Bioinformatics Advance Access originally published online on November 14, 2006
Bioinformatics 2007 23(2):150-155; doi:10.1093/bioinformatics/btl575
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Complementary intron sequence motifs associated with human exon repetition: a role for intragenic, inter-transcript interactions in gene expression
1 Department of Cardiovascular Sciences Leicester, LE3 9Q, UK
2 Department of Biochemistry, University of Leicester Leicester, LE3 9Q, UK
*To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT 1 INTRODUCTION2 METHODS3 RESULTS4 DISCUSSIONREFERENCES |
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Motivation: Exon repetition describes the presence of tandemly repeated exons in mRNA in the absence of duplications in the genome. The regulation of this process is not fully understood. We therefore investigated the entire flanking intronic sequences of exons involved in exon repetition for common sequence elements.
Results: A computational analysis of 48 human single exon repetition events identified two common sequence motifs. One of these motifs is pyrimidine-rich and is more common in the upstream intron, whilst the other motif is highly enriched in purines and is more common in the downstream intron. As the two motifs are complementary to each other, they support a model by which exon repetition occurs as a result of trans-splicing between separate pre-mRNA transcripts from the same gene that are brought together during transcription by complementary intronic sequences. The majority of the motif instances overlap with the locations of mobile elements such as Alu elements. We explore the potential importance of complementary intron sequences in a rat gene that undertakes natural exon repetition in a strain specific manner. The possibility that distant complementary sequences can stimulate inter-transcript splicing during transcription suggests an unsuspected new role for potential secondary structures in endogenous genes.
Availability:
Contact: rd67{at}le.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
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1 INTRODUCTION |
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TOPABSTRACT1 INTRODUCTION2 METHODS3 RESULTS4 DISCUSSIONREFERENCES |
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The majority of human genes are now known to be involved in mRNA alternative splicing suggesting that alternative splicing is one of the most significant processes contributing to the functional complexity of the human genome (Johnson et al., 2003; Maniatis and Tasic, 2002; Modrek and Lee, 2002). Most alternative splicing research to date has focussed on alternative cis-splicing, in which exons located within an individual pre-mRNA are differentially joined to generate mature mRNAs (Black, 2003). This involves joining the exons to be included in the mature transcript in a linear contiguous manner, 5'–3', in the order they are found in the genome. However, the discovery of tandemly repeated exons in mammalian mRNA in the absence of duplications in the genome, a phenomenon termed exon repetition, (or non-linear mRNA) suggested that there is an additional level of alternative splicing complexity in mammals (Caudevilla et al., 1998; Frantz et al., 1999). Exon repetition is allele-specific and operates strictly in cis, meaning that in heterozygotes only mRNA from the susceptible allele contains the repetition (Rigatti et al., 2004). Until recently <10 genes have been discovered, mostly through serendipity, to exhibit exon repetition in mammals. However, our recent genome wide survey of candidate exon repetition events in expressed sequences from multiple species has yielded evidence for the occurrence of this phenomenon in at least 1% of mammalian genes suggesting that this process could contribute to phenotypic variation (Dixon et al., 2005).
Despite the thousands of mRNA alternative splicing events identified to date, the regulation of alternative splicing is still not fully understood (Maniatis and Tasic, 2002). Regulatory sequence elements that direct alternative splicing have been characterized but it is still unclear how the majority of alternative splicing events are regulated (Ladd and Cooper, 2002; Matlin et al., 2005). Superimposed upon this complexity of splicing regulatory elements is the role of the promoter architecture and the rate of RNA transcription procession (Proudfoot et al., 2002). One aspect of uncertain significance is the influence of pre-mRNA secondary structures on splicing activity (Buratti and Baralle, 2004; Eperon et al., 1988).
