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1.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 68(Pt 11): 1341-5, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-23143245

ABSTRACT

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions of de novo pyrimidine biosynthesis: glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Šresolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a=82.1, b=159.3, c=61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.


Subject(s)
Aspartate Carbamoyltransferase/chemistry , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/chemistry , Dihydroorotase/chemistry , Aspartate Carbamoyltransferase/biosynthesis , Aspartate Carbamoyltransferase/isolation & purification , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/isolation & purification , Catalytic Domain , Chromatography, Affinity , Chromatography, Gel , Crystallization , Crystallography, X-Ray , Dihydroorotase/biosynthesis , Dihydroorotase/isolation & purification , Escherichia coli , Humans , Light , Protein Structure, Quaternary , Scattering, Radiation
2.
Gene ; 462(1-2): 18-25, 2010 Aug 15.
Article in English | MEDLINE | ID: mdl-20451592

ABSTRACT

In most prokaryotes and many eukaryotes, synthesis of carbamoylphosphate (CP) by carbamoylphosphate synthetase (CPSase; E.C. 6.3.5.5) and its allocation to either pyrimidine or arginine biosynthesis are highly controlled processes. Regulation at the transcriptional level occurs at either CPSase genes or the downstream genes encoding aspartate carbamoyltransferase (E.C. 2.1.3.2) or ornithine carbamoyltransferase (E.C. 2.1.3.3). Given the importance of pyrimidine and arginine biosynthesis, our lack of basic knowledge regarding genetic regulation of these processes in plants is a striking omission. Transcripts encoding two CPSase small subunits (MtCPSs1 and MtCPSs2), a single CPSase large subunit (MtCPSl), ACTase (MtPyrB), and OCTase (MtArgF) were characterized in the model legume Medicago truncatula. Quantitative real-time PCR data provided evidence (i) that the accumulation of all CPSase gene transcripts, as well as the MtPyrB transcript, was dramatically reduced following seedling incubation with uridine; (ii) exogenously supplied arginine down regulated only MtArgF; and (iii) mRNA levels of both CPSase small subunits, MtPyrB, and MtArgF were significantly increased after supplying plants with ornithine alone or in combination with uridine or arginine compared to plants treated with only uridine or arginine, respectively (P< or =0.05). A proposed novel, yet simple regulatory scheme employed by M. truncatula more closely resembles a prokaryotic control strategy than those used by other eukaryotes.


Subject(s)
Aspartate Carbamoyltransferase/biosynthesis , Genes, Fungal , Pyrimidines/biosynthesis , Arginine/biosynthesis , Arginine/genetics , Arginine/metabolism , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , Down-Regulation , Eukaryota , Genes, Regulator/physiology , Medicago truncatula/genetics , Medicago truncatula/metabolism , Plants/genetics , Plants/metabolism , Prokaryotic Cells/metabolism , Pyrimidines/metabolism , Uridine/biosynthesis , Uridine/genetics , Uridine/metabolism
3.
Nucleic Acids Res ; 33(16): 5190-8, 2005.
Article in English | MEDLINE | ID: mdl-16155188

ABSTRACT

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.


Subject(s)
Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Gene Silencing , Repressor Proteins/metabolism , Transcription Factors/metabolism , Aspartate Carbamoyltransferase/biosynthesis , Binding Sites , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Cell Cycle , Cell Hypoxia , Cell Line , Cells, Cultured , Deferoxamine/pharmacology , Dihydroorotase/biosynthesis , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Iron Chelating Agents/pharmacology , Promoter Regions, Genetic , RNA, Messenger/metabolism , Response Elements , Transcription, Genetic
4.
Yeast ; 19(5): 449-57, 2002 Mar 30.
Article in English | MEDLINE | ID: mdl-11921093

ABSTRACT

Computational analysis predicted three potential hydrophobic transmembrane alpha-helices within the Ura2 multidomain protein of Saccharomyces cerevisiae, the C-terminal subdomain of which catalyses the second step of uridine-monophosphate biosynthesis by its L-aspartate carbamoyltransferase activity (EC 2.1.3.2). The fourth step of pyrimidine biosynthesis is catalysed by dihydro-orotate dehydrogenase (Ura1 protein; EC 1.3.99.11), which was similarly characterized as a peripheral membrane protein. Ex situ, the activities of the investigated enzymes were associated both with isolated yeast membranes, fractionated by differential centrifugation to remove intact nuclei, and with soluble cytoplasmic proteins.


