Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands1
Author for correspondence: Barend Kraal. Tel: +31 71 5274770. Fax: +31 71 5274340. e-mail: B.kraal{at}chem.leidenuniv.nl
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ABSTRACT |
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Keywords: rpsB operon, tsf regulation, GTPase switch protein, guanine-nucleotide exchange factor, ribosomal protein S2
Abbreviations: EF, elongation factor
The GenBank accession numbers for the sequences of the S. coelicolor rpsBtsf operon and the S. ramocissimus tsf gene determined in this work are AF034101 and AF130345, respectively.
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INTRODUCTION |
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In Escherichia coli, EF-Tu is the most abundant cytoplasmic protein and occurs at a 5- to 10-fold molar excess over ribosomes. It is encoded by two tuf genes, tufA and tufB, with their gene products differing only in the C-terminal amino acid residue. tufA is located in the rpsL operon, immediately downstream of fus, the gene for EF-G, which in turn is preceded by the ribosomal protein genes rpsL and rpsG (Lindahl & Zengel, 1986 ). tufB is the distal gene in an operon with four tRNA genes (Lee et al., 1981
; Miyajima et al., 1981
). Consequently, while transcription of tufA is subject to the same regulatory controls as ribosomal protein genes (Jinks-Robertson & Nomura, 1987
), tufB transcription is regulated in the same way as stable RNA genes (van Delft et al., 1987
). Nevertheless, expression of tufA and tufB is coordinately regulated in E. coli, as shown by the almost constant 1:1 ratio of EF-TuA and EF-TuB across a range of growth rates (van der Meide et al., 1982
). Similar to fus and tufA, the single gene for EF-Ts in E. coli, tsf, is also located in a ribosomal protein operon, rpsBtsf, which forms a single transcription unit.
The three-dimensional structure of the E. coli EF-Tu·EF-Ts complex was determined to a resolution of 2·5 (Kawashima et al., 1996
). From the crystal structure it appears that domain I of EF-Tu (encompassing the first 200 aa, including the guanine-nucleotide-binding site) interacts predominantly with the N-terminal half of EF-Ts, while EF-Tu domain III (encompassing the last 100 aa) interacts with the C-terminal part of the EF-Ts core (i.e. residues 140180 and 230260). Mutational analysis of EF-Ts confirmed that residues in the N-terminal domain and in subdomain C of the EF-Ts core are crucial for this interaction (Zhang et al., 1998
). The driving force behind GDP release from EF-Tu is probably the dislocation of Mg2+ from the molecule, which is catalysed by EF-Ts (Kawashima et al., 1996
).
In most Gram-positive bacteria, the high concentration of EF-Tu in the cytoplasm is maintained by a single tuf gene. Interestingly, multiple tuf genes are found in members of the genus Streptomyces, a Gram-positive soil bacterium with a complex mycelial life cycle that includes sporulation (Hopwood et al., 1995 ). For example, three tuf genes occur in Streptomyces ramocissimus, the producer of kirromycin, an antibiotic that binds specifically to EF-Tu. The S. ramocissimus tuf genes, designated tuf1, tuf2 and tuf3, encode EF-Tus that are surprisingly heterogeneous: EF-Tu3 shows only about 65% amino acid identity with EF-Tu1 and EF-Tu2 (Vijgenboom et al., 1994
). Streptomyces coelicolor A3(2) and other streptomycetes contain two tuf genes (van Wezel, 1994
), designated tuf1 and tuf3 by reference to the corresponding genes in S. ramocissimus. Streptomyces tuf1 encodes the major EF-Tu and, like E. coli tufA, is located in the rpsL operon; it is transcribed at a very high level during exponential growth (van Wezel et al., 1995
) from the rpsL operon promoter and a promoter specific for tuf1 (Tieleman et al., 1997
). The roles of tuf2 and tuf3 are unclear; under normal growth conditions, the gene products could not be detected. Interestingly, tuf3 transcription was shown to be subject to positive stringent control, and is also induced by other stress conditions, including heat shock (van Wezel et al., 1995
; G. P. van Wezel, unpublished results).
