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• W2124016711 abstract "CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1- and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase 1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) catalyzes the final step in the pyrimidine biosynthetic pathway (1.Evans D.R. Guy H.I. J. Biol. Chem. 2004; 279: 33035-33038Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). The end product CTP is required for the synthesis of nucleic acids and membrane phospholipids (2.Stryer L. Biochemistry. Fourth Ed. W.H. Freeman and Company, New York1995Google Scholar). Thus, CTP synthetase is an essential enzyme for the growth and metabolism of all organisms (2.Stryer L. Biochemistry. Fourth Ed. W.H. Freeman and Company, New York1995Google Scholar). In eukaryotes, CTP synthetase activity regulates the balance of nucleotide pools (3.Aronow B. Ullman B. J. Biol. Chem. 1987; 262: 5106-5112Abstract Full Text PDF PubMed Google Scholar, 4.Robert de Saint Vincent B. Buttin G. Biochim. Biophys. Acta. 1980; 610: 352-359Crossref Scopus (34) Google Scholar, 5.Meuth M. L'Heureux-Huard N. Trudel M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6505-6509Crossref PubMed Scopus (89) Google Scholar, 6.Ozier-Kalogeropoulos O. Fasiolo F. Adeline M.-T. Collin J. Lacroute F. Mol. Gen. Genet. 1991; 231: 7-16Crossref PubMed Scopus (47) Google Scholar, 7.Ozier-Kalogeropoulos O. Adeline M.-T. Yang W.-L. Carman G.M. Lacroute F. Mol. Gen. Genet. 1994; 242: 431-439Crossref PubMed Scopus (54) Google Scholar, 8.Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Crossref PubMed Scopus (42) Google Scholar, 9.Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and influences the pathways by which membrane phospholipids are synthesized (9.Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 10.Hatch G.M. McClarty G. J. Biol. Chem. 1996; 271: 25810-25816Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11.McDonough V.M. Buxeda R.J. Bruno M.E.C. Ozier-Kalogeropoulos O. Adeline M.-T. McMaster C.R. Bell R.M. Carman G.M. J. Biol. Chem. 1995; 270: 18774-18780Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The importance of understanding the mode of action and regulation of CTP synthetase is further highlighted by the fact that elevated CTP synthetase activity is a common property of several cancers in humans (12.van den Berg A.A. van Lenthe H. Busch S. de Korte D. Roos D. van Kuilenburg A.B.P. van Gennip A.H. Eur. J. Biochem. 1993; 216: 161-167Crossref PubMed Scopus (54) Google Scholar, 13.van den Berg A.A. van Lenthe H. Kipp J.B. de Korte D. Van Kuilenburg A.B. van Gennip A.H. Eur. J. Cancer. 1995; 31A: 108-112Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 14.Verschuur A.C. van Gennip A.H. Muller E.J. Voute P.A. Van Kuilenburg A.B. Adv. Exp. Med. 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Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 22.Anderson P.M. Biochemistry. 1983; 22: 3285-3292Crossref PubMed Scopus (62) Google Scholar, 23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar), Saccharomyces cerevisiae (8.Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Crossref PubMed Scopus (42) Google Scholar, 24.Nadkarni A.K. McDonough V.M. Yang W.-L. Stukey J.E. Ozier-Kalogeropoulos O. Carman G.M. J. Biol. Chem. 1995; 270: 24982-24988Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and rat liver (25.Thomas P.E. Lamb B.J. Chu E.H.Y. Biochim. Biophys. Acta. 1988; 953: 334-344Crossref PubMed Scopus (24) Google Scholar). In addition, crystal structures for the Escherichia coli (26.Endrizzi J.A. Kim H. Anderson P.M. Baldwin E.P. Biochemistry. 2004; 43: 6447-6463Crossref PubMed Scopus (90) Google Scholar) and Thermus thermophilus (27.Goto M. Omi R. Nakagawa N. Miyahara I. Hirotsu K. Structure (Camb.). 2004; 12: 1413-1423Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) enzymes have been solved. The enzymological properties of CTP synthetase enzymes from various sources are similar, although some differences have been identified (23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar). The enzyme catalyzes a complex set of reactions involving the ATP-dependent transfer of the amide nitrogen from glutamine (i.e. glutaminase reaction) to the C-4 position of UTP to generate CTP (Fig. 1) (21.Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 28.Liberman I. J. Biol. Chem. 1956; 222: 765-775Abstract Full Text PDF PubMed Google Scholar). GTP activates the glutaminase reaction by accelerating the formation of a covalent glutaminyl enzyme intermediate (21.Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 29.Levitzki A. Koshland Jr., D.E. Biochemistry. 1972; 11: 241-246Crossref PubMed Scopus (99) Google Scholar). CTP synthetase exhibits positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (8.Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Crossref PubMed Scopus (42) Google Scholar, 21.Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar, 24.Nadkarni A.K. McDonough V.M. Yang W.-L. Stukey J.E. Ozier-Kalogeropoulos O. Carman G.M. J. Biol. Chem. 1995; 270: 24982-24988Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 29.Levitzki A. Koshland Jr., D.E. Biochemistry. 1972; 11: 241-246Crossref PubMed Scopus (99) Google Scholar, 30.Levitzki A. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 1121-1128Crossref PubMed Scopus (354) Google Scholar, 31.Levitzki A. Koshland Jr., D.E. Biochemistry. 1971; 10: 3365-3371Crossref PubMed Scopus (89) Google Scholar, 32.Lewis D.A. Villafranca J.J. Biochemistry. 1989; 28: 8454-8459Crossref PubMed Scopus (44) Google Scholar, 33.von der Saal W. Anderson P.M. Villafranca J.J. J. Biol. Chem. 1985; 260: 14993-14997Abstract Full Text PDF PubMed Google Scholar). The positive cooperative kinetics toward UTP and ATP is attributed to the nucleotide-dependent tetramerization of the enzyme (8.Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Crossref PubMed Scopus (42) Google Scholar, 21.Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 34.Levitzki A. Koshland Jr., D.E. Biochemistry. 1972; 11: 247-252Crossref PubMed Scopus (81) Google Scholar, 35.Pappas A. Yang W.-L. Park T.-S. Carman G.M. J. Biol. Chem. 1998; 273: 15954-15960Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Indeed, the CTP synthetase tetramer is the active form of the enzyme (8.Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Crossref PubMed Scopus (42) Google Scholar, 21.Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar, 24.Nadkarni A.K. McDonough V.M. Yang W.-L. Stukey J.E. Ozier-Kalogeropoulos O. Carman G.M. J. Biol. Chem. 1995; 270: 24982-24988Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 29.Levitzki A. Koshland Jr., D.E. Biochemistry. 1972; 11: 241-246Crossref PubMed Scopus (99) Google Scholar, 30.Levitzki A. Koshland Jr., D.E. Proc. Natl. Acad. Sci. U. S. A. 1969; 62: 1121-1128Crossref PubMed Scopus (354) Google Scholar, 31.Levitzki A. Koshland Jr., D.E. Biochemistry. 1971; 10: 3365-3371Crossref PubMed Scopus (89) Google Scholar, 32.Lewis D.A. Villafranca J.J. Biochemistry. 1989; 28: 8454-8459Crossref PubMed Scopus (44) Google Scholar, 33.von der Saal W. Anderson P.M. Villafranca J.J. J. Biol. Chem. 1985; 260: 14993-14997Abstract Full Text PDF PubMed Google Scholar, 35.Pappas A. Yang W.-L. Park T.-S. Carman G.M. J. Biol. Chem. 1998; 273: 15954-15960Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The enzyme may also utilize dUTP for the synthesis of dCTP (23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar, 36.Pappas A. Park T.-S. Carman G.M. Biochemistry. 1999; 38: 16671-16677Crossref PubMed Scopus (14) Google Scholar). 2The major pathway for dCTP synthesis is the reaction sequence CTP → CDP → dCDP → dCTP (83.Traut T.W. Crit. Rev. Biochem. 1988; 23: 121-169Crossref PubMed Scopus (20) Google Scholar). An important mode of CTP synthetase regulation is feedback inhibition by CTP (8.Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Crossref PubMed Scopus (42) Google Scholar, 21.Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar, 24.Nadkarni A.K. McDonough V.M. Yang W.-L. Stukey J.E. Ozier-Kalogeropoulos O. Carman G.M. J. Biol. Chem. 1995; 270: 24982-24988Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 25.Thomas P.E. Lamb B.J. Chu E.H.Y. Biochim. Biophys. Acta. 1988; 953: 334-344Crossref PubMed Scopus (24) Google Scholar). CTP inhibits CTP synthetase activity by increasing the positive cooperativity of the enzyme for UTP (8.Yang W.-L. McDonough V.M. Ozier-Kalogeropoulos O. Adeline M.-T. Flocco M.T. Carman G.M. Biochemistry. 1994; 33: 10785-10793Crossref PubMed Scopus (42) Google Scholar, 21.Long C.W. Pardee A.B. J. Biol. Chem. 1967; 242: 4715-4721Abstract Full Text PDF PubMed Google Scholar, 24.Nadkarni A.K. McDonough V.M. Yang W.