Molecular cloning, characterization, and chromosomal assignment of porcine cationic amino acid transporter-1

Genomics 85 (2005) 352 – 359 www.elsevier.com/locate/ygeno

Molecular cloning, characterization, and chromosomal assignment of $ porcine cationic amino acid transporter-1 Zhaoqiang Cuia, Sergey Zharikova, Shen-Ling Xiaa, Susan I. Andersonb, Andy S. Lawb, Alan L. Archibaldb, Edward R. Blocka,c,* a Department of Medicine, University of Florida, Gainesville, FL 32610, USA Genetics and Genomics, Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, Scotland, UK c Research Service, Malcolm Randall Veterans Affairs Medical Center, Gainesville, FL 32608, USA

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Received 26 August 2004; accepted 10 November 2004 Available online 18 December 2004

Abstract We have cloned and characterized the gene encoding the porcine cationic amino acid transporter, member 1 (CAT-1) (HGMW-approved gene symbol SLC7A1) from porcine pulmonary artery endothelial cells. The porcine SLC7A1 encodes 629 deduced amino acid residues showing a higher degree of sequence similarity with the human counterpart (91.1%) than with the rat (87.3%) and mouse (87.6%) counterparts. Confocal microscopic examination of porcine CAT-1–GFP-expressing HEK293 cells revealed that porcine CAT-1 localizes on the plasma membrane. Amino acid uptake studies in Xenopus oocytes injected with cRNA encoding this protein demonstrated transport properties consistent with system y+. Radiation hybrid mapping data indicate that the porcine SLC7A1 maps to the distal end of the short arm of pig chromosome 11 (SSC11). This map location is consistent with the known conservation of genome organization between human and pig and provides further confirmation that we have characterized the porcine orthologue of the human SLC7A1. D 2004 Elsevier Inc. All rights reserved. Keywords: l-Arginine; Cationic amino acid transporter; CAT-1; SLC7A1; System y+; Nitric oxide; Endothelial nitric oxide synthase; Endothelium

Pulmonary artery endothelial cells (PAEC) are a rich source of nitric oxide (NO), a nitrogen-centered free radical with multiple and unique physiologic bioregulatory activities [1,2]; e.g., NO functions as a signaling molecule and plays a major role in the regulation of pulmonary vascular tone, leukocyte and platelet adhesion to endothelium, and pulmonary vascular smooth muscle proliferation. A number of lung diseases associated with primary or secondary pulmonary hypertension are characterized by impaired vascular production of NO [3–5]. As such, modulation of NO generation has the potential to become a novel strategy in the prevention and treatment of a number of lung diseases.

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Sequence data from this article have been deposited with the GenBank Data Library under Accession No. AY371320. * Corresponding author. Fax: +1 352 338 9884. E-mail address: [email protected] (E.R. Block). 0888-7543/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2004.11.006

PAEC generate NO from l-arginine (l-Arg) via the catalytic action of an NADPH-requiring, Ca2+/calmodulindependent constitutive NO synthase [2]. Synthesis requires O2 as a cosubstrate and results in NO and the coproduct lcitrulline. Cofactors required include NADPH, BH4, flavin adenine dinucleotide, and flavin mononucleotide. l-Arg is the exclusive precursor of NO, and NOS-mediated formation of NO is critically dependent upon an adequate and continuing supply of l-Arg [6–8]. The l-Arg content of endothelial cells is derived primarily from plasma membrane-dependent transport of extracellular l-Arg, although l-Arg can also be synthesized from l-citrulline [9–12]. The importance of transport to the maintenance of intracellular lArg content is clear from work demonstrating that removal of l-Arg from the medium results in rapid depletion of intracellular l-Arg in cultured endothelial cells [13,14]. Transport of l-Arg is mediated by several independent transport activities in mammalian cells [15]. The distribution

