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1
Eguae, Samuel Iyamu. "Pyrimidine nucleotide metabolism in Rhizobium meliloti: purification of aspartate transcarbamoylase from a pyrimidine auxotroph." Thesis, University of North Texas, 1990. https://digital.library.unt.edu/ark:/67531/metadc332674/.
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Rhizobium aspartate transcarbamoylase (ATCase; EC 2.1.3.2) was previously believed to be similar to the Pseudomonas ATCase which has been studied extensively. To facilitate the study of the Rhizobium ATCase a pyrimidine-requiring mutant of R. meliloti was isolated and used in the purification of the enzyme.
2
Stewart, John E. B. (John Edward Bakos). "Characterization of Aspartate Transcarbamoylase in the Archaebacterium Methanococcus Jannaschii." Thesis, University of North Texas, 1996. https://digital.library.unt.edu/ark:/67531/metadc935724/.
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Asparate transcarbamoylase catalyzes the first committed step in the de novo synthesis of pyrmidine nucleotides UMP, UDP, UTP, and CTP. The archetype enzyme found in Escherichia coli (310 kDa) exhibits sigmodial substrate binding kinetics with positive control by ATP and negative control with CTP and UTP. The ATCase characterized in this study is from the extreme thermophilic Archaebacterium, Methanococcus jannaschii. The enzyme was very stable at elevated temperatures and possessed activity from 20 degrees Celsius to 90 degrees Celsius. M. Jannaschii ATCase retained 75% of its activity after incubation at 100 degrees Celsius for a period of 90 minutes. No sigmodial allosteric response to substrate for the enzyme was observed. Velocity substrate plots gave Michaelis-Menten (hyperbolic) kinetics. The Km for aspartate was 7 mM at 30 degrees Celsius and the KM for carbamoylphosphate was .125 mM. The enzyme from M. jannaschii had a broad pH response with an optimum above pH 9. Kinetic measurements were significantly affected by changes in pH and temperature. The enzyme catalyzed reaction had an energy of activation of 10,300 calories per mole. ATCase from M. jannaschii was partially purified. The enzyme was shown to have a molecular weight of 110,000 Da., with a subunit molecular weight of 37,000 Da. The enzyme was thus a trimer composed of three identical subunits. The enzyme did not possess any regulatory response and no evidence for a regulatory polypeptide was found, DNA from M. jannaschii did hybridize to probes corresponding to genes for both the catalytic and regulatory subunits from E. coli. Analysis of DNA sequences for the M. jannaschii ATCase genes showed that the gene for the catalytic subunits shares significant homology with the pyrB genes from E. coli, and maximum homology amongst known ATCase genes to pyrB from Bacillus. An unlinked gene homologous to E. coli pyrl encoding the regulatory subunit was identified, though its expression and true function remain uncharacterized.
3
So, Ngar-chung Nellie. "Pyrimidine nucleotide biosynthesis in adult angiostrongylus Cantonensis (Nematoda : Metastrongyloidea) /." [Hong Kong : University of Hong Kong], 1993. http://sunzi.lib.hku.hk/hkuto/record.jsp?B13637745.
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蘇雅頌 and Ngar-chung Nellie So. "Pyrimidine nucleotide biosynthesis in adult angiostrongylus Cantonensis (Nematoda : Metastrongyloidea)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1993. http://hub.hku.hk/bib/B3123320X.
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Brichta, Dayna Michelle. "Construction of a Pseudomonas aeruginosa Dihydroorotase Mutant and the Discovery of a Novel Link between Pyrimidine Biosynthetic Intermediates and the Ability to Produce Virulence Factors." Thesis, University of North Texas, 2003. https://digital.library.unt.edu/ark:/67531/metadc4344/.
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The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.
6
Entezampour, Mohammad. "Characterization of pyrimidine biosynthesis in Acinetobacter calcoaceticus using wild type and mutant strains." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc798038/.
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Pyrimidine nucleotide biosynthesis was studies in Acinetobacter calcoaceticus ADP-1. Pyrimidine auxotrophic mutants were isolated and characterized for this purpose. One such Pyr mutant, strain ADP-1-218 was chosen for further study.
7
Vickrey, John F. (John Fredrick) 1959. "Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosa." Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc278859/.
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The Pseudomonas aeruginosa ATCase was cloned and sequenced to determine the correct size, subunit composition and architecture of this pivotal enzyme in pyrimidine biosynthesis. During the course of this work, it was determined that the ATCase of Pseudomonas was not 360,000 Da but rather present in a complex of 484,000 Da consisting of two different polypeptides (36,000 Da and 44,000 Da) with an architecture similar to that of E. coli ATCase, 2(C3):3(r2). However, there was no regulatory polypeptide found in the Pseudomonas ATCase.
8
AsFour, Hani. "Effector Response of the Aspartate Transcarbamoylase From Wild Type Pseudomonas Putida and a Mutant with 11 Amino Acids Deleted at the N-terminus of PyrB." Thesis, University of North Texas, 2002. https://digital.library.unt.edu/ark:/67531/metadc3163/.
