Structure and Expression of the Murine Phosphatidylserine Synthase-1 Gene
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m009776200
ISSN1083-351X
AutoresBénédicte Sturbois-Balcerzak, Scot J. Stone, Avula Sreenivas, Jean E. Vance,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoIn mammalian cells, phosphatidylserine is synthesized by two different enzymes, phosphatidylserine synthase (PSS)-1 and -2, via a base exchange reaction in which the head group of a phospholipid (phosphatidylcholine or phosphatidylethanolamine) is replaced by l-serine. Since the amino acid sequences of PSS1 and PSS2 are only ∼30% identical, it is likely that they are encoded by different genes. We have screened a murine liver genomic DNA library, included in bacterial artificial chromosomes, with full-length murine PSS1 cDNA and isolated a clone containing the majority of the PSS1 gene. This gene spans ∼35 kilobases and contains 13 exons and 12 introns. The sizes of the exons range from 44 to 1035 base pairs. The gene was localized to chromosome 13 in region B-C1. According to reverse transcriptase-mediated polymerase chain reaction, PSS1 and PSS2 mRNAs were expressed in all murine tissues examined. The mRNA encoding PSS1 was most abundant in kidney, brain, and liver, whereas PSS2 mRNA was most highly expressed in testis. In general agreement with the levels of mRNA expression, the choline exchange activity (contributed by PSS1, but not PSS2) was highest in brain, whereas serine and ethanolamine exchange activities were highest in testis and kidney. The transcriptional initiation site for PSS1 was identified 111 base pairs upstream of the ATG specifying the start of translation. The putative 5′-proximal promoter region of the gene contained no TATA or CAAT box, but did have a high GC content. Isolation of the murine PSS1 gene is a step toward generation of genetically modified mouse models that will help to understand the functions of PSS1 and PSS2 in animal biology. In mammalian cells, phosphatidylserine is synthesized by two different enzymes, phosphatidylserine synthase (PSS)-1 and -2, via a base exchange reaction in which the head group of a phospholipid (phosphatidylcholine or phosphatidylethanolamine) is replaced by l-serine. Since the amino acid sequences of PSS1 and PSS2 are only ∼30% identical, it is likely that they are encoded by different genes. We have screened a murine liver genomic DNA library, included in bacterial artificial chromosomes, with full-length murine PSS1 cDNA and isolated a clone containing the majority of the PSS1 gene. This gene spans ∼35 kilobases and contains 13 exons and 12 introns. The sizes of the exons range from 44 to 1035 base pairs. The gene was localized to chromosome 13 in region B-C1. According to reverse transcriptase-mediated polymerase chain reaction, PSS1 and PSS2 mRNAs were expressed in all murine tissues examined. The mRNA encoding PSS1 was most abundant in kidney, brain, and liver, whereas PSS2 mRNA was most highly expressed in testis. In general agreement with the levels of mRNA expression, the choline exchange activity (contributed by PSS1, but not PSS2) was highest in brain, whereas serine and ethanolamine exchange activities were highest in testis and kidney. The transcriptional initiation site for PSS1 was identified 111 base pairs upstream of the ATG specifying the start of translation. The putative 5′-proximal promoter region of the gene contained no TATA or CAAT box, but did have a high GC content. Isolation of the murine PSS1 gene is a step toward generation of genetically modified mouse models that will help to understand the functions of PSS1 and PSS2 in animal biology. phosphatidylserine phosphatidylcholine phosphatidylethanolamine phosphatidylserine synthase bacterial artificial chromosome(s) polymerase chain reaction reverse transcriptase-mediated polymerase chain reaction rapid amplification of cDNA ends fluorescence in situ hybridization 4,6-diamidino-2-phenylindole kilobase(s) base pair(s) Phosphatidylserine (PtdSer)1 is an important amino phospholipid that accounts for 5–10% of animal cell membrane phospholipids. In addition to a presumed structural role in membranes, PtdSer is an activator of protein kinase C (1Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4231) Google Scholar) and is involved in progression of the blood coagulation cascade (2Schroit A.J. Zwaal R.F.A. Biochim. Biophys. 