From the
Carbamoyl-phosphate synthetase II (CPSase II), aspartate
transcarbamoylase (ATCase), and dihydroorotase (DHOase) catalyze the
first three steps of de novo pyrimidine nucleotide
biosynthesis, respectively. In mammalian species, these three enzyme
activities exist in the cytosol in liver and other tissues as a
multifunctional complex on a single polypeptide called
carbamoyl-phosphate synthetase-aspartate
transcarbamoylase-dihydroorotase (CAD) in the order of
NH
Two different types of carbamoyl-phosphate synthetases
(CPSases)
In ureoosmotic
elasmobranch fishes (sharks, skates, and rays), carbamoyl phosphate
formation in the urea cycle is catalyzed by a different type of CPSase,
CPSase III (Anderson, 1980, 1991, 1995a). The properties of CPSase III
are very similar to those of CPSase I except that glutamine is utilized
instead of ammonia as the nitrogen-donating substrate (Anderson, 1981).
We have recently reported that the predicted amino acid sequence based
on the nucleotide sequence of the CPSase III cDNA from spiny dogfish
(Squalus acanthias), a representative elasmobranch (hereafter
referred to as shark), has a high degree of identity to the amino acid
sequences of rat, human, and frog CPSase I (Hong et al.,
1994). In addition to the ureoosmotic elasmobranchs, CPSase III
activity is present in certain teleost fishes and in invertebrates, and
it has been suggested that CPSase III is an evolutionary intermediate
between type II CPSases of lower eukaryotes and CPSase I of higher
eukaryotes (Mommsen and Walsh, 1989; Campbell and Anderson, 1991; Hong
et al., 1994; Anderson, 1995a, 1995b).
In addition to the
unique difference from the CPSases related to the urea cycle in higher
vertebrates, our previous studies with spiny dogfish have indicated
that the CPSase activity related to pyrimidine biosynthesis in
elasmobranchs may also be different from the corresponding CPSase II
activity in higher vertebrates (Anderson, 1989). The enzyme activities
catalyzing the first three steps of the pyrimidine pathway (CPSase II,
ATCase, and DHOase, respectively) along with glutamine synthetase are
present in the cytosol of extracts of extra-hepatic tissues such as
spleen and testes, but they elute as three separate entities during gel
filtration chromatography, suggesting that the three genes may not be
expressed as a single mRNA. In addition, the apparent absence of ATCase
activity in liver extracts suggests that CPSase II activity and the
pyrimidine biosynthetic pathway may be expressed only in extra-hepatic
tissues (Anderson, 1989). CPSase II activity has also not been observed
in liver extracts.
The plasmids with three
different inserts were sequenced using primers specific for plasmid
sequence flanking the inserts. Sequencing was carried out from one end
of each strand toward the internal region of the insert. The primer
walking strategy was used to obtain (as necessary) additional internal
sequence until the overlapping sequence of the two complementary
strands was obtained. Several clones were sequenced to reduce the
possibility of incorrectly identifying mutations incorporated by
Taq DNA polymerase as part of the sequence. Sequencing was
carried out using the Sequenase version 2.0 DNA sequencing kit (U. S.
Biochemical Corp.) and employing
A more sensitive
determination of expression was accomplished using RT-PCR. The
synthesis of cDNA from liver or testis poly(A)
To obtain cDNA
sequence toward the extreme 5` end of the mRNA, 5` RACE PCR was
employed. First strand testis cDNA was synthesized using 2 µg of
testis mRNA as template and 25 pmol of primer 16 (designed based on the
sequence obtained from piece 7-8) as primer following the
procedure described above. The final volume was about 50 µl.
Purified first strand cDNA (46 µl) was added along with 12 µl
of 5
The PCR product (piece
14-15) was purified from the gel by Wizard PCR Preps DNA
purification system (Promega) and directly sequenced by the cycle
sequencing method using fmol DNA sequencing system (Promega); the
manufacturer's protocol was followed. Primer 14 and sequencing
primers (not listed) nested to primer 15 were used in the cycle
sequencing and labeled using [
To obtain the remaining sequence of shark CAD cDNA,
the 5.1-kb PCR product (piece 12-18, Fig. 1) was purified
from the gel by Gelase (Epicentre Technologies, Madison, WI) digestion
and then precipitation by addition of ethanol following the
manufacturer's instructions. The cycle sequencing method was
applied as described above. The sequencing primers (not listed) nested
to primers 12 and 18 were used in the cycle sequencing to obtain
sequence from both strands and the primer walking strategy (primers not
listed) was used to advance the sequencing until the overlapping
sequence of the two complementary strands was obtained.
The sequence
data were analyzed using the GCG sequence analysis software package
version 7 of the Wisconsin Genetics Computer Group.
