (15), suggesting that the cis-DNA
elements that control promoter activity in H441 cells also control its
activity in MLE-12 cells.
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DISCUSSION |
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SP-B mRNA is expressed in a highly cell type-specific manner by the alveolar type II and bronchiolar epithelial cells of the lung (20, 29). Although analysis of SP-B promoter expression by transfection studies has indicated that promoter fragments of human (4, 27), rabbit (15), and mouse (6) SP-B genes contain the cis-DNA elements necessary for cell-specific expression, their role in conferring lung cell-specific expression has not been investigated. Furthermore, the regulatory DNA regions that control developmental expression of the SP-B gene have not been identified.
Our laboratory previously determined (15) that rabbit SP-B
5'-flanking DNA sequences within 2,176 bp are capable of expressing the CAT reporter gene in NCI-H441 cells and that a minimal promoter sequence comprising
236/+39 bp was necessary and sufficient for high-level expression of the CAT gene. In the present study, we analyzed SP-B promoter activity in transgenic mice to identify the DNA
regions necessary for cell-specific and developmental regulation of
expression. Transgene expression was not detected in lung and other
tissues of several independent lines of mice harboring the
2,176/+39-bp region of the SP-B gene. The lack of expression of the
transgene may not be related to the site of integration or the copy
number of the transgene because similar results were found in mice
derived from seven independent lines. Although we did not determine the
site of integration of the transgene in each line, it is highly likely
that each line had a unique integration site because the microinjected
DNA integrates randomly in individual embryos (19).
Southern blot analysis did not indicate modification of the injected
DNA by degradation or rearrangement. Taken together, these data
indicated that the
2,176/+39-bp sequence of the SP-B gene is not
capable of expressing the transgene.
The inability of the 2,176/+39-bp region of the SP-B gene to express
CAT suggests the presence of silencer elements within the
2,176-bp
sequence. These DNA elements can bind transcriptional repressors to
promote closed chromatin structure, leading to the inhibition of gene
transcription. Our previous transfection study (15) showed
that deletion of the
2,176-bp fragment of SP-B 5'-flanking DNA to
730 and
236 bp increased CAT expression by nearly twofold
(15), suggesting the existence of putative silencer elements in the upstream region. The results from transgenic mice are
consistent with the data from transfection studies that indicate the
presence of putative silencer elements within the
2,176/
730-bp region of SP-B 5'-flanking DNA.
Because the 2,176/+39-bp region of SP-B 5'-flanking DNA failed to
express the CAT gene in the transgenic mice, we sought to
determine if shorter fragments of SP-B 5'-flanking DNA,
730/+39 and
236/+39 bp, were capable of expressing CAT gene in the
transgenic mice. Our laboratory showed in a previous study
(15) that a minimal promoter region comprising
236/+39
bp of the SP-B gene supported high-level expression of the CAT reporter
gene in H441 cells. CAT expression was detected in a tissue-specific
manner in the lungs of all three independent lines of transgenic mice harboring the
730/+39-bp region. These data indicated that
cis-acting DNA elements within the
730/+39-bp region of
SP-B gene 5'-flanking DNA are capable of directing tissue-specific
expression of the CAT gene in the lung. Because all three independent
lines of transgenic mice expressed CAT specifically in the lung, the
findings cannot be related to the integration site or the copy number
of the transgene.
The 236/+39-bp SP-B region expressed CAT at a significantly lower
level than the
730/+39-bp region. Consistent with these data,
immunohistochemical staining showed diminished CAT expression in
alveolar type II and Clara cells of
236/+39-bp transgenic mice. As in
the case of the
730/+39-bp construct, CAT activity was not detected
in any tissue examined except lung, indicating that strict
tissue-specific control of expression was maintained. Further deletion
of the SP-B promoter to
136 bp resulted in no expression of CAT.
These data are consistent with the results of our previous cell
transfection study (15) that demonstrated the presence of
the cis-DNA elements necessary for lung cell-specific expression within the
236/+39-bp region of the SP-B gene.
SP-B promoter function was regulated similarly in both H441 cells and
MLE-12 cells, suggesting that the same cis-DNA elements control cell-specific expression of the SP-B promoter in alveolar type
II and Clara cells. This conclusion is further supported by the
deletion mapping studies of SP-B promoter function in transgenic mice.
The markedly lower expression of CAT in mice harboring the 236/+39-bp
construct compared with the
730/+39-bp construct suggested that
sequences within the
730/
236-bp region contain enhancer elements
necessary for the optimal expression of SP-B promoter activity. The
lack of CAT expression from the
136/+39-bp region indicates that
upstream sequences contain important cis-DNA elements and is
consistent with the results of in vitro transfection studies that
showed that the
236/
136-bp region contains functional cis-DNA elements for thyroid transcription factor-1 and
Sp1/Sp3 binding (14). cis-DNA elements within
236/
136- and
136/
27-bp regions are necessary for SP-B promoter
activity in H441 cells in vitro (14, 15).