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Like its enteric counterpart, aspartate transcarbamoylase (ATCase) from Pseudomonas putida is a dodecamer of two different polypeptides. Unlike the enterics, the Pseudomonas ATCase lacks regulatory polypeptides but employs instead inactive dihydroorotases for an active dodecamer. Previous work showed that PyrB contains not only the active site but also the effector binding sites for ATP, UTP and CTP at its N-terminus. In this work, 11 amino acids were deleted from the N-terminus of PyrB and the ATCase with the truncated protein was expressed in E. coli pyrB- and purified. The wild type enzyme was similarly treated. Velocity-substrate plots without effectors gave Michaelis-Menten kinetics in all cases. Deleting 11 amino acids did not affect dodecameric assembly but altered effector responses. When carbamoylphosphate was varied, the mutant enzyme was inhibited by UTP while the wild type enzyme was activated 2-fold. When the aspartate was varied, CTP had no effect on the mutant enzyme but strongly inhibited the wild type enzyme.
9
Kumar, Alan P. "Structure-Function Studies on Aspartate Transcarbamoylase and Regulation of Pyrimidine Biosynthesis by a Positive Activator Protein, PyrR in Pseudomonas putida." Thesis, University of North Texas, 2003. https://digital.library.unt.edu/ark:/67531/metadc4362/.
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The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate.
10
Hammerstein, Heidi Carol. "Isolation of a Pseudomonas aeruginosa Aspartate Transcarbamoylase Mutant and the Investigation of Its Growth Characteristics, Pyrimidine Biosynthetic Enzyme Activities, and Virulence Factor Production." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc4704/.
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The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.
11
Ruley, Jill R. (Jill Rosanne). "Creation and characterization of an Escherichia coli and Pseudomonas putida hybrid aspartate transcarbamoylase." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc798137/.
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Aspartate transcarbamoylase (ATCase) is encoded by the pyrBI genes in E. coli. Expression of these genes is reduced four-fold by attenuation when grown on uracil. Using plasmid, pRO1727. the pyrB and the pyrBI genes from E. coli were cloned into a P. putida pyrB auxotroph. A recombinant pyrB gene was recovered that encoded a functional hybrid ATCase with a molecular weight of 470 kDa.
12
Kim, Hyunju. "Multiple Activities of Aspartate Transcarbamoylase in Burkholderia cepacia: Requirement for an Active Dihydroorotase for Assembly into the Dodecameric Holoenzyme." Thesis, University of North Texas, 2010. https://digital.library.unt.edu/ark:/67531/metadc33176/.
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The aspartate transcarbamoylase (ATCase) was purified from Burkholderia cepacia 25416. In the course of purification, three different ATCase activities appeared namely dodecameric 550 kDa holoenzyme, and two trimeric ATCases of 140 kDa (consists of 47 kDa PyrB subunits) and 120 kDa (consists of 40 kDa PyrB subunits) each. The 120 kDa PyrB polypeptide arose by specific cleavage of the PyrB polypeptide between Ser74 and Val75 creating an active polypeptide short by 74 amino acids. Both the 40 and 47 kDa polypeptides produced active trimers. To compare the enzyme activity of these trimers, an effector assay using nucleotides was performed. The 140 kDa trimer showed inhibition while the 120 kDa polypeptide showed less inhibition. To verify the composition of the pyrBC holoenzyme complex, B. cepacia dihydroorotase (DHOase, subunit size of 45 kDa) was purified by the pMAL protein fusion and purification system and holoenzyme reconstruction was performed using purified ATCase and DHOase. Both the 140 kDa and the 120 kDa trimers could produce holoenzymes of 550 kDa and 510 kDa, respectively. The reconstructed ATCase holoenzyme from cleaved ATCase showed better reconstruction compared to that from uncleaved ATCase in the conventional ATCase activity gel assay. To characterize the relationship between pyrimidine pathway and virulence factor production, motility tests and biofilm assays were conducted using pyrC- mutant. Even though no significant difference in growth rates was observed, there were significant differences between the wild type and mutant in the production of biofilm and virulence factors. This study will help us to understand the structure and regulation of ATCase holoenzyme with DHOase, and facilitate the use of B. cepacia as an applicable bio-tool. Additionally, we can potentially pursue more efficient drug targets for B. cepacia.
13
Fong, Yuen Ting. "Quantitative structure retention relationships on using high-performance liquid chromatography." HKBU Institutional Repository, 2003. http://repository.hkbu.edu.hk/etd_ra/426.
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Linscott, Andrea J. (Andrea Jane). "Regulatory Divergence of Aspartate Transcarbamoylase from the Pseudomonads." Thesis, University of North Texas, 1996. https://digital.library.unt.edu/ark:/67531/metadc277625/.
