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cDNA cloning and genomic organization of the murine MRP7, a new ATP-binding cassette transporter
Gene 286 (2002) 299–306 www.elsevier.com/locate/gene
cDNA cloning and genomic organization of the murine MRP7, a new ATP-binding cassette transporter q Hsin-hsin Kao a,b, Jin-ding Huang b, Ming-shi Chang c,* a
b
Department of Food Nutrition, Chung-Hwa College of Medical Technology, Tainan, Taiwan Department of Pharmacology, Medical College, National Cheng-Kung University, Tainan 701, Taiwan c Department of Biochemistry, Medical College, National Cheng-Kung University, Tainan 701, Taiwan Received 17 October 2001; received in revised form 17 December 2001; accepted 29 January 2002 Received by E.Y. Chen
Abstract Cellular resistance to cytotoxic drugs is a major obstacle to the treatment of disseminated cancers. Multidrug resistance protein (MRP) subfamily is a member of the ATP-binding cassette transporters which has been shown to cause multidrug resistance, except for Pglycoprotein. A new MRP subfamily gene, mrp7A (Abcc10), and its splicing variant, mrp7B, were isolated from mouse. The lengths of the open reading frames of mouse mrp7A and mrp7B are 4383 and 4506 bp, respectively. Estimated polypeptide sequences of mrp7A and mrp7B are 1460 and 1501 amino acids. The mouse mrp7 gene consists of at least 21 exons and 20 introns spanning around 20 kb that is almost the same as the one in human MRP7 gene, but different with the other MRP subfamily genes. The promoter region was isolated from the genomic clone and shown to support the luciferase activity seven fold over the promoterless negative control and two fold activity higher than the positive control of SV40 promoter. The analysis of tissue expression of mrp7A and mrp7B showed that these two transcripts express differentially in specific tissues. q 2002 Published by Elsevier Science B.V. Keywords: Multidrug resistance; ATP-binding cassette transporter; Murine mrp7; Genomic structure
1. Introduction The multidrug resistance-associated protein (MRP) gene is a subfamily of ATP-binding cassette (ABC) transporters originally cloned from multidrug-resistance human lung cancer cells and identified as an integral membrane glycoprotein of about 190 kDa (Cole et al., 1992). Both P-glycoprotein (Pgp) and MRP are members of ABC transporters subfamily, but they share less than 15% identity in amino acid (Cole et al., 1992), except for the two highly conserved ATP-binding domains. The amino acid sequences of the ATP-binding domain (Walker A and B domains and C or signature motif) distinguish ABC genes from other ATPbinding proteins such as kinases (Higgins, 1992). Seven Abbreviations: aa, amino acid(s); ABC, ATP-binding cassette; bp, base pairs; cDNA, DNA complementary to RNA; kDa, kilodalton(s); ORF, open reading frame; TMD0, transmembrane domain; MSD, membrane spanning domain; NBD, nucleotide binding domain q The nucleotide sequences reported in the paper have been submitted to the GenBank TM/EBI Data Bank with accession number AF406642 and AF417121. * Corresponding author. Tel.: 1886-6-235-3535 ext. 5677; fax: 1886-6274-1694. E-mail address: [email protected] (M. Chang). 0378-1119/02/$ - see front matter q 2002 Published by Elsevier Science B.V. PII: S 0378-111 9(02)00461-4
human MRP subfamily genes, MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, and MRP7, are isolated (Juliano and Ling, 1976; Bu¨chler et al., 1996; Taniguchi et al., 1996; Kool et al., 1997, 1999; Hopper et al., 2001; Kao et al., 2002). MRP 1 is characterized to be a primary active ATP-dependent transporter of amphiphilic anions such as glutathions, leukotriene C4 (LTC4), and activated aflatoxin B1 (Jedlitschky et al., 1996; Leier et al., 1994, 1996; Loe et al., 1996, 1997; Muller et al., 1994). MRP2 is involved in the hepatocytes transport and excretion of compounds across the bile canalicular membrane (Gatmaitan and Arias, 1995; Ito et al., 1997) and related to Dubin-Johnson syndrome in humans (Dubin and Johnson, 1954). MRP3 prefers glucuronate conjugates as substrate over GSH conjugates (Hirohashi et al., 1999). MRP4 and MRP5 both act as cellular efflux pumps for the nucleoside analogues (McAleer et al., 1999; Schuetz et al., 1999; Wijnholds et al., 2000). The potential involvement of MRP6 and MRP7 in drug resistance and the transport of different molecules in humans is still unclear. In order to use mouse as a model for studies on the physiological functions of the MRP subfamily members, it is essential to isolate the murine cDNA clones encoding these
