Structure and Expression of the Murine Phosphatidylserine Synthase-1 Gene*

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.

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.

EXPERIMENTAL PROCEDURES
Materials-Restriction endonucleases, the random primer labeling kit, and the end labeling kit were obtained from Life Technologies, Inc. (Burlington, Ontario, Canada). The radioisotopes ([␣-32 P]CTP and [␥-32 P]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).
DNA Isolation and Southern Analysis-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 ϩ nylon membranes (Amersham Pharmacia Biotech) in 10ϫ SSC (SSC ϭ 0.15 M 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 6ϫ SSC, 5ϫ Denhardt's solution, 0.5% SDS, 50% formamide, 100 g/ml salmon sperm DNA (Sigma), and 1.5 ϫ 10 6 cpm/ml probe, as required. Membranes were washed under conditions of low (2ϫ SSC and 0.1% SDS at room temperature) or high (0.1ϫ SSC and 0.1% SDS at 37 or 55°C) stringency and then scanned using a Storm 540 PhosphorImager (Molecular Dynamics, Inc.).
Cloning of the Gene-Using the full-length cDNA encoding murine liver PSS1 (GenBank TM /EBI accession number AF042731) (30), 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.
Gene Structure-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. RNA Extraction and RT-PCR Analysis-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 g of RNA from tissues were incubated at 70°C for 10 min in the presence of 1 l of oligo(dT) primer (10 M). After GCCTGTTTCGGCTCTGATTCCT Promoter a brief centrifugation, 4 l of "first strand buffer," 2 l of dithiothreitol (0.1 M), and 1 l of dNTP mixture (10 mM) were added and incubated at 42°C for 2 min prior to addition of Superscript reverse transcriptase (1 l) 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, TGGAGTCACACAAGC-CAAAGAC; 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).
Identification of the Transcriptional Initiation Site Using 5Ј-RACE-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 l of ATP (1 mM), 5 l of buffer, and 1 l 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 l 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, GACTC-GAGTCGACATCGATTTTTTTTTTTTTTTTT) 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.
3Ј-Untranslated Region of PSS1 cDNA and Its Location on the PSS1 Gene-Total RNA was prepared from 129/J mouse liver using Trizol. RNA (2 g) 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 Table I) 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.
Analysis of the Putative Proximal Promoter-The 5Ј-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 g of BAC DNA. The sequence of the putative promoter region was evaluated using the Transfac search engine.
Chromosome Localization-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 Ϫ5 M ␤-mercaptoethanol. After 44 h, the cultured lymphocytes were treated for 14 h with 0.18 mg/ml 5Ј-bromo-2Ј-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.) (33). The procedure for FISH detection was as described previously (33,34). 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 2ϫ 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 (33). 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 (34).
Measurement of PSS Enzymatic Activity-PSS activity was determined as previously described (24). Briefly, frozen tissues were homogenized in HEPES buffer (pH 7.5) containing 0.25 M sucrose. Homoge-

RESULTS AND DISCUSSION
Cloning and Exon-Intron Organization of the Murine PSS1 Gene-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 fulllength 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 3Ј-end of the PSS1 cDNA. None of the other four clones contained the entire 3Ј-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 3Ј-end of the PSS1 gene by performing PCR amplification using oligonucleotide primers (1920S and 2312AS; Table I) specific for the 3Ј-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 3Ј-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 Tables II and III). The PSS1 gene is ϳ35 kb in length, which is ϳ15 times longer than the PSS1 cDNA. The gene is composed of 13 exons inter-rupted by 12 introns (Fig. 1 and Table II). Exon 1 contains the ATG codon of the translational start site (Fig. 1A). We have designated the first nucleotide of exon 1 of the PSS1 gene as nucleotide ϩ1. The sizes of the exons range from 44 to 1035 bp. Exon 13 contains the stop codon (TGA) and the 3Ј-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 Ͼ5 kb ( Fig. 1A and Table III). 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.
