Molecular Biology and Evolution 18:1841-1848 (2001)
© 2001 Society for Molecular Biology and Evolution
Intergenic Transcripts Containing a Novel Human Cytochrome P450 2C Exon 1 Spliced to Sequences from the CYP2C9 Gene
Center for Nutrition and Toxicology, Department of Biosciences at NOVUM, Karolinska Institute, Huddinge, Sweden
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Abstract |
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The cytochrome P450 2C (CYP2C) gene locus was found to include a novel exon 1 sequence with high similarity to the canonical exon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) and PCR amplifications of human liver cDNA revealed the presence of several intergenic species containing the CYP2C18 exon 1–like sequence spliced to different combinations of exonic and intronic sequences from the CYP2C9 gene. One splice variant was found to have an open reading frame starting at the canonical translation initiation codon of the CYP2C18 exon 1–like sequence. Another variant consisted of the nine typical CYP2C9 exons spliced after the CYP2C18 exon 1–like sequence through a segment of CYP2C9 5' flanking sequences. Moreover, analysis of bacterial artificial chromosome (BAC) clones revealed that the CYP2C18 exon 1–like sequence was located in the intergenic region between the CYP2C19 and CYP2C9 genes. The finding that a solitary exon is spliced with sequences from a neighboring gene may be interpreted as representing a general evolutionary mechanism aimed at using the full expression potential of a cell's genomic informational content.
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The cytochrome P450 (CYP) superfamily of proteins are heme-containing enzymes that play important roles in the synthesis and degradation pathways of various biomolecules, as well as being involved in the metabolism and detoxification of numerous xenobiotics (Nelson et al. 1996
; Chang and Kam 1999
). In all known cases, genes of the same subgroup (CYP subfamily) are genomically arranged in clusters, although individual clusters belonging to the same CYP family may be located on different chromosomes (Nelson et al. 1996
). A total of around 50 genes belonging to the cytochrome P450 superfamily have been identified in humans (Nelson 1999
). The human CYP2C subfamily is composed of four members containing nine exons with well-conserved intron/exon boundaries (Finta and Zaphiropoulos 2000a
). The members of the CYP2C subfamily have been mapped to a cluster on chromosome 10q24 with the order CYP2C18, CYP2C19, CYP2C9, CYP2C8 (Gray et al. 1995
).
Pre-mRNAs, the primary transcripts in eukaryotic cells, undergo a number of posttranscriptional modifications, including the excision of noncoding intronic sequences by large protein/RNA complexes called spliceosomes (Smith and Valcárcel 2000
). Conserved sequences necessary for the identification of exon/intron boundaries include two intronic dinucleotides, in most cases GT and AG, located, respectively, at the 5' and 3' splice sites (Burset, Seledtsov, and Solovyev 2000
; Smith and Valcárcel 2000
). A branch site sequence containing a conserved A nucleotide located close to the 3' splice site is also required for exon/intron recognition (Smith and Valcárcel 2000
).
There is increasing evidence that pre-mRNA splicing cannot be explained by the simple mechanism of the spliceosome scanning 5' to 3', removing introns, and joining exons in the order they are found in the primary transcript. The existence of low-abundant, nontypical mRNA molecules has been documented in numerous studies (reviewed in Finta and Zaphiropoulos 2000c
). RNA species containing scrambled exons, where consensus splice sites have been used to join exons in an order different from that of the genomic DNA (Nigro et al. 1991
; Cocquerelle et al. 1992
; Caldas et al. 1998
; Chao et al. 1998
; Marcucci et al. 1998
; Crawford et al. 1999
; Surono et al. 1999
; Takahara et al. 2000
), and exon repetition in the form of tandem repeats of exons which are not the result of gene duplications (Caudevilla et al. 1998
; Akopian et al. 1999
; Frantz et al. 1999
; Finta and Zaphiropoulos 2000a
) are examples of phenomena that indicate that pre-mRNA splicing is indeed a highly complex process. In addition, evidence of the existence of RNA molecules possessing properties of circular molecules has been presented (Capel et al. 1993
; Cocquerelle et al. 1993
; Pasman, Been, and Garcia-Blanco 1996
; Li and Lytton 1999
). Furthermore, nontypical splicing events may result in the joining of canonical exons with sequences, both exonic and intronic, from pseudogenes or neighboring genes belonging to the same gene family (Benson, Nguyen, and Maas 1995
; Zaphiropoulos 1999
; Finta and Zaphiropoulos 2000b
), as well as unrelated nearby sequences (Shimamura et al. 1998
; Rogalla et al. 2000
).