Subject(s)
Aspartate Carbamoyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Multienzyme Complexes/metabolism , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Aspartate Carbamoyltransferase/biosynthesis , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Cell Membrane/enzymology , Cytoplasm/enzymology , Dihydroorotate Dehydrogenase , Fluorescent Dyes/chemistry , Indoles/chemistry , Molecular Sequence Data , Multienzyme Complexes/biosynthesis , Oxidoreductases/biosynthesis , Protein Structure, Secondary , Saccharomyces cerevisiae/physiology
5.
J Bacteriol ; 183(9): 2785-94, 2001 May.
Article in English | MEDLINE | ID: mdl-11292797

ABSTRACT

The four genes pyrR, pyrP, pyrB, and carA were found to constitute an operon in Lactococcus lactis subsp. lactis MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon, pyrB and carA, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (pyrB) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (carA) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The carA gene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the pyrRPB-carA operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.


Subject(s)
Dioxygenases , Lactococcus lactis/genetics , Multigene Family/genetics , Oxygenases/genetics , Pentosyltransferases/genetics , Pyrimidines/biosynthesis , Repressor Proteins/genetics , Amino Acid Sequence , Aspartate Carbamoyltransferase/biosynthesis , Aspartate Carbamoyltransferase/genetics , Bacterial Proteins/genetics , Lactococcus lactis/metabolism , Membrane Transport Proteins/biosynthesis , Membrane Transport Proteins/genetics , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Open Reading Frames , Operon , Oxygenases/biosynthesis , Sequence Alignment , Transcription, Genetic
6.
Mol Carcinog ; 27(2): 84-96, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10657901

ABSTRACT

Although the Myc family of transcription factors is upregulated in many human tumors, it is unclear which genes are targets for the deregulated Myc. Previous studies suggest that hamster and rat carbamoyl phosphate synthase, aspartate transcarbamylase, dihydroorotase Cad genes are regulated by c-Myc. In fact, of all putative target genes thought to be activated by c-Myc, only the Cad gene showed loss of growth regulation in rat cells nullizygous for c-Myc. However, it was unknown whether upregulation of CAD, which performs the first three rate-limiting steps of pyrimidine biosynthesis, contributes to c-Myc's role in human neoplasia. To explore this possibility, we cloned the human cad promoter. We found that c-Myc could bind to an E box in the human cad promoter in gel shift assays and that growth regulated transcription from the human cad promoter was dependent on this c-Myc binding site. However, the increased amount of c-Myc found in Burkitt's lymphoma cell lines did not lead to increased cad mRNA levels. Thus, we suggest that although c-Myc is clearly important for the normal transcriptional control of the cad promoter, it is unlikely that increased levels of CAD are important mediators of c-Myc-induced neoplasia. Therefore, an understanding of the mechanism by which overexpressed c-Myc contributes to the development of Burkitt's lymphoma requires the identification of additional c-Myc target genes.