We established and sequenced the exon-intron boundaries (Table III). The boundary sequences at the 5Ј-and 3Ј-ends of all the introns, except intron VII, are GT and AG, respectively. These are consensus sequences for pre-mRNA splicing donor and acceptor sites (35). Clone 595 F12 was digested separately with BamHI, EcoRI, HindIII, Xba, and KpnI, and the resulting fragments were analyzed by Southern blotting with probes specific for individual exons (Fig. 1B and Table I). Using this strategy, we isolated several overlapping clones, which were subcloned into Bluescript. Clone 1 (BamHI, 5.2 kb) contains the first exon with 900 bp of the first intron; clone 2 (Xba, 2.3 kb) contains the first exon with 700 bp of the first intron; and clone 3 (KpnI, 5 kb) contains the first exon with 4.1 kb of the first intron (Fig. 1B). The remainder of the gene was similarly mapped. Some of these clones will be used for targeted disruption of the murine PSS1 gene.
Identification of the Transcriptional Initiation Site-The transcriptional initiation site of the PSS1 gene was identified by 5Ј-RACE PCR. Total RNA was isolated from 129/J mouse liver and reverse-transcribed using primer 913AS (Table I) with Superscript reverse transcriptase. The resulting cDNA product was tailed with dATP. This template was amplified by PCR with the primer pair Race dT and 396AS (Table I and Fig.  2A). A single PCR product of 420 bp was generated (Fig. 2B,  upper). Southern blot analysis of this PCR product, which hybridized with a probe corresponding to the 5Ј-end of PSS1 cDNA (primer 85S; Table I), confirmed that the PCR product was derived from the PSS1 gene (Fig. 2B, lower). To identify the transcriptional initiation site, the 420-bp PCR product was subcloned into the pCR2.1 vector and sequenced in both directions using primers corresponding to the pCR2.1 vector. As shown in Fig. 2C, the transcriptional start site was identified 25 bp upstream of the start of exon 1 and 111 bp upstream of the translational start codon, ATG. The 5Ј-RACE PCR was performed three additional times using different preparations of total RNA from 129/J mouse liver, with the same result. Transcription begins with an adenine residue, which is the most common transcription-initiating nucleotide.
Analysis of the Putative Proximal Promoter Region-Clone 1 (Fig. 1B), which consists of a 5.2-kb BamHI digestion fragment from the PSS1 gene, was identified by Southern blotting using the exon 1-specific primer 85S (Table I) as a probe. Clone 1 contains ϳ4300 bp of the 5Ј-natural flanking region of the PSS1 gene, all of exon 1 (264 bp), and 700 bp of intron I. We sequenced 800 bp of the DNA upstream of the transcriptional initiation site, shown as position ϩ1 in Fig. 3, using primers 1U, 2U, 3U, and 4U (Table I). This region of the PSS1 gene did not contain either TATA or CAAT boxes, but did have a high GC content (Fig. 3) (36, 37); 75% of the nucleotides in the first 600 bp were G or C.
Using computer analysis of the 5Ј-natural flanking region, a number of potential transcription factor-binding sites were revealed, as indicated in Fig. 3. A total of three Sp1-binding sites (GC boxes) were located at positions Ϫ12, Ϫ70, and Ϫ310. Sp1-binding sites have been shown to be widespread in promoters of cellular and viral genes (38) and are usually located close to the transcriptional start site. The promoter of the murine CTP:phosphocholine cytidylyltransferase-␣ gene, which encodes the rate-limiting enzyme of the CDP-choline pathway for PtdCho synthesis, also contains Sp1-binding sites (39). In the murine PSS1 gene, the Sp1 site at position Ϫ310 overlaps with the upstream stimulatory factor site and the N-Myc site. Two GATA-1 and two GATA-2 sites were also identified. One GATA-1 site and one GATA-2 site overlap at position Ϫ375. A heat shock factor-2 site was found at position Ϫ361, and an AML-1a (acute myeloid leukemia factor-1a) site was at position Ϫ548.