The present study was directed at investigating splice variants within the human CYP2C subfamily, specifically species encompassing a novel CYP2C18 exon 1–like sequence. Moreover, with the use of six independent bacterial artificial chromosome (BAC) clones, the gene locus containing the CYP2C18 exon 1–like sequence was characterized.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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cDNA Synthesis
cDNA was synthesized from human liver total RNA using random hexamers or oligo dT primers. A 20-µl reaction mixture containing M-MLV Reverse Transcriptase buffer (Promega), 1 mM dNTPs, 20 U RNasin (Promega), 30 pmol random hexamer primer (Promega) or 10 pmol oligo dT primer (Promega), 10 µg RNA (Clontech), and 200 U M-MLV Reverse Transcriptase RNase H (-) Point Mutant (Promega) was incubated at 42°C for 1.5 h.
Isolation of BAC DNA
BAC clone RP11-400G3 was obtained from BACPAC Resources. BAC clones CIT-342L6, RP11-622P11, CIT-2061B9, CIT-2339N22, and CIT-3052K18 were supplied by Research Genetics. BAC DNA for PCR analysis of the six BAC clones for the presence of CYP2C19 exon 9, CYP2C9 exon 1, and the CYP2C18 exon 1–like sequence was isolated using the JetQuick Plasmid Miniprep kit (Genomed), but with phenol/chloroform extractions and an ethanol precipitation instead of column purification. BAC DNA for the Southern blot was obtained using the Concert High Purity Plasmid Maxiprep System (Life Technologies) with an isopropanol precipitation included before column loading.
PCR Amplifications
PCR amplifications were performed using either the Perkin Elmer 480 thermal cycler or the MJ Research PTC-200 DNA Engine. All reactions were carried out for 30 cycles, unless stated otherwise, in a total volume of 50 µl using the hot start method. For the nested rapid amplification of cDNA ends (RACE) and intergenic PCR amplifications, 1 µl of the initial amplification products was used. Gene-specific primers (table 1
) were synthesized by Cybergene AB (Huddinge, Sweden).
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3' RACE Amplifications
The RACE reactions were performed on 5 µl Marathon-Ready human liver cDNA (Clontech) using the Expand Long Template PCR System (Boehringer Mannheim) and the universal primers supplied with the Marathon cDNA Amplification Kit (Clontech). The nested gene-specific primer (table 1 ) was designed to include a SalI site to be used for subsequent cloning together with the NotI site in the nested universal primer. Amplifications were carried out in a reaction mixture containing Expand buffer 1, 350 µM dNTPs, 90 pmol gene-specific primer (table 1 ), 10 pmol of the universal primer, and 3.5 U of Expand enzyme. Denaturation was at 94°C for 1 min, annealing at 60°C (initial amplification) or 70°C (nested amplification) for 1 min, and extension at 72°C for 1–3 min (+4 s/cycle).