The CPSase II domain extends from 1
to 1462. Like other CPSases (Evans, 1993; Anderson, 1995b), it can be
further divided into a glutaminase domain(1-365) and a synthetase
domain
(2) with a linker region(366-397) in between. In
the glutaminase domain, Cys
The DHOase domain begins immediately after the synthetase
domain, extending from Met
The ATCase domain begins at Leu
The results of these studies clarify two unresolved issues
about the enzymes catalyzing the first three steps of pyrimidine
nucleotide biosynthesis in spiny dogfish. Our earlier observation that
the first three enzymes of the pathway apparently exist in extracts as
separate polypeptide chains suggested that the genes for these enzymes
in elasmobranchs may not be expressed as a single transcript, which is
translated to give the multifunctional protein CAD as is observed for
most other higher eukaryote species (Anderson, 1989). However, the
results here clearly show that the genes for the three enzymes are, in
fact, expressed as a single transcript analogous to CAD. This does not
eliminate the possibility that the integrity of the multifunctional
complex may not be essential for normal function in some species and
that the transcript is translated to give a multifunctional protein
(CAD) which is subject to rapid post-translational proteolysis of the
interdomain regions in vivo to give the three enzymes as
separate entities. If the multifunctional structural integrity of the
CAD transcriptional product was essential for normal physiological
function in spiny dogfish and this structure was maintained in
vivo, complete proteolysis of the interdomain linker regions
during extract preparation prior to or during gel filtration
chromatographic analysis seems unlikely for reasons previously
discussed (Anderson, 1989). The susceptibility of the polypeptide
linker regions between domains in hamster CAD to proteolysis have been
well characterized and each of the enzymatic domains can be readily
separated from each other by limited proteolysis of the
protease-sensitive interdomain linker sequences and each can function
in the absence of the other (Evans, 1986; Kim et al., 1992;
Davidson et al., 1993; Evans et al., 1993).
Complementation studies involving expression of each of the separate
functional domains of CAD have shown that each domain can fold into its
catalytically active conformation independent of the other portions of
the CAD protein (Davidson et al., 1993). In vitro and
in vivo studies with mammalian CAD have failed to provide
evidence for substrate channeling (Mally et al., 1980;
Christopherson and Jones, 1980; Otsuki et al., 1982). Davidson
et al.(1993) have suggested that an alternative explanation
for the evolution of CAD as a multifunctional entity may be the
selective advantage of coordinate expression of enzymes catalyzing
steps in a particular pathway, which would preserve the advantage of
the analogous structure in bacteria, i.e. the operon. These
considerations suggest that there may not be a functional need to
preserve the multifunctional nature of CAD after translation and that
specific proteolysis at the interdomain linker regions may normally
occur in some situations, giving three separate enzymes as observed in
spiny dogfish. These three enzymes have been reported to exist as
separate entities in a few other eukaryote species, e.g. frog
(Kent et al., 1975) and some protozoal parasites (Tampitag and
O'Sullivan, 1986; Aoki and Oya, 1987; Mukherjee et al.,
1988). It has been reported that a proteolytic fragment of the yeast
difunctional protein which contains the ATCase and CPSase II activities
may exist in vivo (Denis-Duphil et al., 1981).
Retention in the shark CAD of the two serine residues and the
associated phosphorylation consensus sequences that are known to be
subject to phosphorylation in hamster CAD (Carrey et al.,
1985; Carrey and Hardie, 1988; Carrey, 1992, 1993) may indicate that
shark CAD (or the separate enzymes) is subject to regulation by
phosphorylation. It has been reported that the effect of
phosphorylation on hamster CAD is to activate CPSase II, and in
particular to relieve the feedback inhibition by UTP (Carrey et
al., 1985).
The results of Northern blot analysis and RT-PCR
indicate that 1) the CPSase III gene is expressed in liver but not in
an extra-hepatic tissue such as testes and that 2) the CAD gene is not
expressed in liver, but is expressed in testes. The latter result
confirms earlier observations (Anderson, 1989) of the presence of the
CAD enzyme activities as well as glutamine synthetase in the cytosol of
extra-hepatic tissues such as spleen and testes but the apparent
absence of detectable aspartate transcarbamoylase activity in liver,
suggesting that pyrimidine nucleotide biosynthesis does not occur in
liver of spiny dogfish (Anderson, 1989). The absence of the pyrimidine
pathway in liver may be related to the exclusive mitochondrial
localization of glutamine synthetase in liver of shark where it is
coupled with the glutamine-dependent CPSase III to provide an efficient
system for uptake of ammonia and its conversion into glutamine and then
into carbamoyl phosphate for urea synthesis. Thus, glutamine formed in
the mitochondria may be utilized entirely for urea synthesis and may
not be available for CPSase II or any other amidotransferase activity
in the cytosol. This suggests that other pathways usually present in
liver that involve cytosolic amidotransferases, such as the purine
biosynthetic pathway, may also not exist in liver of shark. It might be
expected then that the source of purine and pyrimidines in liver would
be diet, an active salvage pathway, and/or other tissues via the
circulatory system. In spiny dogfish and other elasmobranchs, glutamine
synthetase in extra-hepatic tissues is localized in the cytosol where
glutamine formed can be utilized for CPSase II and other
amidotransferase activities (Campbell and Anderson, 1991). The absence
of the pyrimidine pathway in liver is probably unique to the
ureoosmotic elasmobranch fishes and the coupled mitochondrial glutamine
synthetase-CPSase III system for assimilating ammonia for urea
synthesis; glutamine synthetase and the pyrimidine pathway enzymes are
present in liver and are localized in the cytosol in several teleost
fishes (Cao et al., 1991; Anderson and Walsh, 1995).