Sequence of the 3Ј-Untranslated Region of PSS1 cDNA-3Ј-RACE PCR using adapter primer Ad68 (see "Experimental Procedures") and primer 620S (Table I) was performed to obtain the sequence of the 3Ј-untranslated region of the PSS1 cDNA (Fig. 4A). This procedure yielded a 1820-bp product, the partial sequence of which is shown in Fig. 4B. The 3Ј-untranslated region, from the stop codon (TGA) to the site of polyadenylation (ATTAAA), consists of 929 bp (Fig. 4B). An unusual polyadenylation signal sequence (ATTAAA) is located 14 bp upstream of the site at which the poly(A) tail is attached (40). As depicted in Fig. 4C, the 3Ј-untranslated region is directly attached to exon 13 of the PSS1 gene, without an intervening intron. The final exon, exon 13, contains 1035 bp and is the largest in the PSS1 gene. Clone 595 F12 lacks 437 bp at the  Table I, and that of primer Race dT is shown under "Experimental Procedures." B, 1.5% agarose gel (upper) and Southern blot analysis of the PCR product using 32 P-labeled primer 85S (Table I)  3Ј-end of the cDNA. However, this portion of the gene does not appear to be interrupted by any introns because (i) the PCR product obtained using genomic DNA as a template and primers specific for these 437 bp is exactly the same size as the PCR product obtained when the cDNA was used as a template; and (ii) according to the sequence obtained from the NCBI Database (accession number AC068091; 966284529-3551-12509), the human PSS1 gene does not contain any introns within the 437 bp at the 3Ј-end of the cDNA.
Tissue Distribution of Murine PSS1 and PSS2-Although several tissues (e.g. brain (11) and liver (23,24)) have been shown to possess PSS activity, it is unclear what proportion of the total PSS activity in tissues is contributed by PSS1 and PSS2. Our previous Northern blot experiments using a commercially supplied mRNA blot of several murine tissues suggested that PSS1 and PSS2 are expressed to different extents in different tissues (31). We therefore analyzed more carefully the tissue distribution of PSS1 and PSS2 mRNAs using RT-PCR. In addition, we measured the serine, ethanolamine, and choline exchange activities in several tissues. These studies allowed us to determine whether or not a correlation existed between PSS1 and PSS2 mRNA expression and enzymatic activity.
For analysis of mRNA levels, we performed RT-PCR using mRNAs isolated from several tissues of 129/J mice. To avoid cross-reactivity between PSS1 and PSS2 primers, we performed the RT-PCR with primers specific for PSS1 and PSS2 (Fig. 5A) and the internal control cyclophilin. The amount of the cyclophilin control product was very similar in the different tissues, showing that comparable amounts of template cDNA had been used for analysis of each tissue. PSS1 mRNA was most highly expressed in kidney, liver, brain, heart, lung, and testis. However, all tissues examined, including adipose and spleen, contained detectable PSS1 mRNA (Fig. 5B). In contrast, PSS2 mRNA was most abundant in testis, with high expression also in kidney and brain, whereas PSS2 mRNA expression was almost undetectable in white adipose and spleen (Fig. 5B).
Cellular homogenates of several murine tissues were also  (Table I)

FIG. 5. Expression of PSS1 and PSS2 mRNAs in murine tissues.