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proteins. In vitro and in vivo mrp1 knockout models had generated and its function was studied (Lorico et al., 1996, 1997; Rappa et al., 1997; Wijnholds et al., 1997). Mrp1 contributes to the transport of LTC4 (Saptarsgu et al., 1996) and can protect the normal tissues from the toxicity of the anticancer agent etoposide in the mrp1 knockout system (Rappa et al., 1999). It is conceivable that the results obtained in the mrp1 knockout systems can be extrapolated to humans. Human MRP7A (GenBank accession BAA92227; Hopper et al., 2001) and MRP7A (Kao et al., 2002) were isolated from spleen and small intestine, respectively, and their physiological roles in different tissues remain to be identified. To understand the mechanism of biological functions of MRP7, the generation of transgenic mice or gene knockout would be very useful. Therefore, isolation of the mouse mrp7 is essential. In this paper we report the cloning of two alternative spliced variants of novel murine MRP7 cDNA clones, mrp7A, and mrp7B, its genomic organization, promoter activity, and expression levels in different murine tissues. 2. Materials and methods 2.1. Cloning of the full-length cDNAs of mrp7A, and mrp7B A homology screening of mouse high throughput genomes database using the human MRP7 cDNA sequence as the query was carried out by basic BLAST search. The mouse genomic sequences (accession number AC078884) were identified and used for designing primers to clone the cDNAs of mrp7A and mrp7B. The primers used to clone the overlapping cDNA fragments are as the following: MS5: 5 0 CCTCCTTTCCATCTTCCCACTGCT-3 0 , MR11: 5 0 -CCAGCAAAGTTGAGCAGCCTCTC-3 0 , MS8: 5 0 -GAGAGGCTGCTCAACTTTGCTGG-3 0 ; MR7: 5 0 -GCATAGGAAGCAAACTGGCACCTC-3 0 ; MS3: 5 0 -CTCCGGGCTGTGCTCTTTGC-3 0 , MR12: 5 0 -GAGGAAAACATAGACCAGCAGCCCT-3 0 , and MS22: 5 0 -ATGGAGGGGCTCCTGGCCCAG-3 0 . Polymerase chain reaction (PCR) was performed using the mouse spleen, heart, and kidney QUICK-Clonee cDNAs (Clontech) as the templates. 5 0 RACE were performed to obtain the full-length cDNA clones. 2.2. Quantitative PCR The PCR fragments containing the nucleotide sequences located at nt 2491–123 upstream of mrp7A and at nt 1–246 upstream of mrp7B were amplified by PCR. Different mouse tissues cDNAs (Clontech) were used as the templates. The primers used were an antisense primer common to both mrp7A and mrp7B, MR9 (nt 106–123 for mrp7A; nt 229– 246 for mrp7B), and the specific primers located at the 5 0 end of mrp7A (MS9, nt 2491 to 2467 for mrp7A) and mrp7B (MS22, nt 1–21 for mrp7B), respectively. The nested PCR were performed with a nested antisense primer (MR10, nt 70–94 for mrp7A; nt 200–217 for mrp7B) and the same
sense primers specific to mrp7A (MS9) and mrp7B (MS22) by the 50 times diluted first PCR products as the templates. Both the translation initiation sites of mrp7A and mrp7B are referred to the first nucleotides. The lengths of the nested PCR products are 585 bp for mrp7A and 217 bp for mrp7B. The PCR was amplified for 20 cycles. The quantification of the PCR product was performed by ImageMaster w VDS (Pharmacia biotech). The primer sequences for quantitative PCR are as the following: MR9: 5 0 -CTAGCAGTGGGAAGATGGAAAGGAGG-3 0 , MS9: 5 0 -GAG-GAAATAGGCAGAACTGAAAGAA-3 0 , MS22: 5 0 -ATGGAGGGGCTCCTGGCCCAG-3 0 , and MR10: 5 0 -ATGCT-GCGAGTCGGAGGC-3 0 . 2.3. Isolation of promoter region and analysis of promoter activity A PCR fragment containing nucleotide segments located at nucleotide 245 to 21905 upstream of translation initiate site was amplified form the mouse genomic clone AC078884. This 1.86-kb fragment was cloned into pGL3Enhancer vector (Promega). The construct was used along with the promoterless pGL3-Enhancer DNA in the transfection of the MDCK cells. pGL3-Promoter vector with the SV40 promoter was used as the positive control. Cells at a density of 3 £ 10 5/well in a six-well plate were transfected with 4 mg of plasmid DNAs using 5 ml of lipofectAMINE 2000 reagent (GIBCOL BRL). After transfection (5 h), the medium was replaced with fresh growth medium. The cells were collected and the luciferase activity was analyzed according to the protocol of the luciferase assay system after transfected for 24 h (Promega). 2.4. Analysis of tissue distribution with MTN northern blot A MTN northern blot containing 1 mg of poly(A) 1 RNA per mouse tissue was purchased from Clontech (Clontech). The membrane was prehybridized in ExpressHyb solution (Clontech) at 608C for 2 h and hybridized at 608C for 18 h in the same buffer with the 32P-labeled cDNA probe that were prepared by a random primed labeling method (Rediprime; Amersham). A 511-bp cDNA fragment located at nt 2956– 3466 of mouse mrp7A (the start codon, ATG, were designated as the first nucleotide) was amplified from the heart cDNA clone. The hybridized membrane was washed in 2 £ SSC and 0.1% SDS at room temperature for 20 min, followed by washing in 2 £ SSC and 0.1% SDS at 608C for 20 min. The membrane were exposed to a BioMAX MS film (Amersham) at 2808C for 27 h. 3. Results and discussion 3.1. Isolation of the full-length cDNA sequences of mrp7A and mrp7B To isolate the cDNA sequence of mouse homologue of