Intergenic PCR
For the set of experiments performed to identify intergenic products containing the CYP2C18 exon 1–like sequence, 1 µl human liver cDNA synthesized as described above was the template in all cases. The reaction mixture for amplifications using Taq DNA polymerase (Promega) contained Taq buffer, 2 mM MgCl2, 200 µM dNTPs, 90 pmol of each primer (table 1
), and 5 U Taq polymerase. With Expand polymerase, the reaction mixture was as described above. For the Perkin Elmer 480 thermal cycler, denaturation was at 94°C for 1 min, annealing at the appropriate temperature (1–4°C below the calculated melting temperature) for 1 min, and extension at 72°C for 1–3 min (+4 s/cycle). When using the MJ Research PTC-200 DNA Engine, amplifications were performed with 10 s at 92°C, 30 s at the appropriate annealing temperature, and 2 min at 72°C.
Genomic PCR
The BAC clones were characterized by PCR using Taq DNA polymerase essentially as described above. Denaturation was at 92°C for 10 s, annealing at the appropriate temperature for 20 s, and extension at 72°C for either 40 s or 1–3 min (+4 s/cycle). Longer fragments of the RP11-400G3 BAC clone were amplified using the Expand Long Template PCR System with Expand buffer 3, 500 µM dNTPs, 20 pmol of each primer (table 1 ), and 3.5 U of Expand enzyme. The reactions were carried out with denaturation at 92°C for 10 s and annealing at the appropriate temperature for 20 s. The extension step was performed for 5–10 cycles of 10 min at 68°C, followed by 15–20 cycles at the same temperature for 10 min + 20 s/cycle.
Cloning and Sequencing
The PCR products from the RACE experiments were purified directly using the JetQuick Gel Extraction Spin Kit (Genomed), digested with the restriction enzymes NotI and SalI, and ligated to a pGEM-5 vector (Promega). PCR products from all other experiments were directly cloned in a pGEM-T vector (Promega). Electrocompetent Escherichia coli XL1-Blue cells were transformed and clones containing plasmids with inserts were identified using blue/white selection. Plasmid DNA was isolated from overnight cultures of selected clones using the Jetquick Plasmid Miniprep Spin Kit (Genomed). Analysis of PCR products and plasmid DNA was carried out on 0.7%–2% agarose gels. Sequencing of clones was performed using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). Electrophoresis of the sequenced samples was carried out at KISeq (Centre for Genomics Research, Karolinska Institute, Solna, Sweden) or Cybergene AB (Huddinge, Sweden). DNA sequences were analyzed using the NCBI BLAST similarity search tool (Altschul et al. 1997
).
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The CYP2C18 Exon 1–like Sequence
Data from the Human Genome Project have revealed the presence of a CYP2C18 exon 1–like sequence at the CYP2C locus (GenBank entry AL133513, BAC clone RP11-400G3). Furthermore, in an earlier experiment, a species containing the CYP2C18 exon 1–like sequence spliced to a CYP2C9 5' flanking sequence was detected in human liver (Zaphiropoulos 1999
). At the time, however, it was assumed that the differences in sequence between exon 1 of this species and the canonical CYP2C18 exon 1 represented a polymorphism and not a novel exon 1. This exon 1–like sequence is very similar to the canonical CYP2C18 exon 1 from which it originates (fig. 1
), probably as the result of a duplication event. To obtain a more detailed analysis of the CYP2C18 exon 1–like sequence and its flanking regions, a 13-kb sequence from the AL133513 GenBank entry containing the CYP2C18-like sequence was compared with the corresponding region of the CYP2C18 gene from GenBank entry AL157835. The resulting alignment showed that a significant similarity (92.5%) between the CYP2C18 and CYP2C18-like sequences extends about 4 kb upstream and 400 bp downstream of the initiation codon. Based on a divergence rate of 3.5 x 10-9/bp/year (Li 1997
, p. 181), the duplication of the 5' end of the CYP2C18 gene is estimated to have occurred about 21 MYA.
