From the Department of Medicine and the Canadian Institutes for Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
Received for publication, October 26, 2000
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ABSTRACT |
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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
(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 (1) and is involved in progression of the blood coagulation cascade (2, 3). PtdSer exposure on
the external leaflet of the plasma membrane, via an amino-phospholipid
translocase (4, 5) and scramblase (6-8), is also a signal for
recognition and removal of apoptotic cells by macrophages (5, 9). In
mammalian cells, PtdSer is synthesized by a
calcium-dependent base exchange reaction catalyzed by
PtdSer synthase (PSS) activity that exchanges L-serine for
the polar head group of phosphatidylcholine (PtdCho) and
phosphatidylethanolamine (PtdEtn) (10, 11). The pathway for PtdSer
synthesis in mammals is different from that in bacteria (12) and
Saccharomyces cerevisiae (13, 14), 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 (15) and a base exchange reaction (16).
The existence of two mammalian PSSs, PSS1 and PSS2, was deduced from
studies with mutant Chinese hamster ovary cell lines (17-19). PSS1 and
PSS2 differ in their phospholipid substrate specificities (17, 18), but
the reason why mammalian cells possess two distinct PSSs is not clear
(20). In intact cells, PSS1 uses PtdCho as donor of the phosphatidyl
group, whereas PSS2 uses PtdEtn (21). 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 (22, 23) and is enriched 2-4-fold in an
endoplasmic reticulum subfraction, the mitochondrion-associated
membranes, compared with the bulk of the endoplasmic reticulum
(24-27). 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 (24, 25, 28, 29). 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 (26).
The cDNAs encoding PSS1 and PSS2 from murine liver (30, 31) and
CHO-K1 cells (18, 32) 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 (18, 31), 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 (19))
(30). 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 (30). 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.
Materials--
Restriction endonucleases, the random primer
labeling kit, and the end labeling kit were obtained from Life
Technologies, Inc. (Burlington, Ontario, Canada). The radioisotopes
([ 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+ nylon
membranes (Amersham Pharmacia Biotech) in 10× SSC (SSC = 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 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.).
Cloning of the Gene--
Using the full-length cDNA encoding
murine liver PSS1 (GenBankTM/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.). 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.
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 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 (0.1 M), 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).
Identification of the Transcriptional Initiation Site Using
5'-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 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.
3'-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 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 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 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.
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 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. 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 mM
CaCl2 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.
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 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 Tables
II 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.
1A). 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. 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 5'- and 3'-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
5'-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 5'-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 5'-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 ~4300 bp of the 5'-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 +1 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 5'-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 Sequence of the 3'-Untranslated Region of PSS1
cDNA--
3'-RACE PCR using adapter primer Ad68 (see
"Experimental Procedures") and primer 620S (Table I) was performed
to obtain the sequence of the 3'-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 3'-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 3'-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 3'-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 3'-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 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 ~35
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 PtdSer synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]CTP and [
-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).
5
M
-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.) (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 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
(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).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Sequences of primers used for PCR amplification and sequencing
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Fig. 1.
Structure of the murine PSS1 gene.
A, shown is a schematic representation of the murine PSS1
gene. Solid horizontal lines and solid boxes
represent introns and exons, respectively. Numbers (Roman
numerals) and sizes (in kilobases; Arabic
numbers) of introns are indicated. In the lower part of
A, exon numbers and their positions in the PSS1 cDNA are
shown. The translational start site of the protein (ATG) is in exon 1, and the stop codon (TGA) is in exon 13 at position 1505 of the
cDNA. B, restriction sites are indicated as follows:
B, BamHI; H, HindIII;
K, KpnI; X, Xba; and
E, EcoRI. The positions of clones 1-3 used for
sequence analysis are shown.
Positions and sizes of exons in the 129/J mouse PSS1 gene
Exon-intron boundaries of the 129/J mouse PSS1 gene
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Fig. 2.
Determination of the transcriptional
initiation site of the murine PSS1 gene. A, strategy of
5'-RACE PCR using 129/J murine liver RNA. The sequences of primers
396AS and 913AS are shown in 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 32P-labeled primer 85S (Table I) (lower).