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human MRP7, we take the approach of identifying its genome sequence first. A mouse genomic clone was obtained by BLAST search using human MRP7 cDNA as the query. A mouse genomic clone (accession number AC078884) containing several homology regions with human MRP7 cDNA was isolated. A full-length 4565-bp cDNA sequence encoding a 4383-bp open reading frame (ORF) and 69-bp 3 0 untranslated region (UTR) of mouse mrp7A (accession number AF406642) was obtained from three overlapping cDNA fragments amplified by PCR using the mouse spleen cDNAs as a template and the primers designed according to the mouse genomic clone sequence. Mouse mrp7B (accession number AF417121), an alternatively spliced transcript homologous to the human spleen MRP7 (accession number BAA92227; Hopper et al., 2001), which has two extra exons at 5 0 end (Kao et al., 2002), was also isolated from mouse heart and spleen cDNA library. It contains 4575-bp nucleotide sequence including 4506-bp ORF and 69-bp 3 0 UTR. The lengths of the deduced polypeptide sequences of mrp7A and mrp7B are 1460 and 1501 amino acids, respectively. The estimated molecular weight of mrp7A and mrp7B are 159.7 and 163.7 kDa, respectively. The percentage of amino acid identity between mrp7A mouse cDNA and human MRP7A cDNA is 82%, similar to the identity between mrp7B mouse cDNA and MRP7 human cDNA (BAA92227), 84%. The significant three conserved regions of ABC family members, Walker A (GAGCKST), Walker B (DEATSALD) and signature motif (C motif; LSGGQQ) are also located in the deduced amino acid sequences of mrp7A and mrp7B. The genomic structure of the mouse mrp7A is almost the same as its human homologue, MRP7 (Kao et al., 2002; Fig.
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1). Each nucleotide binding domain was encoded by four exons, i.e. exons 5–8 for NBD1 and exons 16–19 for NBD2, which is similar to NBD1 in MRP4 and MRP5. In contrast, the NBD1 is encoded by three exons in MRP1 and MRP2 (Tsujii et al., 1999; Grant et al., 1997). The suggested model for the membrane topology of mouse mrp7A using TMpred program also shows a 5 1 5 1 6 configuration of transmembrane helices, like human MRP7, within the three membrane spanning domains (Fig. 2). It possesses the Nterminal membrane spanning domain which is absent from the MRP5, but exists in MRP1 (Borst et al., 1999). MRP2, MRP3, and MRP6 have a similar structure as MRP1 with the extra N-terminal extension with five transmembrane regions (TMD0) connected to a PgP-like core by a cytoplastic linker (Bakos et al., 1996; Tusnady et al., 1997; Borst et al., 1999). MRP5 and MRP4 both lack the TMD0 domain (Bakos et al., 1998; Borst et al., 1999). It is notably that sequence homology on TMD0 domain is lowest in the MRP subfamily members. 3.2. Quantitative PCR The differential expression of the mrp7A and mrp7B was detected by quantitative PCR by using the common antisense primers, MR9 and MR10, and the splice variant specific primers, MS9 and MS22, located at the 5 0 end of mrp7A and mrp7B, respectively (Fig. 3). Both mouse mrp7A and mrp7B are expressed in all the tissues examined, including liver, brain, embryo, heart, kidney, and spleen. The expression levels of mrp7A and mrp7B are similar in the tissues of liver, brain, and heart. However, the expression level of mrp7B is much lower (about 50%) than that of mrp7A in spleen. In contrast, the expression
Fig. 1. Genomic organization of mouse mrp7A gene and its alignment with mrp7 mRNA.
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Fig. 2. Amino acid sequence comparison of mouse mrp7 and the other MRP subfamily proteins. Amino acid sequences are aligned by the PILEUP and PRETTY programs from the University of Wisconsin Genetics Group (GCG) package. The identical amino acids are shown in uppercase letters. Walker A and B motifs and active transport family signature sequence C are indicated by the characters italic and boxed. The O-glycosylation and N-glycosylation sites are indicated by boxed and underlined symbols, respectively. The transmembrane segments are boxed and shaded. The following accession numbers were used: mrp7B; mrp1, AF022908; mrp2, AF227274.