-L. Stukey J.E. Ozier-Kalogeropoulos O. Carman G.M. J. Biol. Chem. 1995; 270: 24982-24988Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 25.Thomas P.E. Lamb B.J. Chu E.H.Y. Biochim. Biophys. Acta. 1988; 953: 334-344Crossref PubMed Scopus (24) Google Scholar). dCTP does not substitute for CTP as a feedback inhibitor of CTP synthetase activity using dUTP or UTP as a substrate (23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar, 36.Pappas A. Park T.-S. Carman G.M. Biochemistry. 1999; 38: 16671-16677Crossref PubMed Scopus (14) Google Scholar). A defect in CTP feedback inhibition results in abnormally high intracellular levels of CTP and dCTP (4.Robert de Saint Vincent B. Buttin G. Biochim. Biophys. Acta. 1980; 610: 352-359Crossref Scopus (34) Google Scholar, 9.Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 37.Trudel M. van Genechten T. Meuth M. J. Biol. Chem. 1984; 259: 2355-2359Abstract Full Text PDF PubMed Google Scholar), resistance to nucleotide analog drugs used in cancer chemotherapy (38.Meuth M. Goncalves O. Thom P. Somat. Cell Genet. 1982; 8: 423-432Crossref PubMed Scopus (14) Google Scholar, 39.Aronow B. Watts T. Lassetter J. Washtien W. Ullman B. J. Biol. Chem. 1984; 259: 9035-9043Abstract Full Text PDF PubMed Google Scholar, 40.Kaufman E.R. Mutat. Res. 1986; 161: 19-27Crossref PubMed Scopus (22) Google Scholar, 41.Chu E.H.Y. McLaren J.D. Li I.-C. Lamb B. Biochem. Genet. 1984; 22: 701-715Crossref PubMed Scopus (20) Google Scholar), and an increased rate of spontaneous mutations (5.Meuth M. L'Heureux-Huard N. Trudel M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6505-6509Crossref PubMed Scopus (89) Google Scholar, 39.Aronow B. Watts T. Lassetter J. Washtien W. Ullman B. J. Biol. Chem. 1984; 259: 9035-9043Abstract Full Text PDF PubMed Google Scholar, 41.Chu E.H.Y. McLaren J.D. Li I.-C. Lamb B. Biochem. Genet. 1984; 22: 701-715Crossref PubMed Scopus (20) Google Scholar). Studies on the URA7-encoded enzyme from S. cerevisiae have revealed that CTP synthetase activity is regulated by phosphorylation. The yeast enzyme is phosphorylated on multiple serine residues in vivo (42.Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In vitro studies have shown that CTP synthetase is a substrate for protein kinases A (43.Yang W.-L. Carman G.M. J. Biol. Chem. 1996; 271: 28777-28783Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and C (42.Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 44.Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). These phosphorylations result in the stimulation of CTP synthetase activity by a mechanism that increases catalytic turnover (42.Yang W.-L. Carman G.M. J. Biol. Chem. 1995; 270: 14983-14988Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 43.Yang W.-L. Carman G.M. J. Biol. Chem. 1996; 271: 28777-28783Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 44.Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In addition, phosphorylation facilitates the nucleotide-dependent tetramerization of the enzyme (35.Pappas A. Yang W.-L. Park T.-S. Carman G.M. J. Biol. Chem. 1998; 273: 15954-15960Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and causes a decrease in the sensitivity of the enzyme to feedback inhibition by CTP (43.Yang W.-L. Carman G.M. J. Biol. Chem. 1996; 271: 28777-28783Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 44.Yang W.-L. Bruno M.E.C. Carman G.M. J. Biol. Chem. 1996; 271: 11113-11119Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Genes encoding CTP synthetase have been isolated from a variety of bacteria (23.Wadskov-Hansen S.L. Willemoes M. Martinussen J. Hammer K. Neuhard J. Larsen S. J. Biol. Chem. 2001; 276: 38002-38009Abstract Full Text Full Text PDF PubMed Google Scholar, 45.Weng M. Makaroff C.A. Zalkin H. J. Biol. Chem. 1986; 261: 5568-5574Abstract Full Text PDF PubMed Google Scholar, 46.Trach K. Chapman J.W. Piggot P. Lecoq D. Hoch J.A. J. Bacteriol. 1988; 170: 4194-4208Crossref PubMed Google Scholar, 47.Tipples G. McClarty G. J. Biol. 