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Several potential binding sites for transcription factors have been identified in the promoter region of the CYP2C18 gene (de Morais et al. 1993
). Therefore, approximately 1 kb of genomic sequence upstream of the initiation codon of both the CYP2C18 and the CYP2C18-like sequences were compared with respect to the presence of putative transcription-factor-binding sites (fig. 1
). Several binding sites located within about 650 bp upstream of the ATG of the CYP2C18-like sequence were found to be disrupted, among them a TATA box. This TATA box, located 56 bp upstream of the initiation codon, is conserved in all four members of the human CYP2C family, as well as in the rat CYP2C genes. By analogy with the defined transcription start sites in rat CYP2C11, CYP2C12, and CYP2C13 (Morishima et al. 1987
; Zaphiropoulos et al. 1990
; Eguchi et al. 1991
), it is expected that the major transcription start site would be about 25 bases downstream of that TATA box. Further upstream in the comparison between the CYP2C18 and CYP2C18-like genes, in a region proposed to contain a number of liver-enriched transcription-factor-binding sites, the similarity between the two genes is considerably higher, with the potential binding sites being intact.
3' RACE Amplifications of the CYP2C18 Exon 1–like Sequence
With the purpose of acquiring more information concerning the expression of the CYP2C18 exon 1–like sequence, 3' RACE amplifications were performed on human liver cDNA using forward primers specific for the CYP2C18 exon 1–like sequence. Cloning and sequencing of the products from the 3' RACE experiments resulted in the detection of five species (fig. 2A
). Three splice variants were identified, with the CYP2C18 exon 1–like sequence spliced either to sequences from the CYP2C9 gene or to a sequence originating from the CYP2C locus. In all cases, splicing had occurred via recognition of the canonical GT-AG dinucleotides. This provides evidence that the PCR products analyzed represent preexisting RNA molecules and do not originate from in vitro recombination events during PCR (Zaphiropoulos 1998
). The two additional species were found to comprise the CYP2C18 exon 1–like sequence followed by contiguous intronic sequences. The results of the 3' RACE experiments would seem to indicate that the CYP2C18 exon 1–like sequence is used for splicing only to sequences from the CYP2C9 gene and not to other members of the gene cluster.
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CYP2C18 Exon 1–like/CYP2C9 Intergenic Species
After establishing that splicing of CYP2C9 sequences to CYP2C18 exon 1–like sequences takes place, experiments using forward primers for the CYP2C18 exon 1–like sequence and reverse primers for exon 9 of CYP2C9 were carried out in order to selectively focus on CYP2C18 exon 1–like/CYP2C9 intergenic species. Cloning and subsequent sequencing of the PCR products from the nested experiments revealed a mixture of species containing combinations of intronic and exonic sequences spliced at conserved GT-AG dinucleotides (fig. 2B ). The CYP2C9 exons were always spliced in a pattern that followed their genomic order. A species containing exons 6–9 of CYP2C9 joined to the CYP2C18 exon 1–like sequence was, however, the only splice variant with an open reading frame starting at the ATG codon of the novel exon 1–like sequence and ending at the canonical termination codon of CYP2C9 exon 9. Moreover, an additional species was found to contain the nine typical exons of the CYP2C9 gene spliced to a 5' sequence composed of the CYP2C18 exon 1–like sequence and a segment of the 5' flank of CYP2C9.
The CYP2C18 Exon 1–like Sequence Is Positioned in the CYP2C19/CYP2C9 Intergenic Region
The GenBank entry AL133513 corresponding to the BAC clone RP11-400G3 contains, in addition to the CYP2C18 exon 1–like sequence, exons 6–9 of CYP2C19 and exons 1–5 of CYP2C9. In order to investigate whether the BAC clone encompasses any further sequences from the CYP2C19 and CYP2C9 genes, efforts were undertaken to amplify exon 1 of CYP2C19 and exon 9 of CYP2C9 from the BAC clone. These experiments did not result in the detection of any PCR product, implying that the BAC clone contains only the 3' end of CYP2C19 and the 5' end of CYP2C9.