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Fig. 2. (continued)
level of mrp7A is much lower (about 25%) than that of mrp7B in kidney. In embryo, the expression levels of both transcripts are much lower compared to most tissues examined. Whether the difference in expression level of two
Fig. 3. PCR analysis of the expression of mouse mrp7A and mrp7B in different mouse tissues. The primers used in PCR are shown in materials and methods. cDNAs from different mouse tissues (QUICK-Clonee cDNAs (Clontech) were used as the templates. Lanes 1 and 2: nuclease free water (negative control); Lanes 3 and 4: liver; Lanes 5 and 6: brain; Lanes 7 and 8: embryo; Lanes 9 and 10: heart; Lanes 11 and 12: kidney; and Lane 13 and 14: spleen. Primers specific for mrp7A, MR9, MR10 and MS9, were used in the lanes of odd numbers. Primers specific for mrp7B, MR9, MR10 and MS22, were used in the lanes of even numbers. PCR products were separated by 1.5% agarose electrophoresis with 1 kb plus marker (GIBCOL BRL), lane M.
splice variants has any significance in their biological functions awaits further study. 3.3. Genomic structure of mrp7 The mouse genomic structure of mouse mrp7 gene is similar to that of human MRP7gene, except some differences in the lengths of some introns (Fig. 4). The sequences of the intron-exon junctions of the mrp7A and mrp7B are summarized in Fig. 4. There are totally 20 exons for mrp7A and 21 exons for mrp7B (Fig. 4). Compared to other members of MRP family, mrp7 seems to contain least numbers of exons. The significant difference in exons number may suggest that mrp7 are derived from a different ancestral gene. The average sizes of internal exons encoding the cDNAs are 219 bp for mrp7A and 215 bp for mrp7B, ranging from 78 bp (exon 9) to 1260 bp (exon 1). All intron-exon boundaries conform to the consensus splice junction sequences for eukaryotic cells (Shapiro and Senapathy, 1987; Grant et al., 1997). The 9 bp of the consensus 5 0 donor site ((A/C)AG/gt(a/g)agt are conserved in mouse mrp7A/mrp7B in 95/95, 74/75, 84/85, 100/100, 100/100, 89/90, 58/55, 84/85, 32/35%, respectively. The 4 bp of the 3 0 acceptor site ((c/a)/ag/(A/G)) are conserved in mouse mrp7A/mrp7B in 89/90, 100/100, 100/100, 79/80, respectively. The genomic organization of mouse mrp7 gene is also quite different from that of other mrp subfamily members at the first exon (Fig. 5). Compared with the other isolated mrp subfamily members,
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Fig. 4. Sequences of the intron-exon junctions of the mrp7 genes. (A) Sequences of the intron-exon junctions of the mrp7A. (B) Sequences of the intron-exon junctions of the mrp7B. Exon sequences are shown in uppercase letters, and intron sequences in lowercase letters. The encoded amino acid and its position are indicated below the exon sequences. The lengths of the introns are shown in parentheses. The latter 1222-bp sequence of the exon 2 of mrp7B is corresponding to the latter 1222-bp nucleotide sequence of the exon 1 of mrp7A. The genomic structure of mrp7B is identical to that of mrp7A except the exon1.
it has quite large exon 1, similar to its human homologue. ABC transporters contain the four structural domains including MSD2, MSD3, NBD1, and NBD2 (Stride et al., 1996; Bakos et al., 1996; Tusnady et al., 1997; Deeley
and Cole, 1997). Each nucleotide binding domain of mrp7 was encoded by four exons, i.e. exons 5–8 for NBD1 and exons 16–19 for NBD2. The extra N-terminal membrane spanning domain (MSD1) in mrp7 shows that mrp7
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Fig. 5. Comparison of genomic structures of mouse mrp7A, and mrp7B. The locations of the translation initiate sites are indicated by vertical lines and Ms. Walker A and B motifs and active transport family signature are denoted A, B and C, respectively.
belongs to the subgroup including MRP1, MRP2, MRP3, and MRP6. 3.4. Analysis of the promoter activity
Fig. 6. Analysis of the mouse mrp7 promoter activity. The promoterless pGL3-Enhancer vector and the pGL3-promoter vector with SV40 were used as the negative and the positive controls, respectively.
A 1.86-kb DNA fragment upstream of the translation initiation site was isolated from the genomic clone (accession number AC078884). This DNA fragment was cloned into pGL3-Enhancer vector. The MDCK cells were transfected with mouse mrp7 promoter and the luciferase activities were analyzed. This 1.86-kb fragment supports the luciferase activity seven fold over the promoterless negative control and two fold activity higher than the positive control of SV40 promoter (Fig. 6). The strong promoter activity demonstrated by this region suggests that mouse mrp7 gene may be well regulated in vivo to execute its biological function. 3.5. Tissue distribution of mrp7 Tissue distribution of mouse mrp7 was analyzed by northern hybridization on a mouse Multiple Tissue Northern blot with a cDNA probe. A signal around 5.5 kb was observed in the tissue of heart, liver, skeletal muscle, and kidney (Fig. 7). In summary, we describe that the mouse mrp7 has two spliced transcripts, mrp7A and mrp7B. The biological significance of these two spliced variants awaits for further study. Their genomic organization including the intronexon boundaries, the promoter activity and differential expression pattern were presented. Study of gene knockout model or transgenic mice of mrp7 may shed some light on the mechanism of the physiological functions of MRP7. References
Fig. 7. Tissue distribution of mouse mrp7 transcripts. Membrane containing 1 mg of poly(A) 1 RNA per tissue was hybridized with a mrp7 cDNA fragment as the probe. Lane 1: heart; Lane 2: brain; Lane 3: spleen; Lane 4: lung; Lane 5: liver; Lane 6: skeletal muscle; Lane 7: kidney; and Lane 8: testis.
