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Genomics 90 (2007) 595–609 www.elsevier.com/locate/ygeno

Identification of six putative human transporters with structural similarity to the drug transporter SLC22 family ⁎ Josefin A. Jacobsson, Tatjana Haitina, Jonas Lindblom, Robert Fredriksson

Department of Neuroscience, Unit of Pharmacology, Uppsala University, BMC, Uppsala SE 75124, Sweden Received 5 February 2007; accepted 24 March 2007 Available online 22 August 2007

Abstract

The solute carrier family 22 (SLC22) is a large family of organic cation and anion transporters. These are transmembrane proteins expressed predominantly in kidneys and liver and mediate the uptake and excretion of environmental toxins, endogenous substances, and drugs from the body. Through a comprehensive database search we identified six human proteins not yet cloned or annotated in the reference sequence databases. Five of these belong to the SLC22 family, SLC22A20, SLC22A23, SLC22A24, SLC22A25, and SPNS3, and the sixth gene, SVOPL, is a paralog to the synaptic vesicle protein SVOP. We identified the orthologs for these genes in mouse and rat and additional homologous proteins and performed the first phylogenetic analysis on the entire SLC22 family in human, mouse, and rat. In addition, we performed a phylogenetic analysis which showed that SVOP and SV2A-C are, in a comparison with all vertebrate proteins, most similar to the SLC22 family. Finally, we performed a tissue localization study on 15 genes on a panel of 30 rat tissues using quantitative real-time polymerase chain reaction. © 2007 Elsevier Inc. All rights reserved.

Keywords: Drug transporter; SLC22; Solute carrier; Anion; Cation

The solute carrier family 22 (SLC22) is a large family of intracellular loop between the sixth and the seventh transmem- organic ion transporters that belongs to the major facilitator brane regions [4]. The family can be divided into three sub- superfamily (MFS). The MFS is one of the two largest families groups according to substrate specificity and function: the of membrane transporters comprising uniporters, symporters, organic cation transporters (OCTs), the organic cation/carnitine and antiporters from mammals, lower eukaryotes, bacteria, and transporters (OCTNs), and the organic anion transporters plants [1,2]. The first member of the SLC22 family, the rat (OATs) [3]. Moreover, there are several members of the organic transporter Slc22a1 (Oct1), was identified by expression SLC22 family with unidentified substrate specificity or function. cloning in 1994 by Gründemann et al. [3]. Today it is known that OCTs function as uniporters that mediate facilitated diffusion members of the SLC22 family transport a variety of compounds in either direction and are responsible for translocation of (1) including drugs, environmental toxins, and endogenous meta- organic cations, such as tetraethylammonium (TEA) and 1- bolites across the cell membrane [1,2]. Many SLC22 genes are methyl-4-phenylpyridinium (MPP), (2) endogenous amines, such expressed in liver, kidneys, and intestine where they play im- as dopamine, (3) therapeutic drugs, such as cimetidine and portant roles in absorption and excretion of drugs while they are morphine, and (4) cationic xenobiotics, such as antihistamines also found in other organs such as brain and heart [4]. The [1,2]. Three OCTs encoded by SLC22 have been identified, SLC22 proteins have been predicted to have 12 transmembrane SLC22A1-3 (OCT1-3) [3,5,6]. These are expressed in various (TM) α-helices, one extended extracellular loop between the tissues but have their primary expression in liver and kidneys. first and the second transmembrane regions, and one extended SLC22A2 and SLC22A3 are also expressed in brain where they are known to transport cationic neurotoxins and neurotransmitters [7]. + ⁎ Corresponding author. Fax: +46 18 51 15 40. OCTNs function as Na /L-carnitine cotransporters but are E-mail address: [email protected] (R. Fredriksson). also known to transport cations such as TEA [2]. Three OCTNs

0888-7543/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2007.03.017 596 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609

Fig. 1. Phylogenetic analysis of the known human, mouse, and rat SLC22 members together with the novel human, mouse, and rat genes, and SVOP and SV2A-C. The topology was obtained by maximum parsimony analysis using Phylip 3.6 [52], and maximum likelihood branch lengths were mapped onto that topology using Tree- Puzzle [35]. The prefixes m and r correspond to mouse and rat members. Members of the SLC22 family with known ligands are marked with shadings, anion transporters with dark gray and cation transporters with light gray. The novel human genes are marked with black boxes and mouse and rat genes with gray boxes. J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 597 encoded by SLC22 have been identified, SLC22A4-5 (OCTN1- involved in influx and efflux of drugs. Interestingly, many of 2) [8,9] and mouse Slc22a21 (Octn3) [8–10], which are all these transporters have now also been identified in the brain. The important for the maintenance of an appropriate level of central nervous system (CNS) contains cellular barriers that carnitine. maintain homeostasis by eliminating toxic metabolites generated OATs function as anion exchangers and are primarily in the brain. The primary interfaces between the CNS and the responsible for elimination of organic anions such as glutarates, peripheral circulation are the blood–brain barrier (BBB) and the salicylates, and prostaglandins which is mainly performed by blood–serum and cerebrospinal fluid barrier. Drugs acting in the the liver and kidneys [2]. The OAT family encoded by SLC22 brain have to overcome these barriers to achieve clinically consists of seven members: SLC22A6-8 (OAT1-3) [11–16], significant concentration in the brain [7]. Three SLC22 family SLC22A11 (OAT4) [17], SLC22A12 (URAT1, RST) [18,19], members, SLC22A5 (OCTN2), SLC22A8 (OAT3), and rodent Slc22a19 (Oat5) [20], and mouse Slc22a20 (Oat6) [21]. SLC22A12 (URAT1, RST), have been shown to be involved in These genes are abundantly expressed in kidneys but some are the efflux transport across the BBB [25,26]. In addition, some also expressed in other tissues such as liver, placenta, and brain SLC22s are of pharmaceutical interest due to their involvement in [22–24]. tumorigenesis. SLC22A16 (CT2) is a mediator of doxorubicin Most studies on SLC22 members have focused on their uptake in cancer cells [27], and SLC22A18 (ORCTL2) is thought function in peripheral tissues where they are known to be to be associated with breast cancer as well as Beckwith–