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Aspartate transcarbamoylase (ATCase) was purified from 16 selected bacterial species including existing Pseudomonas species and former species reassigned to new genera. An enormous diversity was seen among the 16 enzymes with each class of ATCase being represented. The smallest class, class C, with a catalytically active homotrimer, at 100 kDa, was found in Bacillus and other Gram positive bacteria. In this report, the ATCases from the Gram negatives, Shewanella putrefaciens and Stenotrophomonas maltophilia were added to class C membership. The enteric bacteria typify class B ATCases at 310 kDa, with a dodecameric structure composed of two catalytic trimers coupled to three regulatory dimers. A key feature of class B ATCases is the dissociability of the holoenzyme into regulatory and catalytic subunits which were enzymatically active. In this report, the ATCase from Pseudomonas indigofera was added to class B ATCases. The largest class, at 480 kDa, class A, contains the fluorescent Pseudomonas including most members of the 16S rRNA homology group I. Two polypeptides are produced from overlapping pyrBC' genes. The former, pyrB, encodes a 34 kDa catalytic polypeptide while pyrC' encodes a 45 kDa dihydroorotase-like polypeptide. Two non active trimers are made from six 34 kDa chains which are cemented by six 45 kDa chains to form the active dodecameric structure. Dissociation of the holoenyzme into its separate active subunits has not been possible. In this report, the ATCases from Comamonas acidovorans and C. testosteroni, were added to the class A enzymes. An even larger class of ATCase than class A at 600 kDa was discovered in Burkholderia cepacia. Stoichiometric measurements predict a dodecamer of six 39 kDa polypeptides and six 60 kDa polypeptides. Unlike other large pseudomonads ATCases, the enzyme from B. cepacia was dissociable into smaller active forms. Both the holoenzyme and its dissociated forms were regulated by nucleotide effectors. A new class of ATCase was proposed for B. cepacia type enzymes.
15
Barron, Vincent N. (Vincent Neal). "Comparison of Aspartate Transcarbamoylase and Pyrimidine Salvage in Sporosarcina urea, Sprolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus." Thesis, University of North Texas, 1994. https://digital.library.unt.edu/ark:/67531/metadc278938/.
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The enzyme that catalyzes the committed step in pyrimidine biosynthesis, aspartate transcarbamoylase, has been compared in selected endospore-forming organisms and in morphologically similar control organisms. The ATCases and pyrimidine salvage from Sporosarcina ureae, Sporolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus were compared to those of Bacillus subtilis. While the ATCases from Sporosarcina ureae, Sporolactobacillus inulinus, and L. fermentum were found to exhibit characteristics to that of Bacillus with respect to molecular weight and kinetics, M. luteus ATCase was larger at approximately 480 kDa. Furthermore, pyrimidine salvage in Sporosarcina ureae and M. luteus was identical to those of B. subtilis, while pyrimidine salvage of Sporolactobacillus inulinus and L. fermentum resembled that of the pseudomonads.
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Hongsthong, Apiradee 1970. "Assembly of Pseudomonas putida Aspartate Transcarbamoylase and Possible Roles of the PyrC' Polypeptide in the Folding of the Dodecameric Enzyme." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc278618/.
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Aspartate transcarbamoylase (ATCase) of Pseudomonas putida consists of two different polypeptides, PyrB and PyrC' (Schurr et al, 1995). The role of the PyrC' and the assembly of PyrB and PyrC' have been studied. The ATCase made in vitro of P.putida PyrB with P.putida PyrC', and of E.coli PyrB with P.putida PyrC ' were generated under two different conditions, denaturation and renaturation, and untreated. It was found that PyrC' plays a role in the enzymatic regulation by ATP, CTP and UTP. In addition to playing a role in substrate binding, the PyrB polypeptide is also involved in effector binding (Kumar et al., manuscript in preparation). The most energetically preferred form of the P.putida WT is a dodecamer with a molecular mass of 480 kDa. The ratio between the PyrB and the PyrC' is 1:1. In studies of nucleotide binding, it was discovered that the P.putida PyrB was phosphorylated by a protein kinase in the cell extract. In the presence of 20 mM EDTA, this phosphorylation was inhibited and the inhibition could be overcome by the addition of divalent cations such as Zn2+ and Mg2+. This result suggested that the phosphorylation reaction required divalent cations. In the CAD complex of eukaryotes, phosphorylations of the CPSase and the linker region between ATCase and DHOase did not occur in the presence of UTP and it was hypothesized (Carrey, 1993) that UTP and phosphorylation(s) regulated the conformational change in the enzyme complex. Therefore, the same idea was approached with P.putida ATCase, where it was found that 1.0 mM UTP inhibited the phosphorylation of PyrB by more than 50%. These results suggested that the regulation of the conformational change of the P.putida ATCase might be similar to that of CAD. Furthermore, peptide mapping for phosphorylation sites was performed on P.putida ATCase WT, WT --11 amino acids and WT --34 amino acids from the N-terminus of the PyrB polypeptide. The results showed that the phosphorylation sites were located on the fragment that contained amino acid number-35 to amino acid number-112 from the N-terminus of the PyrB polypeptide.
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Dill, Michael T. "Characterization of the Aspartate Transcarbamoylase that is Found in the pyrBC Complex of Bordetella Pertussis." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc3057/.