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and relative contribution of each of these transport activities to the total l-Arg uptake by a particular cell type vary widely due to cell-specific expression of the corresponding genes. Under physiologic conditions l-Arg transport in PAEC is mediated by two transport agencies [9,16–18]: system y+, a Na+-independent system encoded by the SLC7A gene family, and system BO,+, a Na+-dependent cotransport system encoded by the ATBO,+ gene [19,20]. Previous studies from our laboratory using plasma membranes from porcine PAEC indicate that 94% of saturable lArg transport in these cells is mediated by system y+ [18]. System y+ has been characterized in porcine PAEC by our lab [18,21] and its properties in this cell type are similar to those described for many others. These properties include Na+-independent transport that is responsive to membrane potential, nonobligatory exchange, and, at neutral pH and physiological substrate concentrations, specificity restricted to amino acids with net positive charge. At present, four members of a family of cationic amino acid transporters (CAT-1, CAT-2A, CAT-2B, CAT-3) have exhibited system y+ activity [15]. The function of a more distantly related isoform, CAT-4, has remained elusive [22]. Although the deduced amino acid sequences of these proteins are similar and they share common substrate specificity, the tissue-specific expression of these proteins differs. The distribution of CAT-1 expression appears to be nearly ubiquitous, with the notable exception of quiescent liver, in keeping with its identification as the gene encoding constitutive system y+ activity. Under physiological conditions, CAT-3 exhibits a brain-specific expression [23]. Using immunohistochemistry, we documented the expression of CAT-1, but not CAT-2 or -2a, within the plasma membrane of porcine PAEC [24]. Because of the major role of CAT-1 in l-Arg transport, regulation of CAT-1 expression or activity represents a potential target for modulating cellular NO generation. During the past decade, our lab has defined the functional characteristics and localization of porcine CAT-1 in porcine PAEC [12,16–18,21,24–33]. Such information is not available in other species. So, knowing the sequence of the porcine SLC7A1 gene would allow for correlation of function of the product and structure of the gene that cannot be offered in other species. Therefore, the goal of the present study was to clone and characterize further the porcine SLC7A1 gene.

Results Molecular cloning of porcine SLC7A1 As described under Materials and methods, a 959-bp porcine SLC7A1 cDNA was obtained from pig PAEC RNA using RT-PCR amplification. The PCR primers were designed based on the conserved regions of the following known mammalian SLC7A1 cDNAs: human, rat, and mouse. Analysis of the sequence of the 959-bp fragment

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demonstrated that it shared a high degree of sequence identity with the human, rat, and mouse sequences, indicating it was the porcine homologue of SLC7A1. Based on this sequence, two gene-specific primers were synthesized and 3V/5V rapid amplifications of cDNA ends (RACE) were performed. 3V RACE (~4.8 kb) and 5V RACE (~2.7 kb) products were cloned into the pCR4-TOPO vector and sequenced. The sequence of the 3V RACE cDNA fragment (4783 bp) overlaps the 5V RACE cDNA fragment by 452 bp. Despite trying several different methods we were unable to identify about 600 bp of the most distal portion of the 5V end of the cDNA. Finally, 6416 bp of the cDNA was assembled from the overlapping 3V (4783 bp) and 5V RACE (2085 bp) fragments (GenBank Accession No. AY371320). Identification and characterization of porcine SLC7A1 Sequence analysis of the porcine SLC7A1 cDNA revealed (1) an ORF of 1890 bp that would encode a protein of 629 residues with a calculated molecular mass of 67.8 kDa, (2) 366 bp of 5V untranslated region (UTR), and (3) 4160 bp of 3V UTR with a consensus AATAAA polyadenylation signal at 21–26 nt upstream of a poly(A) stretch. BLASTn or BLASTp analysis demonstrated that the porcine sequence shares a high degree of sequence identity, both in the nucleotide sequences, especially in coding sequence (CDS) regions (86, 83, and 82%), and in the deduced amino acid sequences (91, 87, and 87%), with the human (Accession No. AF078107), rat (Accession No. AF245000), and mouse (Accession No. NM_007513) SLC7A1, respectively. Furthermore, unlike rat and mouse CAT-1, which are shorter than human CAT-1 by 5 and 7 aa, respectively, the porcine CAT-1 is the same length as human CAT-1. Hydrophobicity prediction suggests 14 putative membrane-spanning domains within porcine CAT-1, similar to other mammalian CATs. Consistent with the results of homologous comparison, phylogenetic analysis shows that the divergence of porcine SLC7A1 and human SLC7A1 in evolution seems later than that of rat and mouse SLC7A1. To determine whether there is more than one transcript encoded by the porcine SLC7A1 gene, total RNA from PAEC was subjected to Northern blot analysis and probed with a 1.376-kb porcine SLC7A1 cDNA. Unlike other known mammalian SLC7A1, only one transcript (7.0 kb) was identified in porcine PAEC. Furthermore, this transcript can be upregulated by lipopolysaccharide (LPS)/interferong (IFN-g) activation (Fig. 1). This is consistent with our previous functional studies demonstrating that LPS/interferon-g activated l-Arg uptake in porcine PAEC (unpublished data). Sequencing shows that porcine SLC7A1 possesses a lengthy 3V UTR (4.0 kb) within which a few copies of ATTTA pentamer motifs exist. Since this consensus is characteristic of unstable mRNA [34,35], we next studied the porcine SLC7A1 mRNA half-life using real-time QRTPCR and the total RNA from porcine PAEC treated for 0–6