To explore the possibility that exon repetition is regulated by specific sequences in the exons and their flanking introns, we have analysed a set of 48 human single exon repetition events. These are events that comprise repetition of a single exon sequence (e.g. exon 2-2) within an expressed sequence from GenBank. Computational analysis of the entire exons and intronic sequences flanking these events was undertaken with algorithms that identify common motifs in unaligned sequences. This approach has revealed two motifs, a pyrimidine rich motif that is more predominant in the upstream introns and a purine rich motif that is more predominant in the downstream introns. Interestingly, these sequence motifs are complementary suggesting that base pairing interactions between these motifs could contribute to an RNA secondary structure that is conducive to the phenomenon of exon repetition taking place. This hypothesis is investigated by sequence analysis of the rat Sa gene in two different strains, one that exhibits exon repetition and one that does not (Frantz et al., 1999; Rigatti et al., 2004).
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2 METHODS |
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TOPABSTRACT1 INTRODUCTION2 METHODS3 RESULTS4 DISCUSSIONREFERENCES |
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2.1 Detection of candidate exon repetition events in expressed sequences
The dataset of single exon repetition events used in this study were derived from a computational genome wide survey as implemented and described in detail previously (Dixon et al., 2005). The current survey in human was implemented exactly as mentioned previously (Dixon et al., 2005) except for the use of more recent EST and mRNA sequence archives. Human mRNA sequences were downloaded from the UCSC genome browser (UCSC hg17, May 2004, NCBI 35 assembly), (http://hgdownload.cse.ucsc.edu/goldenPath/hg17/bigZips/). Human EST sequences were downloaded from NCBI (February 2006), (ftp://ftp.ncbi.nih.gov/blast/db/FASTA/).
2.2 Intron and exon sequence retrieval
The intron and exon sequences from the 48 human single exon repetition events that were used in this study were obtained from Ensembl (v29.35b, NCBI 35 assembly), (http://www.ensembl.org/). The flanking intron sequences were obtained by using a series of Perl scripts incorporating the BioPerl module ‘intron.pm’ (http://doc.bioperl.org/releases/bioperl-current/bioperl-live/Bio/SeqFeature/Gene/Intron.html) that utilized the Ensembl Perl API. As a background intron sequence data set we used the above Perl programs to obtain the entire upstream and downstream introns of a random set of 1038 exons selected from a list of all Ensembl human exons, which had been filtered to remove those genes for which we have evidence for involvement in exon repetition. Random sampling was undertaken using a Perl program with a random number generator.
2.3 Identification of common motifs and sequence analysis
Two separate programs that search for common sequence motifs in unaligned sequences were used, MEME (http://meme.sdsc.edu/meme/intro.html) (Bailey and Elkan, 1994) and the Gibbs motif sampler (http://bayesweb.wadsworth.org/gibbs/gibbs.html) (Lawrence et al., 1993). These programs were run on the exon sequences, the entire upstream and downstream intron sequences on a local Linux machine, with the default parameters when the length of the searched motif was limited to 20 nt, incorporated with a background dataset of 1038 upstream and downstream introns from a random set of exons that are not currently associated with exon repetition. The identified motifs in MEME were searched in the different datasets using the program MAST (http://meme.sdsc.edu/meme/mast-intro.html) (Bailey and Gribskov, 1998). The identified motifs were represented as weight matrices and presented in a graphical format with the Pictogram program (http://genes.mit.edu/pictogram.html) (Lim and Burge, 2001). The program Einverted from the EMBOSS package (version 3.0) was used to search for inverted repeats sequences in intronic sequences with default parameters (http://emboss.sourceforge.net/) (Rice et al., 2000). The program Needle from the EMBOSS package (version 3.0) was used to undertake global sequence alignments with default parameters (http://emboss.sourceforge.net/) (Rice et al., 2000). The program RNAcofold from the Vienna RNA package (version 1.6.1) was used with default parameters to predict the RNA secondary structure of two complementary RNA sequences (http://www.tbi.univie.ac.at/~ivo/RNA/) (Bernhart et al., 2006). The locations and sequences of transposable elements in the human genome was obtained from the RepeatMasker track of the UCSC genome browser (Karolchik et al., 2003). These are the output from RepeatMasker [http://www.repeatmasker.org]. The Rat Sa gene sequences were also analysed for transposable elements using the RepeatMasker web server.