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An aspartate transcarbamoylase (ATCase) gene from Bordetella pertussis was amplified by PCR and ligated into pT-ADV for expression in Escherichia coli. This particular ATCase (pyrB) was an inactive gene found adjacent to an inactive dihydroorotase (DHOase) gene (pyrC'). This experiment was undertaken to determine whether this pyrB gene was capable of expression alone or if it was capable of expression only when cotransformed with a functional pyrC'. When transformed into E. coli TB2 pyrB-, the gene did not produce any ATCase activity. The gene was then co-transformed into E. coli TB2 pyrB- along with a plasmid containing the pyrC' gene from Pseudomonas aeruginosa and assayed for ATCase activity. Negative results were again recorded.
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Kim, Seongcheol. "Purification and Characterization of Proteolytic Aspartate Transcarbamoylase (ATCase) from Burkholderia cepacia 25416 and Construction of a pyrB1 Knock-out Mutant." Thesis, University of North Texas, 2004. https://digital.library.unt.edu/ark:/67531/metadc4694/.
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Burkholderia cepacia is a common soil bacterium of significance in agriculture and bioremediation. B. cepacia is also an opportunistic pathogen of humans causing highly communicable pulmonary infections in cystic fibrosis and immunocompromized patients. The pyrB gene encoding ATCase was cloned and ATCase was purified by the glutathione S-transferase gene fusion system. The ATCase in B. cepacia has been previously classified as a class A enzyme by Bethell and Jones. ATCase activity gels showed that B. cepacia contained a holoenzyme pyrBC complex of 550 kDa comprised of 47 kDa pyrB and 45 kDa pyrC subunits. In the course of purifying the enzyme, trimeric subunits of 140 kDa and 120 kDa were observed as well as a unique proteolysis of the enzyme. The 47 kDa ATCase subunits were cleaved to 40 kDa proteins, which still demonstrated high activity as trimers. The proteolysis site is between Ser74 and Val75 residues. To confirm this, we converted the Ser74 residue to an Ala and to an Arg by site-directed mutagenesis. After this primary sequence changed, the proteolysis of ATCase was not observed. To further investigate the characteristics of B. cepacia pyrB gene, a pyrB knock-out (pyrB-) was constructed by in vitro mutagenesis. In the assay, the 550 kDa holoenzyme and 140 kDa and 120 kDa trimers disappeared and were replaced with a previously unseen 480 kDa holoenzyme pyrB- strain. The results suggest that B. cepacia has two genes that encode ATCase. ATC1 is constitutive and ATC2 is expressed only in the absence of ATC1 activity. To check for the virulence of these two strains, a eukaryotic model virulence test was performed using Caenorhabditis elegans (C. elegans). The pyrB1+pyrB2+ (wild type) B cepacia killed the nematode but pyrB1-pyrB2+ B. cepacia had lost its virulence against C. elegans. This suggests that ATC1 (pyrB1) is involved in virulence in B.cepacia and ATC2 (pyrB2) is not.
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Higginbotham, Leah. "Aspartate Transcarbamoylase of Aeromonas Hydrophila." Thesis, University of North Texas, 2000. https://digital.library.unt.edu/ark:/67531/metadc5840/.
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This study focused on the enzyme, aspartate transcarbamoylase (ATCase) from A. hydrophila, a Gram-negative bacterium found in fresh water. The molecular mass of the ATCase holoenzyme from A. hydrophila is 310 kDa. The enzyme is likely composed of 6 catalytic polypeptides of 34 kDa each and 6 regulatory polypeptides of 17 kDa each. The velocity-substrate curve for A. hydrophila ATCase is sigmoidal for both aspartate and carbamoylphosphate. The Km for aspartate was the highest to date for an enteric bacterium at 97.18 mM. The Km for carbamoylphosphate was 1.18 mM. When heated to 60 ºC, the specific activity of the enzyme dropped by more than 50 %. When heated to 100 ºC, the enzyme showed no activity. The enzyme's activity was inhibited by ATP, CTP or UTP.
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Hooshdaran, Massoumeh Ziba. "Comparative Biochemistry and Evolution of Aspartate Transcarbamoylase from Diverse Bacteria." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc500380/.