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Fig. 1. Northern blot analysis of porcine SLC7A1. Total RNA (20 Ag each) from control (lane 1) and LPS/IFN-g (LPS 100 Ag/ml, IFN-g 50 units/ml for 24 h)-activated PAEC (lane 2) was hybridized, respectively, with a 1.376-kb porcine SLC7A1 cDNA located in CDS (+68 to +1443 nt) and a 0.651-kb porcine GAPDH cDNA (347 to 997 nt, Accession No. AF017079) that had been labeled using [32P]dCTP, according to the instructions for the Prime-a-Gene Labeling System (Promega, Madison, WI, USA). The detailed manipulations for Northern blotting are as described under Materials and methods. Only one porcine SLC7A1 transcript (7.0 kb) was observed and could be significantly induced by LPS/IFN-g.

h with actinomycin D (Act D; 10 mg/ml). A 125-min t 1/2 was observed (Fig. 2). To verify the bioinformatics analysis indicating that the cloned cDNA is porcine SLC7A1, we examined the amino acid transport properties of the putative porcine CAT-1 in Xenopus laevis oocytes 2 days after injection with 25 ng porcine SLC7A1 cRNA or water alone. After a 3-h incubation, porcine SLC7A1 cRNA-injected oocytes accumulated twofold more l-Arg than water-injected oocytes. lArg uptake was not diminished significantly by isotonic substitution of Li+ for Na+ but was competitively inhibited by another cationic amino acid, l-lysine (l-Lys) (Fig. 3). These transport properties are consistent with the transport properties of system y+.

Fig. 3. Effects of porcine CAT-1 on l-Arg uptake in X. laevis oocytes. The oocytes were injected with 25 ng porcine SLC7A1 cRNA (0.5 ng/nl) or water alone as control. The l-Arg uptake studies were carried out after a 2day incubation at 168C in modified Barth’s saline solution (MBS) postinjection and a subsequent 3-h incubation at 168C in the different uptake buffers (Buffer 1, MBS containing 50 AM unlabeled l-Arg plus 10 ACi/ml l-[3H]Arg; Buffer 2, Li+/Na+ equivalently replaced Buffer 1; Buffer 3, 50 mM unlabeled l-Lys-containing Buffer 1). Controls 1–3 corresponded to Buffers 1–3 but the oocytes in the controls were injected with water alone. All measurements of l-Arg uptake were normalized by subtracting the nonspecific component of uptake. Bars represent means F SE (n = 6). Statistical analysis was performed using paired t test. Buffer 1 vs Control 1, p = 0.01; Buffer 2 vs Control 2, p b 0.01; Buffer 3 vs Control 3, not significant.