The
size and sequence, and consequently the probable domain organization,
of the shark CAD are very similar to CAD from other species. As shown
in Fig. 4, the shark CAD sequence has 77% identity to the
published hamster CAD sequence. This similarity also applies to the
relationship between the CPSase domain and other CPSases as pointed out
under ``Results'' and as noted in the relationship of the
CPSase III sequence to other CPSases (Hong, et al., 1994).
Comparison of the alignments of the shark and other reported CAD CPSase
II sequences with the reported CPSase I and the shark CPSase III
sequences has revealed a sequence of about seven amino acids in all
CPSase I and III sequences that is absent in all CAD CPSase II
sequences (between Leu
The sequences
for primers used in polymerase chain reactions and cDNA synthesis are
listed in the table below. I represents inosine. Several nucleotides
within the parentheses represent the degeneracy of nucleotides at one
position. (T)
The
nucleotide sequence(s) reported in this paper has been submitted to the
GenBank
-CPSase II-DHOase-ATCase-COOH. Previous studies provided
evidence that in Squalus acanthias (spiny dogfish) these
enzymes are not expressed in liver and that they exist as separate
entities in the cytosol of extra-hepatic tissues such as testes and
spleen (Anderson, P. M.(1989) Biochem. J. 261, 523-529).
Here we report that the genes for these three enzymes are expressed in
testes as a single transcript analogous to CAD in mammalian species and
that these genes are not expressed in liver at levels that can be
detected by Northern blots or by the polymerase chain reaction. The
absence of the pyrimidine pathway in the liver may be related to the
exclusive localization of glutamine synthetase in the mitochondrial
matrix which provides for efficient assimilation of ammonia as
glutamine for urea synthesis in these ureoosmotic species; thus
glutamine may not be available for CPSase II or other amidotransferase
activities in the cytosol. The amino acid sequence deduced from the
nucleotide sequence of the shark CAD cDNA reported here is very similar
to CAD from other species; alignment with the hamster CAD sequence
shows 77% identical residues.
(
)
catalyze formation of carbamoyl
phosphate, which is utilized in two major metabolic pathways in
ureotelic terrestrial vertebrates (Jones, 1980; Evans, 1986; Anderson,
1991, 1995b). CPSase I catalyzes carbamoyl phosphate formation as the
first step of the urea cycle. CPSase I is present only in liver and
small intestine, is localized in the mitochondrial matrix, utilizes
ammonia as the nitrogen-donating substrate, and requires the presence
of N-acetyl-L-glutamate as a positive allosteric
effector for activity. Carbamoyl phosphate formation as the first step
of the pyrimidine nucleotide pathway is catalyzed by CPSase II. CPSase
II, in contrast to CPSase I, is present in liver as well as most other
tissues, is localized in the cytosol, utilizes glutamine rather then
ammonia as the nitrogen-donating substrate, does not require the
presence of N-acetyl-L-glutamate for activity (and
activity is not affected by the presence of
N-acetyl-L-glutamate), and is subject to feedback
inhibition by UTP and activation by 5-phosphoribosyl-1-pyrophosphate.
Furthermore, the CPSase II activity is physically linked at its
C-terminal end to the third and second enzymes in the pyrimidine
pathway, DHOase and ATCase, to form a multifunctional enzyme called
CAD. The apparently typical hamster CAD is a single 243-kDa polypeptide
with separate functional domains in the sequence NH
-CPSase
II-DHOase-ATCase-COOH (Evans et al., 1993).
(
)
However, these studies are
not conclusive, since 1) although precautions were taken to minimize
proteolysis of linker regions between the domains of a CAD-like
multifunctional enzyme, the presence of the three enzyme activities in
extracts as separate polypeptide chains as a result of very active
and/or specific protease activity cannot be excluded, 2) the presence
of a CPSase II activity in liver may be difficult to detect because of
the very high level of CPSase III activity in liver compared with the
expected very low level of CPSase II activity, and 3) inability to
detect ATCase in crude extracts does not definitively establish that
the activity is not present. The study reported here was initiated to
establish the nature of the CPSase II activity in spiny dogfish
relative to that in higher eukaryotes. To our knowledge,
characterization of the mRNA for any of the first three enzymes of the
pyrimidine pathway has not been reported previously for any fish
species. The results show that 1) the first three enzymes of the
pyrimidine pathway are encoded as a single mRNA, which has a predicted
amino acid sequence analogous to CAD; 2) the spiny dogfish CAD
transcript is expressed in testes but not in liver; and 3) the
transcript for CPSase III, as expected, is expressed in liver but not
in testes.
Isolation of Poly(A)
Freshly excised liver and testis tissue (1 g for
each preparation) were immediately dropped into liquid nitrogen and
then stored at -70 °C. Poly(A)RNA
RNA was
isolated using the FastTrack mRNA isolation kit from Invitrogen Corp.