Subject(s)
Aspartate Carbamoyltransferase/genetics , Burkitt Lymphoma/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic , Genes, myc , Multienzyme Complexes/genetics , Neoplasm Proteins/genetics , 3T3 Cells , Animals , Aspartate Carbamoyltransferase/biosynthesis , Base Sequence , Burkitt Lymphoma/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Cloning, Molecular , Cricetinae , Dihydroorotase/biosynthesis , G1 Phase/genetics , Gene Targeting , Humans , Mice , Molecular Sequence Data , Multienzyme Complexes/biosynthesis , Neoplasm Proteins/biosynthesis , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Regulatory Sequences, Nucleic Acid , S Phase/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Tumor Cells, Cultured
7.
Protein Expr Purif ; 17(2): 312-23, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10545281

ABSTRACT

We have constructed an expression system for heterologous proteins which uses the molecular machinery responsible for the high level production of bacteriorhodopsin in Halobacterium salinarum. Cloning vectors were assembled that fused sequences of the bacterio-opsin gene (bop) to coding sequences of heterologous genes and generated DNA fragments with cloning sites that permitted transfer of fused genes into H. salinarum expression vectors. Gene fusions include: (i) carboxyl-terminal-tagged bacterio-opsin; (ii) a carboxyl-terminal fusion with the catalytic subunit of the Escherichia coli aspartate transcarbamylase; (iii) the human muscarinic receptor, subtype M1; (iv) the human serotonin receptor, type 5HT2c; and (v) the yeast alpha mating factor receptor, Ste2. Characterization of the expression of these fusions revealed that the bop gene coding region contains previously undescribed molecular determinants which are critical for high level expression. For example, introduction of immunogenic and purification tag sequences into the C-terminal coding region significantly decreased bop gene mRNA and protein accumulation. The bacteriorhodopsin-aspartate transcarbamylase fusion protein was expressed at 7 mg per liter of culture, demonstrating that E. coli codon usage bias did not limit the system's potential for high level expression. The work presented describes initial efforts in the development of a novel heterologous protein expression system, which may have unique advantages for producing multiple milligram quantities of membrane-associated proteins.


Subject(s)
Bacteriorhodopsins/genetics , Gene Expression Regulation, Bacterial , Halobacterium salinarum/genetics , Muscle Proteins , Amino Acid Sequence , Animals , Aspartate Carbamoyltransferase/biosynthesis , Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/isolation & purification , Blotting, Western , Cloning, Molecular , DNA, Recombinant/genetics , DNA-Binding Proteins , Electrophoresis, Polyacrylamide Gel , Escherichia coli/enzymology , Genetic Vectors , Halobacterium salinarum/chemistry , Halobacterium salinarum/ultrastructure , Humans , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Molecular Sequence Data , Protein Conformation , Purple Membrane/chemistry , Rats , Receptors, Mating Factor , Receptors, Muscarinic/biosynthesis , Receptors, Muscarinic/genetics , Receptors, Muscarinic/isolation & purification , Receptors, Peptide/biosynthesis , Receptors, Peptide/genetics , Receptors, Peptide/isolation & purification , Receptors, Serotonin/biosynthesis , Receptors, Serotonin/genetics , Receptors, Serotonin/isolation & purification , Recombinant Fusion Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Transcription Factors/genetics , Yeasts/chemistry
8.
Microbiology (Reading) ; 144 ( Pt 5): 1435-1441, 1998 May.
Article in English | MEDLINE | ID: mdl-9611817

ABSTRACT

The aspartate carbamoyltransferase (ATCase) genes of psychrophilic Vibrio strain 2693 were cloned by complementation in Escherichia coli and the enzyme was partly characterized. The genes constitute a pyrBI operon homologous to the cognate structure in E. coli where pyrB and pyrI respectively encode the catalytic and the regulatory chains of ATCase. The strong sequence similarities noted between Vibrio and E. coli ATCases include extensive conservation of residues involved in interactions between subunits, suggesting that the two enzymes have very similar tertiary and quaternary structures. Vibrio ATCase is, however, not activated by ATP and not synergistically inhibited by CTP and UTP. It is also much more thermolabile than E. coli ATCase. With respect to Pyrococcus abyssi and E. coli ATCases, Vibrio ATCase presents marked differences in composition which could be related to its psychrophilic character. The results of these structural and functional comparisons indicate that Vibrio 2693 ATCase is a suitable model for biochemical studies on structure-function relationships in a 'cold' allosteric enzyme. The operon is expressed from a promoter which is immediately followed by a pyrimidine-rich leader ORF terminating within a putative transcription attenuator. These genetic and enzymic data strengthen the evolutionary relationship already noted between Vibrionaceae and Enterobacteriaceae.