The multiplicity of tuf genes in various Streptomyces species, and the high level of divergence between the gene products, raised the question as to whether the latter interact with a single EF-Ts species. This prompted investigation into the number of tsf genes in streptomycetes and the homology of the gene products to other known EF-Ts species. In this paper we present the sequences of the single tsf genes in S. coelicolor A3(2) and S. ramocissimus, and provide a transcriptional analysis of the S. coelicolor rpsBtsf operon both in vivo and in vitro.
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METHODS |
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Liquid cultures were grown in TSBS medium [3% (w/v) Difco tryptone soy broth with 10% (w/v) sucrose], in yeast extract/malt extract medium (YEME) supplemented with 0·5% (w/v) glycine (Hopwood et al., 1985 ), or in minimal medium (MM) with 1% (w/v) glucose as carbon source, and were inoculated at a density of approximately 5x106 c.f.u. ml-1 and grown at 30 °C with vigorous shaking (300 r.p.m. min-1). Cell growth was measured as increase in optical density at 600 nm.
PCR conditions.
PCRs were performed in a minicycler (MJ Research), using Pfu polymerase (Stratagene) and the buffer provided by the supplier, in the presence of 5% (v/v) DMSO and 200 µM dNTP. No additional Mg2+ was added to the reaction mixture. The following PCR programme was used: 30 cycles of 45 s melting at 94 °C, 1 min annealing at 54 °C and 90 s extension at 72 °C, followed by an additional 10 min at 72 °C.
DNA manipulation and sequencing.
DNA cloning, isolation and gel electrophoresis were performed by standard procedures (Sambrook et al., 1989 ). DNA was sequenced using the T7 DNA polymerase sequencing kit (Pharmacia). For sequencing of rpsB and tsf, we used subclones in pUC18 as templates, and where necessary oligonucleotides were designed and used to fill in the gaps.
Southern hybridization.
Genomic DNAs used for Southern analysis were isolated as described by Hopwood et al. (1985) . For high-resolution hybridization experiments to investigate the presence of tsf in S. coelicolor and S. ramocissimus, genomic DNA was digested with the appropriate enzymes and separated electrophoretically on a 0·7% agarose gel in TAE buffer, using the Gibco-BRL 1 kb ladder as DNA size markers. Agarose gels were pretreated and subsequently blotted on Hybond-N+ nylon membranes (Amersham) using 20xSSC as the transfer buffer, basically according to Sambrook et al. (1989)
. Hybridization and washing conditions were as described previously (van Wezel et al., 1991
).
Probes.
As a probe for screening the M145 cosmid library we used Tsf2 (Fig. 1a), a fragment generated by PCR of S. coelicolor chromosomal DNA using oligonucleotides TSF1 and TSF2. The latter are multiply degenerated oligonucleotides designed to match highly conserved regions from known tsf genes and correspond to the sequences encoding amino acid residues 2027 and 7582 of E. coli EF-Ts, respectively. For Southern hybridization experiments we used Tsf3, a 1 kb BglII fragment from pUSCTs-1 harbouring the entire S. coelicolor tsf gene (Fig. 1a
). Both probes were 32P-labelled by random priming (Feinberg & Vogelstein, 1983
).
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Promoter-probe experiments.
The promoterless S. coelicolor redD gene present in pIJ2587 (G. P. van Wezel, J. White & M. J. Bibb, unpublished results) was used as a reporter gene for screening of in vivo promoter activity. Insertion of the 1·3 kb SmaIPstI fragment, containing the start of rpsB and upstream sequences (Fig. 1a), into pIJ2587 resulted in pIJ2587-rpsB. To screen the rpsBtsf intergenic region for possible promoter activity, we cloned the 0·73 kb SacI fragment into pIJ2587, with the start of tsf oriented towards the start of redD, resulting in pIJ2587-tsf. M512 transformants containing pIJ2587, pIJ2587-rpsB or pIJ2587-tsf were grown on R2YE and MM in the presence of 10 µg thiostrepton ml-1. Undecylprodigiosin production was assessed visually.
RNA isolation.