In order to provide evidence for the exact positioning of the CYP2C18 exon 1–like sequence relative to the 3' end of CYP2C19 and the 5' end of CYP2C9, additional BAC clones containing sequences from this region of the CYP2C locus were identified using the TIGR Human BAC Ends database (Institute for Genomic Research, http://www.tigr.org). Five BAC clones were selected and analyzed for the presence of exon 9 of CYP2C19 and exons 1 of CYP2C9 and CYP2C18-like (fig. 3 ). These results, taken together with the finding of a contiguous sequence containing CYP2C9 exons 1–5 in two independent GenBank entries (AL133513 and AL359672), provide unambiguous evidence that the CYP2C18 exon 1–like sequence is located in the CYP2C19/CYP2C9 intergenic region.
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With the aim of obtaining an independent assessment that the CYP2C18 exon 1–like region is not rearranged in the BAC clones, three of these clones (RP11-400G3, CIT-2339N22, and CIT-3052K18) were also analyzed by Southern hybridization. The clones all produced the same expected pattern of fragments when digested with the four restriction endonucleases BamHI, BglII, HindIII, and NcoI and probed with a CYP2C18 exon 1–like oligonucleotide (data not shown), providing evidence that these BAC clones accurately represent that genomic region.
Attempts were also made to link the CYP2C18 exon 1–like sequence to the CYP2C9 and CYP2C19 genes by carrying out PCR amplifications of the BAC clone RP11-400G3. The use of a CYP2C18 exon 1–like forward primer in combination with a CYP2C9 exon 1 reverse primer did not result in any product. In parallel with this experiment, amplification of exons 1–5 of CYP2C9 was used as a positive control and produced the expected 10.5-kb species. A second amplification using a CYP2C18 exon 1–like reverse primer together with a CYP2C19 exon 9 forward primer was performed in order to try to connect the exon 1–like sequence to the CYP2C19 gene. This experiment also failed to detect the desired species, implying an extensive CYP2C19/CYP2C9 intergenic region, hence the failure of long-range PCR to detect products. These findings, together with the results of the analysis of the six BAC clones, support the most recent update of the AL133513 entry (version of March 20, 2001; GI: 13396316), which reveals an 86-kb CYP2C19/CYP2C9 intergenic region with the CYP2C18 exon 1–like sequence located 33 kb upstream of CYP2C9. Furthermore, this update provides evidence that the sequence from the CYP2C locus identified in one of the RACE splice variants (fig. 2A ) originates from the CYP2C18 exon 1–like/CYP2C9 intergenic region.
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Discussion |
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The Role of "Pseudogenes" in the Expression Patterns of Genomes
The results presented in this report indicate that upstream of the CYP2C9 gene there is a solitary CYP2C exon 1 sequence with the capability to be expressed in combination with exons from the CYP2C9 gene. This exon has only five nucleotide differences in its coding region from the canonical exon 1 of CYP2C18 and was originally interpreted as representing a polymorphic variant of that gene (Zaphiropoulos 1999
). However, 3' RACE experiments unambiguously show that this exon is not spliced with the canonical CYP2C18 exons, but instead with sequences originating from the CYP2C9 gene. Moreover, analysis of six independent BAC clones provides solid evidence that the CYP2C18 exon 1–like sequence is positioned in the intergenic region of the CYP2C19 and CYP2C9 genes.
The discovery that exon-like sequences located in intergenic regions do not necessarily represent inactive pseudogenes has previously been reported in the case of the CYP3A7 gene in chromosome 7q21–q22.1. An alternatively spliced CYP3A7 mRNA form was detected where two "pseudogene" exons provide the coding information for a variant carboxyl terminus (Finta and Zaphiropoulos 2000b
). However, in the present report, the CYP2C18 exon 1–like sequence is under the control of its own promoter and is expressed in combination with exons from the neighboring CYP2C9 gene. Interestingly, even sequences from the 5' flanking region of the CYP2C9 gene have been captured as spliced exons in transcripts originating from the promoter of the CYP2C18 exon 1–like sequence.
