Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

Yamauchi, Masatake; Yamauchi, Noriko; Meuth, Mark

doi:10.1002/j.1460-2075.1990.tb07377.x

Cited by 46 publications

(18 citation statements)

References 26 publications

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“…The most frequent mutations in the clustered region of the enzyme were glutamate to lysine and histidine to lysine (47). The clustered sites are in a highly conserved region of the CTP synthetases from human cells (48), E. coli (49), Chlamydia trachomatis (50), Bacillus subtilis (51), and S. cerevisiae (7,8). Based on this information, we hypothesized that the two amino acids that are most frequently mutated in Chinese hamster ovary cells (47) would be involved in the regulation of the S. cerevisiae CTP synthetases by CTP.…”

Section: Resultsmentioning

confidence: 97%

Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae

Ostrander¹,

O'Brien²,

Gorman³

et al. 1998

Journal of Biological Chemistry

125

142

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CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu 161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu 161 3 Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4-and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6 -15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1.3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADPforming)) is a cytosolic-associated glutamine amidotransferase that catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP (1, 2). This enzyme plays an essential role in the synthesis of all membrane phospholipids in eukaryotic cells (3, 4). Its reaction product CTP is the direct precursor of the activated, energyrich phospholipid pathway intermediates CDP-DG 1 (5), CDPcholine (6), and CDP-ethanolamine (6) (Fig. 1). CDP-DG is the source of the phosphatidyl moiety of PS, PE, and PC synthesized by the CDP-DG pathway as well as PI, phosphatidylglycerol, and cardiolipin (3, 4). CDP-choline and CDP-ethanolamine are the sources of the hydrophilic head groups of PC and PE synthesized by the Kennedy pathways, respectively (3, 4). Our laboratory utilizes the yeast Saccharomyces cerevisiae as a model eukaryote to study the regulation of CTP synthetase and its impact on phospholipid metabolism. CTP synthetase is encoded by ...

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Section: Resultsmentioning

confidence: 97%

Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae

Ostrander¹,

O'Brien²,

Gorman³

et al. 1998

Journal of Biological Chemistry

125

142

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“…1A). Similarly, UMPS and CTPS encode the enzymes UMP synthase (Suttle et al 1988) and CTP synthase (Yamauchi et al 1990), which are essential for the production of CTP nucleotides used in the synthesis of DNA. These two genes are positively correlated in the three populations (r CEPH-Utah = 0.69; r ASN = 0.64; r YRI = 0.68; test of homogeneity, P = 0.80), and the weighted average correlation is 0.67 (Fig.…”

Section: Gene Coexpression Networkmentioning

confidence: 99%

Coexpression network based on natural variation in human gene expression reveals gene interactions and functions

et al. 2009

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Genes interact in networks to orchestrate cellular processes. Analysis of these networks provides insights into gene interactions and functions. Here, we took advantage of normal variation in human gene expression to infer gene networks, which we constructed using correlations in expression levels of more than 8.5 million gene pairs in immortalized B cells from three independent samples. The resulting networks allowed us to identify biological processes and gene functions. Among the biological pathways, we found processes such as translation and glycolysis that co-occur in the same subnetworks. We predicted the functions of poorly characterized genes, including CHCHD2 and TMEM111, and provided experimental evidence that TMEM111 is part of the endoplasmic reticulum-associated secretory pathway. We also found that IFIH1, a susceptibility gene of type 1 diabetes, interacts with YES1, which plays a role in glucose transport. Furthermore, genes that predispose to the same diseases are clustered nonrandomly in the coexpression network, suggesting that networks can provide candidate genes that influence disease susceptibility. Therefore, our analysis of gene coexpression networks offers information on the role of human genes in normal and disease processes.

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“…We showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in S. cerevisiae and could rescue the lethal phenotype of the yeast ura7⌬ ura8⌬ double mutant that lacked CTP synthetase activity. In previous studies, CTPS1 and CTPS2 were identified as genes encoding human CTP synthetase enzymes by genetic complementation of mutant (mouse or E. coli) cells defective in CTP synthetase activity (49,50). However, the direct gene-enzyme relationship for the human CTP synthetases had not been established.…”

Section: Discussionmentioning

confidence: 99%

“…Genes encoding CTP synthetase have been isolated from a variety of bacteria (23,(45)(46)(47)(48), yeast (6,7), and human (49,50). Owing to the relatively high degree of deduced amino acid sequence identity (ϳ53%) between the yeast and human enzymes, we examined the hypothesis that the human CTPS1 and CTPS2 genes are functionally expressed in S. cerevisiae.…”

mentioning

confidence: 99%

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A

Han

Sreenivas

Choi

et al. 2005

Journal of Biological Chemistry

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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 ura7⌬ ura8⌬ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7⌬ ura8⌬ 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 32 P i -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). The end product CTP is required for the synthesis of nucleic acids and membrane phospholipids (2). Thus, CTP synthetase is an essential enzyme for the growth and metabolism of all organisms (2). In eukaryotes, CTP synthetase activity regulates the balance of nucleotide pools (3-9) and influences the pathways by which membrane phospholipids are synthesized (9 -11). 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)(13)(14)(15)(16)(17)(18)(19)(20).CTP synthetase has been purified and characterized from bacteria (21-23), Saccharomyces cerevisiae (8, 24), and rat liver (25). In addition, crystal structures for the Escherichia coli (26) and Thermus thermophilus (27) enzymes have been solved. The enzymological properties of CTP synthetase enzymes from various sources are similar, although some differences have been identified (23). 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, 28). GTP activates the glutaminase reaction by accelerating the formation of a covalent glutaminyl enzyme intermediate (21,29). CTP synthetase exhibits positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP (8, 21, 23, 24, 29 -33). The positive c...

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Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

Cited by 46 publications

References 26 publications

Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae

Effect of CTP Synthetase Regulation by CTP on Phospholipid Synthesis in Saccharomyces cerevisiae

Coexpression network based on natural variation in human gene expression reveals gene interactions and functions

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A

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