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Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis. Bacterial ATCases are divided into three classes, A, B and C. Class A ATCases are largest at 450-500, are. dodecamers and represented by Pseudomonas ATCase. The overlapping pyrBC' genes encode the Pseudomonases ATCase, which is active only as a 480 kDa dodecamer and requires an inactive pyrC'-encoded DHOase for ATCase activity. ATCase has been studied in two non-pathogenic members of Mycobacterium, M. smegmatis and M. phlei. Their ATCases are dodecamers of molecular weight 480 kDa, composed of six PyrB and six PyrC polypeptides. Unlike the Pseudomonas ATCase, the PyrC polypeptide in these mycobacteria encodes an active DHOase. Moreover, the ATCase: DHOase complex in M. smegmatis is active both as the native 480 kDa and as a 390 kDa complex. The latter lacks two PyrC polypeptides yet retains ATCase activity. The ATCase from M. phlei is similar, except that it is active as the native 480 kDa form but also as 450,410 and 380 kDa forms. These complexes lack one, two, and three PyrC polypeptides, respectively. By contrast,.ATCases from pathogenic mycobacteria are active only at 480 kDa. Mycobacterial ATCases contain active DHOases and accordingly. are placed in class A1 . The class A1 ATCases contain active DHOases while class A2 ATCases contain inactive DHOases. ATCase has also been purified from Burkholderia cepacia and from an E. coli strain in which the cloned pyrB of B. cepacia was expressed. The B. cepacia ATCase has a molecular mass of 550 kDa, with two different polypeptides, PyrB (52 kDa) and PyrC of (39 kDa). The enzyme is active both as the native enzyme at 550 kDa and as smaller molecular forms including 240 kDa and 165 kDa. The ATCase synthesized by the cloned pyrB gene has a molecular weight of 165 kDa composed of three identical PyrB and no PyrC polypeptides. Nucleotide effectors ATP, CTP, and UTP inhibited all forms of enzymes. Because of its size and its activity as a trimer and smaller than native forms, the B. cepacia enzyme is placed in a new class.
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Fields, Christopher J. "Comparative biochemistry and genetic analysis of nucleoside hydrolase in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas fluorescens." Thesis, University of North Texas, 2002. https://digital.library.unt.edu/ark:/67531/metadc3290/.
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The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation.
As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The
physiological relevance of this is explained.
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Wei, Shuang. "Modifications du metabolisme des nucleotides en relation avec la differenciation et en reponse a une irradiation dans des cellules tumorales humaines (doctorat : structure et fontionnement des systemes biologiques integres)." Paris 11, 1998. http://www.theses.fr/1998PA114846.
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Hooshdaran, Sahar. "Characterization of Moraxella bovis Aspartate Transcarbamoylase." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc3012/.
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Aspartate transcarbamoylase (ATCase) catalyzes the first committed step in the pyrimidine biosynthetic pathway. Bacterial ATCases have been divided into three classes, class A, B, and C, based on their molecular weight, holoenzyme architecture, and enzyme kinetics. Moraxella bovis is a fastidious organism, the etiologic agent of infectious bovine keratoconjunctivitis (IBK). The M. bovis ATCase was purified and characterized for the first time. It is a class A enzyme with a molecular mass of 480 to 520 kDa. It has a pH optimum of 9.5 and is stable at high temperatures. The ATCase holoenzyme is inhibited by CTP > ATP > UTP. The Km for aspartate is 1.8 mM and the Vmax 1.04 µmol per min, where the Km for carbamoylphosphate is 1.05 mM and the Vmax 1.74 µmol per min.
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Patel, Seema R. "A Study of the Pyrimidine Biosynthesis Pathway and its Regulation in Two Distinct Organisms: Methanococcus jannaschii and Pseudomonas aeruginosa." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc3038/.
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Methanococcus jannaschii is a thermophilic methane producing archaebacterium. In this organism genes encoding the aspartate transcarbamoylase (ATCase) catalytic (PyrB) and regulatory (PyrI) polypeptides were found. Unlike Escherichia coli where the above genes are expressed from a biscistronic operon the two genes in M. jannaschii are separated by 200-kb stretch of genome. Previous researchers have not been able to show regulation of the M. jannaschii enzyme by the nucleotide effectors ATP, CTP and UTP. In this research project we have genetically manipulated the M. jannaschii pyrI gene and have been able to assemble a 310 kDa E. coli like enzyme. By using the second methionine in the sequence we have shown that the enzyme from this organism can assemble into a 310 kDa enzyme and that this enzyme is activated by ATP, CTP and inhibited by UTP. Thus strongly suggesting that the second methionine is the real start of the gene. The regulation of the biosynthetic pathway in Pseudomoans aeruginosa has previously been impossible to study due to the lack of CTP synthase (pyrG) mutants. By incorporating a functional uridine (cytidine) kinase gene from E. coli it has been possible to isolate a pyrG mutant. In this novel mutant we have been able to independently manipulate the nucleotide pools and study its effects on the enzymes in the biosynthetic pathway. The enzyme asapartate transcarbamoylase was repressed 5-fold when exogenous uridine was high and cytidine was low. The enzyme dihydroorotate was repressed 9-fold when uridine was high. These results suggest that a uridine compound may be the primary repressing metabolite for the enzymes encoded by pyrB and pyrC. This is the first study to be done with the proper necessary mutants in the biosynthetic pathway of P aeruginosa. In the past it has been impossible to vary the internal UTP and CTP pools in this organism.
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Schurr, Michael J. (Michael John). "Molecular and Kinetic Characterization of the Aspartate Transcarbamoylase Dihydroorotase Complex in Pseudomonas putida." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc277575/.