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cDNA cloning and genomic organization of the murine MRP7, a new ATP-binding cassette transporter q Hsin-hsin Kao a,b, Jin-ding Huang b, Ming-shi Chang c,* a
b
Department of Food Nutrition, Chung-Hwa College of Medical Technology, Tainan, Taiwan Department of Pharmacology, Medical College, National Cheng-Kung University, Tainan 701, Taiwan c Department of Biochemistry, Medical College, National Cheng-Kung University, Tainan 701, Taiwan Received 17 October 2001; received in revised form 17 December 2001; accepted 29 January 2002 Received by E.Y. Chen
Abstract Cellular resistance to cytotoxic drugs is a major obstacle to the treatment of disseminated cancers. Multidrug resistance protein (MRP) subfamily is a member of the ATP-binding cassette transporters which has been shown to cause multidrug resistance, except for Pglycoprotein. A new MRP subfamily gene, mrp7A (Abcc10), and its splicing variant, mrp7B, were isolated from mouse. The lengths of the open reading frames of mouse mrp7A and mrp7B are 4383 and 4506 bp, respectively. Estimated polypeptide sequences of mrp7A and mrp7B are 1460 and 1501 amino acids. The mouse mrp7 gene consists of at least 21 exons and 20 introns spanning around 20 kb that is almost the same as the one in human MRP7 gene, but different with the other MRP subfamily genes. The promoter region was isolated from the genomic clone and shown to support the luciferase activity seven fold over the promoterless negative control and two fold activity higher than the positive control of SV40 promoter. The analysis of tissue expression of mrp7A and mrp7B showed that these two transcripts express differentially in specific tissues. q 2002 Published by Elsevier Science B.V. Keywords: Multidrug resistance; ATP-binding cassette transporter; Murine mrp7; Genomic structure
1. Introduction The multidrug resistance-associated protein (MRP) gene is a subfamily of ATP-binding cassette (ABC) transporters originally cloned from multidrug-resistance human lung cancer cells and identified as an integral membrane glycoprotein of about 190 kDa (Cole et al., 1992). Both P-glycoprotein (Pgp) and MRP are members of ABC transporters subfamily, but they share less than 15% identity in amino acid (Cole et al., 1992), except for the two highly conserved ATP-binding domains. The amino acid sequences of the ATP-binding domain (Walker A and B domains and C or signature motif) distinguish ABC genes from other ATPbinding proteins such as kinases (Higgins, 1992). Seven Abbreviations: aa, amino acid(s); ABC, ATP-binding cassette; bp, base pairs; cDNA, DNA complementary to RNA; kDa, kilodalton(s); ORF, open reading frame; TMD0, transmembrane domain; MSD, membrane spanning domain; NBD, nucleotide binding domain q The nucleotide sequences reported in the paper have been submitted to the GenBank TM/EBI Data Bank with accession number AF406642 and AF417121. * Corresponding author. Tel.: 1886-6-235-3535 ext. 5677; fax: 1886-6274-1694. E-mail address: [email protected] (M. Chang). 0378-1119/02/$ - see front matter q 2002 Published by Elsevier Science B.V. PII: S 0378-111 9(02)00461-4
human MRP subfamily genes, MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, and MRP7, are isolated (Juliano and Ling, 1976; Bu¨chler et al., 1996; Taniguchi et al., 1996; Kool et al., 1997, 1999; Hopper et al., 2001; Kao et al., 2002). MRP 1 is characterized to be a primary active ATP-dependent transporter of amphiphilic anions such as glutathions, leukotriene C4 (LTC4), and activated aflatoxin B1 (Jedlitschky et al., 1996; Leier et al., 1994, 1996; Loe et al., 1996, 1997; Muller et al., 1994). MRP2 is involved in the hepatocytes transport and excretion of compounds across the bile canalicular membrane (Gatmaitan and Arias, 1995; Ito et al., 1997) and related to Dubin-Johnson syndrome in humans (Dubin and Johnson, 1954). MRP3 prefers glucuronate conjugates as substrate over GSH conjugates (Hirohashi et al., 1999). MRP4 and MRP5 both act as cellular efflux pumps for the nucleoside analogues (McAleer et al., 1999; Schuetz et al., 1999; Wijnholds et al., 2000). The potential involvement of MRP6 and MRP7 in drug resistance and the transport of different molecules in humans is still unclear. In order to use mouse as a model for studies on the physiological functions of the MRP subfamily members, it is essential to isolate the murine cDNA clones encoding these
