©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nucleotide Sequence and Tissue-specific Expression of the Multifunctional Protein Carbamoyl-phosphate Synthetase-Aspartate Transcarbamoylase-Dihydroorotase (CAD) mRNA in Squalus acanthias(*)

Jin Hong , Wilmar L. Salo , Paul M. Anderson(§)

From the (1) Department of Biochemistry and Molecular Biology, University of Minnesota, Duluth, Minnesota 55812

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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-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.


INTRODUCTION

Two different types of carbamoyl-phosphate synthetases (CPSases)() 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).

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.() 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.


MATERIALS AND METHODS

Isolation of Poly(A)RNA

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 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.

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 S as a label (5`-[-S]dATP, 1000 Ci/mmol, purchased from Amersham Corp.).

Preparation of P-Labeled Probes

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; 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.

A more sensitive determination of expression was accomplished using RT-PCR. The synthesis of cDNA from liver or testis poly(A) 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.

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 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.

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 [-P]ATP (3000 Ci/mmol, 10 mCi/ml, Amersham). The sequence from both strands of DNA was obtained.

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.


RESULTS

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).

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 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).

The DHOase domain begins immediately after the synthetase domain, extending from Met 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).

The ATCase domain begins at Leu 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).




DISCUSSION

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 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.

  
Table: Sequences of primers

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) represents a 16-mer of deoxyoligothymidine.



FOOTNOTES

*
This research was supported by National Science Foundation Grant DCB-9105797. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U18868.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 10 University Dr., University of Minnesota, Duluth, MN 55812-2487. Tel.: 218-726-7921; Fax: 218-726-8014; E-mail: panderso@d.umn.edu.

The abbreviations used are: CPSase, carbamoyl-phosphate synthetase; DHOase, dihydroorotase; ATCase, aspartate transcarbamoylase; PCR, polymerase chain reaction; RT, reverse transcription; RACE, rapid amplification of cDNA ends; UTR, untranslated region; CAD, carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase; kb, kilobase(s).

P. M. Anderson, unpublished observations.

W. L. Salo, unpublished observation.

W. L. Salo, J. J. Korte, and P. M. Anderson, unpublished observations.


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