Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.5.5 (CPS)
 1,262 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced. This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A). The inferred amino acid sequence of the T. cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S. cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T. cutaneum argA gene. The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification. The T. cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi. A major transcript of around 4 kb was found in Northern (RNA) blots. The T. cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter. A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed. Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half. These domains are highly conserved among the known CPS-A sequences from S. cerevisiae, E. coli, and the rat. From these results we conclude that the T. cutaneum argA gene encodes the large subunit of CPS. This is the first gene to be identified and analyzed in the T. cutaneum DSM 70698 strain.
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PMID:Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations. 818 3

The human CGL-1/cytotoxic serine protease B gene (CSP-B; also known as granzyme B) is transcriptionally activated during cytotoxic T-lymphocyte maturation. Activation can be mimicked in the PEER T-cell leukemia cell line by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP). In this report, we show that a consensus AP-1 element and a consensus cAMP response element (CRE) located 5' to the CSP-B transcriptional start site are both required for transcriptional activation of the CPS-B promoter in TPA + bt2cAMP-stimulated PEER cells. A 94-bp fragment containing both elements activates a heterologous promoter in an orientation-independent fashion. Several single nucleotide substitutions in the AP-1 site abolish activity of the 94-bp fragment. Several point mutations in the consensus CRE substantially reduce promoter activity, but one CRE mutation increases activity fourfold. Replacement of the CRE with a second copy of the AP-1 site results in a level of transcriptional activity comparable with that of the wild-type sequence, but replacement of the AP-1 site with a CRE abolishes activity. Neither the AP-1 site nor the CRE can be effectively replaced with an SP-1 site. Deletions between the AP-1 site and the CRE retain full activity only if helical spacing is preserved, suggesting that synergism between these two elements is either the result of cooperative binding of factors to the DNA or of cooperative binding of DNA-bound factors to another protein.
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PMID:Consensus AP-1 and CRE motifs upstream from the human cytotoxic serine protease B (CSP-B/CGL-1) gene synergize to activate transcription. 821 27

1. Carbamoyl-phosphate synthetase (EC 6.3.5.5.) catalyses the synthesis of carbamoyl phosphate, the immediate precursor of arginine and pyrimidine biosynthesis, and is highly conserved throughout evolution. The large subunit of all carbamoyl-phosphate synthetases sequenced to date comprises two highly homologous halves, the product of a proposed ancestral gene duplication. The sequences of the enzymes of Escherichia coli, Drosophila melanogaster, Saccharomyces cerevisiae, rat and Syrian hamster all have duplications, suggesting that this event occurred in the progenote predating the separation of the major phylae. Until now, only limited data on carbamoyl-phosphate synthetase were available for the primitive eukaryote Dictyostelium discoideum and for the Archaea Methanosarcina barkeri MS. The DNA sequence of the D. discoideum carbamoyl-phosphate gene and additional sequence for the carbamoyl-phosphate synthetase gene of M. barkeri MS have been determined, and a duplicated structure for both is clearly demonstrated. 2. Genes with ancient duplications provide unique information on their evolution. A study of the intron/exon organization of the rat carbamoyl-phosphate synthetase I gene and the carbamoyl-phosphate synthetase hamster II gene in the CAD multi-gene complex shows that at least some of their introns are very old. Evidence is provided that some introns must have been present in the ancestral precursor before its duplication. 3. The human carbamoyl-phosphate synthetase I gene has been isolated and characterized. A human liver cDNA library was constructed and probed for carbamoyl-phosphate synthetase I. A human genomic DNA cosmid library was also probed for the carbamoyl-phosphate synthetase I gene. The cDNA sequence of the human carbamoyl-phosphate synthetase I gene has been determined, and work has been initiated to confirm that at least part of this gene is contained within two cosmids spanning 46kb. This will enable future studies to be made on mutations in this gene in the rare autosomal recessive deficiency of carbamoyl-phosphate synthetase I.
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PMID:Molecular studies on an ancient gene encoding for carbamoyl-phosphate synthetase. 838 76