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Owing to the relatively high degree of deduced amino acid sequence identity (∼53%) between the yeast and human enzymes, we examined the hypothesis that the human CTPS1 and CTPS2 genes are functionally expressed in S. cerevisiae. We showed in this study that the CTPS1 or CTPS2 genes complemented the lethal phenotype of an ura7Δ ura8Δ mutant lacking CTP synthetase. In addition, expression of the CTPS1-encoded human CTP synthetase in yeast revealed that the enzyme was phosphorylated and regulated by protein kinase A. Materials—All chemicals were reagent grade. Growth medium supplies were purchased from Difco Laboratories. Restriction endonucleases, modifying enzymes, and recombinant Vent DNA polymerase were purchased from New England Biolabs. Plasmid DNA purification and DNA gel extraction kits and Ni2+-NTA 3The abbreviations used are: Ni2+-NTA, nickel-nitrilotriacetic acid; SC, synthetic complete; 5FOA, 5-fluoroorotic acid; PVDF, polyvinylidene difluoride. agarose resin were purchased from Qiagen. Oligonucleotides were prepared by Genosys Biotechnologies, Inc. The Yeast Maker yeast transformation kit was purchased from Clontech. Invitrogen was the source of the DNA size ladder used for agarose gel electrophoresis. Radiochemicals were purchased from PerkinElmer Life Sciences. Nucleotides, 5FOA, phenylmethylsulfonyl fluoride, benzamidine, aprotinin, leupeptin, pepstatin, tricarboxyethylphosphine, lyticase, bovine serum albumin, and standard phosphoamino acids were purchased from Sigma. Protein assay reagents, electrophoretic reagents, and protein standards were purchased from Bio-Rad. Mouse monoclonal anti-His6 antibodies were from Cell Signaling Technology. Alkaline phosphatase-conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from Pierce. Protein kinase A catalytic subunit (bovine heart) was purchased from Promega. Hybond-P PVDF membrane and the enhanced chemifluorescence Western blotting detection kit were purchased from GE Healthcare. An Ni2+-NTA column was obtained from Novagen. The Poros HQ column was purchased from Applied Biosystems. Cellulose thin layer glass plates were from EM Science. Scintillation counting supplies were purchased from National Diagnostics. Strains and Growth Conditions—The strains used in this work are listed in TABLE ONE. Yeast cells were grown in SC medium containing 2% glucose at 30 °C as described previously (51.Rose M.D. Winston F. Heiter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar, 52.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For selection of cells bearing plasmids, appropriate amino acids were omitted from the SC medium. Cells were also grown in YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or YEPA medium (1% yeast extract, 2% peptone, 2% acetate). Plasmid maintenance and amplifications were performed in E. coli strain DH5α. E. coli cells were grown in LB medium (1% Tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) at 37 °C. Ampicillin (100 μg/ml) was added to the growth medium for E. coli-carrying plasmids. Media were supplemented with either 2% (yeast) or 1.5% (E. coli) agar for growth on plates. Yeast cell numbers in liquid media were determined spectrophotometrically at A600 nm. For purification of the human CTP synthetase 1 from E. coli, BL21(DE3) cells bearing plasmid pET-28b(+)-hCTPS1 were grown in 6 liters of LB medium at 37 °C to a cell density of A600 = 0.15. The cells were then slowly cooled to 15 °C over a 2-h period until they reached a cell density of A600 = 0.3. At this point, the expression of human CTP synthetase 1 was induced by adding 0.5 mm β-isopropylthioglactoside to the culture. Maximum expression occurred after 16 h at 15 °C. Under these growth conditions, ∼50% of human CTP synthetase 1 protein was in the soluble fraction of the cell lysate.TABLE ONEStrains used in this workStrainRelevant characteristicsSource or Ref.E. coliDH5αF-, ϕ80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk- mk+), phoA, supE44, l- thi-1, gyrA96, relA1(52.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar)BL21(DE3)F- ompT hsdSB (rB- mB-) gal dcm (DE3)NovagenS. cerevisiaeSDO195MATa leu2-3, 112 trp1-289 ura3-52 ura7Δ::TRP1 ura8Δ::hisG [pDO134](9.Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar)GHY52MATa leu2-3, 112 trp1-289 ura3-52 ura7Δ::TRP1 ura8Δ::hisG [pDO134] [pDO105]This studyGHY53MATa leu2-3, 112 trp1-289 ura3-52 ura7Δ::TRP1 ura8Δ::hisG [pDO134] [pDO105-hCTPS1]This studyGHY54MATa leu2-3, 112 trp1-289 ura3-52 ura7Δ::TRP1 ura8Δ::hisG [pDO134] [pDO105-hCTPS2]This studyGHY55MATa leu2-3, 112 trp1-289 ura3-52 ura7Δ::TRP1 ura8Δ::hisG [pDO105-hCTPS1]This studyGHY56MATa leu2-3, 112 trp1-289 ura3-52 ura7Δ::TRP1 ura8Δ::hisG [pDO105-hCTPS2]This study Open table in a new tab DNA Manipulations, Amplification of DNA by PCR, and DNA Sequencing—Standard methods were used to prepare genomic and plasmid DNA, to digest DNA with restriction enzymes, and to ligate DNA (52.