(San Diego, CA); the protocol provided with the kit was followed. The
final concentrations of poly(A)
RNA were measured by
absorbance at 260 nm.
cDNA Synthesis
Shark poly(A) RNA
(1 µg of liver or testis) in 4 µl of water was heated for 3 min
at 75 °C in a 1.5-ml microcentrifuge tube to disrupt the secondary
structure and then rapidly cooled on ice and centrifuged. The denatured
poly(A)
RNA was then transferred to another 1.5-ml
microcentrifuge tube containing a 15-µl mixture of buffer, dNTPs,
and oligo(dT) primer (Promega, Madison, WI). To this mixture was added
1 µl (200 units) of Moloney murine leukemia virus reverse
transcriptase (Promega). The final composition of the reaction mixture
was 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl
10 mM dithiothreitol, 0.5 mM dNTPs,
0.025 µg/µl of oligo(dT) primer, and 10 units/µl of Moloney
murine leukemia virus reverse transcriptase. The reaction mixture was
incubated at 37 °C for 40 min and at 42 °C for an additional 30
min. Then 2 units of RNase H (Promega) were added, and the mixture was
incubated for an additional 20 min at 37 °C. After heating at 95
°C for 5 min to inactivate the enzymes, the total volume was
adjusted to 50 µl with water and the cDNA purified by passage
through a Sephadex G-50 spin column (Sambrook et al., 1989).
After centrifugation, the effluent (about 50 µl) was collected. All
the water and tubes used in the cDNA synthesis were sterile and
RNase-free.
Strategy for Acquiring Specific Probes for Shark CPSase
II, ATCase, and DHOase mRNA
Three sets of consensus primers were
designed based on the reported conserved amino acid sequences for
CPSase (Simmer et al., 1990), ATCase (Simmer et al.,
1989), and DHOase (Quinn et al., 1991): primers 1 and 2 for
CPSase II, primers 3 and 4 for DHOase, and primers 5 and 6 for ATCase.
The primers were synthesized using a PCR-Mate 391 DNA synthesizer
(Applied Biosystems, Foster City, CA). The sequences of these primers
are listed in . These primers were used for the PCRs; the
50-µl reaction mixtures contained 50 mM KCl, 10
mM Tris-HCl, pH 9.0 (at 25 °C), 0.1% Triton X-100, 0.2
mM of each of the four dNTPs, 1.5 mM
MgCl, 50 pmol of each primer, 1 µl of the purified
shark testis cDNA as template, and 2.5 units of Taq DNA
polymerase (Perkin-Elmer). The program for amplifying the targeted
CPSase II and ATCase sequences was: 1 cycle of 5 min at 94 °C
(denaturation), 1 min at 42 °C (annealing), and 1 min at 72 °C
(extension); then 29 cycles of 1 min at 94 °C, 1 min at 42 °C,
and 1 min at 72 °C. For amplifying the targeted DHOase sequence,
touchdown PCR (Don et al., 1991) was used in order to limit
spurious priming. The first cycle included 5 min at 94 °C, 1 min at
55 °C, and 1 min at 72 °C. In each succeeding cycle, the
denaturation step was maintained at 94 °C for 1 min, but the
annealing temperature was decreased by 2 degrees per cycle to a
touchdown temperature of 45 °C, where it remained for 25 more
cycles. The extension time (at 72 °C) was increased by 2 s/cycle
throughout the entire PCR. All the PCRs were performed using a DNA
thermal cycler (Perkin-Elmer). The PCR products were separated by gel
electrophoresis using 4% Nusieve agarose (FMC BioProducts, Rockland,
ME) with Biomarker Low (BioVentures, Murfreesboro, TN) as standard. The
PCR products of the appropriate sizes were cut out from the gel and
cleaned with Wizard PCR Preps DNA purification system (Promega)
following the manufacturer's protocol. The cleaned PCR products
were then cloned into both pT7 Blue (R)-T vector (Novagen, Madison, WI)
and pCR-Script SK(+) cloning vector (Stratagene, La Jolla, CA)
following the manufacturer's instructions. Novagen's vector
has a T-overhang which is suitable for cloning PCR products with an
A-overhang. On the other hand, Stratagene's vector is for cloning
the blunt-end PCR products. Depending on the percentage of A-overhang
(or blunt-end) PCR product in each reaction, it would be expected that
one vector would work better than the other.
S as a label
(5`-[
-
S]dATP, 1000 Ci/mmol, purchased from
Amersham Corp.).
Preparation of
Three sets of specific primers were designed and
synthesized based on the partial sequences obtained for CPSase II,
ATCase, and DHOase. One set of primers specific for CPSase III has been
described (Hong et al., 1994). One µl of purified testis
cDNA was used as template in each PCR reaction with primers specific
for CPSase II, ATCase, and DHOase, respectively. One µl of purified
liver cDNA was used as template in the PCR with primers specific for
CPSase III. The PCR reactions were carried out as described above with
consensus primers, except that the annealing temperature was increased.
After confirming by agarose gel electrophoresis that only one major
product of the correct predicted size was obtained in each reaction,
the entire remaining reaction mixture was subjected to agarose gel
electrophoresis, the desired band cut from the gel, and the DNA
purified using the Wizard PCR Preps DNA purification system (Promega).
The products were used as templates and reamplified by the PCR to give
P-Labeled
Probes
P-labeled probes; the dCTP concentration in the reaction
mixtures was reduced to 0.016 mM, and 10 µl of
5`-[
-
P]dCTP (10 mCi/ml, 3000 Ci/mmol,
Amersham) was added. The
P-labeled probes were purified by
centrifugation through a Sephadex G-50 spin column.