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The pathway for PtdSer synthesis in mammals is different from that in bacteria (12Okada M. Matsuzaki H. Shibuya I. Matsumoto K. J. Bacteriol. 1994; 176: 7456-7461Crossref PubMed Google Scholar) andSaccharomyces cerevisiae (13Nikawa J. Tsukagoshi Y. Kodaki T. Yamashita S. Eur. J. Biochem. 1987; 167: 7-12Crossref PubMed Scopus (48) Google Scholar, 14Letts V.A. Klig L.S. Bae-Lee M. Carman G.M. Henry S.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7279-7283Crossref PubMed Scopus (105) Google Scholar), in both of which PtdSer is synthesized through reaction of l-serine with CDP-diacylglycerol. Interestingly, plants make PtdSer from both the CDP-diacylglycerol pathway (15Moore T.S. Annu. Rev. Plant Physiol. 1982; 33: 235-259Crossref Google Scholar) and a base exchange reaction (16Vincent P. Maneta-Peyret L. Sturbois-Balcerzak B. Duvert M. Cassagne C. Moreau P. FEBS Lett. 1999; 464: 80-84Crossref PubMed Scopus (26) Google Scholar). The existence of two mammalian PSSs, PSS1 and PSS2, was deduced from studies with mutant Chinese hamster ovary cell lines (17Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Abstract Full Text PDF PubMed Google Scholar, 18Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Abstract Full Text PDF PubMed Google Scholar). PSS1 and PSS2 differ in their phospholipid substrate specificities (17Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Abstract Full Text PDF PubMed Google Scholar, 18Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), but the reason why mammalian cells possess two distinct PSSs is not clear (20Vance J.E. Trends Biochem. Sci. 1998; 23: 423-428Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In intact cells, PSS1 uses PtdCho as donor of the phosphatidyl group, whereas PSS2 uses PtdEtn (21Saito K. Nishijima M. Kuge O. J. Biol. Chem. 1998; 273: 17199-17205Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In in vitro enzymatic assays, however, PSS1 uses both PtdCho and PtdEtn. In Chinese hamster ovary cells and rat liver, serine exchange activity is associated with the endoplasmic reticulum (22Jelsema C.J. Morré D.J. J. Biol. Chem. 1978; 253: 7960-7971Abstract Full Text PDF PubMed Google Scholar, 23Vance J.E. Vance D.E. J. Biol. Chem. 1988; 263: 5898-5908Abstract Full Text PDF PubMed Google Scholar) and is enriched 2–4-fold in an endoplasmic reticulum subfraction, the mitochondrion-associated membranes, compared with the bulk of the endoplasmic reticulum (24Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar, 25Shiao Y.-J. Lupo G. Vance J.E. J. Biol. Chem. 1995; 270: 11190-11198Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 26Stone S.J. Vance J.E. J. Biol. Chem. 2000; 275: 34534-34540Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 27Saito K. Kuge O. Akamatsu Y. Nishijima M. FEBS Lett. 1997; 395: 262-266Crossref Scopus (35) Google Scholar). Mitochondrion-associated membranes have been proposed to consist of a specialized domain of the endoplasmic reticulum that is juxtaposed with mitochondria, and mitochondrion-associated membranes have been proposed to mediate the import of newly synthesized PtdSer into mitochondria (24Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar, 25Shiao Y.