Table 1 A summary of human SLC22, SVOP, and SV2 members Symbol Alias Chromosomal Length (aa) Exons Expression Substrate Closest relative position identities % SLC22A1 OCT1 6q25.3 554 11 Liver, spleen, brain Organic cations SLC22A2 67% SLC22A2 OCT2 6q25.3 555 11 Kidney, placenta, brain Organic cations SLC22A1 67% SLC22A3 OCT3 6q25.3 556 11 Placenta, liver, kidney, heart, lung, Organic cations SLC22A2 51% brain, skeletal muscle SLC22A4 OCTN1 5q23.3 551 10 Kidney, skeletal muscle, placenta, Organic cations SLC22A5 75% heart, prostate SLC22A5 OCTN2 5q23.3 557 10 Kidney, skeletal muscle, placenta, Organic cations SLC22A4 75% heart, prostate, brain, liver, lung (carnitine) SLC22A6 OAT1 11q12.3 563 10 Kidney, brain, placenta, Organic anions SLC22A8 52% skeletal muscle SLC22A7 OAT2, NLT 6p21.1 546 10 Liver, kidney Organic anions SLC22A8 37% SLC22A8 OAT3 11q12.3 542 10 Kidney, brain, eye, liver, Organic anions SLC22A6 52% skeletal muscle SLC22A9 UST3, OAT4 11q12.3 553 10 Liver Not determined SLC22A25 73% SLC22A10 OAT5 11q12.3 531 9 Liver Not determined SLC22A25 55% SLC22A11 OAT4 11q13.1 550 10 Placenta, kidney Organic anions SLC22A12 46% SLC22A12 URAT1, RST 11q13.1 553 10 Kidney, brain Organic anions SLC22A11 46% (urate) SLC22A13 ORCTL3, OCTL1 3p22.2 551 10 Kidney, small intestine, colon Not determined SLC22A14 36% SLC22A14 ORCTL4, OCTL2 3p22.2 594 10 Kidney, colon, small intestine Not determined SLC22A13 36% SLC22A15 Flipt1 1p13.1 535 12 Kidney, skeletal muscle, placenta, Not determined SLC22A4 37% heart, lung, spleen, liver, brain SLC22A16 CT2, FLIPT2, 6q21 577 8 Kidney, testis, liver, bone marrow, Organic cations SLC22A1 33% OCT6 leukocytes (L-carnatine) SLC22A17 BOCT, hBOIT 14q11.2 366 7 Not determined Not determined SLC22A23 33% SLC22A18 ORCTL2, IMPT1 11p15.4 424 10 Kidney, liver, placenta, intestine Not determined SPNS3 25% SLC22A20 OAT6 11q13.1 573 9 Not determined Not determined SLC22A6 46% SLC22A23 None 6p25.2 405 7 Not determined Not determined SLC22A17 33% SLC22A24 None 11q12.3 322 4 Not determined Not determined SLC22A9 51% SLC22A25 None 11q12.3 547 9 Not determined Not determined SLC22A9 73% SPNS3 None 17p13.2 512 12 Not determined Not determined SLC22A18 25% SVOPL None 7q34 491 13 Not determined Not determined SVOP 39% (SLC22A15 29%) SVOP None 12q24.11 548 8 Brain Not determined SVOPL 39% (SLC22A1 30%) SV2A None 1q21.2 741 13 Brain Not determined SV2B 68% (SLC22A3 24%) SV2B None 15q26.1 683 12 Brain Not determined SV2C 69% (SLC22A15 23%) SV2C None 5q13.3 727 11 Brain Not determined SV2B 69% (SLC22A3/15 26%) Novel members are marked in boldface type. 598 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609

Table 2 Summary of mouse and rat Slc22, Svop, and Sv2 members Symbol Alias Chromosomal Length Exons Expression Substrate position (aa) Mouse Slc22a1 Oct1, Orct1 17qA1 556 11 Liver, kidney, colon, skin, spleen, small intestine Organic cations Slc22a2 Oct2, Orct2 17qA1 553 12 Kidney, brain Organic cations Slc22a3 Oct3, Orct3 17qA1 551 11 Placenta, brain, heart, uterus, skeletal muscle Organic cations Slc22a4 Octn1 11qB1.3 553 10 Kidney Organic cations Slc22a5 Octn2 11qB1.3 557 10 Kidney Organic cations (carnitine) Slc22a6 Oat1, Orctl1 19qA 545 10 Kidney, skeletal muscle, placenta, brain Organic anions Slc22a7 Oat2, NLT 17qB3 540 6 Liver, kidney, lung, small intestine Organic anions Slc22a8 Oat3, Roct 19qA 537 10 Kidney, brain, liver, skeletal muscle Organic anions Slc22a12 OAT4L, RAT1 19qA 553 10 Kidney Organic anions (urate) Slc22a13 OCTL1, CTL3 9qF3 551 10 Not determined Not determined Slc22a13b None 9qF3 548 10 Not determined Not determined Slc22a14 None 9qF3 629 10 Not determined Not determined Slc22a15 None 3qF2.2 233 5 Not determined Not determined Slc22a16 CT2, FLIPT2, OCT6 18qB1 649 8 Not determined Organic cations (L-carnitine) Slc22a17 Boct, Boit, 14qC1 401 7 Not determined Not determined Slc22a18 Orctl2, Impt1 7qF5 409 10 Liver, kidney, placenta, intestine Not determined Slc22a19 OAT-5, Oat5 19qA 551 10 Kidney Organic anions Slc22a20 Oat6, OAT1-L 19qA 556 10 Olfactory mucosa, testis Organic anions Slc22a21 Octn3, Slc22a9 19qB1.3 564 10 Testis, kidney Organic cations (carnitine) Slc22a22 None 15qD1 554 10 Not determined Not determined Slc22a23 None 19qA3.3 689 9 Not determined Not determined Slc22a24 None 19qA 509 9 Not determined Not determined Slc22a26 None 19qA 551 10 Not determined Not determined Spns3 None 11qB4 514 12 Not determined Not determined Svopl None 6qB1 494 14 Not determined Not determined Svop None 5qF 548 15 Brain Not determined Sv2a None 3qF2.1 497 8 Brain Not determined Sv2b None 7qD1 683 11 Brain Not determined Sv2c None 13qD1 727 12 Brain Not determined