RNase H Mapping
Oligomer-directed RNase H cleavage
as described by Berger(1987) was used for mapping the shark testis
mRNA. Shark testis poly(A) RNA (18 µg) was heated
at 75 °C for 3 min to disrupt secondary structure and then
immediately cooled on ice. After a brief centrifugation to collect the
liquid in the bottom of the centrifuge tube, 1 µl of a 25-base
deoxyoligonucleotide (15 pmol/µl, Primer 10 in )
complementary to the DHOase mRNA sequence followed by 2 µl of 10
Multicore Buffer (Promega) was added. After 5 min on ice, 2
µl of RNase H (1.5 units/µl, Promega) was added; this mixture
was incubated at 37 °C for 60 min and then subjected to Northern
blot analysis.
Northern Blot Analysis
Shark testis
poly(A) RNA (5.4 µg), shark liver
poly(A)
RNA (5.4 µg), and RNase H-digested testis
poly(A)
RNA (9 µg) were electrophoresed in gel
containing 0.8% formaldehyde (Sambrook et al., 1989) with
standard RNA molecular weight markers (0.36-9.49 kb, Promega) and
then transferred to nitrocellulose membrane BioBlot-NC (Costar,
Cambridge, MA) (Sambrook et al., 1989). Each membrane was
prehybridized in 25 ml of 6
SSC, 5
Denhardt's
reagent, 50% formamide, 0.5% SDS, and 50 µg/ml salmon sperm DNA at
42 °C for 90 min. The
P-labeled probe (about 50
µl) was heated with 125 µl of salmon sperm DNA (10 mg/ml) at
100 °C for 5 min and then rapidly cooled on ice. The probe was then
added to the prehybridization solution and hybridized at 42 °C
overnight. After hybridization, the membranes were washed twice (15 min
each time) with 2
SSC, 0.5% SDS solution at room temperature,
and then twice (8 min each time) with 0.1
SSC, 0.1% SDS
solution at 55 °C before autoradiography.
Determining Tissue-specific Expression of CAD and CPSase
III
Tissue-specific expression of CAD and CPSase III genes in
shark liver and testes was determined in two ways: Northern blot
analysis and by reverse transcription-PCR (RT-PCR). Northern blotting
was carried out as described above. CAD expression was monitored using
probes for CPSase II, DHOase, and ATCase. CPSase III expression was
monitored using a probe for CPSase III.
RNA was
carried out as described above. Primer sets were chosen to amplify long
specific segments of CPSase III and CAD cDNA, but under conditions that
would not amplify any possibly contaminating and much longer
intron-laden genomic DNA. The primer set for CAD consisted of an
upstream CPSase II-specific primer and a downstream ATCase-specific
primer (primers 18 and 12, respectively, ), designed to
yield a 5.1-kb PCR product. The primer set for CPSase III consisted of
upstream and downstream primers (primers 20 and 21, respectively,
), designed to yield a 4.5-kb PCR product. Equal quantities
of liver and testis cDNA (1 µl from 50 µl of effluent as
described above) were used as templates for both the CPSase
III-specific primer set and the CAD-specific primer set. Since other
less sensitive methods have shown no expression of CAD gene in liver,
it was important to determine what level of dilution of testis
poly(A)
RNA could be detected using the CAD-specific
primers. To determine this, 1 µl of liver cDNA was mixed with 1
µl of a serial dilution of testis cDNA, from 1:10 to 1:100,000, and
these mixtures were used as templates for the PCR employing
CAD-specific primers. Some modifications in the PCR described by
Barnes(1994) to amplify long sequences of DNA (Barnes, 1994) were
utilized here; 5 units of Taq DNA polymerase (Perkin-Elmer)
and 1 unit of recombinant Pfu DNA polymerase (Stratagene) were included
in each 50-µl reaction mixture. For the PCRs using the CAD-specific
primer set, the program was: 1 cycle of 5 min at 94 °C, 1 min at 54
°C, and 10 min at 72 °C; 28 cycles of 0.5 min at 94 °C, 1
min at 54 °C, and 10 min at 72 °C with an increase of 10 s each
cycle; 1 cycle of 0.5 min at 94 °C, 1 min at 54 °C, and 30 min
at 72 °C. The program for the PCRs using the CPSase III-specific
primer set was similar except that the annealing temperature was
increased to 60 °C.
Sequencing Strategy
The sequencing strategy for
the entire shark CAD cDNA is shown in Fig. 1. The primers used
are listed in . Primers 8-13 and 18 are specific
primers based on regions of DNA sequenced as described above (segments
1-2, 3-4, and 5-6 as shown in Fig. 1). Primers
7 and 9 are consensus primers based on the conserved regions apparent
from the three known CAD sequences from hamster (Simmer et
al., 1990; Bein et al., 1991), Drosophila (Freund and Jarry, 1987), and Dictyostelium (Faure et
al., 1989). Touchdown PCR was carried out to obtain DNA between
primers 7 and 8 and between primers 9 and 10. One µl of purified
shark testis cDNA was used as template in each reaction. The segment
between primers 11 and 12 was amplified by standard PCR procedures. The
cDNA corresponding to the 3`-UTR and part of the ATCase sequence of
shark CAD mRNA (piece 13-14 in Fig. 1) was amplified by 3`
rapid amplification of cDNA ends (RACE) PCR. The template for 3` RACE
PCR was 1 µl of testis cDNA synthesized from primer 19, the
lock-docking primer (Borson et al., 1992) following the same
procedures as described above. The primers for 3` RACE PCR were primers
13 and 14 (a specific primer for the multiple cloning site of primer
19).