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proteins. In vitro and in vivo mrp1 knockout models had generated and its function was studied (Lorico et al., 1996, 1997; Rappa et al., 1997; Wijnholds et al., 1997). Mrp1 contributes to the transport of LTC4 (Saptarsgu et al., 1996) and can protect the normal tissues from the toxicity of the anticancer agent etoposide in the mrp1 knockout system (Rappa et al., 1999). It is conceivable that the results obtained in the mrp1 knockout systems can be extrapolated to humans. Human MRP7A (GenBank accession BAA92227; Hopper et al., 2001) and MRP7A (Kao et al., 2002) were isolated from spleen and small intestine, respectively, and their physiological roles in different tissues remain to be identified. To understand the mechanism of biological functions of MRP7, the generation of transgenic mice or gene knockout would be very useful. Therefore, isolation of the mouse mrp7 is essential. In this paper we report the cloning of two alternative spliced variants of novel murine MRP7 cDNA clones, mrp7A, and mrp7B, its genomic organization, promoter activity, and expression levels in different murine tissues. 2. Materials and methods 2.1. Cloning of the full-length cDNAs of mrp7A, and mrp7B A homology screening of mouse high throughput genomes database using the human MRP7 cDNA sequence as the query was carried out by basic BLAST search. The mouse genomic sequences (accession number AC078884) were identified and used for designing primers to clone the cDNAs of mrp7A and mrp7B. The primers used to clone the overlapping cDNA fragments are as the following: MS5: 5 0 CCTCCTTTCCATCTTCCCACTGCT-3 0 , MR11: 5 0 -CCAGCAAAGTTGAGCAGCCTCTC-3 0 , MS8: 5 0 -GAGAGGCTGCTCAACTTTGCTGG-3 0 ; MR7: 5 0 -GCATAGGAAGCAAACTGGCACCTC-3 0 ; MS3: 5 0 -CTCCGGGCTGTGCTCTTTGC-3 0 , MR12: 5 0 -GAGGAAAACATAGACCAGCAGCCCT-3 0 , and MS22: 5 0 -ATGGAGGGGCTCCTGGCCCAG-3 0 . Polymerase chain reaction (PCR) was performed using the mouse spleen, heart, and kidney QUICK-Clonee cDNAs (Clontech) as the templates. 5 0 RACE were performed to obtain the full-length cDNA clones. 2.2. Quantitative PCR The PCR fragments containing the nucleotide sequences located at nt 2491–123 upstream of mrp7A and at nt 1–246 upstream of mrp7B were amplified by PCR. Different mouse tissues cDNAs (Clontech) were used as the templates. The primers used were an antisense primer common to both mrp7A and mrp7B, MR9 (nt 106–123 for mrp7A; nt 229– 246 for mrp7B), and the specific primers located at the 5 0 end of mrp7A (MS9, nt 2491 to 2467 for mrp7A) and mrp7B (MS22, nt 1–21 for mrp7B), respectively. The nested PCR were performed with a nested antisense primer (MR10, nt 70–94 for mrp7A; nt 200–217 for mrp7B) and the same
sense primers specific to mrp7A (MS9) and mrp7B (MS22) by the 50 times diluted first PCR products as the templates. Both the translation initiation sites of mrp7A and mrp7B are referred to the first nucleotides. The lengths of the nested PCR products are 585 bp for mrp7A and 217 bp for mrp7B. The PCR was amplified for 20 cycles. The quantification of the PCR product was performed by ImageMaster w VDS (Pharmacia biotech). The primer sequences for quantitative PCR are as the following: MR9: 5 0 -CTAGCAGTGGGAAGATGGAAAGGAGG-3 0 , MS9: 5 0 -GAG-GAAATAGGCAGAACTGAAAGAA-3 0 , MS22: 5 0 -ATGGAGGGGCTCCTGGCCCAG-3 0 , and MR10: 5 0 -ATGCT-GCGAGTCGGAGGC-3 0 . 2.3. Isolation of promoter region and analysis of promoter activity A PCR fragment containing nucleotide segments located at nucleotide 245 to 21905 upstream of translation initiate site was amplified form the mouse genomic clone AC078884. This 1.86-kb fragment was cloned into pGL3Enhancer vector (Promega). The construct was used along with the promoterless pGL3-Enhancer DNA in the transfection of the MDCK cells. pGL3-Promoter vector with the SV40 promoter was used as the positive control. Cells at a density of 3 £ 10 5/well in a six-well plate were transfected with 4 mg of plasmid DNAs using 5 ml of lipofectAMINE 2000 reagent (GIBCOL BRL). After transfection (5 h), the medium was replaced with fresh growth medium. The cells were collected and the luciferase activity was analyzed according to the protocol of the luciferase assay system after transfected for 24 h (Promega). 2.4. Analysis of tissue distribution with MTN northern blot A MTN northern blot containing 1 mg of poly(A) 1 RNA per mouse tissue was purchased from Clontech (Clontech). The membrane was prehybridized in ExpressHyb solution (Clontech) at 608C for 2 h and hybridized at 608C for 18 h in the same buffer with the 32P-labeled cDNA probe that were prepared by a random primed labeling method (Rediprime; Amersham). A 511-bp cDNA fragment located at nt 2956– 3466 of mouse mrp7A (the start codon, ATG, were designated as the first nucleotide) was amplified from the heart cDNA clone. The hybridized membrane was washed in 2 £ SSC and 0.1% SDS at room temperature for 20 min, followed by washing in 2 £ SSC and 0.1% SDS at 608C for 20 min. The membrane were exposed to a BioMAX MS film (Amersham) at 2808C for 27 h. 3. Results and discussion 3.1. Isolation of the full-length cDNA sequences of mrp7A and mrp7B To isolate the cDNA sequence of mouse homologue of