Subject(s)
Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/metabolism , Vibrio/enzymology , Amino Acid Sequence , Aspartate Carbamoyltransferase/biosynthesis , Aspartate Carbamoyltransferase/chemistry , Cloning, Molecular , Cold Temperature , DNA, Bacterial , Escherichia coli/enzymology , Escherichia coli/genetics , Genes, Bacterial , Molecular Sequence Data , Operon , Promoter Regions, Genetic , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Structure-Activity Relationship , Temperature , Transcription, Genetic , Transformation, Bacterial , Vibrio/chemistry , Vibrio/genetics , Vibrio/growth & development
10.
FEBS Lett ; 422(2): 170-4, 1998 Jan 30.
Article in English | MEDLINE | ID: mdl-9489999

ABSTRACT

In Saccharomyces cerevisiae, the first two reactions of pyrimidine biosynthesis are catalyzed by the multifunctional protein Ura2 carrying both carbamyl-phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) enzyme activities. In order to study how UTP regulates both of these activities mutant strains were constructed: one strain which expressed the Ura2 protein fused to the green fluorescent protein, and two strains expressed truncated Ura2 proteins. These strains exhibited a phenotype associated with a modified regulation of the pyrimidine pathway. Results presented in this report provide arguments in favor of a single UTP binding site located on the CPSase domain, and support a model in which ATCase activity is inhibited by UTP only when it can interact with the CPSase domain.


Subject(s)
Aspartate Carbamoyltransferase/metabolism , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/metabolism , Multienzyme Complexes/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Uridine Triphosphate/pharmacology , Aspartate Carbamoyltransferase/antagonists & inhibitors , Aspartate Carbamoyltransferase/biosynthesis , Binding Sites , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Green Fluorescent Proteins , Kinetics , Luminescent Proteins/biosynthesis , Luminescent Proteins/metabolism , Multienzyme Complexes/biosynthesis , Phenotype , Pyrimidines/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development
11.
Biochem Biophys Res Commun ; 219(1): 249-55, 1996 Feb 06.
Article in English | MEDLINE | ID: mdl-8619816

ABSTRACT

A human CAD cDNA encoding a trifunctional enzyme of carbamoylphosphate synthetase-aspartate transcarbamoylase-dihydroorotase, which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis, was cloned from a human fibroblast cell line of TIG-1-20 by polymerase chain reaction (PCR). The predicted open reading frame encodes a protein of 2,225 amino acids with a deduced molecular weight (Mr) OF 242,913. The deduced amino acid sequence exhibits 95.3 and 76.1% identity with the CAD sequences of hamster and Squalus acanthias. The DNA fragment of 6,679 bp containing the full-length coding sequence was amplified by nested PCR using the first-strand cDNA of human cell lines of TIG-1-20 and COLO205 as a template. Southern blot analysis suggested that the CAD gene exists as a single copy in the human genome.


Subject(s)
Aspartate Carbamoyltransferase/biosynthesis , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Dihydroorotase/biosynthesis , Multienzyme Complexes/biosynthesis , Amino Acid Sequence , Animals , Aspartate Carbamoyltransferase/genetics , Base Sequence , Blotting, Southern , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Cell Line , Cloning, Molecular , Cricetinae , DNA Primers , DNA, Complementary , Dihydroorotase/genetics , Dogfish , Fibroblasts , Humans , Molecular Sequence Data , Molecular Weight , Multienzyme Complexes/genetics , Open Reading Frames , Polymerase Chain Reaction , Pyrimidines/biosynthesis , Restriction Mapping , Sequence Homology, Amino Acid
12.
J Biol Chem ; 271(3): 1285-94, 1996 Jan 19.
Article in English | MEDLINE | ID: mdl-8576114