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3 RESULTS |
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TOPABSTRACT1 INTRODUCTION2 METHODS3 RESULTS4 DISCUSSIONREFERENCES |
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3.1 Detection of candidate exon repetition events in expressed sequences
To maximize the number of exon repetition events available for analysis, we extended our previous genome wide survey (Dixon et al., 2005) for candidate single exon repetition events in human expressed sequences from Genbank. From this search a total of 54 human expressed sequences from 48 human genes were identified as candidate single exon repetition events (Supplementary Table A). We chose to utilize only the single exon repetition events for sequence motif analyses as these present a more simplified system of exon repetition to analyse than multiple exon repetition events.
3.2 Features of the single exon repetition events and the flanking intron sequences
We examined the nucleotide composition of the repeated exons and their flanking introns, to elucidate whether they deviate from the control sequences (exons and introns that are not associated with exon repetition). As shown in Supplementary Table 1, the composition of the repeated exons was not significantly different from that of the control exons. In both repeated and control exons, guanine and cytosine residues were over-represented. The introns flanking the repeated exons were not significantly different from the control introns, both sets being enriched in adenine and thymine residues. The lengths of the repeated exons and their flanking introns were compared as shown in Table 1. Our null hypothesis that the lengths of the introns from the control and exon repetition groups were not different was rejected. Also, the exons involved in exon repetition are significantly longer than the control exons.
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3.3 Intron and exon sequence motif search
We examined the exons and flanking introns of the 48 human single exon repetition events that we identified in the genome wide survey described above. The common motifs identified by MEME in the exon sequences were of low informational value (<10 bits), compared to the identified motifs in both the upstream and downstream intron sequences that were of high information content (>30 bits; Fig. 1). We cannot rule out the existence of exonic motifs that regulate exon repetition, however they are probably much shorter than the 20 base window of our search and may be less well defined. It has been shown previously (Miriami et al., 2003) that using a 20 base window with MEME is appropriate for detecting intron sequence motifs associated with alternative splicing. Therefore we decided to concentrate on the intronic motifs that we discovered. Our results from the MEME program were supported by application of the Gibbs sampler program, which produced the same results for the intron sequences. As can be seen in Figure 1a the upstream intron motif is highly enriched in pyrimidines and the downstream intron motif (Fig. 1b) is highly enriched in purines. Remarkably these two consensus motifs are
100% complementary (Fig. 2).
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The program MAST was used to locate the two identified motifs in various datasets requiring an E-value of <10–5. All positive matches with the MAST program at this E-value exhibited at least 15 out of 20 base matches with the consensus motif sequence. Indeed 261 out of the 269 motif instances uncovered by MAST (Supplementary Figure 1), showed a base match score of 18–20 out of 20 bases with the relevant consensus motif sequence. In the training data set, we found an instance of at least one of the motifs in 43 out of 48 (90%) of the flanking introns from the single exon repetition events. Further analysis revealed that all of these 43 events contained this pair of motifs in a pattern where at least one of the pyrimidine rich and one of the purine rich motifs appeared in a complementary manner in the flanking introns. The locations and instances of these motifs within the flanking introns are summarized in Supplementary Figure 1. Of these 43 events, 13 contain the two motifs in both flanking introns of the repeated exons. It is evident that some introns are more enriched with these motifs than others. However, each of the 43 single exon repetition events has at least one pyrimidine rich motif in the upstream intron and at least one purine rich motif in the downstream intron to create the pair of motifs. If these motifs were indeed associated with exon repetition, then we would not expect to find them frequently in our background flanking intron data set of 1038 exons from genes not known to be involved in exon repetition. We found that only 11.6% (121 out of 1038) of these random exons contained the pair of motifs in their flanking introns in a complementary manner (at least one motif per flanking intron). We also searched the flanking introns of the exons we found to be involved in multiple exon repetition from our previous genome wide survey (Dixon et al., 2005). Of these 156 multiple exon repetition events, we found that 86% (134 out of 156) contained the pair of motifs in a complementary manner with the combination of upstream intron of the 3'-exon in a non-linear splice junction and the downstream intron of the 5'-exon involved in the non-linear exon splice junction (for example, in a 2-3-2-3 multiple exon repetition event, the upstream intron of exon 2 and the downstream intron of exon 3). The presence of these motifs in a complementary manner in other intron combinations from the data set of multiple exon repetition events was much lower, <40% in all combinations. We also observed the pair of intronic motifs in a complementary manner in introns 1 and 3 of the human Sp1 gene (Takahara et al., 2005), which is the most studied human gene exhibiting this phenomenon and has been shown to exhibit exon repetition of exons 2 and 3 (2-3-2-3).