Rat Slc22a1 Oct1, Orct1, Roct1 1q11 556 11 Liver, kidney, intestine, skin, colon, spleen, Organic cations brain, skeletal muscle, adrenal gland Slc22a2 Oct2, Oct2r 1q11 555 11 Kidney, thymus, brain, skeletal muscle Organic cations Slc22a3 Oct3 1q11 481 11 Placenta, brain, heart, kidney, skin, thymus, Organic cations intestine, adipose tissue, liver, heart Slc22a4 Octn1 10q22 529 6 Liver, kidney, intestine, brain, heart, placenta Organic cations Slc22a5 Octn2, Ust2 10q22 557 10 Kidney, testis, colon, ovary, heart, lung Organic cations (L-carnitine) Slc22a6 Oat1, Orctl1, Roat1 1q43 551 10 Kidney Organic anions Slc22a7 Oat2 9q12 528 9 Liver Organic anions Slc22a8 Oat3 1q43 536 10 Kidney, brain, liver, skeletal muscle Organic anions Slc22a9 Ust1r, Ust1 1q43 552 10 Kidney, skeletal muscle, liver, brain, testis Organic anions Slc22a12 Rst 1q43 553 10 Kidney Organic anions Slc22a13 None 8q32 551 10 Not determined Not determined Slc22a13b None 8q32 543 10 Epididymis, brain, adrenal gland, pineal gland, Not determined skeletal muscle, heart, kidney, ovary, uterus Slc22a14 None 8q32 406 5 Not determined Not determined Slc22a15 None 2q34 640 15 Not determined Not determined Slc22a17 Boct 15p13 516 9 Not determined Not determined Slc22a18 None 1q43 412 10 Liver, intestine, skeletal muscle, brain, kidney, Not determined eye, ovary, uterus, adipose tissue Slc22a19 Oat5 1q43 551 10 Kidney Organic anions Slc22a20 None 1q43 599 10 Brain, eye, adipose tissue, liver Not determined Slc22a22 None 7q32 555 10 Not determined Not determined Slc22a23 None 17p12 690 9 Brain, spinal cord, kidney, liver, eye, adipose Not determined tissue, lung, epididymis, adrenal gland, pineal gland, skeletal muscle, heart, spleen, thymus, ovary, uterus, testis, epididymis Slc22a24 None 1q43 319 5 Liver, skeletal muscle, brain Not determined Spns3 None 10q24 514 12 Brain, kidney, liver, spleen, thymus, testis, uterus, Not determined ovary, lung, heart, adipose tissue, skeletal muscle, pineal gland, adrenal gland, spinal cord, eye J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 599

Table 2 (continued) Symbol Alias Chromosomal Length Exons Expression Substrate position (aa) Svopl None 4q22 368 10 Brain, liver, kidney, skeletal muscle,epididymis, Not determined testis, ovary, thymus, spinal cord Svop None 12q16 536 15 Brain, eye, spinal cord, adrenal gland, heart, Not determined pineal gland, intestine, kidney, liver, lung, spleen, thymus, uterus, adipose tissue, skeletal muscle, testis, epididymis Sv2a None 2q34 489 8 Brain, eye, spinal cord, adrenal gland, heart, Not determined pineal gland, kidney, lung, spleen, thymus, uterus, adipose tissue, skeletal muscle, epididymis, ovary Sv2b None 1q31 673 11 Brain, eye, spinal cord, pineal gland, intestine, Not determined kidney, spleen, thymus, ovary Sv2c None 2q12 728 12 Brain, eye, spinal cord, adrenal gland, ovary, uterus Not determined The prefixes m and r correspond to mouse and rat, respectively. Novel members are marked in boldface type.

Wiedemann syndrome [28,29]. Furthermore, SLC22A4 remained that were not present in the seeding set. Four of these, (OCTN1) and SLC22A5 (OCTN2) are both thought to be SVOP and SV2A-C, are not members of the SLC22 family but associated with Crohn's disease [30,31], and SLC22A12 showed relative high similarity to the SLC22 family. In (URAT1, RST) is associated with idiopathic renal hypouricemia addition, from BLASTP searches in the NCBI databases these [18]. were shown to be, of all vertebrate proteins, most similar to the Through a comprehensive database search we identified six SLC22 family. Also, from searches in the Conserved Domain human proteins not yet cloned or annotated in the reference Database (CDD) they were shown to fit the sugar transporter sequence databases. Five of these belong to the SLC22 family, model, the same model that the SLC22 family of proteins SLC22A20, SLC22A23, SLC22A24, SLC22A25, and SPNS3, matches. Due to this, SVOP and SV2A-C are included in the and the sixth gene, SVOPL, is a paralog to the synaptic vesicle phylogenetic and tissue analysis presented below. The remain- protein SVOP. We identified the orthologs for these genes in ing 6 sequences were considered novel and subsequently sent to mouse and rat and additional homologous proteins and the HUGO Gene Nomenclature Committee (HGNC). The performed the first phylogenetic analysis on the entire SLC22 HGNC confirmed the novelty of these sequences and allocated family in human, mouse, and rat. In addition, we performed a the following symbols to them: SLC22A20, SLC22A23, phylogenetic analysis which showed that SVOP and SV2A-C SLC22A24, SLC22A25, SPNS3, and SVOPL. Below we are most closely related to the SLC22 family. Finally, we present the origin and database status of each of these 10 performed a tissue localization study on 15 genes on a panel of sequences. 30 rat tissues using quantitative real-time polymerase chain reaction. SLC22A20

Results This sequence was not annotated as SLC22A20 in the human RefSeq database but has previously been assigned this symbol A dataset consisting of 18 SLC22 sequences, SLC22A1– by the HGNC to, by us, an unknown group. The sequence was SLC22A18, was obtained by downloading all SLC22 present in the currated RefSeq (http://www.ncbi.nlm.nih.gov/ sequences present in the SLC- database at http://www. RefSeq/) dataset as FLJ16331, but this annotation contained bioparadigms.org/slc/menu.asp. Of these, the protein version only the first five of the nine coding exons and was hence only of SLC22A10 was annotated in the Third Party Annotation partial. SLC22A20 was represented in GenBank by two partial (TPA) database at NCBI while all other SLC22 sequences were mRNA sequences, AK131327 and AK094565, which together included in the human RefSeq database. This dataset was contained eight of the nine coding exons. The sequence was considered the seeding set and used to construct a sequence- found to be supported by 26 expressed sequence tag (EST) based hidden Markov model (HMM). Four different databases, sequences which, when combined, provided support for all RefSeq, Ensembl peptides, Ensembl AbInitio, and the human coding exons. subset of the Mammalian Gene Collection (MGC), were all searched against the HMM, and all hits were extracted. The hits SLC22A23 were filtrated by BLAST searches against the human RefSeq database, possible pseudo genes were removed, and the This sequence was present in the RefSeq database as sequences were manually corrected with methods similar to C6orf85 and consists of nine coding exons. The sequence was those that we have used before for new genes with complicated supported by one full-length (BC038748) and one partial genomic structures [32–34]. After this process 10 sequences (AK172770) mRNA from GenBank and 13 ESTs. 600 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609