Figure 1:
Strategy
for sequencing the complete cDNA of shark CAD. The arrangement of the
5`-UTR, CPSase II, DHOase, and ATCase coding regions and 3`-UTR of
shark CAD mRNA is shown in the bar above. The region between
DHOase and ATCase represents the sequence of the interdomain linker.
The cDNA made from CAD mRNA was used as template for the PCRs. The
position and direction of each primer for PCR or cDNA synthesis is
represented by an arrowhead. Each primer is numbered and the
sequence of every primer is listed in Table I. The line between two primers represents the PCR product amplified by the
primers. The overlap of lines represents the overlap of two strands of
DNA sequence.
The PCR products (pieces 7-8, 9-10, 11-12,
and 13-14) were purified from the gel, cloned, and sequenced as
described above for pieces 1-2, 3-4, and 5-6. One
clone containing each PCR product was sequenced.
terminal deoxynucleotidyl transferase buffer (500
mM cacodylate (pH 6.8), 5 mM CoCl
, 0.5
mM dithiothreitol), 2 µl 10 mM dATP, and 2 µl
of terminal deoxynucleotide transferase (20 units/µl, Promega).
After incubation at 37 °C for 15 min and then at 65 °C for 15
min to denature the enzyme, the reaction mixture was applied to and
eluted from a Sephadex G-50 column. One µl of the effluent was used
as template in the first round of 5` RACE PCR. Lock-docking primer 19
(50 pmol) and 50 pmol of primer 17, which is nested to primer 16 and
designed on the basis of the sequence of piece 7-8, were used as
primers. The program was: 1 cycle of 5 min at 94 °C, 1 min at 37
°C, and 1 min and 15 s at 72 °C (the ramp time between 37 and
72 °C was set at 1 min to minimize the detachment of the
lock-docking primer); then 24 cycles of 1 min at 94 °C, 1 min at 37
°C, and 1 min and 15 s at 72 °C (the ramp time between 37 and
72 °C remained 1 min, and the extension time increased 3 s every
cycle). For the second round of 5` RACE PCR, 1 µl of the first
round product was used as template, and 50 pmol each of primers 14 and
15 (a primer nested to primer 17 and designed based on the sequence of
piece 7-8) were used as primers.
-
P]ATP (3000
Ci/mmol, 10 mCi/ml, Amersham). The sequence from both strands of DNA
was obtained.
Probes Specific for CPSase II, ATCase, and
DHOase
Using shark testis cDNA as template for the PCR, products
of 580, 590, and 220 base pairs were obtained using consensus primers
for CPSase II, DHOase, and ATCase, respectively. The size of the
products corresponded to the respective predicted sizes based on the
reported cDNA sequences between the respective consensus primers for
these enzymes from other species. The base sequences of these products
were determined, and the predicted amino acid sequences were found to
compare favorably with the corresponding regions of the previously
published sequences. For example, these sequences were found to have
83, 71, and 81% identity with the corresponding sequences of hamster
CAD CPSase II, DHOase, and ATCase, respectively. However, in each case,
unique sequences not found in any of the other enzymes were present in
each product. The shark CPSase II region shares high identity (67%)
with the corresponding region of shark CPSase III, but the fact that
they are different excludes the possibility that the product was
derived from CPSase III mRNA. These results strongly indicate that the
products of the PCR using the consensus primers represent partial
sequences for shark CPSase II, DHOase, and ATCase, respectively. Based
on these sequences, P-labeled probes specific for shark
CPSase II, DHOase, and ATCase were then prepared by the PCR using
testis cDNA as template and three different sets of specific primers.
These probes were then used for Northern blot analysis and RNase H
mapping.
CPSase, DHOase, and ATCase in Testes Are Encoded by a
Single Transcript
As shown in Fig. 2A (lane
2), B (lane 1), and C (lane
2), Northern blot analysis revealed a predominant band of the same
size when poly(A) RNA from shark testis was hybridized
with either of the three different probes specific for shark CPSase II,
DHOase, and ATCase, respectively. The large size of the transcript
(about 8.8 kb) is similar to that of the 7.5-kb hamster CAD transcript
(Bein et al., 1991). These results provide strong evidence
that these three enzymes are expressed as a single transcript analogous
to CAD. We also observed that the ATCase- and CPSase II-specific probes
hybridized to a single transcript of about 14 kb in one Northern blot
experiment using mRNA from another individual shark.
(
)
The explanation for this observed difference in transcript
size is not known but could be the result of multiple transcription
termination sites, alternative splicing, or RNA degradation.
Figure 2:
Northern blot analysis. Results from using
CPSase II-, DHOase-, and ATCase-specific probes are shown in
A, B, and C, respectively. Lane 1 of both A and C were loaded with 9 µg each
of DHOase-specific oligomer-directed RNase H-digested shark testis
poly(A) RNA. Lane 2 of A and C and lane 1 of B were loaded with 5.4 µg each of
testis poly(A)
RNA. Lane 3 of A and
C and lane 2 of B were loaded with 5.4
µg each of liver poly(A)
RNA. The indicated sizes
of 8.8, 4.8, and 3.7 kb were determined in reference to RNA molecular
weight standards subjected to electrophoresis on the same
gel.