3.4 Further analysis of the identified intron motifs
A sequence comparison of the intronic motifs with known human transposable elements revealed that the purine-rich motif is identical to a region of the Alu consensus sequence. An analysis of the location of the 269 motif instances in Supplementary Figure 1 and their overlap with known transposable elements has revealed that the majority (88%) of these motif instances are the result of transposable element insertions (Supplementary Table 2). Indeed, the Alu family elements represent the largest fraction (79%), with the LINE family elements representing 9%. Of the 43 exon repetition events depicted in Supplementary Figure 1, there are 15 that display a single instance of each motif in the flanking introns. Of these 15 events, 9 are the result of Alu insertions in both flanking introns, to produce an inverted Alu pair. Interestingly, 4 of the 43 exon repetition events in Supplementary Figure 1 (designated with ‘w’) have single instances of the motifs in both flanking introns in which one of them is a weak motif (15–17 out of 20 bases match with the consensus motif), yet for 3 out of these 4 events the sequence complementarities between the motifs is maintained (>90%). These weak motif instances do not coincide with the location of a transposable element. Moreover, for the 15 exon repetition events that display a single instance of each motif in the flanking introns, 14 of them exhibit >90% sequence complementarity between the pair of motifs.
3.5 The potential role of intron motifs in exon repetition
The most apparent pattern that we observed from the distribution of our two intron motifs (Supplementary Figure 1) was that, of the 43 single exon repetition events that contained a >75% sequence match to both consensus motif sequences, all contained at least one instance of each motif in the flanking introns. A number of these events contained only one instance of the motifs in their entire intron sequence, suggesting that they are not involved in causing stem-loop structures within the same intron. Also, in instances where there are multiple copies of both motifs in the flanking introns, the order and the distance from the exon of the pyrimidine-rich and purine-rich motifs is not conserved suggesting that these motifs are not involved in creating a stem-loop structure with both flanking introns to form a loop containing the exon. Indeed, we found no evidence within the Alternative Splicing Database (Stamm et al., 2006) that any of these 43 exons are skipped in linear mRNA spliceforms transcribed from their respective genes. These observations suggest a model in which the complementary intron sequence motifs could be involved in base-pairing between separate pre-mRNA transcripts. In Figure 2, a model for the possible role of these intron motifs in exon repetition is illustrated for a gene that exhibits non-linear splicing of exon 2, producing a duplicated exon 2 in the resultant mRNA. Folding these two intron sequence motifs by the RNAcofold program reveals a predicted RNA duplex secondary structure with free energy of –42 kcal/mol.