SLC22A24 AB018279, BC000776, BX537754, and BC045111) and one partial mRNA (AK075480). We also found over 100 ESTs from The SLC22A24 sequence contained four coding exons and this transcript. was present in the RefSeq database as MGC34821. It was supported by one mRNA (BC034394) that covered the entire SV2B coding region. In addition, four ESTs provided support for the last two exons where one of these suggested one additional The sequence was annotated as SV2B and was supported by intron in the most C-terminal exon. three full-length mRNAs (BC030011, AB018278, and CR457086) identical to the RefSeq sequence and one partial mRNA SLC22A25 (AF052188), lacking 4 of the 12 exons. SV2B was supported by 31 EST sequences. This sequence was present in the RefSeq database as LOC387601. It was also supported by seven mRNAs in SV2C GenBank (AY437532,BC101314,BC101317,AK127610, AK091990,AX747296, andAY517500). SLC22A25 contains The sequence was present in the RefSeq database as SV2C. nine coding exons in the version presented here, but four of the We found five identical mRNAs containing all 11 exons mRNA sequences suggested one additional exon between exon (AK094917, BC100824, BC100825, BC100826, and 8 and 9, clearly indicating that SLC22A25 has at least one BC100827) and two mRNAs lacking the first 2 exons additional splice variant. In addition, 11 ESTs were present (AB028977 and AL707553). These two sequences could for this sequence that, when combined, provided support for potentially represent a shorter splice variant of SV2C. In all coding exons, including the alternative exon mentioned addition to the mRNA sequences we found four ESTs for this above. transcript. We retrieved mouse and rat sequences by translating mRNA SPNS3 and EST sequences and by manually assembling them from genomic data. Orthologs were identified for all novel human SPNS3 was present in the RefSeq database as MGC29671 genes except for SLC22A25 which seems to lack rodent and consists of 12 coding exons. We found six full-length orthologs. We also retrieved the rat Slc22a20 sequence and (CR606228, AK172832, CR614611, CR593280, BC023646, identified two additional homologous proteins present in mouse and CR618257), and four partial (CR611034, CR624976, and rat. All assembled sequences are present in the supplemen- AK093870, and AX748345) mRNAs. The sequence was also tary material. supported by 47 ESTs. We constructed a phylogenetic tree with several SLC families (Supplementary Fig. 1) which divides the SLC22 SVOPL family into six subgroups. The previously known SLC22A18 and the novel SPNS3 formed one subgroup distant from the rest The version of SVOPL presented here consists of 13 coding of the family, and an alignment of all human SLC22 members exons. The sequence was present in the RefSeq dataset as (data not shown) showed that SLC22A18 and SPNS3 had low LOC136306, but this annotation did not contain the last three sequence similarity with the rest of the family. coding exons that contain the last amino acids in TM12 and the A more comprehensive phylogenetic analysis was performed entire C terminus. SVOPL was covered by two mRNAs on the SLC22 family, along with SVOP and SV2, which also (BX648789 and BC036796) of which the later one (BC036796) clearly divides the family into several subgroups. The tree contained an alternative C terminus. We found 18 ESTs from topology was calculated using the maximum parsimony method SVOPL and when combined, all coding exons were supported. on an amino acid alignment consisting of all transmembrane No ESTs supporting the alternative C terminus from BC036796 regions and the interspacing loops. The length of the alignment was found. was 593 amino acids, and of these 441 (74.3%) were parsimony informative. On this topology, maximum likelihood branch SVOP lengths were subsequently mapped using Tree-Puzzle [35]. The phylogenetic tree (SLC22A18 and SPNS3 are excluded) is The sequence was annotated as SVOP and contains eight presented in Fig. 1. exons. The entire coding region was supported by two identical The novel human SLC22A24, its rodent orthologs, and mRNAs (BC094722 and BC033587) and one partial mRNA SLC22A25 clustered together with previously known SLC22 in (AL359592). We found 35 ESTs that together supported seven a clearly delimited cluster, where several of the proteins are of the eight exons. known to transport anions. SLC22A25 was shown to lack rodent orthologs but proved to be a paralog to SLC22A9 which SV2A also lacks rodent orthologs. SVOPL clustered together with SVOP and SV2A-C, and the analysis also showed that these The sequence was present in the RefSeq database as SV2A. were most phylogenetically related to SLC22A1-3. Rodent We found five identical full-length mRNAs (BC034038, Slc22a23 was confirmed to be orthologs of human SLC22A23 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 601 which together with SLC22A17 constituted a new cluster Slc22a22 nor orthologs of mouse Slc22a26 in either human or relatively distant from the rest of the members. SLC22A20 and rat. Mouse and rat Slc22a13b were orthologs of the human rat Slc22a20 proved to be orthologs of the previously known SLC22A13 and duplication of mouse and rat Slc22a13, mouse Slc22a20 which clustered together with SLC22A6 and respectively. A summary of the previously known human, SLC22A8. Additional homologous proteins in rodents, mouse, and rat SLC22 members together with the new genes Slc22a13b, Slc22a22, and Slc22a26, were also identified. We and SVOP and SV2A-C are presented in Tables 1 and 2, where, were, however, not able to identify any human ortholog to symbol, alias, chromosomal position, length, number of exons,

Fig. 2. Amino acid sequence alignment of known human SLC22 members, SVOP, and SV2A-C along with the novel genes. The alignment was made using MEGAlign from the Lasergene package (DNASTAR). The N and C termini along with the loops are replaced with dots. The black boxes mark putative TM regions while the gray boxes mark conserved motifs. Gray shades indicate conserved amino acids (N50% identity). The borders of the TM regions were assigned after the TM regions of SLC22A1 [1]. 602 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609