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human MRP7, we take the approach of identifying its genome sequence first. A mouse genomic clone was obtained by BLAST search using human MRP7 cDNA as the query. A mouse genomic clone (accession number AC078884) containing several homology regions with human MRP7 cDNA was isolated. A full-length 4565-bp cDNA sequence encoding a 4383-bp open reading frame (ORF) and 69-bp 3 0 untranslated region (UTR) of mouse mrp7A (accession number AF406642) was obtained from three overlapping cDNA fragments amplified by PCR using the mouse spleen cDNAs as a template and the primers designed according to the mouse genomic clone sequence. Mouse mrp7B (accession number AF417121), an alternatively spliced transcript homologous to the human spleen MRP7 (accession number BAA92227; Hopper et al., 2001), which has two extra exons at 5 0 end (Kao et al., 2002), was also isolated from mouse heart and spleen cDNA library. It contains 4575-bp nucleotide sequence including 4506-bp ORF and 69-bp 3 0 UTR. The lengths of the deduced polypeptide sequences of mrp7A and mrp7B are 1460 and 1501 amino acids, respectively. The estimated molecular weight of mrp7A and mrp7B are 159.7 and 163.7 kDa, respectively. The percentage of amino acid identity between mrp7A mouse cDNA and human MRP7A cDNA is 82%, similar to the identity between mrp7B mouse cDNA and MRP7 human cDNA (BAA92227), 84%. The significant three conserved regions of ABC family members, Walker A (GAGCKST), Walker B (DEATSALD) and signature motif (C motif; LSGGQQ) are also located in the deduced amino acid sequences of mrp7A and mrp7B. The genomic structure of the mouse mrp7A is almost the same as its human homologue, MRP7 (Kao et al., 2002; Fig.
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1). Each nucleotide binding domain was encoded by four exons, i.e. exons 5–8 for NBD1 and exons 16–19 for NBD2, which is similar to NBD1 in MRP4 and MRP5. In contrast, the NBD1 is encoded by three exons in MRP1 and MRP2 (Tsujii et al., 1999; Grant et al., 1997). The suggested model for the membrane topology of mouse mrp7A using TMpred program also shows a 5 1 5 1 6 configuration of transmembrane helices, like human MRP7, within the three membrane spanning domains (Fig. 2). It possesses the Nterminal membrane spanning domain which is absent from the MRP5, but exists in MRP1 (Borst et al., 1999). MRP2, MRP3, and MRP6 have a similar structure as MRP1 with the extra N-terminal extension with five transmembrane regions (TMD0) connected to a PgP-like core by a cytoplastic linker (Bakos et al., 1996; Tusnady et al., 1997; Borst et al., 1999). MRP5 and MRP4 both lack the TMD0 domain (Bakos et al., 1998; Borst et al., 1999). It is notably that sequence homology on TMD0 domain is lowest in the MRP subfamily members. 3.2. Quantitative PCR The differential expression of the mrp7A and mrp7B was detected by quantitative PCR by using the common antisense primers, MR9 and MR10, and the splice variant specific primers, MS9 and MS22, located at the 5 0 end of mrp7A and mrp7B, respectively (Fig. 3). Both mouse mrp7A and mrp7B are expressed in all the tissues examined, including liver, brain, embryo, heart, kidney, and spleen. The expression levels of mrp7A and mrp7B are similar in the tissues of liver, brain, and heart. However, the expression level of mrp7B is much lower (about 50%) than that of mrp7A in spleen. In contrast, the expression
Fig. 1. Genomic organization of mouse mrp7A gene and its alignment with mrp7 mRNA.
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Fig. 2. Amino acid sequence comparison of mouse mrp7 and the other MRP subfamily proteins. Amino acid sequences are aligned by the PILEUP and PRETTY programs from the University of Wisconsin Genetics Group (GCG) package. The identical amino acids are shown in uppercase letters. Walker A and B motifs and active transport family signature sequence C are indicated by the characters italic and boxed. The O-glycosylation and N-glycosylation sites are indicated by boxed and underlined symbols, respectively. The transmembrane segments are boxed and shaded. The following accession numbers were used: mrp7B; mrp1, AF022908; mrp2, AF227274.