Having verified that the cloned cDNA behaves functionally like porcine SLC7A1, we next sought to identify the cellular localization of the gene product in porcine PAEC using green fluorescent protein (GFP) tagging. However, the transfection efficiency of porcine CAT-1– GFP was too low in porcine PAEC. Therefore, we employed HEK293 cells to investigate the cellular localization of the porcine CAT-1–GFP fusion protein in mammalian cells. The expressed porcine CAT-1–GFP localizes to the plasma membrane at 12 h after transfection. Peak expression time was at 72 h (Fig. 4), but the signal could be detected at least 6 days after transfection using laser confocal microscopy. Chromosomal mapping

Fig. 2. Kinetic characteristics of porcine SLC7A1 mRNA decay. The halflife (t 1/2) of porcine SLC7A1 mRNA was determined using real-time QRTPCR. Total RNA was obtained from PAEC treated with Act D for 0–6 h. 18S rRNA expression levels served as the internal control. Serial twofold dilutions of total RNA (2 to 0.125 Ag for porcine SLC7A1, 80 to 5 pg for 18S rRNA) were used as a reference for the standard curve calculation. The fluorescence threshold values were calculated using the iCycler iQ system software, and porcine SLC7A1 mRNA t 1/2 was determined by fitting an exponential decay curve of the percentage of porcine SLC7A1 mRNA remaining and decay time. Porcine SLC7A1 mRNA t 1/2, 125 F 4min (mean F SE, n = 7).

Chromosomal location of porcine SLC7A1 was assigned by PCR screening of a whole genome porcine/hamster radiation hybrid panel (Roslin Institute, Edinburgh, UK) as described under Materials and methods. Porcine SLC7A1specific amplification was achieved. The resulting code (000000000010000000000010110111010000001001001000011110011001001101110100000111001111000000101001) was loaded into the Roslin Radiation Hybrid Database. Two-point analyses revealed that the porcine SLC7A1 gene maps close to the anonymous DNA marker S0385 (17.8 cR3000rad; lod score 15.74). The anonymous DNA marker S0385 has been mapped previously to the distal end of the

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Fig. 4. Cellular localization of porcine CAT-1–GFP fusion protein detected by confocal microscopy. HEK293 cells transfected with the porcine CAT-1–GFP construct (pCAT-1-GFP) or control pEGFP-N1 were observed using laser scanning confocal microscopy (Zeiss LSM 510) 48 h after the transfection. The fluorescence at the midsection of cells is demonstrated. Most of the porcine CAT-1–GFP fusion protein targets to the plasma membrane 48 h after transfection, whereas GFP distributes evenly in the cell. (Original magnification 40, section 1.2 Am.)

short arm of pig chromosome 11 (SSC11) by linkage analysis [36] and analysis of somatic cell hybrids [37].

Discussion In this study, we cloned and characterized the porcine SLC7A1 gene. A 6416-bp cDNA was obtained by the RACE on the basis of conserved sequences of other known mammalian SLC7A1 cDNAs. The identity of this cDNA as porcine SLC7A1 was demonstrated by (1) sequencing and homologous comparison with other known mammalian SLC7A1, (2) computer deduction of protein molecular features, (3) observation of GFP-fusion protein localization in HEK293 cells, (4) characterization of the functional properties in X. laevis oocytes injected with its cRNA, and (5) comparative chromosomal mapping with human SLC7A1. Furthermore, phylogenetic analysis demonstrates that the phylogeny of porcine SLC7A1 and human SLC7A1 is more related than that of rat and mouse SLC7A1, which probably underlies our previous finding that porcine CAT-1 and human CAT-1 are similar in function and that the lArg:NO pathway in porcine PAEC is similar to that in human PAEC. The number and size of transcripts that SLC7A1 genes can express seem to depend on species and tissues. For example, mouse SLC7A1 expresses two large transcripts of 7.0 and 7.9 kb [38,39]; in contrast, a single transcript of approximately 7.9 kb was observed in Northern blots of RNA from rat intestine, kidney, and spleen, as well as in fetal rat liver [39–41], whereas various cultured rat cells demonstrate two transcripts of 3.4–4 and 7.9–8 kb [39,41]. We found only one porcine SLC7A1 mRNA transcript of