Fig. 3. The column charts with standard deviations display the relative expression of mRNA (%±SD %) in rat tissues relative to rat genomic DNA for (A) Slc22a1-3, Slc22a9, Slc22a13b, Slc22a18, Slc22a20, and Slc22a23 and (B) Slc22a24, Spns3, Svopl, Svop, and Sv2a-c. The abbreviations I–VIII indicate the eight rat brain coronal sections shown in the schematic presentation of the rat brain. J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 603 604 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 expression pattern, and substrates are listed. In addition, the (5.79±0.37), liver (10.10±0.64), and uterus (1.10±0.39). High closest human member within the family is presented in Table 1, levels in sections I and II and no expression in kidney based on local alignment made with BLAST. The new SLC22 corresponded well with the previously reported expression in members showed an amino acid identity of 25–73% with other mouse [21]. Transcript for Slc22a23 was detected in all tested SLC22 members, and SVOPL showed 39 and 29% amino acid tissues with the exception of intestine and with the highest identity to SVOP and SLC22A15, respectively. expression level in liver (255.65±22.). Slc22a24 showed To identify sequence similarities and shared motifs in the highest expression in liver (239.85±3.01) and skeletal muscle novel human genes, known SLC22 members, SVOP, and (154.17±14.20) but was also expressed in all coronal sections SV2A-C, we made an amino acid alignment which is presented (9.03–164.21±1.19–12.24). Expression of Spns3 was detected in Fig. 2. The TM regions are marked with boxes and elements in the majority of tissues examined with highest levels in coronal conserved in the majority (N50% identity) of sequences are sections I (140.64±22.89) and II (66.24±23.93) and lower marked with gray. The borders of the TM regions were assigned levels in sections III–VII (10.95–28.31±0.84–4.83), kidney according to the TM regions of SLC22A1 [4], and the N and C (26.78±5.42), spleen (28.70±1.03), and thymus (50.30 ±1.80). termini together with loops were manually removed from the Svopl was almost exclusively expressed in the coronal sections alignment. with the highest expression levels in sections I (181.73±11.91) To obtain an expression profile over the SLC22 family we and II (88.23±11.93). Svop and Sv2a-c showed similar performed an expression study by analyzing cDNA in expression patterns, with high levels in the CNS and only low quantitative real-time polymerase chain reaction (real-time levels in peripheral tissues, which corresponded to previously PCR). Sixteen rat genes were analyzed: Slc22a1-3, Slc22a9, published data [36–38]. For Svop the highest levels were Slc22a13b, Slc22a18, Slc22a20, Slc22a22, Slc22a23, detected in cerebellum (816.83± 83.91), hindbrain (782.44 ± Slc22a24, Spns3,andSvopl, and Svop and Sv2a-c. The tissues 150.08), and pineal gland (657.53±123.74). For Sv2a, cere- examined were eight coronal sections of rat brain (Fig. 3B), bellum (463.36±56.92) and hindbrain (548.83±27.90) were hindbrain, brainstem, cerebellum, hypothalamus, pituitary, eye, also tissues with high expression levels but the highest level was spinal cord, adrenal gland, pineal gland, skeletal muscle, detected in brainstem (600.59 ±81.42). The highest expression adipose tissue, heart, intestine, kidney, liver, lung, spleen, levels for Sv2b were highest in coronal sections III–VI (290.73– thymus, ovary, uterus, testis, and epididymis. We received 312.84±3.09–44.28) and hindbrain (321.70±22.19). Expres- reliable melting curves for all genes except for Slc22a22. sion for Sv2c was detected in coronal section VIII (419.04 ± Despite three different pairs of primers the real-time PCR data 67.81), brainstem (410.04±25.37), and spinal cord (359.05± continuously showed poor melting curves, and no further 70.07). analysis was performed on this gene. The relative mRNA expression levels for the analyzed genes (% ±SD %) are Discussion presented in Figs. 3A and 3B. The expression patterns for Slc22a1-3 corresponded to previously published data [1,7], We have identified six human and seven mouse and rat except for high levels of expression in skeletal muscle and transporters with structural similarity to the SLC22 family by adipose tissue, which has previously not been shown. Slc22a1 searching the entire human, mouse, and rat genomes using was highly expressed in skeletal muscle (390.75±104.86) and HMMs. The transporters were annotated by the HGNC as liver (227.72±26.61), and Slc22a2 was mainly expressed in SLC22A20; SLC22A23-SLC22A25; SPNS3; and SVOPL.Itis kidney (479.43±0.88) and skeletal muscle (409.41±20.77). likely that these new SLC22 members together with the Slc22a3 had a broad expression pattern which was relatively previously known members represent the entire or almost low compared, to genomic DNA. Highest levels were seen in complete set of genes for the SLC22 family in these species, adipose tissue (627.33±24.97), thymus (197.38±63.56), kid- considering the number of sequences being analyzed and ney (80± 0.87), heart (34.09±0.56), intestine (25.22±3.01), completeness of the current genome assembles. and brain (10.92–163.80±0.84–20.15). Slc22a9 was mainly This paper presents the first phylogenetic study of the known expressed in skeletal muscle (69.09±27.93) and in coronal human, mouse, and rat members of the SLC22 family, and the sections I–VII (6.77–63.15±0.037–14.44). We were, however, overall phylogenetic topology indicates that there exist seven not able to detect any expression in kidney as previously main clusters which fit remarkably well with the known reported [9]. The relative expression levels for Slc22a13b were substrate preferences for the proteins. Six of them are present in highest in epididymis (236.90 ±21.69), and lower levels were Fig. 1. One of the clusters consists of members with anions as detected in coronal section I (133.29 ±12.20) and II (80.46 their substrate for transport while two of the clusters consist of ±5.04). Slc22a18 showed an extremely high expression level members that are able to transport cations and/or carnitine. The compared to genomic DNA in liver (1156±13.53) and lower substrate specificity has not been determined for any members levels in coronal sections I–III (25.33–105.68±0.99–12.15), of the remaining three clusters. We also found, through database VII (24.89± 1.94), VIII (14.26±4.34), hindbrain (64.56±24.68), searches, that the synaptic vesicle proteins SVOP and SV2A-C hypothalamus (105.73±53.60), skeletal muscle (193.47±3.77), are most similar to the SLC22 family of all vertebrate proteins. and intestine (89.28±55.36). Slc22a20 was almost completely It has recently been shown that SV2A has a binding site and is restricted to the coronal sections (13.26–201.68±0.86–12.93) likely a target for the antiepileptic drug levetiracetam [39] and except for low expression in eye (3.49±0.37), adipose tissue that Botulinum neurotoxin A (BoNT/A) binds to SV2 to enter J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 605 neurons [40,41]. The function or substrate specificity for SVOP pairs or cluster with such a pair and that these pair members has not been determined but it has been suggested that it may be exist to facilitate coregulation [44]. Our analysis supports these involved in the development of neurons [42]. SVOP and SV2A- findings, and we have identified a new pair of close paralogs. C are included in the tree (Fig. 1) and form a cluster with the SLC22A10 was previously paired with SLC22A9 but is now novel SVOPL. SVOP was shown to be more closely related to replaced by the novel member SLC22A25. However, some the SLC22 family than SV2, with 30% amino acid identity to members lack close paralogs, SLC22A7, SLC22A15, SLC22A1 (Table 1) but even the more distantly related SV2C SLC22A16, SLC22A17, and the novel SLC22A23, and are show a structural homology to the SLC22 family with 26% phylogenetically isolated from the rest of the family. identity to both SLC22A3 and SLC22A15. SV2A and SV2B SPNS3 has not been confirmed to be a member of the are 24% identical to SLC22A3 and 23% identical to SLC22 family but is most similar to the previously known SLC22A15, respectively, which is an indication that there SLC22A18, which itself is an atypical member. SLC22A18 exists homology between these genes. In addition to the and SPNS3 were not included in the tree presented in Fig. 1 sequence similarities, the synaptic vesicle proteins share the but are, however, other examples that there exist some same conserved domain, the sugar transporter domain, 12 TM members of the family which are phylogenetically isolated. It regions, and several motifs with the SLC22 family. Fig. 2 shows is also interesting to note that human SLC22A24 seems to be that DR()GRRK between TM2 and TM3, PES()RWL between under a more intense evolutionary pressure, compared to TM6 and TM7, ELYPT between TM10 and TM11, and LLPET rodent Slc22a24, due to their relatively extended distance to after TM12 are conserved in all proteins. The STIVTEW(D/N) each other. LVC motif between TM1 and TM2 is conserved in the majority Human and rodents have in general a similar repertoire of of proteins but not in SLC22A17, SLC22A23, and SPNS3. The SLC22 genes, but there are some differences. SLC22A9, lack of this motif may be due to a separate specific functional SLC22A10, and SLC22A11 exist only in human, and Slc22a19 role or possibly there is no common ligand binding site for these and the novel Slc22a22 exist only in rodents. There are also genes between TM1 and TM2. some examples of differences between the rodents. Slc22a9 is The sequence comparison and the putative transmembrane found only in rat, and Slc22a21 and the novel Slc22a26 exist topology also show that the majority of the proteins do have 12 only in mouse. In addition, Slc22a13b exists only in rodents and TM regions, but it is interesting to note that some have a deviant is a duplication of Slc22a13 which is the only example of a topology. The previously known SLC22A17 and the novel duplication event in the SLC22 family. SLC22A23 consist of 9 TM regions, and the novel human Differences in tissue distribution of organic ion transporters SLC22A24 consists of 6 TM regions. Previous studies have may play a role in drug disposition to various tissues. It is shown that mutations in SLC22 members influence the substrate therefore important to obtain comprehensive expression profiles selectivity and it has been suggested that TM4 and TM11 may be for them. Here, we analyzed 15 genes by real-time PCR on a a part of the binding sites and that TM8 may contribute to panel of 30 different rat tissues, which showed that some are substrate recognition [1]. The functional role for the extracellular highly expressed