The
results of RNase H mapping provided confirming evidence that the three
enzymes are encoded in a single transcript. A deoxyoligonucleotide
complementary to a segment of DHOase message was hybridized to shark
testis mRNA, and the hybridized product was subsequently treated with
RNase H, which only hydrolyzes the portion of the RNA strand present as
a double stranded RNA/DNA heteroduplex. Northern blot analysis of the
products of the reaction with RNase H yielded a predominant 4.8-kb band
(Fig. 2A, lane 1) when the P-labeled CPSase
II-specific probe was used for hybridization and a predominant 3.7-kb
band (Fig. 2C, lane 1) when the
P-labeled
ATCase-specific probe was used for hybridization. The sum of these two
products (8.5 kb) is very close to the size of the original transcript
(8.8 kb). In addition to confirming that these three enzymes are
encoded in a single transcript, these results indicate that the region
of the mRNA corresponding to DHOase is located in the middle of the
transcript between CPSase II and ATCase, as found in hamster CAD mRNA
(Simmer et al., 1990).
Sequence of the CAD Transcript
The strategy and
method for obtaining the entire shark CAD cDNA sequence is explained
under ``Materials and Methods.'' The entire sequence of 8781
bases, including the 5`-UTR, the open reading frame, and the 3`-UTR is
shown in Fig. 3. The open reading frame was identified by
comparing the derived amino acid sequence with that of hamster CAD as
shown in Fig. 4. There are four other ATG start codons upstream
of the suggested start codon at 336-338. The first three
potential ATG start codons at 5-7, 19-21, and 51-53
can be excluded, since UGA stop codons would be encountered at
20-22, 52-54, and 159-161, respectively. The fourth
ATG at 309-311 is in frame with the derived amino acid sequence
shown in Fig. 4, but if translation started with this codon, the
amino acid sequence MPGRVVPEE would be added to the N terminus of the
shark CAD amino acid sequence shown in Fig. 4. The ATG at
336-338 is suggested as the translation start codon on the basis
of the sequence alignment with hamster CAD. However the N terminus of
purified shark CPSase II has not been sequenced to verify this
suggestion. The open reading frame terminates at the stop codon UGA at
7062-7064. The 3`-UTR extends from 7065-8781. The
polyadenylation signal AAUAAA is located at 8759-8764, which is
17 residues upstream of the poly(A) addition site.
Figure 3:
Nucleotide sequence of shark CAD cDNA. The
sequence is that of the sense strand. The open reading frame appears as
uppercase letters. The 5`- and 3`-UTR appear as lowercase
letters. The putative start codon ATG and stop codon TGA appear as
bold letters. The other ATG sequences in the 5`-UTR are
underlined. The polyadenylation signal AATAAA sequence is
bold and underlined. (A) indicates the poly(A) tract.
Figure 4:
Aligned amino acid sequences of hamster
and spiny dogfish shark CAD. The identical residues of hamster and
shark CADs are indicated by shaded bases. The proposed
sequences comprising glutaminase, synthetase, DHOase, and ATCase
domains are indicated by``- - - -
and - - - -
''. The conserved
cysteine residue essential for glutaminase activity is identified by
. The positions at which one or both of a pair of cysteine
residues related to the function of acetylglutamate-dependent CPSases I
and III are replaced by other residues in CPSase II are identified by
▾. The putative cyclic AMP-dependent protein kinase
phosphorylation site 1 and site 2 are labeled by a single line and a double line above the alignment, respectively. The
five candidates for zinc-binding residues in DHOase are identified by
an asterisk.
The derived amino
acid sequence has 2242 residues, with a calculated molecular mass of
249 kDa. This sequence has 77, 52, and 58% identity to the published
CAD sequences from hamster (Simmer et al., 1990 and Bein
et al., 1991), Drosophila (Freund and Jarry, 1987)
and Dictyostelium (Faure et al., 1989), respectively.
The identification of the interdomain linker regions between the CPSase
II, DHOase, and ATCase domains of the shark CAD shown in
Fig. 4
is based on the domain structural organization of hamster
CAD (Kim et al., 1992).
can be identified by sequence
alignment with other glutamine-dependent CPSases as the cysteine
residue required for formation of the
-glutamyl thioester
intermediate, a common feature in the mechanism of all
amidotransferases and required for glutamine-dependent CPSase activity
(Zalkin, 1993). Also like other CPSases, the CPSase synthetase domain
of the shark CAD contains two homologous halves (confirmed by dot
matrix analysis; Nyunoya and Lusty, 1983); the N-terminal half extends
from Lys
to Lys
and the C-terminal half
from Pro
to Ser
. Alignment analysis of
these two halves shows 28% identity and 51% similarity in the amino
acid sequences. Two specific cysteine residues in the C-terminal half
of the synthetase domain have been identified as apparently
distinguishing and conserved features of the
N-acetyl-L-glutamate-dependent CPSases I and III
(Hong et al., 1994). As with other CPSases that do not require
N-acetyl-L-glutamate for activity, both of these
cysteine residues are not present in the shark CAD CPSase II; although
one cysteine is retained (Cys
), Val
substitutes for cysteine at the position expected for a cysteine
in CPSases I or III. Two cyclic AMP-dependent protein kinase
phosphorylation sites have been identified in hamster CAD (Carrey and
Hardie, 1988). One is located at the C-terminal end of the CPSase
synthetase domain. The shark CAD sequence Arg-Arg-Leu-Ser at this
location(1410-1413) fits the consensus sequence Arg-Arg-Xaa-Ser
for phosphorylation by cyclic AMP-dependent protein kinases (Cohen,
1985).