ABSTRACT

Electrostatics are central to the function and regulation of Escherichia coli aspartate transcarbamylase, and modeling has suggested that long range electrostatic effects are likely to be important (Glackin, M. P., McCarthy, M. P., Mallikarachchi, D., Matthew, J. B., and Allewell, N. M. (1989) Proteins Struct. Funct. Genet. 5, 66-77; Oberoi, H., Trikha, J., Yuan, X., and Allewell, N. M. (1995) Proteins Struct. Funct. Genet., in press). To investigate this possibility from an experimental standpoint, we have examined the effects both of assembly and of removing ionizable and polar side chains outside the active site (Glu-50, Tyr-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase. The holoenzyme (c6r6) assembles from three regulatory dimers (r2) and two catalytically active trimers (c3). pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of the catalytic subunit (c3) and holoenzyme (c6r6) with carbamyl phosphate are different. In contrast, the Michaelis complex appears similar for both c3 and c6r6, except for pK shifts of approximately 1 pH unit. Results also indicate that the catalytic mechanism of the holoenzyme does not involve reverse protonation, as has recently been proposed for the catalytic trimer (Turnbull, J. L., Waldrop, G. L., and Schachman, H. K. (1992) Biochemistry 31, 6562-6569). The tyrosines at positions 165 and 240 are part of a cluster of interactions that links the catalytic subunits in the T state (the cluc4 interface) and which is disrupted in the T --> R transition. The effects of mutating the two Tyr residues are quite different: Y240F has higher than wild-type activity and affinity over the entire pH range, while Y165F has activity and affinity an order of magnitude lower than wild-type. Removal of the regulatory subunits from Y165F increases activity and affinity and restores the pH dependence of the wild-type catalytic subunit. Like Y165F, E50A has low activity and affinity over the entire pH range. Linkage analysis indicates that there is long range energetic coupling among the active site, the ear subunit interfaces, and residue Y165. The substantial quantitative difference between Y165F and Y240F, both of which are at the c1:c4 interface about 14-16 A from the closest active site, demonstrates specific path dependence, as opposed to general distance dependence, of interactions between this interface and the active site.


Subject(s)
Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/metabolism , Escherichia coli/enzymology , Protein Conformation , Amino Acid Sequence , Aspartate Carbamoyltransferase/biosynthesis , Binding Sites , Escherichia coli/genetics , Hydrogen-Ion Concentration , Kinetics , Macromolecular Substances , Mathematics , Models, Theoretical , Mutagenesis, Site-Directed , Point Mutation , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
13.
Cancer Res ; 56(1): 36-9, 1996 Jan 01.
Article in English | MEDLINE | ID: mdl-8548770

ABSTRACT

Defects in cell cycle control and increased genomic instability, including gene amplification, often occur during cancer development. Cyclin D1 plays a pivotal role in G1, and this gene is frequently amplified and overexpressed in several types of human cancer. This study demonstrates that ectopic overexpression of cyclin D1 in a rat liver epithelial cell line markedly increased the yield of cells containing amplified copies of the CAD gene. This effect was associated with a loss of G1-S checkpoint control, although the cyclin D1-overexpressing cells had a normal p53 gene. The capacity of cyclin D1 to enhance gene amplification may contribute to the process of genomic instability during tumor development.