in peripheral tissues but all are also expressed loop between TM1 and TM2 is not well understood but seems in brain (Figs. 3A and 3B). not to be required for transport due to the fact that transport is Slc22a1 and Slc22a2, as previously described [1,7], are preserved in a splice variant of rat SLC22A1 that is lacking TM1 abundantly expressed in liver and kidneys, respectively, while and TM2 and the N terminus while it seems to contribute to the Slc22a3 has a more broad tissue distribution. Here, we also stability or formation of the binding site [1]. For SLC22A24 report high expression in skeletal muscle and adipose tissues, which lacks TM11 and SLC22A17 and SLC22A23 which lack which has previously not been shown. In addition, real-time the first 3 TM and the large extracellular loop, it is tempting to PCR studies [3,45,46] have shown that SLC22A1-3 are speculate that these proteins have alternative transport mechan- expressed in brain, but at very low levels, which is in agreement isms and binding sites. with our results on these genes. The expression analysis on It has previously been shown for solute carriers that Slc22a9, Slc22a24, Spns3, and Svopl gives an indication of phylogenetically related proteins have the same or similar involvement in some cortical or olfactory functions due to the substrates [43], and it is therefore possible that SLC22A24 and expression in the coronal sections but no or only low expression SLC22A25 function as anion transporters as they cluster with in hindbrain, brainstem, and hypothalamus. The expression members that are known anion transporters (Fig. 1). No pattern for rat Slc22a20 is in agreement with previously substrates or functions have been identified for SLC22A17 reported expression pattern for mouse Slc22a20 [21], which which would otherwise have given an indication of the function also support our suspicion of some olfactory or cortical function of SLC22A23. It is in addition interesting to note that the for Slc22a9, Slc22a24, Spns3, and Svopl. However, these members are well defined in clusters on a substrate-specificity transporters, especially Slc22a24 and Spns3, also seem to have level but also on a chromosomal-position level. All members additional peripheral functions, possibly different from that in that group together are localized on the same chromosome, the brain, due to expression in peripheral tissues. The four except SLC22A17 and SLC22A23, and those that are most synaptic vesicle proteins tested, rat Svop and Sv2a-c, seem to phylogenetically related are also localized on the same have a more general function in the brain, compared to Svopl, chromosomal band (Tables 1 and 2 and Fig. 1). It has also due to their more broad distribution in the brain. A broad been shown that a majority of the human members occur in distribution in general is also seen for Slc22a23, which is an 606 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 indication that this protein is involved in more general was available only as cDNA sequences, and these were batch-translated into processes. The highest expression, compared to genomic proteins using a custom made JAVA program. These protein Fasta files were searched, one database at a time in the order given above, against the HMM DNA, for Slc22a18 was found in liver, which is a strong model using HMMsearch from the HMMER 2.2 package with a cut of at E=10. indication that this protein is involved in the elimination of The sequences for all hits were extracted using bash-scripts and a custom made organic ions. The real-time PCR analysis also revealed that JAVA program and searched against the reference database using BLASTP. The Slc22a13b has its strongest expression in epididymis and seems three best BLAST hits were examined, and at least one of these had to be a to be important for the reproduction system in male rats. SLC22 for that sequence to be included. To remove the previously known sequences all hits were aligned against the human genome using translated In summary, we have identified five novel human genes BLAT. The resulting PSL files were parsed, the BLAT scores were calculated coding for proteins from the SLC22 family and one from the according to [50], and only the highest scoring position was kept. This was done synaptic vesicle protein family. We retrieved orthologs from using a custom made JAVA program. Finally, the sequences from the HMM mouse and rat and identified additional homologous proteins in search were aligned against the genome, and all sequences that aligned to the these species. A comprehensive phylogenetic analysis showed same position in the genome as one of the previously known SLC sequences were automatically removed using a JAVA program. The novel hits were then that the SLC22 family comprises seven subgroups, the members merged into the set of known SLC22 proteins, and the procedure was repeated occur in pairs or cluster with such a pair, and these pair members for the next dataset. The order in which the datasets were used (RefSeq, Ensembl have in general similar expression patterns. Finally, we peptide, MGC, Ensembl AbInitio) ensured that the sequence most likely to be of performed an expression analysis on 15 genes that showed high quality was used. that all of them were expressed in brain. The new human sequences were searched against mouse and rat genomes using BLAT genome search (http://genome.ucsc.edu/). Ortholog sequences from these species were obtained by translating mRNA or EST sequences or, if Materials and methods no such sequences were available, manually assembled from genomic data as previously described [51]. Construction of hidden Markov models Phylogenetic analysis The sequences for all known SLC22 family of proteins were downloaded from the NCBI (http://www.ncbi.nlm.nih.gov/) using the Entrez data retrieval Amino acid sequences of all human, mouse, and rat proteins from the tool based on the accession numbers obtained from the SLC-database (http:// final dataset were combined into a FASTA file and aligned using the UNIX www.bioparadigms.org/slc/menu.asp). This resulted in a dataset with 18 version of ClustalW 1.82 [47]. The default alignment parameters were sequences with the following Accession Nos.: SLC22A1, gi%7C55662276; applied. The alignment was bootstrapped 1000 times using SEQBOOT from SLC22A2, gi%7C56206263; SLC22A3, gi%7C55960727; SLC22A4, gi% the Win32 version of the Phylip 3.6 package [52]. Maximum parsimony 7C20271469; SLC22A5, gi%7C15147378; SLC22A6, gi%7C21707179; trees were calculated on the bootstrapped alignment with PROTPARS from SLC22A7, gi%7C21707474; SLC22A8, gi%7C18490227; SLC22A9, gi% the Win32 version of the Phylip 3.6 package. The trees were unrooted and 7C42542386; SLC22A10, gi%7C52078132; SLC22A11, gi%7C23844; calculated using ordinary parsimony, and the topologies were obtained using SLC22A12, gi%7C31419814; SLC22A13, gi%7C23273554; SLC22A14, gi% the built-in tree-search procedure. Majority-rule consensus trees were 7C4758854; SLC22A15, gi%7C24308161; SLC22A16, gi%7C31542327; constructed using CONSENSE from the Win32 version of the Phylip 3.5 SLC22A17, gi%7C9663117; SLC22A18, gi%7C83288333. Of these, package. For the maximum likelihood tree the topology obtained from the SLC22A10 was present only as a TPA, and we used EST data to extend that maximum parsimony tree was used as a user defined tree in Tree-Puzzle protein, we then used the extended version for all subsequent analysis. The [35], and maximum likelihood branch lengths were estimated in Tree-Puzzle sequences were aligned using ClustalW 1.83 [47] using default settings. From using the following parameters: Type of analysis, Tree reconstruction; Tree the alignments, sequence HMMs were constructed using the HMMER 2.2 search procedure, User defined trees; Compute clocklike branch lengths, No; package [48]. The models were constructed using HMMbuild with default Location of root, Best place (automatic search); Parameter estimates, Exact settings and calibrated using HMMcalibrate. (slow); Parameter estimation uses, 1st input tree; Type of sequence input data, Amino acids; Model of substitution, JTT [35]; Amino acid frequencies, Construction of a reference database Estimate from data set; Model of rate heterogeneity, Mixed (1 invariable+8 Gamma rates); Fraction of invariable sites, Estimate from data set; Gamma A reference database was constructed from the RefSeq dataset version 35 distribution parameter alpha, Estimate from data set; Number of Gamma rate (May 2005 release) (ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/) by first remov- categories, 8. The tree was plotted using TreeView [53]. Alignment statistics ing all SLC proteins that were present in the SLC database (http://www. were obtained using MEGA 3.1 [54]. bioparadigms.org/slc/menu.asp), in total 307 SLC proteins, from the RefSeq datafile using repeated BLASTP searches. The sequence names of all 307 SLC proteins were then modified with a tag (_SLC_) to allow for automatic Transmembrane region alignment identification. The RefSeq file that was lacking all SLC sequences was then merged with the modified SLC database into a Fasta file containing 29,061 The new human protein sequences were aligned with known SLC22 proteins. A protein database was finally constructed from this Fasta file using members, SVOP, and SV2A-C using MEGAlign from the Lasergene package formatdb from the version 2.2.6 BLAST package [49]. (DNASTAR). The ClustalW method was used for multiple alignments with a gap penalty of 10 and a gap length penalty of 0.20. The slow-accurate method Identification of novel members of the SLC22 family was used for the initial pairwise alignments, and the protein weight matrix was Blossom 30. The borders of the TM regions were predicted using Phobius (http://phobius.cgb.ki.se/) and assigned after the TM regions of SLC22A1 [1]. Fasta files that contained the protein versions of the human RefSeq database The N and C termini together with loops were manually removed from the version 35 (May 2005 release) (ftp://ftp.ncbi.nih.gov/%20refseq/H_sapiens/), alignment. the Ensembl human peptide database version 35 (April 2005 release) (ftp://ftp. ensembl.org/pub/current_homo_sapiens/data/fasta/pep/), the human Mamma- lian Gene Collection (MGC) cDNA database (Downloaded 6th of June 2005) Animal handling and tissue isolation (ftp://ftp1.nci.nih.gov/pub/MGC/fasta/), and the Ensembl human AbInitio protein database version 35 (April 2005 release) (ftp://ftp.ensembl.org/pub/ Six Sprague–Dawley rats (Alab, Sweden), four males and two females, were current_homo_sapiens/data/fasta/pep/) were downloaded. The MGC database housed in a temperature-light-, and humidity controlled environment with free J.A. Jacobsson et al. / Genomics 90 (2007) 595–609 607