A, shown is the strategy used for RT-PCR. Total RNA from various tissues of 129/J mice was reverse-transcribed using an oligo(dT) primer. The resulting cDNA was used as a template for PCR using three different primer pairs, one specific for PSS1, one specific for PSS2, and one specific for the internal control cyclophilin. The resulting PCR products were 313 bp for PSS1, 500 bp for PSS2, and 200 bp for cyclophilin. B, the products were analyzed by agarose gel electrophoresis (left panels) and Southern blot analysis (right panels) using oligonucleotide probes 316AS for PSS1 (upper panels), 3S for PSS2 (center panels), and cyclo rev2 (primer specific for internal sequence of cyclophilin) (lower panels). PCR amplifications from individual tissues were performed with the same amounts (50 -100 ng) of template cDNA. Data are representative of three independent experiments with the same results. examined for enzymatic activities of serine, choline, and ethanolamine exchange (Fig. 6). In in vitro enzymatic assays, PSS1 uses choline, ethanolamine, and serine for base exchange, whereas PSS2 uses ethanolamine and serine, but not choline. Choline exchange activity is therefore a marker for the presence of PSS1, whereas ethanolamine and serine exchange activities are contributed by both PSS1 and PSS2. The highest specific activity for serine exchange was in testis and kidney (Fig. 6), corresponding to the high level of expression of PSS1 and PSS2 mRNAs in these tissues (Fig. 5B). Serine exchange activity was also detected in all other tissues examined (brain, liver, lung, skeletal muscle, spleen, and heart). These observations correlate well with the results from the RT-PCR experiments shown in Fig. 5B. The tissue distribution of ethanolamine exchange activity essentially paralleled that of serine exchange activity, with testis and kidney exhibiting the highest ethanolamine exchange activity (Fig. 6), in agreement with the high level of both PSS1 and PSS2 mRNAs detected in these tissues (Fig. 5B). The ethanolamine exchange activity in skeletal muscle, liver, lung, spleen, brain, and heart was 50 -70% lower than that in testis and kidney. The highest specific activity for choline exchange, presumably imparted by PSS1, was in brain (Fig. 6). This tissue also showed one of the highest levels of expression of PSS1 mRNA, according to the RT-PCR analysis depicted in Fig. 5B. The choline exchange activity in the eight tissues examined varied over a 5-fold range, with lung having the lowest specific activity and brain, kidney, and liver having relatively high activity (Fig. 6). These activities are in good agreement with the highest level of expression of PSS1 mRNA being in kidney, brain, liver, and heart (Fig. 5B) (31). The differential tissue expression of PSS1 and PSS2 mRNAs suggests that these two enzymes might have tissue-specific functions.
Chromosome Localization-Clone 595 F12 was used as a probe to determine the chromosome localization of the murine PSS1 gene by the FISH mapping technique (41). Under the conditions used, the detection efficiency was 98% for this probe. Among 100 checked mitotic figures, 98 showed hybridization signals on one pair of chromosomes. DAPI banding was also performed to identify the chromosome on which the PSS1 gene was located. The assignment of signals to mouse chromosome 13 was deduced by examining the overlap between the signal from the PSS1 probe and the DAPI signal. The detailed position of the gene on chromosome 13 was further determined to be in region B-C1 from a combination of 10 photographs (Fig.  7B). Other genes identified on murine chromosome 13 are cytidine-monophospho-N-acetylneuraminic acid hydrolase, coagulation factor II receptor, 3-hydroxy-3-methylglutaryl-CoA reductase, and phosphatidylinositol 3-kinase regulator-1. The human PSS1 and PSS2 genes are localized to chromosomes 8 and 11, respectively (NCBI Database accession number AC068091; RID 966284529-3551-12509).
Conclusion-We have isolated and characterized the murine PSS1 gene and compared its tissue expression with that of the PSS2 gene. The PSS1 gene was localized to mouse chromosome 13 in region B-C1, is ϳ35 kb in length, and consists of 13 exons and 12 introns. PSS1 and PSS2 mRNAs were widely distributed throughout the murine tissues examined. According to RT-PCR analysis, PSS2 mRNA was most highly expressed in testis, in agreement with the specific activities of serine and ethanolamine exchange also being highest in this tissue. Choline exchange activity, contributed by PSS1, was highest in brain and kidney. In experiments presently underway, we are using this information on the PSS1 gene to generate PSS1 gene-disrupted mice with a goal of determining why mammals have (at least) two genes encoding enzymes that catalyze Ptd- Ser synthesis.