These findings interpreted from the perspective of the CYP2C9 gene may also be viewed as follows: CYP2C9 transcription does not necessarily start between the putative TATA box (de Morais et al. 1993
) and the translation start site. Instead, some transcripts may initiate at promoter sequences upstream of the coding segment of the CYP2C18 exon 1–like sequence. The ATA box (fig. 1
) detectable at the position equivalent to the TATA boxes of the CYP2C18, CYP2C9, and CYP2C19 genes may allow transcription, albeit at a lower efficiency than that of the canonical CYP2C18 gene. The CYP2C9 gene might therefore be considered part of a complex transcription unit having more than one promoter and undergoing several alternative splicing events.
Significance of the Transcripts Produced by the CYP2C18-like Promoter
Is there a coding potential in the transcripts generated by the CYP2C18-like promoter? Well, most transcripts contain termination signals in frame with the open reading frame of the exon 1–like sequence. Moreover, no transcript was identified where the canonical exon 1 of CYP2C9 had been replaced with the CYP2C18 exon 1–like sequence, resulting in a typical CYP2C mRNA of nine coding exons, even though CYP2C intergenic transcripts corresponding to a standard-length CYP2C mRNA have been observed in a previous study (Finta and Zaphiropoulos 2000a
). The transcript composed of exons 6–9 of CYP2C9 spliced after the exon 1–like sequence (fig. 2B
) does, however, have an open reading frame that starts at the Met codon of the exon 1–like sequence and ends at the canonical TGA codon of CYP2C9. Moreover, the additional 5' sequence present in the transcript containing the nine typical CYP2C9 exons (fig. 2B
) may have a role in modulating the translational ability of that mRNA (Morris and Geballe 2000
).
The results of the RACE and PCR amplifications clearly indicate that transcription is ongoing at the CYP2C18 exon 1–like locus. The fact that these transcripts are not very abundant may relate to the observation that they do not represent typical CYP2C mRNAs of nine exons. These transcripts are, however, processed RNA molecules; that is, they have undergone splicing events, thereby discriminating them from unprocessed transcripts. Examples of the latter are species originating from the CYP3AP1, the CYP3AP2 (Finta and Zaphiropoulos 2000b
), and the CYP3A43P (GenBank entry AC011904, GI: 8051573, nucleotide positions 113048–112978) (unpublished data) pseudogenes. These transcripts contain only exon-like sequences with the contiguous unspliced intronic sequences. We therefore favor the possibility that the CYP2C18 exon 1–like transcripts represent efforts of the transcriptional/splicing machinery to generate exon combinations that may be useful to the cell (Finta and Zaphiropoulos 2001
). This would be in line with genomewide analyses of transcriptome sequences that suggest the presence of a large number of transcripts deviating from typical mRNAs (Velculescu et al. 1999
; Croft et al. 2000
; Hirosawa et al. 2000
). However, at this point in evolutionary time, no single CYP2C18-like/CYP2C9 combination predominates, as is the case for the CYP3A7 carboxyl terminus variant (Finta and Zaphiropoulos 2000b
). On the other hand, these sequences have the potential to generate functional products, as the cell is continuously experimenting with various possibilities that may result in a "winning" combination.
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This study was supported by grants from the Swedish Natural Science Research Council, the Åke Wibergs Foundation, and the Karolinska Institute.
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Footnotes |
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David Irwin, Reviewing Editor
1 Keywords: pseudogene
alternative promoter
pre-mRNA splicing
gene evolution
Homo sapiens
cytochrome P450 
2 Address for correspondence and reprints: Center for Nutrition and Toxicology, Department of Biosciences at NOVUM, Karolinska Institute, SE-141 57 Huddinge, Sweden. peter.zaphiropoulos{at}cnt.ki.se
.
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