RNA was isolated from M145 according to Hopwood et al. (1985). To remove residual DNA, the RNA was salt-precipitated in 3 M sodium acetate (pH 6·0) and subsequently treated with DNase I (37 °C for 1 h, with 0·1 U DNase I per 50 ml initial culture sample). Samples were then extracted with a 1:1 mixture of phenol and chloroform (saturated with 100 mM Tris pH 7·0) and precipitated in 0·4 M sodium acetate (pH 6·0) with 2-propanol. The RNA was resuspended in water and the concentration determined spectrophotometrically.
Nuclease S1 mapping.
RNA (10 µg) was hybridized with the appropriate DNA probe according to Murray (1986) in TCA buffer (Summerton et al., 1983
). All subsequent steps were carried out as described previously (Strauch et al., 1991
), using an excess of probe.
Northern analysis.
RNA samples (approx. 10 µg) were glyoxylated, run in a 1·2% agarose gel in 20 mM sodium phosphate buffer (pH 6·7) and transferred to Hybond-N+ nylon membranes using 30 mM sodium phosphate (pH 6·7) as the blotting buffer. Hybridization and washing conditions were as described previously (Tieleman et al., 1997 ).
Computer analysis.
The blast search engines blastn, blastp and blastx (Altschul et al., 1997 ) were used to perform database searches; the Wisconsin GCG Package (Devereux et al., 1984
) for sequence alignments, protein analysis and prediction of RNA secondary structures; and rasmol (Sayle & Milner-White, 1995
) for drawing the EF-Tu·EF-Ts structure.
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RESULTS |
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An internal fragment of the tsf gene from S. coelicolor, designated Tsf2, was obtained by PCR, using S. coelicolor A3(2) M145 total DNA as template and as primers oligonucleotides TSF1 and TSF2 (see Methods section and Fig. 1a). Tsf2 was hybridized to the cosmid library of S. coelicolor M145 (Redenbach et al., 1996
), allowing identification of a cosmid clone harbouring the complete tsf gene and flanking regions. tsf was identified on the overlapping cosmids 2E1 (GenBank accession no. AL023797) and 6A9 (AL031035), located between 5 and 6 oclock on the chromosomal map. A 2·1 kb KpnIBglII fragment was isolated from cosmid 2E1 and subcloned into KpnI/BamHI-digested pUC18, resulting in pUSCTs-1. A restriction map is shown in Fig. 1(a
).
To check whether more than one tsf gene is present in S. coelicolor or S. ramocissimus, genomic DNA was isolated from each of these strains, digested with BamHI or PstI and fractionated by agarose gel electrophoresis. The gel was blotted and hybridized with Tsf3 (Fig. 1a) at low stringency to identify all genes with some similarity to tsf. A single hybridizing band was observed in each lane, indicating that only one tsf gene occurs in both organisms. A representive result is shown in Fig. 2
.
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Sequence analysis of the chromosomal region around the rpsBtsf operon in the S. coelicolor A3(2) genome has recently been completed (Sanger Centre, Cambridge, UK; sequencing data can be obtained from ftp://ftp.sanger.ac.uk/pub/S_coelicolor/sequences). The data indicate that the rpsBtsf gene cluster is located downstream of a putative regulatory gene, which in turn is preceded by the developmental sigma factor gene whiG (Chater et al., 1989 ). Downstream of rpsBtsf lies pyrH, a gene involved in pyrimidine biosynthesis.
The S. coelicolor S2 protein consists of 310 aa with sequence identity to approximately 50% of the 241 aa in E. coli S2 (GenBank accession no. P02351) and carries a C-terminal domain significantly larger than that of most other S2 homologues in eubacteria, with an extension consisting of a repetitive amino acid sequence (Fig. 1b). Considering the unusual nature of the C-terminal extension, and the presence of an out-of-frame stop codon immediately in front of it, the sequence of this region is of particular interest. Its accuracy has therefore been confirmed by extensive DNA sequencing carried out by the Sanger Centre on both cosmids 2E1 and 6A9. The only other example of an S2 protein with a repetitive sequence in its C-terminal region was found in Mycobacterium tuberculosis (GenBank accession no. Q10796), but the extension shares no significant homology to that of S. coelicolor EF-Ts.