Subject(s)
Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Cell Cycle/genetics , Cyclins/biosynthesis , Dihydroorotase/genetics , Liver/metabolism , Multienzyme Complexes/genetics , Oncogene Proteins/biosynthesis , Animals , Aspartate Carbamoyltransferase/biosynthesis , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Cells, Cultured , Cyclin D1 , Cyclins/genetics , Dihydroorotase/biosynthesis , Epithelial Cells , Epithelium/metabolism , Gene Dosage , Gene Expression Regulation , Humans , Multienzyme Complexes/biosynthesis , Oncogene Proteins/genetics , Rats
14.
Protein Expr Purif ; 6(5): 679-84, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8535162

ABSTRACT

A procedure has been developed for the overexpression and purification of milligram quantities of the Bacillus subtilis aspartate transcarbamoylase. The plasmid pEK171, carrying the B. subtilis pyrB structural gene under the control of the Escherichia coli pyrBI promoter, was transformed into the E. coli strain EK1104 and the enzyme overexpressed to approximately 50% of total soluble protein under extreme derepression of the pyrimidine pathway. The enzyme was subsequently purified by means of ammonium sulfate fractionation, anionic exchange chromatography using Q-Sepharose Fast Flow resin, negative chromatography on Matrex Gel Red A agarose, and hydrophobic interaction chromatography using Matrex Phenyl Cellufine. The purification yields approximately 60 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties very similar to those of the enzyme purified from B. subtilis cells.


Subject(s)
Aspartate Carbamoyltransferase/genetics , Aspartate Carbamoyltransferase/isolation & purification , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Aspartate Carbamoyltransferase/biosynthesis , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Carbamyl Phosphate/chemistry , Carbamyl Phosphate/metabolism , Chromatography/methods , Cloning, Molecular/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Kinetics , Plasmids/chemistry , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
15.
J Mol Biol ; 246(2): 254-63, 1995 Feb 17.
Article in English | MEDLINE | ID: mdl-7869377

ABSTRACT

In previous studies we have shown that specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) particles that represent the splicing machinery in vivo. The lnRNP particles contain all U small nuclear ribonucleoproteins (snRNPs) required for splicing, as well as several proteins including non-snRNP splicing factors. Here we show that upon addition of EDTA to sucrose gradient-fractionated 200 S particles, part of their components (e.g. part of the U snRNPs) are no longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg2+ is required for the integrity of the 200 S lnRNP particles.


Subject(s)
Magnesium/metabolism , RNA Precursors/metabolism , RNA Precursors/ultrastructure , Ribonucleoproteins, Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/ultrastructure , Animals , Aspartate Carbamoyltransferase/biosynthesis , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Cell Line , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Centrifugation, Density Gradient , Cricetinae , Dihydroorotase/biosynthesis , Edetic Acid/pharmacology , Kinetics , Mesocricetus , Microscopy, Electron , Multienzyme Complexes/biosynthesis , RNA Polymerase II/metabolism , RNA Splicing , RNA, Messenger/biosynthesis , Ribonucleases , Ribonucleoproteins, Small Nuclear/isolation & purification , Transcription, Genetic
16.
Anticancer Res ; 15(1): 189-92, 1995.
Article in English | MEDLINE | ID: mdl-7733632

ABSTRACT

In mammalian cells selected in culture for resistance to PALA the CAD gene is amplified and these cells are a widely used model system to study gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD gene.


Subject(s)
Aspartic Acid/analogs & derivatives , Dipyridamole/pharmacology , Drug Resistance , Phosphonoacetic Acid/analogs & derivatives , Uridine/metabolism , Animals , Aspartate Carbamoyltransferase/biosynthesis , Aspartic Acid/pharmacology , Biological Transport/drug effects , CHO Cells , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Cell Survival/drug effects , Cricetinae , Cricetulus , Dihydroorotase/biosynthesis , Dose-Response Relationship, Drug , Gene Amplification , Kinetics , Multienzyme Complexes/biosynthesis , Mutagenesis , Neoplasm Proteins/biosynthesis , Phosphonoacetic Acid/pharmacology , Uridine/pharmacology
17.
Plant Physiol ; 105(1): 377-84, 1994 May.
Article in English | MEDLINE | ID: mdl-8029359