To investigate the potential role of complementary intron sequences in exon repetition we analysed the rat Sa gene sequence in two different strains, the WKY strain which exhibits exon repetition isoforms 2-2 and 2-3-4-2-3-4 and the SHR strain which does not exhibit exon repetition at all. The sequences of cloned Sa genes from WKY and SHR rats are available from GenBank with accession nos AY456695 [GenBank] (WKY) and AY455861 [GenBank] (SHR). A previous detailed comparative analysis of these sequences (Rigatti et al., 2004) has shown that the major sequence difference between the strains in this gene is a LINE element of 1.4 kb in the SHR intron 1 that is absent in WKY. The program Einverted was used to detect complementary sequences between the introns 1 and 4 in both strains. We found the same complementary sequences between introns 1 and 4 for both strains (Fig. 3a) suggesting that these could be involved in base pairing between separate transcripts. A global alignment of the two sequences using the EMBOSS needle program showed that there was only one single nucleotide difference between the strains in the intronic complementary regions identified in Figure 3a. However, the LINE element insertion in the SHR intron 1 sequence introduces an extra potential base pairing interaction within intron 1, that would cause a large stem-loop structure within intron 1 (free energy of –89 kcal/mol) (Fig. 3a). This intron 1 stem-loop structure would not be present in the WKY strain. We hypothesize (Fig. 3b) that this major sequence difference causes a major secondary structure difference that prevents the intronic base pairing interactions that take place in the WKY strain and are pivotal in enabling exon repetition to take place.
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The contribution made by rodent mobile elements to the complementary sequences depicted in Figure 3a were investigated by scanning the rat Sa gene sequences with RepeatMasker to identify the locations of transposable elements. Only the complementary sequences depicted as a yellow square in Figure 3a coincided with the location of a mobile element, with the yellow square in intron 1 matching a SINE/ID element and the complementary yellow square sequence in intron 4 matching a SINE/Alu element.
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4 DISCUSSION |
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TOPABSTRACT1 INTRODUCTION2 METHODS3 RESULTS4 DISCUSSIONREFERENCES |
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Our computational analysis has enabled us to identify two intron sequence motifs that are associated with human exon repetition events. We found no major motif within the exons of these events. Moreover, it is unlikely that exon repetition is regulated by exon sequences as studies on the rat COT gene (2-2 isoform) have shown that the exon 2 involved is exactly the same sequence in strains that exhibit exon repetition and in those that do not. The distribution and complementary features of the sequence motifs in the flanking introns of single and multiple exon repetition events suggests that partial base pairing between the introns of two separate precursor mRNAs is involved in the natural mechanisms of this phenomenon. This model of mammalian trans-splicing between pre-mRNAs from the same gene has been proposed previously from studies on individual mammalian genes (Eul et al., 1995; Takahara et al., 2000, 2005). Our investigations with the largest collection of human exon repetition events from our genome wide survey support this model. It is intriguing also that studies on the human Sp1 gene (Takahara et al., 2005) have shown that the presence of a long intron promotes trans-splicing, as our collection of exon repetition events are also characterized by long flanking introns. The presence of long introns flanking an exon would increase the time taken for synthesis of the introns by RNA polymerase II and therefore increase the likelihood of the introns from separate pre-mRNA molecules interacting in a RNA–RNA secondary structure. The major difference from conventional trans-splicing is that highly efficient exon repetition in endogenous genes is determined in cis, meaning that transcripts from two alleles do not splice to each other (Rigatti et al., 2004). Instead, it is likely that the splicing takes place between two RNA molecules while they are attached to the same gene (Rigatti et al., 2004). It is consistent with this model that a role for transcriptional pausing (Proudfoot et al., 2002) is evident, as was shown for the human Sp1 gene (Takahara et al., 2005) and the rat Sa gene (J. -H. Jia and A. Sidorov, unpublished work). Other factors such as transcriptional pausing are likely to be involved and could explain why 5 of the 48 human single exon repetition events used in this study did not contain instances of the intron motifs. It has also been shown previously (Konarska et al., 1985) that the efficiency of trans-splicing between pre-mRNA molecules in vitro can be increased by the introduction of complementary intronic sequences to enable a short RNA duplex to occur between the separate molecules. Researchers have also utilized the trans-splicing process as a tool for gene therapy (Puttaraju et al., 1999) to enable the repair of genetic defects such as cystic fibrosis (Liu et al., 2002) or to reprogram gene expression. A crucial step in this process is the introduction of an intronic complementary sequence of 20–150 nt to enable the targeted binding of a synthetic pre-mRNA to the intron region of a target pre-mRNA molecule (Garcia-Blanco, 2003; Mansfield et al., 2003). These studies suggest that complementary intron sequences would contribute to exon repetition via intragenic trans-splicing.