about 7.0 kb, and it was upregulated by LPS/IFN-g, consistent with functional data, demonstrating that LPS/ IFN-g upregulated the l-Arg transport (data not shown). Porcine SLC7A1 mRNA contains a lengthy 3V UTR of 4.16 kb in which a consensus AATAAA polyadenylation signal is present 21–26 nt upstream of a poly(A) stretch. This is a canonical feature of transcripts with a full-length 3V UTR. As a factor of posttranscriptional regulation, the mRNA stability is influenced by a few factors [35]. The majority of papers dealing with mRNA stability determinants have identified mRNA decay signals in the 3V UTR, suggesting that the half-lives of most mRNAs are influenced by this region. Of those signals, ATTTA consensus is characteristic of unstable mRNA [34,35]. Because three copies of ATTTA motifs appear within the 3V UTR of porcine SLC7A1, they are possibly critical determinants of porcine SLC7A1 mRNA stability. To quantify the stability of the porcine SLC7A1 transcript, mRNA decay was followed using real-time quantity RT-PCR after complete blockade of new transcription by Act D. Because Act D also inhibits the transcription of internal control genes, which, depending on the half-life of the internal control, may bias the real value of mRNA half-life, we chose 18S rRNA as the internal control because it has a long half-life (70 h) [42]. A half-life for porcine SLC7A1 mRNA of 125 min was observed, which is longer than that of the long transcript (7.9 kb) of rat SLC7A1. This may be because the latter possesses one more ATTTA consensus and holds other motifs characteristic of mRNA instability [43]. Rat SLC7A1 has been found to possess a special 5V UTR [44,45]. Its length, determined by S1 nuclease protection analysis of RNA, is 270 nt. Unlike most eukaryotic mRNAs, the translation of rat SLC7A1 is via a cap-independent

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IRES-mediated mechanism, allowing for direct recruitment of ribosomes to the initiation codon. A small upstream ORF (uORF) encoding 48 aa is the switch that controls structural remodeling of the mRNA leader of rat SLC7A1. The 5V UTR of porcine SLC7A1 is approximately 1 kb. A 366-nt sequence in the proximal region of the 5V UTR rich in GC prevented further sequencing of the 5V UTR. Although a uORF also exists within the mRNA leader, its position and length are different from those of the uORF of rat SLC7A1. These suggest that the regulation of porcine SLC7A1 expression is probably more complicated than that of rat SLC7A1. We have mapped the porcine SLC7A1 gene to the proximal end of the short arm of pig chromosome 11. The human SLC7A1 gene maps to human chromosome 13q12– q14 [46]. On the basis of the known conservation of genome organization between human and pig [47], we predicted that the porcine SLC7A1 gene would map to pig chromosome 11. The human SLC7A1 gene is located at ca. 28 Mb along human chromosome 13. The nearest gene for which the pig homologue has been mapped is FLT1, which is located at 26.7 Mb on HSA13. Pig FLT1 maps to the telomeric end of pig chromosome 11p [47]. This is where the radiation hybrid mapping data suggest that porcine SLC7A1 maps. Thus, the map location for the porcine SLC7A1 gene is consistent with the known human–pig comparative map. The results of the present study will provide a molecular basis for correlation of function and structure of the porcine SLC7A1 gene and for further investigation into expressional regulation of porcine SLC7A1 and how it modulates cellular NO generation.