Comparison of the S. coelicolor and S. ramocissimus tsf genes with E. coli tsf
The S. ramocissimus tsf gene was obtained by PCR on chromosomal DNA using oligonucleotides based on the sequence of the S. coelicolor rpsBtsf gene cluster. A 1·45 kb DNA fragment containing the S. ramocissimus tsf gene and upstream sequences was amplified using oligonucleotides corresponding to nt positions -529/-510 and +939/+959 with respect to the start of S. coelicolor tsf, respectively. The 0·95 kb BglII segment internal to the PCR-amplified DNA fragment was inserted into BamHI-digested pUC18, resulting in pUSRTs-1, and the DNA sequence of the insert was determined (GenBank accession no. AF130345).
The S. coelicolor and S. ramocissimus tsf genes display high sequence homology and encode a protein of 278 aa. A sequence alignment of the derived EF-Ts gene products of E. coli, S. coelicolor and S. ramocissimus tsf is shown in Fig. 3. The Streptomyces gene products show 93% identical amino acids, and exhibit approximately 40% amino acid sequence identity with E. coli EF-Ts. The N-terminal parts of the proteins share significantly higher similarity than the middle and C-terminal parts. The EF-Ts signature TDFV (underlined in Fig. 3
) is conserved among the three EF-Ts species. Residues reported to be involved in interaction with EF-Tu in the crystallized EF-Tu·EF-Ts complex (Kawashima et al., 1996
) are shown under the alignment. Triangles represent contact residues conserved among all three EF-Ts species, while asterisks represent contact residues that differ between E. coli and Streptomyces EF-Ts (Fig. 3
). Interestingly, while all contact residues conserved in EF-Ts of E. coli and Streptomyces (triangles) correspond to residues in EF-Tu that are conserved in the three different EF-Tu species (EF-Tu1, 2 and 3), those contact residues that are different in the Streptomyces EF-Ts (asterisks) correspond to putative contact residues in EF-Tu1 and EF-Tu3 that are also different.
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The upstream region of S. coelicolor rpsB was cloned as a 1·3 kb SmaIPstI fragment into pIJ2587 digested with the same enzymes, and the rpsBtsf intergenic region was cloned as a 0·73 kb SacI fragment into SacI-digested pIJ2587 with the start of tsf proximal to redD, resulting in pIJ2587-rpsB and pIJ2587-tsf, respectively. Introduction of pIJ2587-rpsB resulted in strong Red production as soon as recombinant colonies could be discerned, indicative of the presence of at least one promoter in the SmaIPstI fragment harbouring the start of rpsB and 980 bp of upstream region. The lack of any trace of Red production by recombinant M512 containing pIJ2587-tsf was similar to the result with control transformants harbouring pIJ2587 without an insert, and suggested that transcription is not initiated in the rpsBtsf intergenic region.
The rpsBtsf gene cluster is transcribed from a single promoter
To establish the level and timing of rpsB transcription in vivo, RNA was isolated from mycelium harvested from S. coelicolor cultures grown in MM with 1% glucose. RNA samples were analysed by high-resolution nuclease S1 mapping, using Rps1 (Fig. 1a) as the probe, and the protected fragment was analysed alongside a DNA sequence ladder produced with oligonucleotide RP1 as sequencing primer. A representative result is shown in Fig. 4
. A band corresponding to rpsB transcripts could be readily identified in RNA isolated at all time points; the signals arising from RNA isolated during early exponential phase (lanes 12) were significantly more intense than those arising from RNA isolated in later growth phases (lanes 36). The rpsB transcript corresponds to a transcriptional start site 161162 nt upstream of the rpsB translational start site. The putative transcriptional start site is preceded by the sequences GTCACC around -35 and TACACT around -10, which are separated by 19 bp (Fig. 1b
). These sequences show low but relevant similarity to the consensus sequence for the major class of eubacterial promoters, TTGACA around -35 and TATAAT around -10 (Hawley & McClure, 1983
). In S. coelicolor, such canonical sequences appear to be recognized by
hrdB (Brown et al., 1992
).