Table 3 Annealing temperatures and primers for two housekeeping genes, histone 3 and ribosomal protein L19, and for the analyzed genes Gene Annealing Forward primer Reverse primer temperature H3f3b 60.0 °C ATT CGC AAG CTC CCC TTT CAG TGG AAG CGC AGG TCT GTT TTG Rpl19 60.0 °C TCG CCA ATG CCA ACT CTTC GTC AGC CCG GGA ATG GAC AGT CAC Slc22a1 55.7 °C GCT GTA CCA GGG TCT CAT C TGA CCA GGA TGA TGA AGG C Slc22a2 58.2 °C GAG TTT CCA GCC GCC TTC TGC TCC TGC CAC CAT ATT TG Slc22a3 54.0 °C GGA CTC ATC GGA GGC AAC C GAA GGC GTC GTC CAA GGC Slc22a9 52.8 °C AAG GGC AAC TGC TTT AGG AG AGG AAG AAG AAG GAC AAC AAG G Slc22a13b 58.2 °C AAC AGT ACC ACA AGG CGA TTC C AGG CAG CAG AGC ACA CAG TA Slc22a18 58.2 °C GCA TCC ATT GAC CCG TAC TGT AG ACC AAT AGG ACA AGC AAG TTC ACC Slc22a20 58.2 °C AGT TCT GCC ACC TGT ATC CTT TG CAC ATT GCG GGT CTC AGT CAG Slc22a22 60.1 °C GAC GCT TTC GGC ACC CTC AAT G TCC ATC CAT CTA GGC AAG GCT CAG – TGA AGG TCT CCA TCC CAA TG GTC CAT CCA TCT AGG CAA GG – CGA TTA GCA TCA GTC CTG TC ATG TTG ATT GTT GCC ACT ATG Slc22a23 55.7 °C TGA CAG CAC CCA TTA TTG AGC TCA GGC AGC AGG AGG ATG Slc22a24 58.2 °C GCA GCC ATT CCC AGT CAT CG TCT TCA GGA GTT TAT CTT GGT TCA GG Spns3 60.1 °C ATG TCC AGA GAG TGT ATC AAA CC GAG GGA GGC AGA CAG TTA CC Svopl 54.0 °C CTG GGG ATT TCT TTT GCC TAT TAC CTC TGA CTC CGA CTT TGA ACC Svop 54.0 °C CTC CTG CTT TCT GCC CAT C ACG CCT GTG CTG TTT GTC Sv2a 58.2 °C TGT GTA CCT GGG CAT GAT GG CGA AGA CGC TGT TGA CTG AG Sv2b 52.8 °C TTC GGC ATC CTC AAT GGA CTG TG CAG AGA AGC AGC AGC CAG AAG G Sv2c 58.2 °C AAC GGG CTT TGG CTT CTT GAA C GGA TAG GGA TTG CTT TGG TGA T GC