Lanes 1 and 2 contain 50 ng of template;
lane 3 contains 100 ng of template. C, deduced
sequence of the 5'-end of the PSS1 gene showing the transcriptional
initiation site at 25 bp, the start of the first exon at +1 bp, and
the ATG translational start site at +86 bp (boldface).
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Fig. 3.
Consensus sequences of transcription
factor-binding elements in the putative 5'-promoter. The sequence
shows exon 1 (264 bp; positive numbers) and 600 bp of the
putative 5'-promoter region (negative numbers). The
transcriptional initiation site is boxed, and the
translational start site (ATG) is in boldface and
underlined. The consensus sequences of transcription
factor-binding elements are underlined. Sp-1,
stimulating factor protein-1; USF, upstream stimulatory
factor; HSF-2, heat shock factor-2; AML-1a, acute
myeloid leukemia factor-1a.
12,
70, and
310. 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
310 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
375. A heat shock factor-2 site was
found at position
361, and an AML-1a (acute
myeloid leukemia factor-1a) site
was at position
548.
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Fig. 4.
3'-Untranslated region of PSS1 cDNA.
A, 3'-RACE PCR strategy using 129/J murine liver RNA, the
adapter primer (shown under "Experimental Procedures"), and primer
620S (Table I). A product of 1812 bp was generated. The position of the
translational stop codon (TGA) is indicated. B, sequence of
the 3'-untranslated region of PSS1 cDNA. The translational stop
codon (TGA) is boxed, and the polyadenylation signal
sequence (ATTAAA) is in boldface and underlined.
The total length of the 3'-untranslated region of the PSS1 gene is
929 bp. C, location of the 3'-untranslated region in exon
XIII of the PSS1 gene. The stop codon (TGA) and the polyadenylation
signal (ATTAAA) are in exon XIII, separated by 910 bp.
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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. Lane 1,
adipose; lane 2, brain; lane 3, heart; lane
4, kidney; lane 5, lung; lane 6, liver;
lane 7, spleen; lane 8, testis.
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Fig. 6.
Serine, ethanolamine, and choline exchange
activities of murine tissues. Base exchange activities were
measured in tissue homogenates using [3-3H]serine,
[methyl-3H]choline, and
[1-3H]ethanolamine. Data are means ± S.D. of
duplicate analyses from three independent experiments. Sk.
muscle, skeletal muscle.
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Fig. 7.
Chromosome localization of the murine PSS1
gene. A, diagrammatic representation of the FISH
mapping results using the PSS1 BAC clone 595 F12 as a probe. Each
dot shown on the right represents an individual analysis
showing double FISH signals detected on mouse chromosome 13. B, example of FISH mapping using BAC clone 595 F12 as a
probe, showing the FISH signals on mouse chromosome 13 (left
panel) and the same mitotic figure stained with DAPI (right
panel).
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ACKNOWLEDGEMENTS |
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We thank Russ Watts and Igor Cvetkovic for excellent technical assistance and Luis Agellon, David Shields, and Vern Dolinsky for helpful discussions.
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FOOTNOTES |
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* The work was supported by an operating grant from the Canadian Institutes for Health Research (formerly the Canadian Medical Research Council) and a postdoctoral fellowship (to B. S.-B.) from the Alberta Heritage Foundation for Medical Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 332 Heritage Medical
Research Centre, University of Alberta, Edmonton, AB T6G 2S2, Canada.
Tel.: 780-492-7250; Fax: 780-492-3383; E-mail: Jean.Vance@ ualberta.ca.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009776200
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ABBREVIATIONS |
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The abbreviations used are: PtdSer, phosphatidylserine; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PSS, phosphatidylserine synthase; BAC, bacterial artificial chromosome(s); PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-mediated polymerase chain reaction; RACE, rapid amplification of cDNA ends; FISH, fluorescence in situ hybridization; DAPI, 4,6-diamidino-2-phenylindole; kb, kilobase(s); bp, base pair(s).
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