We report the identification of Integration Host Factor (IHF) as a new element involved in modulation of P1, the upstream pyrimidine-specific promoter of the Escherichia coli K12 and Salmonella typhimurium carAB operons. Band-shift assays, performed with S-30 extracts of the wild type and a himA, hip double mutant or with purified IHF demonstrate that, in vitro, this factor binds to a region 300 bp upstream of the transcription initiation site of P1 in both organisms. This was confirmed by deletion analysis of the target site. DNase I, hydroxyl radical and dimethylsulphate footprinting experiments allowed us to allocate the IHF binding site to a 38 bp, highly A+T-rich stretch, centred around nucleotide -305 upstream of the transcription initiation site. Protein-DNA contacts are apparently spread over a large number of bases and are mainly located in the minor groove of the helix. Measurements of carbamoyl-phosphate synthetase (CPSase) and beta-galactosidase specific activities from car-lacZ fusion constructs of wild type or IHF target site mutants introduced into several genetic backgrounds affected in the himA gene or in the pyrimidine-mediated control of P1 (carP6 or pyrH+/-), or in both, indicate that, in vivo, IHF influences P1 activity as well as its control by pyrimidines. IHF stimulates P1 promoter activity in minimal medium, but increases the repressibility of this promoter by pyrimidines. These antagonistic effects result in a two- to threefold reduction in the repressibility of promoter P1 by pyrimidines in the absence of IHF binding. IHF thus appears to be required for maximal expression as well as for establishment of full repression. IHF could exert this function by modulating the binding of a pyrimidine-specific regulatory molecule.
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PMID:Integration host factor (IHF) modulates the expression of the pyrimidine-specific promoter of the carAB operons of Escherichia coli K12 and Salmonella typhimurium LT2. 845 62

The gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthetase, ARG2, of Magnaporthe grisea was characterized to examine the basic regulation of biosynthetic genes in this plant pathogen. The transcript of the ARG2 gene contains an upstream open reading frame (uORF) that is similar to uORFs found in the homologous genes of Neurospora crassa (arg-2) and Saccharomyces cerevisiae (CPA1), suggesting that the M. grisea gene is translationally regulated by a mechanism that is conserved in these fungi. Amino acid imbalance leads to elevated levels of ARG2 mRNA, indicating that in addition to translational control, ARG2 is subject to cross-pathway transcriptional control. A DNA-binding activity that has properties similar to those of the global transcriptional regulator mediating cross-pathway control in N. crassa was detected in M. grisea cell extracts. Thus, it appears that both specific regulation of ARG2 by arginine and global regulation of amino acid biosynthesis are present in M. grisea and highly conserved among M. grisea, N. crassa, and S. cerevisiae.
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PMID:Cross-Pathway and Pathway-Specific Control of Amino Acid Biosynthesis in Magnaporthe grisea 907 79

The gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthetase, ARG2, of Magnaporthe grisea was characterized to examine the basic regulation of biosynthetic genes in this plant pathogen. The transcript of the ARG2 gene contains an upstream open reading frame (uORF) that is similar to uORFs found in the homologous genes of Neurospora crassa (arg-2) and Saccharomyces cerevisiae (CPA1), suggesting that the M. grisea gene is translationally regulated by a mechanism that is conserved in these fungi. Amino acid imbalance leads to elevated levels of ARG2 mRNA, indicating that in addition to translational control, ARG2 is subject to cross-pathway transcriptional control. A DNA-binding activity that has properties similar to those of the global transcriptional regulator mediating cross-pathway control in N. crassa was detected in M. grisea cell extracts. Thus, it appears that both specific regulation of ARG2 by arginine and global regulation of amino acid biosynthesis are present in M. grisea and highly conserved among M. grisea, N. crassa, and S. cerevisiae.
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PMID:Cross-pathway and pathway-specific control of amino acid biosynthesis in Magnaporthe grisea. 912 16

Since various secretory stimuli regulate not only secretion but also protein, RNA, and DNA syntheses in salivary glands, we evaluated the effect of secretory stimuli on the phosphorylation state of CREB (cAMP response element-binding protein). Isoproterenol, forskolin, and CPS-cAMP markedly stimulated the phosphorylation of CREB in parotid acinar cells, and PKA inhibitors H-8 and H-89 dose-dependently inhibited it. In contrast, carbachol (CCH) and A23187 decreased CREB phosphorylation, but CCH did not decrease it in the absence of extracellular Ca2+. Although protein phosphatase inhibitor calyculin A alone markedly increased the phosphorylation, it could not prevent CCH-induced dephosphorylation of CREB. CaM kinase IV, a putative protein kinase for CREB in response to Ca2+ elevation, was undetectable in parotid acinar cells.
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PMID:Regulation of CREB phosphorylation by cAMP and Ca2+ in parotid acinar cells. 935 75