-J. Lupo G. Vance J.E. J. Biol. Chem. 1995; 270: 11190-11198Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 28Vance J.E. J. Biol. Chem. 1991; 266: 89-97Abstract Full Text PDF PubMed Google Scholar, 29Voelker D.R. J. Biol. Chem. 1993; 268: 7069-7074Abstract Full Text PDF PubMed Google Scholar). Using antibodies raised against PSS1 and PSS2 as well as Myc-tagged murine PSS1 and PSS2 expressed in rat hepatoma cells, we have recently shown that these two proteins are localized almost exclusively to mitochondrion-associated membranes (26Stone S.J. Vance J.E. J. Biol. Chem. 2000; 275: 34534-34540Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). The cDNAs encoding PSS1 and PSS2 from murine liver (30Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 31Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Crossref PubMed Scopus (55) Google Scholar) and CHO-K1 cells (18Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 32Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1991; 266: 24184-24189Abstract Full Text PDF PubMed Google Scholar) have been cloned and expressed. Since the predicted amino acid sequences of PSS1 and PSS2 are only ∼30% identical, with no long continuous stretches of homology (18Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 31Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Crossref PubMed Scopus (55) Google Scholar), PSS1 and PSS2 appear to be encoded by different genes. We have expressed murine PSS1 and PSS2 cDNAs in McArdle rat hepatoma cells and M.9.1.1 cells (a Chinese hamster ovary cell line lacking PSS1 (19Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Abstract Full Text PDF PubMed Google Scholar)) (30Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). When the synthesis of PtdSer was increased by expression of murine PSS1 in hepatoma cells, the cellular content of PtdSer and PtdEtn remained unchanged, whereas the production of PtdEtn via PtdSer decarboxylation increased, and PtdEtn synthesis via the CDP-ethanolamine pathway was reciprocally decreased (30Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These data suggest that the rates of synthesis and degradation of PtdSer and PtdEtn are tightly regulated. We now report the isolation of the murine PSS1 gene. A murine genomic DNA library, contained in bacterial artificial chromosomes (BAC), was screened with full-length cDNA encoding murine PSS1. Examination of positive BAC clones showed that the PSS1 gene resides on murine chromosome 13 in region B-C1 and consists of 13 exons and 12 introns. We also demonstrate, using reverse transcriptase-mediated PCR and enzymatic assays, that PSS1 and PSS2 are widely, but differentially, expressed in murine tissues. Restriction endonucleases, the random primer labeling kit, and the end labeling kit were obtained from Life Technologies, Inc. (Burlington, Ontario, Canada). The radioisotopes ([α-32P]CTP and [γ-32P]ATP) were from Amersham Pharmacia Biotech (Quebec, Canada). Accurase long-range polymerase was from DNamp Ltd. (Farnborough United Kingdom) and Ex-Taq and Rec-Taq polymerases were from Takara Biomedicals, Inc. (Panvera, Madison, WI). The TOPO-TA cloning kit was from Invitrogen (Mississauga, Ontario). All other reagents were obtained from Sigma (Oakville, Ontario) or Fisher (Nepean, Ontario). BAC DNA was isolated using a QIAGEN midiprep kit (tip 100), and plasmid DNA was isolated with a Wizard miniprep or maxiprep kit (Promega, Madison, WI). For Southern blot analysis, DNA was separated by agarose gel electrophoresis and transferred to Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) in 10× SSC (SSC = 0.15m NaCl and 0.015 m trisodium citrate) using a vacuum blotter (Bio-Rad) according to the manufacturer's instructions or by capillary action overnight in 0.8 m NaOH. Prehybridization and hybridization steps were performed using 6× SSC, 5× Denhardt's solution, 0.5% SDS, 50% formamide, 100 μg/ml salmon sperm DNA (Sigma), and 1.5 × 106 cpm/ml probe, as required. Membranes were washed under