The promoter activity of the 236/+39-bp region in transgenic mice was
significantly lower compared with the activity we (15) previously observed in NCI-H441 cells in vitro. The relatively low
level of expression of the
236/+39-bp promoter in transgenic mice
could be due to the loss of upstream enhancer elements that were not
detected in the in vitro cell culture system. Alternatively, the
differences in the promoter activities between the in vitro (transient
expression) and in vivo (transgenic animal) systems may be related to
the state of the introduced DNA. Whereas the DNA was not integrated
into the chromosome under conditions of the transient expression assay,
it was stably integrated into the chromosome in transgenic mice.
Because CAT expression was detected in both alveolar type II and Clara
cells of mice containing the
236/+39-bp region, albeit at lower
levels compared with the mice containing the
730/+39-bp region, the
decrease in promoter activity could not have been due to the loss of
type II cell-specific enhancer elements but rather due to the loss of
enhancer elements that increase expression in both alveolar type II and
Clara cells.
Genomic DNA regions that control cell- or tissue-specific and
developmental regulation of other genes that are expressed in the
pulmonary epithelium, such as Clara cell secretory protein (CCSP),
SP-C, and SP-A, have recently been identified. A 2.25-kb fragment of
the 5'-flanking region of the rat CCSP gene directed lung- and Clara
cell-specific expression of the human growth hormone (hGH) reporter
gene (12). In a separate study (13), a
2,338/+49-bp fragment of 5'-flanking DNA of the rat CCSP gene
directed expression of the CAT reporter gene in the lungs and tracheae
of transgenic mice (25). Although the CCSP genomic DNA
fragment maintained Clara cell-specific expression of the hGH reporter
gene, the developmental expression of the transgene was not maintained
(13). In other studies, it was found that even though the
166-bp region of the mouse CCSP gene was sufficient to direct Clara
cell-specific expression of the transgene, enhancer sequences between
the
803- and
166-bp regions were required for maximal expression of
the transgene (23). As little as 215 bp of human SP-C
5'-flanking DNA were found to be capable of directing alveolar type II
cell-specific expression of CAT gene
(10).
A DNA fragment consisting of 378 bp of 5'-flanking DNA of the rabbit
SP-A gene directed hGH expression in alveolar type II and bronchiolar
epithelial cells of transgenic mouse lungs (1). The DNA
fragment also specified appropriate developmental regulation of the hGH
gene (1). However, ectopic expression of the hGH gene was
detected in heart, thymus, and spleen. Sequences upstream of the 378-bp
region, 4,000/
991 bp, appeared to be required to prevent ectopic
expression while maintaining lung-specific expression of the hGH gene
(1).
In the present study, we found that the developmental expression of CAT
activity was similar or identical to the expression of the endogenous
SP-B mRNA (9). SP-B mRNA was detected in embryonic
day 15 lung, and its levels increased markedly thereafter to
reach maximal levels in embryonic day 18 lung
(6). These data suggested that the SP-B genomic fragment
730/+39 bp contains necessary information to specify appropriate
developmental regulation of CAT expression. CAT expression levels
during development varied within members of a given family. The exact
reasons for the variation in CAT expression levels within a family are
not clear but may be caused by integration of the transgene at more
than one site in the genome.
Immunostaining experiments demonstrated CAT expression in cells localized at the corners of the alveoli and in cells lining the bronchiolar epithelium in a manner similar to the expression of endogenous SP-B. Because alveolar type II cells are predominantly located at the corners of the alveoli (17) and Clara cells (21) make up the majority of cells lining the bronchiolar epithelium in the adult lung, the staining patterns identified cells expressing CAT as most likely alveolar type II and Clara cells.
In summary, our studies have shown that the 236/+39-bp region of the
rabbit SP-B gene contains the cis-DNA elements necessary for
alveolar type II- and bronchiolar (Clara) cell-specific expression and
that the
730/
236-bp region contains elements that enhance the
expression of the gene. Our data have also indicated that the
2,176/
730-bp SP-B region may contain elements that repress promoter
activity. It remains to be determined whether cis-DNA elements and interacting trans-acting factors that have been
identified as important for SP-B promoter function in vitro are also
necessary for specifying spatial and temporal expression of the SP-B
gene in transgenic mice.
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ACKNOWLEDGEMENTS |
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We thank Dr. Kathy Graves and John Ritter for the generation of the transgenic mice and Dr. Carole Mendelson and Meg Smith for helpful discussions.
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FOOTNOTES |
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* C. C. Adams and M. N. Alam contributed equally to this work.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-48048.
Address for reprint requests and other correspondence: V. Boggaram, Dept. of Molecular Biology, Univ. of Texas Health Science Center, 11937 US Highway 271, Tyler, TX 75710 (E-mail: vijay.boggaram{at}uthct.edu).
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. Section 1734 solely to indicate this fact.
Received 17 March 2000; accepted in final form 17 October 2000.
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