3V/5V RACE A 959-bp porcine SLC7A1 cDNA was obtained from 1 Ag total RNA using one-step RT-PCR amplification (PowerScript reverse transcriptase and Advantage 2 PCR enzyme system; BD Biosciences Clontech, Palo Alto, CA, USA) and PCR primers (Forward, 5V-TATGCCTTCGTGGGCTTTGACTGCAT-3V; Reverse, 5V-TGGTCCAGCTGCATCATGAGATAGA-3V) that were designed on the basis of published SLC7A1 cDNA sequences in GenBank from Homo sapiens (Accession No. AF078107), Rattus norvegicus (Accession No. AF245000), and Mus musculus (Accession No. M26687). RT-PCR was performed as described below: 428C for 45 min, 948C for 10 min, followed by 35 cycles of amplification (948C for 30 s, 658C for 30 s, 688C for 1 min). After the PCR product was sequenced and homologically compared to certify that it is porcine SLC7A1, based on this sequence, porcine SLC7A1 gene-specific primers were synthesized and 3V/5V RACE were carried out according to the manufacturerTs instructions (BD Biosciences Clontech). Briefly, the first strand cDNA was generated from 1 Ag total RNA using 3V RACE CDS primer A (3V CDS) and 5V-CDS/SMART II A (Clontech) for 3V RACE and 5V RACE, respectively. For 3V RACE, the amplification reaction was performed for 35 cycles (948C for 30 s, 658C for 40 s, 688C for 7 min) using the forward primer (5V-TGGCCGCCTGTGTGTTGGTCTTA-3V) and the reverse primer (universal primer A mix, UPM; Clontech); for 5V RACE, a similar amplification reaction but a 5-min elongation time was carried out using the forward primer (UPM) and reverse primer (5VCACAAAGATGCTCAGGACGGGAAGTA-3V).

Materials and methods cDNA cloning and sequence analysis Cell culture Porcine PAEC were obtained from the main pulmonary artery of 6- to 7-month-old pigs and were cultured and characterized as described previously [17]. Third to fourth passage cells in monolayer culture were maintained in RPMI 1640 medium containing 4% fetal bovine serum and antibiotics (100 u/ml penicillin, 100 Ag/ml streptomycin, 20 Ag/ml gentamicin, and 2 Ag/ml Fungizone) and were harvested 2 or 3 days after confluence and plated in 100mm dishes for experimentation.

The RACE products were gel-purified and cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA, USA). After transformation into Escherichia coli, the plasmid purifications from the overnight-grown colonies were done and the cloned cDNA was sequenced using a Perkin–Elmer/ Applied Biosystems automated DNA sequencer (DNA Sequencing Laboratory, ICBR, University of Florida). The sequence analyses including sequence comparison and secondary structure prediction were performed using NTI Vector 7.1.

RNA extraction

Northern blotting

Total cellular RNA was isolated from the cultured porcine PAEC using an RNeasy Midi kit and digested by DNase according to the manufacturerTs protocol (Qiagen, Valencia, CA, USA). The concentration of RNA solution was determined spectrophotometrically and the quality was verified by visualization of 2:1 intensity ratio of 28S vs 18S rRNA bands over UV light after electrophoresis through a 1% ethidium bromide stain agarose gel.

Twenty micrograms of total RNA each obtained from control cultured porcine PAEC and those activated with bacterial LPS (100 Ag ml 1)/porcine IFN-g (50 U ml 1) (Sigma, St. Louis, MO, USA) for 24 h were analyzed on a Northern blot. A 1.376-kb porcine SLC7A1 cDNA located in CDS (+68 to +1443 nt) and a 0.651-kb porcine GAPDH cDNA (347 to 997 nt, Accession No. AF017079) were labeled using [32P]dCTP (ICN) according to the instructions

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for the Prime-a-Gene Labeling System (Promega, Madison, WI, USA). The detailed manipulations for Northern blotting were as described elsewhere [48] but the components of the prehybridization and hybridization buffers were slightly modified (i.e., 0.25 M Na2HPO4, pH 7.2, 5% SDS, 1.25 mM EDTA, pH 8.0).

then transfected into the HEK293 cells using Effectene transfection reagent (Qiagen) for the expressional localization of porcine CAT-1 by confocal microscopy.