Full textAbstract:
Aerobic Gram negative bacteria such as Pseudomonas putida were reported to possess class A ATCases and to have a M.W. of 360 kD. The nucleotide sequence of the P. putida pyrBC was determined to answer this question once and for all. The expected regulatory gene was not found. It is shown that the P. putida pyrB gene is overlapped by pyrC by 4 bp. The P.putida pyrB is 1005 bp (335 aa) in length and the pyrC is 1275 bp (425 aa) long. Both of these genes complement E. coli mutants with their respective genotypes. Another finding borne out from the sequence is an effector binding site at the N-terminus of pyrB of P. putIda. The binding site shows that effectors compete with carbamoylphosphate for the active site. In this dissertation, it is shown that the ATCase of P.putida is a trimer of M.W. of 109 kD (3 x 36.4 kD) and that the gene encoding pyrB is overlapped by the pyrC gene which encodes DHOase. It is also shown that the pyrBC encoded enzymes copurify as a dodecameric complex with a M.W. of 484 kD.
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Fowler, Michael A. (Michael Allen) 1961. "Characterization of Aspartate Transcarbamoylase and Dihydroorotase in Moraxella Catarrhalis." Thesis, University of North Texas, 1998. https://digital.library.unt.edu/ark:/67531/metadc277709/.
Full textAbstract:
Bacterial aspartate transcarbamoylases (ATCase's) are divided into three classes that correspond to taxonomic relationships within the bacteria. The opportunistic pathogen Moraxeila catarrhalis has undergone several reclassifications based on traditional microbiological criteria. The previously uncharacterized ATCase from M. catarrhalis was purified to homogeneity and its chemical properties characterized. The ATCase from M. catarrhalis is a class C ATCase with an apparent molecular mass of 480-520 kDa. The M. catarrhalis ATCase is a dodecomer composed of six 35 kDa polypeptides and six 45 kDa polypeptides. The enzyme has an unusually high pH optimum of greater than pH 10. The enzyme exhibited hyperbolic kinetic with a Km for aspartate of 2 mM. A single, separate 78 kDa dihydroorotase from M. catarrhalis was identified and it was not associated with ATCase. These data support the reclassification of M. catarrhalis out of the Neisseriaceae family.
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Cooke, Patrick Alan. "BioInformatics, Phylogenetics, and Aspartate Transcarbamoylase." Thesis, University of North Texas, 2000. https://digital.library.unt.edu/ark:/67531/metadc2580/.
Full textAbstract:
In this research, the necessity of understanding and using bioinformatics is demonstrated using the enzyme aspartate transcarbamoylase (ATCase) as the model enzyme. The first portion of this research focuses on the use of bioinformatics. A partial sequence of the pyrB gene found in Enterococcus faecalis was submitted to GenBank and was analyzed against the contiguous sequence from its own genome project. A BLAST (Basic Local Alignment Search Tool; Atschul, et al., 1990) was performed in order to hypothesize the remaining portion of the gene from the contiguous sequence. This allowed a global comparison to other known aspartate transcarbamoylases (ATCases) and once deduced, a translation of the sequence gave the stop codon and thus the complete sequence of the open reading frame. When this was complete, upstream and downstream primers were designed in order to amplify the gene from genomic DNA. The amplified product was then sequenced and used later in phylogenetic analyses concerning the evolution of ATCase. The second portion of this research involves taking multiple ATCase nucleotide sequences and performing phenetic and phylogenetic analyses of the archaea and eubacter families. From these analyses, ancestral relationships which dictate both structure and function were extrapolated from the data and discussed.
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BAILLON, JEAN. "Les enzymes du metabolisme des nucleotides pyrimidiques comme cibles dans la chimiotherapie antitumorale." Paris 6, 1987. http://www.theses.fr/1987PA066118.
Full text
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Stawska, Agnieszka A. "Purification of Aspartate Transcarbamoylase from Moraxella (Branhamella) catarrhalis." Thesis, University of North Texas, 2001. https://digital.library.unt.edu/ark:/67531/metadc2864/.
Full textAbstract:
The enzyme, aspartate transcarbamoylase (ATCase) from Moraxella (Branhamella) catarrhalis, has been purified. The holoenzyme has a molecular mass of approximately 510kDa, harbors predominantly positive charges and is hydrophobic in nature. The holoenzyme possesses two subunits, a smaller one of 40 kDa and a larger one of 45 kDa. A third polypeptide has been found to contribute to the overall enzymatic activity, having an approximate mass of 55 kDa. The ATCase purification included the generation of cell-free extract, streptomycin sulfate cut, 60 °C heat step, ammonium sulfate cut, dialysis and ion, gel-filtration and hydrophobic interaction chromatography. The enzyme's performance throughout purification steps was analyzed on activity and SDS-PAGE gradient gels. Its enzymatic, specific activities, yield and fold purification, were also determined.
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Liu, Haiyan 1966. "Attenuation of Escherichia Coli Aspartate Transcarbamoylase Expressed in Pseudomonas Aeruginosa Mutant and Wild Type Strains." Thesis, University of North Texas, 1994. https://digital.library.unt.edu/ark:/67531/metadc279106/.