to Arg
. This
domain has been shown to have a zinc-binding site in hamster CAD (Kelly
et al., 1986). Five conserved histidine residues have been
suggested to be candidates for the zinc-binding residues in the DHOase
domain of hamster CAD (Quinn et al., 1991). These residues are
conserved in the shark CAD DHOase domain (His
,
His
, His
, His
, and
His
).
and ends at Phe
. Between the DHOase and ATCase
domains, there is a large interdomain linker region, from Gly
to Leu
. The second phosphorylation site in hamster
CAD has been identified as a His-Arg-Ala-Ser sequence present within
this interdomain linker region (Carrey, 1992); this sequence differs
from the phosphorylation consensus sequence Arg-Arg-Xaa-Ser by the
presence of a histidine residue in place of the more usual arginine
residue at the third position N-terminal to the phosphorylated serine
in hamster CAD (Carrey, 1992). The hamster CAD sequence His-Arg-Xaa-Ser
is conserved in the shark CAD(1874-1877).
Tissue-specific Expression of CAD
Using the same
total amount of poly(A) RNA (5.4 µg), shark CAD
mRNA could be detected by Northern blot analysis of poly(A)
RNA isolated from testes using probes specific for CPSase II,
DHOase, or ATCase (Fig. 2A (lane 2), B (lane 1), and C (lane 2),
respectively), but shark CAD mRNA could not be detected in
poly(A)
RNA isolated from liver, regardless of which
probe was used (Fig. 2A (lane 3), B (lane 2), C (lane 3), respectively).
When the same amount of liver and testis poly(A)
RNA
(5.4 µg) was hybridized with a CPSase III-specific probe, as shown
in Fig. 5, a high concentration of the CPSase III transcript was
detected in liver poly(A)
RNA (even after 1-100
dilution of the liver poly(A)
RNA), as expected, but
CPSase III mRNA could not be detected in testis poly(A)
RNA, also as expected. The size of the CPSase III mRNA is about
6.2 kb, which is in agreement with previous results (Hong et
al., 1994).
Figure 5:
Northern blot analysis. Shark liver or
testis poly(A) RNA was hybridized with the CPSase
III-specific probe. Lanes 1 and 5 were loaded with
5.4 µg each of liver and testis poly(A)
RNA,
respectively. Lanes 2, 3, and 4 were loaded with
1/10, 1/100, and 1/1000 as much liver poly(A)
RNA as
lane 1.
Analysis by the more sensitive PCR as described
under ``Materials and Methods'' provided confirmation of
these results (Fig. 6). With the CAD-specific primers the PCR
gave a product of the correct size (5.1 kb) using testis cDNA as
template (lane 2), but no product was obtained when liver cDNA
at the same concentration was used as template (lane 8). When
1 µl of testis cDNA, serially diluted from 1:10 to 1:100,000, was
added to the liver cDNA reaction mixture, the subsequent PCR with the
CAD-specific primers yielded the 5.1-kb product, even after dilution of
the testis cDNA up to 1:10,000 (lanes 3-6). These
results demonstrate that the PCR method can amplify a concentration of
CAD cDNA as low as 0.01% of that in testis in the presence of total
liver cDNA and indicate that the CAD mRNA is not expressed at
detectable levels in liver. In the reverse of these experiments, with
the CPSase III-specific primer set, the PCR gave a product of the
correct size (4.6 kb) using liver cDNA as template (lane 10),
but no product was obtained with testis cDNA as template (lane
11).
Figure 6:
Assessment of tissue-specific expression
by PCR analysis. PCRs were carried out using a CAD-specific primer set
(lanes 2-9) or a CPSase III-specific primer set
(lanes 10-12). As templates, 1 µl of testis cDNA
(lanes 2 and 11), 1 µl of liver cDNA (lanes 8 and 10), 1 µl of liver cDNA plus 1 µl of serially
diluted testis cDNA (1/10 to 1/100,000, lanes 3-7,
respectively), and 1 µl of HO (lanes 9 and
12) were employed. HindIII-cut
DNA was used as
molecular weight standard (lane 1).
and Lys
in the shark
CPSase III sequence). We have also found this to be true for the CPSase
III and II sequences in the teleost fish Oncorhynchus mykiss (rainbow trout).
(
)
This is of significance,
because it provides an opportunity for selectively detecting the
expression of low levels of CPSase III mRNA in the presence of CPSase
II mRNA by probe-specific hybridization methodologies (e.g. Northern blot analysis or nuclease protection assays) or by the
PCR, yielding products of different size.
represents a 16-mer of deoxyoligothymidine.
/EMBL Data Bank with accession number(s) U18868.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.