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Transformation of yeast (53.Ito H. Yasuki F. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar, 54.Schiestl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1776) Google Scholar) and E. coli (52.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) was performed according to standard protocols. PCR reactions were optimized as described by Innis and Gelfand (55.Innis M.A. Gelfand D.H. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego1990: 3-12Google Scholar). DNA sequencing reactions were performed by the dideoxy method using Taq polymerase (52.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and analyzed with an automated DNA sequencer. Construction of Plasmids—The plasmids used in this work are listed in TABLE TWO. To construct E. coli expression vectors for the human CTP synthetase genes, coding sequences for CTPS1 or CTPS2 were amplified by PCR using cDNA clones (Open Biosystems clones 3355881 and 5268973, respectively) as templates. The forward and reverse primers used in the amplification contained a DraI site and an XhoI site, respectively. Digestion of the PCR products (∼1.8 kb) with DraI and XhoI produced the CTPS1 sequence for codons 2–591 and a linker (Leu-Glu), and the CTPS2 sequence for codons 2–586 and a linker (Leu-Glu). The bacterial expression vector pET-28b(+), which contains the T7 lac promoter, was digested with NcoI, filled with T4 polymerase to provide a start codon, and digested with XhoI to provide the linker. DNA fragments of CTPS1 or CTPS2 were ligated to the linear pET-28b(+) to produce plasmids pET-28b(+)-hCTPS1 or pET-28b(+)-hCTPS2, respectively. The sequences for the human CTPS1 and CTPS2 genes in the bacterial expression vectors were confirmed by DNA sequencing. These plasmids directed the expression of full-length His6tagged (C terminus) versions of human CTP synthetase 1 or human CTP synthetase 2, respectively, in E. coli.TABLE TWOPlasmids used in this workPlasmidRelevant characteristicsSource or Ref.pET-28b(+)E. coli expression vector with the T7 lac promoter and C-terminal His6-tag fusionNovagenpET-28b(+)-hCTPS1CTPS1 derivative of pET-28b(+)This studypET-28b(+)-hCTPS2CTPS2 derivative of pET-28b(+)This studyYEpLac181Multicopy E. coli/yeast shuttle vector with LEU2(84.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar)YEpLac195Multicopy E. coli/yeast shuttle vector with URA3(84.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2528) Google Scholar)pDO105Derivative of YEpLac181 with the ADH1 promoter and multiple cloning sites(9.Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar)pDO134URA7 derivative of YEpLac195(9.Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar)pDO105-hCTPS1CTPS1 derivative of pDO105This studypDO105-hCTPS2CTPS2 derivative of pDO105This study Open table in a new tab To construct yeast expression vectors for the human CTPS1 or CTPS2 genes, their coding sequences were released from pET-28b(+)-hCTPS1 or pET-28b(+)-hCTPS2 by digestion with XbaI and XmnI. Two XbaI/XmnI fragments of similar size were differentiated by EcoRV treatment, which only cleaves the fragment that does not contain CTPS1 or CTPS2 coding sequences. Plasmid pDO105, a yeast expression vector with the ADH1 promoter, was digested with MluI, filled with Klenow, and digested with XbaI. The XbaI/XmnI DNA fragments (∼2.7 kb) containing the CTPS1 or CTPS2 coding sequences were ligated to the linear pDO105 to produce pDO105-hCTPS1 or pDO105-hCTPS2. These plasmids direct the expression of full-length His6-tagged (C terminus) versions of human CTP synthetase 1 or human CTP synthetase 2, respectively, in S. cerevisiae. Construction of S. cerevisiae Strains Expressing CTPS1 and CTPS2—Strain SDO195 is a ura7Δ ura8Δ mutant bearing plasmid pDO134, which contains a wild-type URA7 allele (9.Ostrander D.B. O'Brien D.J. Gorman J.A. Carman G.M. J. Biol. Chem. 1998; 273: 18992-19001Abstract Full Text Full Text PDF PubMed Scopus (100)" @default.
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• W2124016711 title "Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A" @default.
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