The observation that the majority of the human intron motifs coincide with the locations of Alu elements is intriguing. The role of transposable elements such as Alu and LINE elements in the shaping of eukaryotic genomes is becoming more recognized (Batzer and Deininger, 2002). For example, Alu element insertions can alter the transcription or the open reading frame of a gene (Batzer and Deininger, 2002) and affect the splicing of a gene by introducing new splice sites (Lev-Maor et al., 2003; Sorek et al., 2002). Not surprisingly, Alu insertions have been found to account for several human genetic disorders (Batzer and Deininger, 2002). Also, various inherited disorders have been caused by Alu-mediated homologous recombination events between dispersed Alu elements in the genome (Batzer and Deininger, 2002). There is also some evidence that Alu elements that insert into an inverted orientation are more prone than others to illegitimate recombination (Lobachev et al., 2000; Stenger et al., 2001), consequently there is a genomic depletion of inverted Alu repeats that are <1 kb apart due to evolutionary negative selection. Interestingly, research into RNA (adenosine to inosine) editing (Kim et al., 2004), has shown that Alu sequences within mRNA transcripts are major targets for RNA editing. This is likely to be due to the nature of the ADAR (Adenosine Deaminase that Act on RNA) family of RNA editing enzymes, to recognize and bind any dsRNA structure. An explanation would be that the inverted Alu pairs within the pre-mRNA transcript form extended RNA duplexes either within the same transcript or in trans. Our results would suggest that the insertion of mobile elements into the genome that create inverted repeat sequences across different introns (at distances >1 kb) from the same gene could increase the potential of a gene to undertake exon repetition via trans-splicing between separate transcripts from the same gene.
The functional significance of exon repetition has yet to be conclusively established. The absence of exon repetition in Sa mRNA from one strain of rat suggests that it is not essential to the viability of the animal and the functional role of the Sa non-linear transcripts are still not understood (Frantz et al., 1999; Rigatti et al., 2004). More detailed research needs to be undertaken on individual examples of exon repetition to elucidate the effect of this alternative splicing phenomenon on the biological function of the gene. Nevertheless, it is likely that exon repetition is subject to selection in some genes with its subsequent effects contributing to the phenotypic variation of a species. It is also likely that exon repetition is a ubiquitous possibility that is selected against in most genes due to the potential deleterious effects of non-linear spliceforms within the cell. As the number of aberrant splicing processes causing human disease is growing (Buratti et al., 2006), it is likely that some human diseases will in the future be linked to deviant exon repetition events. In this study we have added some further insight into the elucidation of the mechanisms of exon repetition. Our observations should help experimentalists to focus on the involvement of RNA secondary structures in the mechanisms of exon repetition. Only by understanding in further detail how this splicing phenomenon occurs to produce non-linear mRNA transcripts and how most genes seemed to have evolved to avoid it will we understand the fundamentals of gene expression. This line of research could be pivotal in our understanding of how alternative splicing evolved to contribute to the complexity of multi-cellular life.
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Acknowledgments |
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This work was supported by the Wellcome Trust Functional Genomics Initiative in Cardiovascular Genetics and a MRC Cooperative grant on variability, instability and pathology of the human genome.
Conflict of Interest: None declared.
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FOOTNOTES |
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Associate Editor: Martin Bishop
Received on September 5, 2006; revised on October 13, 2006; accepted on November 8, 2006
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