Analysis of porcine SLC7A1 mRNA decay

For functional analysis of l-Arg transport, X. laevis oocyte expression was employed. To prepare porcine SLC7A1 cRNA, a 1906-bp cDNA fragment obtained from the BglII/EcoRV-digested porcine CAT-1–GFP was cloned into the BglII/EcoRV sites of the plasmid pT7TS (a kind gift from Dr. Paul A. Krieg). The resulting plasmid was linearized by XbaI, and porcine SLC7A1cRNA was prepared by the mMESSAGE mMACHINE High Yield Capped RNA transcription kit (Ambion, Austin, TX, USA). Oocyte preparation and injection have been described elsewhere [49]. Briefly, after mature female X. laevis African toads (Nasco, Ft. Atkinson, WI) were anesthetized, oocytes were surgically removed. To remove the follicular cell layer, harvested oocytes were treated with 1.25 mg/ml collagenase (in calcium-free BarthTs solution: 88 mM NaCl, 10 mM Hepes, pH 7.6, 0.33 mM MgSO4, 0.1 mg/ml gentamicin sulfate) for 2 h at room temperature. Thereafter, stage 5 oocytes were isolated and injected with 50 nl (25 ng) of cRNA or water alone as controls on the day following harvesting. The l-Arg uptake studies were carried out as described below after a 2-day incubation at 168C in modified BarthTs saline solution (MBS; 88 mM NaCl, 1 mM KCl, 15 mM Hepes, 0.82 mM MgSO4, 0.32 mM Ca(NO3)2, 0.41 mM CaCl2, 2.38 mM NaHCO3, 10 Ag/ml sodium penicillin, 10 Ag/ml streptomycin sulfate, 50 Ag/ml gentamicin sulfate, pH 7.6) postinjection. Briefly, oocytes were washed twice with fresh MBS at 168C and then were incubated at 168C for 3 h in the different uptake solutions (solution 1, MBS containing 50 AM unlabeled l-Arg plus 10 ACi/ml l[3H]Arg; solution 2, Li+/Na+ equivalently replaced solution 1; solution 3, 50 mM unlabeled l-Lys-containing solution 1). Uptake was terminated by placing the dishes on ice and washing twice with 5 ml ice-cold MBS containing 10 mM unlabeled l-Arg. The oocytes were transferred into scintillation vials and lysed with 30 Al of 1% SDS. After solubilization, 5 ml of scintillation fluid was added, and radioactivity was quantitated by liquid scintillation spectrometry. All measurements of l-Arg uptake were normalized by subtracting the nonspecific component of uptake (uptake in the presence of 10 mM unlabeled l-Arg instead of 50 AM unlabeled l-Arg).