conditions of low (2× SSC and 0.1% SDS at room temperature) or high (0.1× SSC and 0.1% SDS at 37 or 55 °C) stringency and then scanned using a Storm 540 PhosphorImager (Molecular Dynamics, Inc.). Using the full-length cDNA encoding murine liver PSS1 (GenBankTM/EBI accession number AF042731) (30Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), a BAC murine genomic library was screened by Research Genetics (Huntsville, AL). To confirm the presence of the PSS1 gene in isolated clones, BAC DNA was digested using EcoRI or BamHI and subjected to Southern analysis with a radiolabeled full-length murine PSS1 cDNA probe. The integrity of the clone was confirmed by comparison with the restriction enzyme digestion pattern of murine liver genomic DNA using the same probe and also by sequencing using primers specific for murine PSS1 cDNA. BAC DNA containing the PSS1 gene was digested with BamHI, EcoRI, HindIII,Xba, and KpnI. Fragments containing exonic sequences were identified by Southern blotting using radiolabeled murine PSS1 cDNA or exon-specific oligonucleotides as probes. Fragments that hybridized were subcloned into pBluescript II/SK (Stratagene, Aurora, Ontario). Using primers based on the murine PSS1 cDNA sequence, exon-intron boundary sequences were identified by sequencing the BAC DNA directly and aligning the sequence with the corresponding PSS1 cDNA sequence. Primers were synthesized at the DNA Core Facility of the University of Alberta using a Model 394 DNA/RNA synthesizer (Applied Biosystems, Inc.). A typical PCR program was as follows: 95 °C for 3 min; 30 cycles of 95 °C for 1 min, 60 °C for 30 s, and 74 °C for 30 s; and 74 °C for 3 min. For amplification of some large DNA fragments, PCR was performed using Accurase from DNamp Ltd. (Farnborough, United Kingdom) under the following conditions: 94 °C for 2 min; 20 cycles of 94 °C for 20 s, 60 °C for 30 s, and 68 °C for 6 min; 20 cycles of 94 °C for 20 s, 60 °C for 30 s, and 68 °C for 6 min + 20 s/cycle; 68 °C for 7 min; and 4 °C. Murine tissues (adipose, brain, heart, kidney, liver, lung, spleen, and testis) from 129/J mice were snap-frozen in liquid nitrogen, and total RNA was isolated using Trizol (Life Technologies Inc.) according to the manufacturer's instructions. RNA quality was evaluated by performing electrophoresis on 1% formaldehyde gels stained with ethidium bromide. Total RNA was reverse-transcribed using an 18-mer oligo(dT) primer and Superscript II reverse transcriptase (Life Technologies Inc.) following the manufacturer's instructions. Briefly, 5 μg of RNA from tissues were incubated at 70 °C for 10 min in the presence of 1 μl of oligo(dT) primer (10 μm). After a brief centrifugation, 4 μl of "first strand buffer," 2 μl of dithiothreitol (0.1m), and 1 μl of dNTP mixture (10 mm) were added and incubated at 42 °C for 2 min prior to addition of Superscript reverse transcriptase (1 μl) and another incubation at 42 °C for 45 min. The reaction was terminated by incubation at 70 °C for 15 min. PCR was performed on the cDNA products using an antisense primer (396AS; see Table I) and a sense primer (85S; see Table I) spanning the ATG codon. Amplification was performed for 35 cycles of 95 °C for 1 min, 60 °C for 30 s, and 74 °C for 30 s and an extension step at 74 °C for 3 min. Southern blot analysis of the reaction products was performed using a radiolabeled internal probe (276AS; see Table I). The same PCR conditions were used with two primers specific for PSS2 (1S, TGGAGTCACACAAGCCAAAGAC; and 2AS, GTAGGTTGGAATGTTCCAGAGG). The identity of the product was confirmed by Southern blot analysis using oligonucleotide 3S (GGACAAGCTGGATGGCTTTGTT) as a probe. Cyclophilin was used as an internal control (95 °C for 2 min; 30 cycles of 95 °C for 1 min, 61 °C for 30 s, and 74 °C for 30 s and an extension step at 74 °C for 3 min).Table ISequences of primers used for PCR amplification