access to water and R36 food pellets (Labfor, Lactamin, Sweden). After 7 days 5 ng/μl of genomic DNA were included on each plate. Amplifications were the animals were sacrificed by decapitation between 3 and 6 h into the light performed with 0.02 U/ml Taq DNA polymerase (Invitrogen, Sweden) under the period. Different brain regions and peripheral tissues were isolated from the rats, following conditions: initial denaturation at 95 °C for 3 min, followed by 50 and two whole brains were dissected into coronal (cross) sections using a brain cycles of denaturing at 95 °C for 15 s, annealing at 52.8–60.1 °C for 15 s, and matrix (Pelco Int., Canada) as presented in Fig. 3B. The tissues were immersed extension at 72 °C for 30 s. into RNA-later solution (Ambion, USA), kept at room temperature for 1 h, and – thereafter stored at 80 °C until further processed. Data analysis and relative expression calculations

RNA isolation and cDNA synthesis MyiQ software v1.04 (Bio-Rad Laboratories, Sweden) was used to analyze the real-time PCR data. Threshold cycle (Ct) values were derived and the Individual tissue samples were homogenized by sonication in TRIzol melting curves were analyzed to confirm that only one product with the expected reagent (Invitrogen, Sweden) using a Branson sonifier. Chloroform was added to melting point had been amplified. The samples Ct values were further analyzed the homogenate, which then was centrifuged at 13,000 rpm at 4 °C for 15 min. if the difference between those and the negative control was greater then 2; The water phase was transferred to a new tube, and RNA was precipitated with otherwise the transcript was considered not to be expressed. LinRegPCR [57] isopropanol. The pellets were washed with 75% ethanol, air dried at room was used to calculate PCR efficiencies for each sample, and Grubbs' test temperature, and dissolved in RNAse-free water. DNA contamination was (GraphPad, USA) was applied to exclude the outliers and to calculate the removed by treatment with DNAse I (Roche Diagnostics, Sweden) for 4 h at average PCR efficiency for each primer pair. The Ct values were transformed 37 °C, and the DNAse I was thereafter inactivated by heating the samples at into relative quantities using the delta Ct method [58] with standard deviations. 75 °C for 15 min. The absence of genomic DNA was confirmed by PCR with To compensate for differences in cDNA amount, normalized quantities were primers for rat glyceraldehyde-3-phosphate dehydrogenase (NM_017008; calculated for each tissue. The normalization factors were calculated with forward TCC CTC AAG ATT GTC AGC AA and reverse CAC CAC CTT GeNorm [59] on the two housekeeping genes, H3 and RPL19. The genomic CTT GAT GTC ATC) on the DNAse-treated RNA. RNA concentration was DNAwas set to 100% and the normalized quantities were thereafter compared to determined using a Nanodrop ND-1000 Spectrophotometer (NanoDrop the genomic DNA. Technologies, DE, USA). cDNA was synthesized with MMLV reverse transcriptase (GE, Sweden), using random hexamers as primers according to Ethical statement the manufacturers' instructions. All animal procedures were approved by the local ethical committee in Quantitative real-time PCR Uppsala and followed the guidelines of European Communities Council Directive (86/609/EEC). The cDNA was analyzed with quantitative real-time PCR on MyiQ thermal cycler (Bio-Rad Laboratories, Sweden) as previously validated [55,56]. Each real-time PCR with a total volume of 20 μl contained cDNA synthesized from Acknowledgments

25 ng of total RNA, 20 mM Tris/HCl (pH 8.4), 50 mM KCl, 4 mM MgCl2, 0.2 mM dNTP, and SYBR Green (1:50 000). Template concentration was 5 ng/ We thank Helgi B. Schiöth for valuable discussion during μl and the concentration of each primer was 0.25 μM. All primers were designed the course of the project, Olga Stephansson for help with real- with Beacon Designer v4.0 (Premier Biosoft, USA). Annealing temperatures time PCR, Thomas Renström for help with preparation of the and primers for two housekeeping genes, histone 3 (H3; NM_053985) and ribosomal protein L19 (RPL19; NM:031103), and for the analyzed genes are tissue panel, Majd A.I. Mirza for help with creating presented in Table 3. All real-time PCR experiments were performed in alignments, and Linnea Holmén for the preparation of the duplicates, and a negative control for each primer pair and a positive control with brain section figure. R.F. was supported by the Swedish Brain 608 J.A. Jacobsson et al. / Genomics 90 (2007) 595–609

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