The arg2 gene which encodes the small subunit of carbamoyl phosphate synthetase for Trichoderma virens has been cloned and used to develop a homologous transformation system. A genomic clone containing the arg2 gene was isolated from a cosmid library of T. virens based on complementation of an arginine auxotrophic mutant of this fungus. The predicted amino acid sequence of the arg2 gene shows 56-82% identity with homologous polypeptides from other fungi. It also contains an upstream open reading frame which encodes 24 amino acids. As is observed with other gene sequences encoding this polypeptide in filamentous fungi, the N-terminus of the predicted polypeptide showed characteristic features of a mitochondrial signal sequence. The arg2 gene was used for genetic transformation of T. virens in frequencies of up to 370 transformants/microgram of DNA. Heat-shock treatment of T. virens protoplasts increased the transformation frequency by fivefold, but more than 85% of the transformants were abortive. Both single-copy, homologous integration events and ectopic, non-homologous integration events were detected by Southern analyses of genomic DNA from transformed strains.
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PMID:The arg2 gene of Trichoderma virens: cloning and development of a homologous transformation system. 950 76

A 25 kb segment of genomic DNA from Trypanosoma cruzi, the causative agent of Chagas' disease, was sequenced. It contains five genes, pyr1, pyr2, pyr3, pyr4, and pyr6-5, encoding all six enzymes involved in de novo pyrimidine biosynthesis, glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydroorotase, dihydroorotate dehydrogenase, and orotidine-5'-phosphate decarboxylase linked with orotate phosphoribosyltransferase, respectively. The pyr genes constitute a polycistronic transcription unit on an 800 kb chromosomal DNA in the order of pyr1, pyr3, pyr6-5, pyr2, and pyr4 from the 5' terminus, with intervening sequences of 2.2, 0.4, 8.1, and 0.8 kb. The amino acid sequences deduced from the trypanosomatid pyr genes, except for pyr6, showed closer similarities to mammalian and yeast sequences, and less similarity to archaeal and bacterial sequences. The last two enzymes encoded by a single gene, pyr6-5, are covalently linked in the order opposite to mammalian pyr5-6, and possess a putative glycosomal targeting signal tripeptide, serine-lysine-leucine, at the C terminus. The calculated isoelectric points of 9.3 and 9.9 are also diagnostic of the glycosomal localization of these enzymes. We conclude that the T. cruzi pyr gene organization represents an early progenitor in de novo pyrimidine biosynthesis in eukaryotic lineage, and that the independent pyr genes may have evolved before the gene fusion events that resulted in the three mammalian-type genes, pyr1-3-2, pyr4, and pyr5-6, for UMP synthesis. Peculiarities in the trypanosomatid pyr6-5 gene product are discussed.
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PMID:Novel organization and sequences of five genes encoding all six enzymes for de novo pyrimidine biosynthesis in Trypanosoma cruzi. 987 95

Hepatocyte proliferation and differentiation occur simultaneously during late mammalian gestation. We hypothesized that regulation of hepatocyte growth and differentiation would be coordinated in late gestation fetal hepatocyte cultures such that proliferation would be most active in a population of less well-differentiated cells. Cultured fetal hepatocytes (embryonic d 19 and 21; E19 and E21) were studied using double staining immunofluorescent microscopy. Differentiation was assessed as staining for alpha-fetoprotein (AFP), three markers of enzymic differentiation (glucokinase [GK], phosphoenolpyruvate carboxykinase [PEPCK], and carbamoyl phosphate synthase [CPS]), and a hepatocyte cell-cell adhesion molecule (C-CAM). Proliferation was assessed using immunocytochemical detection of proliferating cell nuclear antigen (PCNA) or 5-bromo-2'-deoxy-uridine (BrdU) incorporation into DNA. Fetal hepatocyte cultures consisted of a heterogeneous population of cells, slightly more than half of which were proliferative under defined, growth factor-free conditions. These cultures were heterogeneous for AFP expression. There was no correlation between the expression of AFP and PCNA or AFP and S-phase entry (BrdU staining) during the first 48 h in culture. Similar results were obtained in staining for the enzymic differentiation markers and C-CAM. In addition, the differentiation status of cultured fetal hepatocytes was unrelated to a presumed indicator of mature growth regulation, mitogenic responsiveness to transforming growth factor alpha (TGFalpha), and hepatocyte growth factor (HGF). Finally, absence of any correlation between proliferation and differentiated phenotype was supported by in vivo studies using staining for PCNA, AFP, CPS, and PEPCK in liver sections. These results indicate that the developmental program governing differentiation of late gestation fetal rat hepatocytes is independent from mechanisms controlling proliferation.
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PMID:The relationship between differentiation and proliferation in late gestation fetal rat hepatocytes. 1040 Jan 28


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