and sequencingPrimer5′-Sequence-3′Positions in cDNAExon85AGAAGAACTATGGCGTCCTGCG85–1061106CGGAGGACGCCATAGTTCTTCT106–851276GACTCCGTTCCAGAAGA276–2922298TGTTGTCTTCTGGAACGGAGTC298–2712316CAGGATGCCTCTCCAGATGTTG316–2952376TCGACCTCATCCAGCCTTATGG376–3973396CATAAGGCTGGATGAGGTCGAG396–3753498GGATCCAGCCAATACATCAGGG498–4774510TGCTACACGAGAAGCGGACATC510–5314620GGCCATGAAGGCCTTGTTGATCCGT620–6445630TTCATGGCGGAAGCCCAGAAAT630–6195715GCATCTCCTGCCCAATTTTGCT715–7386845TCCTTGAAGCTTGCCCAGTGGT845–8246855ACCACCACAGGGAAGATCAAGC855–8767913TATGAATGTCCTTGAAGCTTGCCCA913–88871027GTGTTCCAGCCAGCCATCCCT1027–104881091GAGCCGTAATGCAGCCGATGAA1091–107081104GTACTATGCCTACCTCACGGAC1104–112591159CCCAGCACTGTGTCCCTACACG1159–113891170CATTGGTTTCCTGGAAGCTATTG1170–1191101249GCATCACGTAGAGTATCTGGGTC1249–1228101273CCACGTTCCTGTGTCTGTACGG1273–1294111330CCCGGTGACCATATTGCTCTGC1330–1309111357GGCACTTACAGTCCAGAGATCTC1357–1378121400GAGCCTTTCCCATGATGCCAGG1400–1379121486TGGACTTGGAATGCCGGTTCCT1486–1465131920GAGTCGCCTGTCCATTTGATTG1920–1941132312CACTAGCACCTCACATGAGCCA2312–2291131UAGGGCGACATCGTAGACTCTCAPromoter2UTAGGTTGCACGGTCGCTGTGAAPromoter3UTAAAGAGTCTAAGGACTGCGGCTGCPromoter4UGCCTGTTTCGGCTCTGATTCCTPromoterPositions in the forward order indicate that the sequences are identical to those of the cDNA; positions in the reverse order are complementary to cDNA sequences. Open table in a new tab Positions in the forward order indicate that the sequences are identical to those of the cDNA; positions in the reverse order are complementary to cDNA sequences. Total RNA was prepared from liver samples of 129/J mice using Trizol according to the manufacturer's instructions, and RNA quality was evaluated by performing electrophoresis on 1% formaldehyde gels stained with ethidium bromide. Total RNA was reverse-transcribed using a 22-mer primer (913AS; see Table I) and Superscript II reverse transcriptase as described above. The cDNA was washed using the Prep-A-gene DNA purification kit (Bio-Rad) and then tailed using dATP and recombinant terminal deoxynucleotidyltransferase (Life Technologies, Inc.). Briefly, 4 μl of ATP (1 mm), 5 μl of buffer, and 1 μl of enzyme were added to the cDNA and incubated at 37 °C for 15 min. The reaction was terminated by incubation at 70 °C for 10 min. The incubation mixture was diluted to 500 μl with Tris/EDTA buffer (pH 8.0). PCR was performed on the reverse-transcribed tailed products using an antisense primer (396AS; see Table I) and a sense primer (Race dT, GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT) specific for the poly(A) tail. The program used for the PCR was as follows: 95 °C for 2 min; 38 cycles of 95 °C for 1 min, 55 °C for 5 min, 72 °C for 40 min, 95 °C for 45 s, 60 °C for 1 min, and 72 °C for 2 min; and an extension step at 72 °C for 8 min. Southern blot analysis was performed on products of the reaction using a radiolabeled internal probe (85S; see Table I). Hybridizing fragments were subcloned into pCR2.1 using the TOPO-TA kit and sequenced. The sequence was compared with that of murine PSS1 cDNA. Total RNA was prepared from 129/J mouse liver using Trizol. RNA (2 μg) was reverse-transcribed for 3 h at 42 °C using adapter primer Ad68 dT (GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTT TTT) and Superscript II reverse transcriptase. The reaction mixture was diluted to 1 ml with 10 mm Tris buffer (pH 8.0) containing 1 mm EDTA. PCR was performed using adapter primer Ad68 and primer 620S (see TableI) with the following program: 95 °C for 5 min; 55 °C for 3 min; 72 °C for 40 min; 30 cycles of 95 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s; and a final step at 72 °C for 10 min. Southern blot analysis was performed on the reaction products using radiolabeled murine PSS1 cDNA as a probe. The 5′-sequence proximal to the transcriptional initiation site was obtained by "gene walking" using primers 1U, 2U, 3U, and 4U (see Table I) for sequencing