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Fig. 2. (continued)
level of mrp7A is much lower (about 25%) than that of mrp7B in kidney. In embryo, the expression levels of both transcripts are much lower compared to most tissues examined. Whether the difference in expression level of two
Fig. 3. PCR analysis of the expression of mouse mrp7A and mrp7B in different mouse tissues. The primers used in PCR are shown in materials and methods. cDNAs from different mouse tissues (QUICK-Clonee cDNAs (Clontech) were used as the templates. Lanes 1 and 2: nuclease free water (negative control); Lanes 3 and 4: liver; Lanes 5 and 6: brain; Lanes 7 and 8: embryo; Lanes 9 and 10: heart; Lanes 11 and 12: kidney; and Lane 13 and 14: spleen. Primers specific for mrp7A, MR9, MR10 and MS9, were used in the lanes of odd numbers. Primers specific for mrp7B, MR9, MR10 and MS22, were used in the lanes of even numbers. PCR products were separated by 1.5% agarose electrophoresis with 1 kb plus marker (GIBCOL BRL), lane M.
splice variants has any significance in their biological functions awaits further study. 3.3. Genomic structure of mrp7 The mouse genomic structure of mouse mrp7 gene is similar to that of human MRP7gene, except some differences in the lengths of some introns (Fig. 4). The sequences of the intron-exon junctions of the mrp7A and mrp7B are summarized in Fig. 4. There are totally 20 exons for mrp7A and 21 exons for mrp7B (Fig. 4). Compared to other members of MRP family, mrp7 seems to contain least numbers of exons. The significant difference in exons number may suggest that mrp7 are derived from a different ancestral gene. The average sizes of internal exons encoding the cDNAs are 219 bp for mrp7A and 215 bp for mrp7B, ranging from 78 bp (exon 9) to 1260 bp (exon 1). All intron-exon boundaries conform to the consensus splice junction sequences for eukaryotic cells (Shapiro and Senapathy, 1987; Grant et al., 1997). The 9 bp of the consensus 5 0 donor site ((A/C)AG/gt(a/g)agt are conserved in mouse mrp7A/mrp7B in 95/95, 74/75, 84/85, 100/100, 100/100, 89/90, 58/55, 84/85, 32/35%, respectively. The 4 bp of the 3 0 acceptor site ((c/a)/ag/(A/G)) are conserved in mouse mrp7A/mrp7B in 89/90, 100/100, 100/100, 79/80, respectively. The genomic organization of mouse mrp7 gene is also quite different from that of other mrp subfamily members at the first exon (Fig. 5). Compared with the other isolated mrp subfamily members,
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Fig. 4. Sequences of the intron-exon junctions of the mrp7 genes. (A) Sequences of the intron-exon junctions of the mrp7A. (B) Sequences of the intron-exon junctions of the mrp7B. Exon sequences are shown in uppercase letters, and intron sequences in lowercase letters. The encoded amino acid and its position are indicated below the exon sequences. The lengths of the introns are shown in parentheses. The latter 1222-bp sequence of the exon 2 of mrp7B is corresponding to the latter 1222-bp nucleotide sequence of the exon 1 of mrp7A. The genomic structure of mrp7B is identical to that of mrp7A except the exon1.
it has quite large exon 1, similar to its human homologue. ABC transporters contain the four structural domains including MSD2, MSD3, NBD1, and NBD2 (Stride et al., 1996; Bakos et al., 1996; Tusnady et al., 1997; Deeley
and Cole, 1997). Each nucleotide binding domain of mrp7 was encoded by four exons, i.e. exons 5–8 for NBD1 and exons 16–19 for NBD2. The extra N-terminal membrane spanning domain (MSD1) in mrp7 shows that mrp7
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Fig. 5. Comparison of genomic structures of mouse mrp7A, and mrp7B. The locations of the translation initiate sites are indicated by vertical lines and Ms. Walker A and B motifs and active transport family signature are denoted A, B and C, respectively.
belongs to the subgroup including MRP1, MRP2, MRP3, and MRP6. 3.4. Analysis of the promoter activity
Fig. 6. Analysis of the mouse mrp7 promoter activity. The promoterless pGL3-Enhancer vector and the pGL3-promoter vector with SV40 were used as the negative and the positive controls, respectively.
A 1.86-kb DNA fragment upstream of the translation initiation site was isolated from the genomic clone (accession number AC078884). This DNA fragment was cloned into pGL3-Enhancer vector. The MDCK cells were transfected with mouse mrp7 promoter and the luciferase activities were analyzed. This 1.86-kb fragment supports the luciferase activity seven fold over the promoterless negative control and two fold activity higher than the positive control of SV40 promoter (Fig. 6). The strong promoter activity demonstrated by this region suggests that mouse mrp7 gene may be well regulated in vivo to execute its biological function. 3.5. Tissue distribution of mrp7 Tissue distribution of mouse mrp7 was analyzed by northern hybridization on a mouse Multiple Tissue Northern blot with a cDNA probe. A signal around 5.5 kb was observed in the tissue of heart, liver, skeletal muscle, and kidney (Fig. 7). In summary, we describe that the mouse mrp7 has two spliced transcripts, mrp7A and mrp7B. The biological significance of these two spliced variants awaits for further study. Their genomic organization including the intronexon boundaries, the promoter activity and differential expression pattern were presented. Study of gene knockout model or transgenic mice of mrp7 may shed some light on the mechanism of the physiological functions of MRP7. References
Fig. 7. Tissue distribution of mouse mrp7 transcripts. Membrane containing 1 mg of poly(A) 1 RNA per tissue was hybridized with a mrp7 cDNA fragment as the probe. Lane 1: heart; Lane 2: brain; Lane 3: spleen; Lane 4: lung; Lane 5: liver; Lane 6: skeletal muscle; Lane 7: kidney; and Lane 8: testis.
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