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We also analysed tuf1 transcription in S. coelicolor by nuclease S1 mapping, using the 200 bp tuf1 probe. Comparison of rpsBtsf and tuf1 transcription (Fig. 4) shows that timing of expression is similar, although rpsB transcripts reach a maximum somewhat earlier than tuf1. Two tuf1 transcripts were identified, one originating from a promoter with a transcription start site 125 nt upstream of the start of the gene (designated tuf1p), and one corresponding to full-length protection of the probe, indicative of a distal promoter, and most likely the rpsL operon promoter (rpsLp). While both promoters show the same growth-phase-dependence, tuf1p apparently contributes more to tuf1 transcription under these conditions than rpsLp. This regulation is similar to that observed for the S. ramocissimus rpsL operon (Tieleman et al., 1997
). Transcription probably starts at the G residue at position -125 relative to the start of tuf1, which is 2 nt downstream of the transcription start site predicted by Tieleman et al. (1997)
on the basis of sequence homology to S. ramocissimus tuf1.
In E. coli, rpsB is expressed more abundantly than tsf, resulting in a 3:1 ratio of the respective gene products, a phenomenon possibly due to terminator sequences immediately downstream of rpsB (An et al., 1981 ). To establish whether such a transcriptional regulation occurs in S. coelicolor, the same RNA as used for nuclease S1 mapping experiments was analysed by Northern blotting using Rps2 (Fig. 1a
) as the probe. The experiment confirmed the growth-phase-dependent regulation of the S. coelicolor rpsBtsf operon (Fig. 5
). Interestingly, two bands could be detected in all lanes, corresponding to transcripts with sizes of approximately 1100 and 2100 nt. Since we have identified the exact location of the rpsB promoter (Fig. 1b
), we can conclude that the smaller band corresponds to the monocistronic rpsB transcript, while the larger band corresponds to the bicistronic rpsBtsf transcript. The approximately equal intensity of the bands suggests a 2:1 molar ratio of rpsB transcripts versus tsf transcripts. The apparent termination immediately downstream of rpsB may be explained by the presence in this region of a perfect inverted repeat, consisting of two elements of 16 bp, followed by an unusually A+T-rich region (Fig. 1b
).
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DISCUSSION |
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The regular EF-Tu1 is an abundant cytoplasmic protein occurring at a considerable molar excess over ribosomes. This implies that expression of the distal tuf1 in the rpsL operon in Streptomyces is individually regulated, since the other three genes express ribosomal proteins S12 and S7 and elongation factor G at levels about equimolar to that of ribosomes. Indeed, we showed that tuf1 is transcribed not only from the rpsL promoter for full operon transcription, but also from a second promoter located in the intergenic region between fus and tuf1 of S. ramocissimus (Tieleman et al., 1997 ) and S. coelicolor (Fig. 4
). In the case of S. coelicolor, tuf1p contributes at least as much to transcript levels during normal growth as rpsLp. For the rpsB operon, the opposite situation prevails since transcription of the distal tsf is expected to be significantly weaker than that of rpsB. In our in vivo promoter probing assays, the Red+ phenotype of S. coelicolor M512 transformants containing pIJ2587-rpsB revealed a promoter within 980 nt upstream of the rpsB translational start site, and the failure of transformants containing pIJ2587-tsf to synthesize Red indicated that promoter activity was absent from the rpsBtsf intergenic region.
Transcription analysis by nuclease S1 mapping of the rpsB start region detected rpsB operon transcripts in S. coelicolor RNA isolated from all growth phases, with the highest transcript levels during early exponential growth (Fig. 4). This suggests that rpsBtsf transcription shows growth-phase-dependent regulation typical of genes for translational components (Lindahl & Zengel, 1986
). Similar analysis of tuf1 transcription (the latter located in the rspL operon) showed that both operons are transcribed in a similar way, although rpsB operon transcripts reached peak levels earlier (at an OD600 below 0·2) than tuf1 transcripts (OD600 0·4).