directly from 5 μg of BAC DNA. The sequence of the putative promoter region was evaluated using the Transfac search engine. The chromosome location of the murine PSS1 gene was determined by DNA Biotech Inc. at the University of Toronto (Toronto, Canada). Lymphocytes were isolated from mouse spleen and cultured at 37 °C in RPMI 1640 medium supplemented with 15% fetal calf serum, 3 mg/ml concanavalin A, 10 mg/ml lipopolysaccharide, and 5 × 10−5m β-mercaptoethanol. After 44 h, the cultured lymphocytes were treated for 14 h with 0.18 mg/ml 5′-bromo-2′-deoxyuridine. The synchronized cells were washed and re-cultured at 37 °C for 4 h in minimal essential medium containing thiamine (2.5 mg/ml). Chromosome identification slides were prepared by hypotonic treatment, fixation, and air drying. For the DNA probe, we used a pure preparation of BAC clone 595 F12 isolated using a QIAGEN midiprep kit (tip 100). The DNA probe was biotinylated with dATP using the Bio Nick labeling kit (15 °C, 1 h; Life Technologies, Inc.) (33Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar). The procedure for FISH detection was as described previously (33Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar, 34Heng H.H.Q. Tsui L.-C. Chromosoma ( Berl. ). 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). Briefly, slides were baked at 55 °C for 1 h and then treated with RNase A. The material on the slides was denatured with 70% formamide in 2× SSC for 2 min at 70 °C, followed by dehydration with absolute ethanol. Probes were denatured at 75 °C for 5 min in a hybridization mixture consisting of 50% formamide and 10% dextran sulfate and then prehybridized for 15 min at 37 °C. After an overnight hybridization reaction with the probes, the slides were washed, and hybridization was detected and amplified (33Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar). FISH signals and the DAPI banding patterns were recorded separately, and the assignment of the FISH mapping data to chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes (34Heng H.H.Q. Tsui L.-C. Chromosoma ( Berl. ). 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar). PSS activity was determined as previously described (24Vance J.E. J. Biol. Chem. 1990; 265: 7248-7256Abstract Full Text PDF PubMed Google Scholar). Briefly, frozen tissues were homogenized in HEPES buffer (pH 7.5) containing 0.25 msucrose. Homogenates were centrifuged for 10 min at 1000 ×g to pellet nuclei and cellular debris, and PSS activity was measured in the supernatant. Typically, 50–100 μg of protein were incubated with [3-3H]serine (50 μCi/μmol, 0.4 mm), [1-3H]ethanolamine (20 μCi/μmol, 0.2 mm), or [methyl-3H]choline (50 μCi/μmol, 0.2 mm) in 25 mm HEPES buffer (pH 7.4) containing 4 mm hydroxylamine and 10 mmCaCl2 in a final volume of 200 μl for 20 min at 37 °C. The reaction was terminated by addition of 5 ml of chloroform/methanol (2:1, v/v). Water (1.5 ml) was added for phase separation, after which the lower phase was washed three times with 1.5 ml of methanol/water (1:1, v/v), and radioactivity was measured. Five BAC clones were isolated from a 129/J murine genomic library by PCR screening and Southern blot analysis using radiolabeled exon-specific probes and radiolabeled full-length PSS1 cDNA. One of these genomic clones, 595 F12, contained the majority of the murine PSS1 gene. However, this clone lacked 437 base pairs at the 3′-end of the PSS1 cDNA. None of the other four clones contained the entire 3′-end of the cDNA. The integrity and identity of the cloned PSS1 gene were confirmed by comparison of the restriction enzyme fragmentation patterns of genomic DNA and the BAC clone (data not shown). One restriction fragment derived from genomic DNA was not the same size as any of those derived from the BAC clone. We confirmed that this fragment corresponded to the 3′-end of the PSS1 gene by performing PCR amplification