Full textAbstract:
No apparent repression of pyr gene expression in Pseudomonas aeruginosa is observed upon addition of exogenous pyrimidines to the growth medium. Upon introduction of the subcloned Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) into a P. aeruginosa pyrB mutant strain, repression was observed in response to exogenously fed pyrimidine compounds. The results proved that it is possible to bring about changes in pyrimidine nucleotide pool levels and changes in transcriptional regulation of gene expression as a result. Thus, the lack of regulatory control in P. aeruginosa pyr gene expression is not due to an inability to take up and incorporate pyrimidine compounds into metabolic pools, or to an inability of the RNA polymerase to respond to regulatory sequences in the DNA but is probably due to a lack of specific regulatory signals in the promoter of the genes themselves.
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Boussiengui-Boussiengui, Gino. "Analysis of the role of relative nucleotide concentrations on the regulation of carbohydrate in higher plants." Thesis, Stellenbosch : Stellenbosch University, 2010. http://hdl.handle.net/10019.1/5473.
Full textAbstract:
Thesis (PhD (Genetics))--Stellenbosch University, 2010.<br>ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively.
Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis.
Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production.
Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines.
Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines.<br>AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is.
Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig.
Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met
die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie.
Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog.
Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie.<br>The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work
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Simpson, Luci N. "Cassette Systems for Creating Intergeneric Hybrid ATCases." Thesis, University of North Texas, 1999. https://digital.library.unt.edu/ark:/67531/metadc2237/.
Full textAbstract:
Cassette systems for creating intergeneric hybrid ATCases were constructed. An MluI restriction enzyme site was introduced at the carbamoylphosphate binding site within the pyrB genes of both Pseudomonas putida and Escherichia coli. Two hybrids, E. coli pyrB polar domain fused with P. putida pyrB equatorial domain and P. putida pyrB polar domain fused with E. coli pyrB equatorial domain, are possible. The intergeneric E. coli-P. putida hybrid pyrB gene was constructed and found to encode an active ATCase which complemented an E. coli Pyr- strain. These hybrids are useful for kinetic and expression studies of ATCase in E. coli.
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Shen, Weiping. "Regulation of Escherichia coli pyrBI Gene Expression in Pseudomonas fluorescens." Thesis, University of North Texas, 1995. https://digital.library.unt.edu/ark:/67531/metadc278188/.
Full textAbstract:
Pseudomonas fluorescens does not appear to regulate the enzymes of de novo
pyrimidine biosynthesis at the level of gene expression. Little or no apparent repression of pyr gene expression is observed upon addition of exogenous pyrimidines to the growth medium. The Escherichia coli pyrBI genes for aspartate transcarbamoylase (ATCase) were sized down and cloned into the broad host range plasmid, pKT230. Upon introduction into a P.fluorescenspyrB mutant strain, ATCase showed repression in response to exogenously fed pyrimidine compounds. Thus, it was possible to bring about changes in pyrimidine nucleotide pool levels and in transcriptional regulation of gene expression at the same time.
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Bean, Heather D. "Prebiotic synthesis of nucleic acids." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/28259.
Full textAbstract:
Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2008.<br>Committee Chair: Hud, Nicholas V.; Committee Member: Fox, Ronald F.; Committee Member: Lynn, David G.; Committee Member: Powers, James C.; Committee Member: Wartell, Roger M.; Committee Member: Williams, Loren D.
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Barbosa, Sara Isabel Cadinha. "Compostos que interferem no metabolismo dos purina- e pirimidina-nucleótidos: utilização como agentes terapêuticos." Master's thesis, [s.n.], 2015. http://hdl.handle.net/10284/5160.
Full textAbstract:
Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas<br>O conteúdo deste trabalho será desenvolvido em dois temas principais, um referente à
utilização de compostos que interferem no metabolismo dos purina- e pirimidinanucleótidos
como agentes antineoplásicos e outro referente à sua utilização como
agentes antivirais.
A síntese dos nucleótidos envolve a construção de ácidos nucleicos e a inserção dos
derivados de nucleótidos noutras vias bioquímicas, sendo responsável por inúmeras
funções do metabolismo celular.
Existem patologias que envolvem enzimas essenciais do metabolismo dos nucleótidos,
o que levou à síntese de novos fármacos. As doenças oncológicas continuam a matar
milhares de pessoas e um tratamento eficaz e com sucesso tem sido um desafio. O
mesmo se passa com algumas infeções virais, nomeadamente infeções provocadas pelo
HIV.
Para contornar os obstáculos enfrentados na terapia destas doenças têm sido usados
análogos de nucleótidos e/ou nucleósidos como agentes terapêuticos. Estes têm o
propósito de inibir a síntese de novo dos nucleótidos em determinadas etapas, estando
envolvidos na replicação e síntese do RNA e DNA nas células em divisão. Atuam por
inibição específica de enzimas no metabolismo dos nucleótidos/nucleósidos ou ainda
por incorporação no DNA ou no RNA. This study will be developed into two main subjects; one related to the use of
compounds which interfere with the metabolism of purine- and pyrimidine- nucleotides
as antineoplastic agents; another related to their use as antiviral agents.