ABSTRACT

We cloned cDNAs encoding two different pea (Pisum sativum L.) aspartate transcarbamoylases (ATCases) by complementation of an Escherichia coli delta pyrB mutant. The two cDNAs, designated pyrB1 and pyrB2, encode polypeptides of 386 and 385 amino acid residues, respectively, both of which exhibit typical chloroplast transit peptide sequences. Wheat germ ATCase antibody recognizes a 36.5-kD polypeptide in pea leaf and root tissues that is similar in size to other plant ATCase polypeptides and to the catalytic polypeptides of bacterial ATCases. Northern analyses indicate that the pyrB1 and pyrB2 transcripts are 1.6 kb in size and are differentially expressed in pea tissues. The small transcript size and data from biochemical studies indicate that plant ATCases are simple homotrimers of 36- to 37-kD catalytic subunits, rather than part of a multifunctional enzyme containing glutamine-dependent carbamoylphosphate synthetase and dihydroorotase activities, as is seen in other eukaryotes. In the pea ATCases, the carbamoylphosphate- and aspartate-binding domains are highly homologous to those of other prokaryotic and eukaryotic ATCases and critical active-site residues are completely conserved. The pea ATCases also exhibit a putative pyrimidine-binding site, consistent with the known allosteric regulation of plant ATCases by UMP in vitro.


Subject(s)
Aspartate Carbamoyltransferase/genetics , Fabaceae/enzymology , Fabaceae/genetics , Genes, Plant , Plants, Medicinal , Amino Acid Sequence , Aspartate Carbamoyltransferase/biosynthesis , Bacillus subtilis/enzymology , Blotting, Northern , Blotting, Southern , Chloroplasts/enzymology , Cloning, Molecular , DNA/analysis , Escherichia coli/enzymology , Gene Expression , Humans , Molecular Sequence Data , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Restriction Mapping , Sequence Homology, Amino Acid , Triticum/enzymology
20.
J Biol Chem ; 269(3): 2252-7, 1994 Jan 21.
Article in English | MEDLINE | ID: mdl-7905000

ABSTRACT

Transcription of the carbamoyl-phosphate synthase (glutamine-hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase (CAD) gene from the Syrian hamster, Mesocricetus auratus, starts at a single major site. We characterized the cis-acting elements that position RNA polymerase II at the correct start site in the CAD promoter. Sequence alignment showed that the CAD promoter lacks a TATA box, but contains a consensus initiator. Mutational analysis of the CAD promoter demonstrated that the sequences between -81 and +26 were sufficient for accurate and efficient transcription in vitro and in vivo; binding sites for the transcription factor Sp1 around -70 and -49 were necessary for transcriptional activity. The binding site at -49 directed initiation about 50 base pairs downstream. A ubiquitous activator protein, Honk, bound to the CAD promoter between -30 and -12, but did not participate in start site selection. The sequences around +1, which contain the consensus initiator, contributed to promoter activity; however, the presence of a consensus initiator in this region was neither necessary nor sufficient for transcription. We concluded from these results that the Sp1 binding site at -49 substituted for the missing TATA box and played a major role in start site selection at the CAD promoter.


Subject(s)
Aspartate Carbamoyltransferase/genetics , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/genetics , Dihydroorotase/genetics , Mesocricetus/genetics , Multienzyme Complexes/genetics , Promoter Regions, Genetic , 3T3 Cells , Animals , Aspartate Carbamoyltransferase/biosynthesis , Base Sequence , Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)/biosynthesis , Cell Line , Cell Nucleus/metabolism , Consensus Sequence , Cricetinae , DNA/metabolism , Dihydroorotase/biosynthesis , Liver/metabolism , Male , Mice , Molecular Sequence Data , Multienzyme Complexes/biosynthesis , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Plasmids , Rats , Rats, Sprague-Dawley , Restriction Mapping , Sequence Homology, Nucleic Acid , T-Lymphocytes/metabolism , TATA Box , Transfection
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