As described above, porcine PAEC were harvested 2 or 3 days after confluence and subcultured in 100-mm dishes. At 60% confluence, the cells were treated with 10 Ag/ml Act D until the time points indicated. The half-life of porcine SLC7A1 mRNA was estimated by real-time QRT-PCR using the Bio-Rad iCycler iQ system (Bio-Rad, Hercules, CA, USA) and Brilliant SYBR Green QRT-PCR Master Mix Kit, One-Step (Stratagene, La Jolla, CA, USA). Expression levels of 18S rRNA served as the internal controls. The following primers were used for the real-time QRT-PCR: porcine SLC7A1 forward, 5V-GGTTATATGATTTTGTGTTTCTTGAT-3V, reverse, 5V-CCATGCTGAAAGGCACAACT-3V; 18S rRNA forward, 5V-TTCCAGCTCCAATAGCGTAT-3V, reverse, 5V-GATCCAACTACGAGCTTTTTAA-3V. Serial twofold dilutions of total RNA (2 to 0.125 Ag for porcine SLC7A1, 80 to 5 pg for 18S rRNA) were used as a reference for the standard curve calculation. All real-time RT-PCRs were performed in a 25-Al volume containing 1 SYBR QRT-PCR Master Mix, 75 nM primers, 0.0625 Al of StrataScript RT/RNase block enzyme mixture, and 200 ng (for porcine SLC7A1) or 20 pg (for 18S rRNA) of total RNA. The fluorescence threshold values were calculated using the iCycler iQ system software, and porcine SLC7A1 mRNA t 1/2 was determined based on the regression equation of the percentage of porcine SLC7A1 mRNA remaining and decay time. Transient expression of porcine SLC7A1 in HEK293 cells For cellular localization studies, the plasmid encoding the porcine CAT-1–GFP fusion protein was constructed as described below. Briefly, a 1919-bp porcine SLC7A1cDNA containing the full-length CDS of porcine SLC7A1 was obtained using one-step RT-PCR amplification and the following PCR primers (Forward, 5V-ATTAATAGATCTCGCCACCATGGGGTGCAAGATGCTG-3V, Reverse, 5V-TCAGATATCGAATTCTCTTGCAATGGTCCAGGTTAC-3V). The reaction conditions of RT-PCR were 428C for 45 min, 948C for 10 min, followed by 35 cycles of amplification (948C for 30 s, 608C for 45 s, 688C for 2 min). After BglII/EcoRI digestion and gel purification, the PCR product was inserted in the BglII/EcoRI sites of pEGFP-N1 (BD Biosciences Clontech). The resulting plasmid was termed pCAT-1-GFP. After transformation and amplification in E. coli, purified plasmids from the E. coli colonies were sequenced. The clone without the mutations by PCR was further amplified in E. coli and

Expression of porcine SLC7A1 cRNA and L -Arg uptake in X. laevis oocytes

Chromosomal mapping A whole genome porcine/hamster radiation hybrid panel (Roslin Institute) was employed for the chromosomal mapping based on PCR screening as described below. Briefly, PCRs were performed with the Advantage 2 PCR

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enzyme system (Clontech) in a volume of 50 Al containing 30 ng of genomic DNA from 1 to 94 clones in the panel, a positive control (porcine genomic DNA), and a negative control (hamster genomic DNA), 0.2 AM porcine SLC7A1specific primers (forward, 5V-AGCAAGACCAAACTCTCCTTC-3V; reverse, 5V-CACAAAGATGCTCAGGACGGGAAGTA-3V). The PCRs were 948C for 5 min, followed by 35 cycles of amplification (948C for 30 s, 608C for 50 s, 688C for 1 min). Ten microliters of the PCR products were run on 1% agarose gel in 1 TAE. The results were loaded into the Roslin Radiation Hybrid Database (http://databases.roslin.ac.uk/radhyb/intro.py), extracted, and tested in two0point analyses with the rh2pt program in RHMAP version 3.0 (http://iubio.bio.indiana.edu:7780/archive/00000471/ftp://ftp.ebi.ac.uk/pub/software/linkage_and_mapping/RHMAP_SIMLINK [50]) for evidence of cosegregation with other sequence-tagged sites that have been scored in the RH panel.

Acknowledgments This study was funded in part by NHLBI Grant HL52136 (MERIT) and by the Medical Research Service, Department of Veterans Affairs (for E.R.B). We gratefully acknowledge Dr. Roger Papke (Pharmacology and Therapeutics, University of Florida) for assistance with Xenopus laevis oocyte manipulation, Dr. Paul Krieg for kindly providing the pT7TS vector, Dr. Leonid Belayev for technical efforts, and Mr. Bert Herrera for cell culture work. A.L.A. and A.S.L. were supported by the UK Biotechnology and Biological Sciences Research Council.

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