The putative -35 and -10 sequences of the rpsBtsf promoter (GTCACC and TACACT, separated by 19 bp) show 3 and 4 out of 6 nt matches, respectively, with consensus sequences for the major class of eubacterial promoters (TTGACA and TATAAT, separated by 1618 bp; Hawley & McClure, 1983 ). Many Streptomyces promoters show relatively low similarity to the consensus promoter (Strohl, 1992
), especially around the -35 sequence, and the timing of expression of the rpsBtsf operon suggests that its regulation is similar to other operons for proteins involved in translation.
Further transcription analysis by nuclease S1 mapping of the rpsBtsf intergenic region revealed only a single band corresponding to read-through from an upstream promoter (data not shown). As in E. coli (Bendiak & Friesen, 1981 ), this indicates that there is no promoter immediately upstream of tsf, and that rpsB and tsf are transcribed from a single promoter upstream of rpsB.
An obvious way of regulating transcription and enhancing expression of rpsB in comparison to tsf could be found in the presence of an additional terminator in the rpsBtsf intergenic region. Indeed, Northern blotting of the S. coelicolor M145 RNA used for nuclease S1 mapping with the Rps2 probe revealed two bands with sizes corresponding to the full operon length and an additional rpsB transcript (Fig. 5). Such an attenuation mechanism had already been postulated by An et al. (1981)
for the rpsBtsf operon in E. coli on the basis of a long but imperfect inverted repeat in the intergenic region. In the intergenic region of the S. coelicolor rpsBtsf operon we see a perfect inverted repeat consisting of two 16 bp elements, possibly allowing Rho-independent termination of the RNA polymerase. A different attenuation mechanism was proposed for the rpsBtsf operon of Spiroplasma citri, where a DNA-binding protein was shown to interact with the region immediately downstream of rpsB (Le Dantec et al., 1998a
, b
). Processing of the rpsBtsf transcript at the inverted repeat is unlikely since S1 nuclease mapping by means of the Tsf1 probe gave only one signal corresponding to full-length protection.
While most eubacterial S2 proteins contain approximately 240 aa residues, for example 241 aa in E. coli (GenBank accession no. P02351), 246 aa in Bacillus subtilis (P21464) and 251 aa in Haemophilus influenzae (P44371), sequence comparison showed S. coelicolor S2 to be a 310 aa protein with a 70 aa C-terminal extension. The highly repetitive 46 aa in the C-terminal region of S. coelicolor S2 consist almost exclusively of Ala, Glu and Pro residues. The S2 homologue of the related actinomycete M. tuberculosis (286 aa; Q10796) also has a repetitive sequence, but it shows no relevant similarity to the extension of S. coelicolor S2. The function of the repetitive C-terminal extension, which may be limited to members of the actinomycete family, is unknown.
The so-called EF-Ts signature sequence TDFV is strictly conserved and is essential for interaction with EF-Tu. From the crystal structure of the E. coli EF-Tu·EF-Ts complex (Fig. 6), Kawashima et al. (1996)
concluded that these residues are involved in dislocating the magnesium ion from EF-Tu, forcing GDP to dissociate from the molecule. Residues known to be involved in the direct interaction between EF-Tu (backbone represented in light blue) and EF-Ts (yellow) are highlighted in Fig. 6
. Amino acids coloured black in the guanine-nucleotide-binding domain of the EF-Tu moiety represent interaction residues also conserved in Streptomyces EF-Tu1, 2 and 3, and the black ones in the EF-Ts moiety represent the contact residues also conserved in S. coelicolor and S. ramocissimus EF-Ts. Interestingly, when interaction residues in the EF-Tu moiety of the complex show divergence among the three Streptomyces species, i.e. aa 323, 349 and 351 in the E. coli numbering (indicated in purple), it transpires that the corresponding residues in EF-Ts, namely aa 167, 170, 234 and 235 in E. coli numbering (red) are also changed.
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ACKNOWLEDGEMENTS |
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We would like to dedicate this paper to Professor Sir David Hopwood on the occasion of his retirement.
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Received 8 March 1999;
accepted 4 May 1999.