using oligonucleotide primers (1920S and 2312AS; Table I) specific for the 3′-end of murine PSS1 cDNA, with genomic DNA and full-length PSS1 cDNA as templates. Although clone 595 F12 lacked 437 base pairs at the 3′-end of the cDNA, this portion of the gene did not appear to contain any introns since (i) the PCR product obtained using murine liver genomic DNA as a template was the same size as the PCR product that would be expected from PSS1 cDNA, and (ii) the sequence of the human PSS1 gene obtained from the NCBI Database (accession number AC068091; 966284529-3551-12509) did not reveal any introns in this region of the human gene. Radiolabeled full-length cDNA encoding murine PSS2 was also used in Southern blot analysis to confirm that the clones with which PSS1 cDNA hybridized did not contain the PSS2 gene. These results demonstrate that murine PSS1 and PSS2 are encoded by different genes. On the basis of these results, clone 595 F12 was used to study the organization of the PSS1 gene. A combination of Southern blotting and sequencing using specific primers identified the exonic sequences and facilitated determination of exon and intron sizes as well as sequences at the exon-intron splice junctions (Fig. 1 and TablesII and III). The PSS1 gene is ∼35 kb in length, which is ∼15 times longer than the PSS1 cDNA. The gene is composed of 13 exons interrupted by 12 introns (Fig. 1 and Table II). Exon 1 contains the ATG codon of the translational start site (Fig.1 A). We have designated the first nucleotide of exon 1 of the PSS1 gene as nucleotide +1. The sizes of the exons range from 44 to 1035 bp. Exon 13 contains the stop codon (TGA) and the 3′-natural flanking region. To determine the sizes of the introns, two main strategies were used. First, we performed PCR amplification using primers flanking the exon-intron boundaries. Second, we mapped the region of the gene that contained an intron and then subcloned this fragment into pBluescript. Since some of the introns (e.g.introns I, VI, and IX) were large, we used the enzyme Accurase, which allowed more accurate amplification of large DNA fragments. The introns range in size from 0.3 to >5 kb (Fig. 1 A and TableIII). We noted that the organization of the mouse PSS1 gene is similar to that of the human gene. For example, exon-intron boundaries of the human PSS1 gene are at exactly the same positions as in the murine gene; and, consequently, the sizes of the exons are the same in the two species. Our data base search of the human gene showed that the sizes of introns VII and XI are 2.3 and 2.2 kb, respectively. We estimated that the sizes of introns VII and XI of the murine gene are 1.6 and 2 kb, respectively.Table IIPositions and sizes of exons in the 129/J mouse PSS1 geneExonPositions in the putative 129/J murine cDNACodon sequences interruptedAmino acids at sequence interruption11–264 (264)AG/GArg602265–356 (92)G/GTGly903357–401 (45)G/GTGly1054402–526 (125)ATG/GAGMet145/Glu1465527–685 (159)GAG/CTGGlu198/Leu1996686–836 (151)AAG/GACLys250/Asp2517837–975 (139)TGG/CAGTrp296/Gln2978976–1092 (117)AG/AArg33591093–1157 (65)GG/GGly357101158–1259 (102)GTG/GCCVal390/Ala391111260–1327 (68)AAG/ACCLys414/Thr415121328–1397 (70)G/GTGly438131398–2432 (1035)TT/CPhe637Numbers in parentheses indicate sizes of exons in base pairs. Open table in a new tab Table IIIExon-intron boundaries of the 129/J mouse PSS1 gene5′-Intron boundaryIntron (approximate size in kb)3′-Intron boundaryGCCTCACCAG/gtcagtctggcI (>5)cgccttcaccag/GGATGACTCCGTGCATTCCCCCAATG/gtgagtcatgtII (0.3)/GTCCATTTACTCGTGTTTTTG/gtgatcccattIII (3)tgcttttttgtcag/GTCTCAGTGTGTTGACATCATG/gtatgtacttIV (0.8)tatgttaaccag/GAGTATGCTGGAGCTGACAGAG/gtaagacatgV (2.8)tctgtgttcctcag/CTTTCTTCATGCAAAGCTTCAAG/gtgagtccttcttVI (4)attctctttccctag/GACATCCATCATATGG/gtaatcttataaataVII (2)ctcttctctt/CAGCTGACTGAGCAGTGAG/gt
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