The nucleotides’ synthesis involves the construction of nucleic acids and the
introduction of the nucleotides’ derivatives into other biochemical pathways and it is
responsible for numerous functions of cellular metabolism.
There are pathologies involving key enzymes from the nucleotides’ metabolism, which
led to the synthesis of new drugs. Cancer is a disease that continues killing thousands of
people, an effective and successful treatment has been a challenge. The same happens
with some viral infections, mainly infections caused by HIV.
To overcome the obstacles faced in the therapy of these diseases it has been used
nucleotide and/or nucleoside analogues as therapeutic agents. These agents have the
purpose of inhibiting the de novo nucleotide synthesis in certain steps, by being
involved in RNA and DNA replication and synthesis in dividing cells. They act by
specific enzymes inhibition in nucleotide/nucleoside metabolism and by incorporation
into DNA or RNA.
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Azad, Kamran Nikkhah. "Pyrimidine Enzyme Specific Activity at Four Different Phases of Growth in Minimal and Rich Media, and Concomitant Virulence Factors Evaluation in Pseudomonas aeruginosa." Thesis, University of North Texas, 2005. https://digital.library.unt.edu/ark:/67531/metadc4918/.
Full textAbstract:
Pseudomonas aeruginosa is a Gram-negative rod, aerobic, non-fermenting, oxidase positive, pigment producing, and nutritionally versatile bacterium. Infections by P. aeruginosa are the most important cause of morbidity and mortality in immunocompromised patients, given virulence factor production that suppresses antibiotic therapy and promotes persistent infection. This research is the first comprehensive report of the pyrimidine biosynthetic pathway for all phases of growth in minimal and rich media coupled with the evaluation of virulence factor production of P. aeruginosa in comparison to four other bacterial species (Pseudomonas putida, Pseudomonas fluorescens, Burkholderia cepacia, and Escherichia coli wild-type strains). Cellular growth and passing genetic information to the next generation depend on the synthesis of purines and pyrimidines, the precursors of DNA and RNA. The pyrimidine biosynthetic pathway is essential and found in most organisms, with the exception of a few parasites that depend upon the pyrimidine salvage pathway for growth. Both the pyrimidine biosynthetic and salvage enzymes are targets for chemotherapeutic agents. In our laboratory, research on pyrimidine auxotrophic mutants showed the role of the pyrimidine biosynthetic pathway and its intermediates on P. aeruginosa metabolism and impaired virulence factors production. The present research shows that pyrimidine enzymes are active in all phases of growth, including the production of two forms of ATCase in the late log phase in P. aeruginosa. This finding may be explained by the displacement of the inactive PyrC' by the active PyrC or PyrC2 to form a new and larger pyrBC encoded ATCase. Pseudomonas aeruginosa wild-type appears to produce by far the most virulence factors, haemolysin, iron chelation, rhamnolipid, adherence, and three types of motility (swimming, swarming, and twitching) investigated in this study, when compared to the other four wild-type strains. Growth analysis was carried out as typically done in minimal medium but also in rich medium to simulate conditions in the blood and lung tissues of humans as P. aeruginosa infections develop.
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Rayala, Ramanjaneyulu. "Design and Synthesis of Novel Nucleoside Analogues: Oxidative and Reductive Approaches toward Synthesis of 2'-Fluoro Pyrimidine Nucleosides." FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/2172.
Full textAbstract:
Fluorinated nucleosides, especially the analogues with fluorine atom(s) in the ribose ring, have been known to exert potent biological activities. The first part of this dissertation was aimed at developing oxidative desulfurization-fluorination and reductive desulfonylation-fluorination methodologies toward the synthesis of 2'-mono and/or 2',2'-difluoro pyrimidine nucleosides from the corresponding 2'-arylthiopyrimidine precursors. Novel oxidative desulfurization-difluorination methodology was developed for the synthesis of α,α-difluorinted esters from the corresponding α-arylthio esters, wherein the arylthio group is present on a secondary internal carbon. For the reductive desulfonylation studies, cyclic voltammetry was utilized to measure the reduction potentials at which the sulfone moiety of substrates can be cleaved.
The 5-bromo pyrimidine nucleosides and 8-bromo purine nucleosides act as crucial intermediates in various synthetic transformations. The second part of the present dissertation was designed to develop a novel bromination methodology using 1,3-dibromo-5,5-dimethylhydantoin (DBH). Various protected and deprotected pyrimidine and purine nucleosides were converted to their respective C5 and C8 brominated counterparts using DBH. The effect of Lewis acids, solvents, and temperature on the efficiency of bromination was studied. Also, N-bromosuccinimide (NBS) or DBH offered a convenient access to 8-bromotoyocamycin and 8-bromosangivamycin.
Third part of this research work focuses on the design and synthesis of 6-N-benzylated derivatives of 7-deazapurine nucleoside antibiotics, such as tubercidin, sangivamycin and toyocamycin. Target molecules were synthesized by two methods. First method involves treatment of 7-deazapurine substrates with benzylbromide followed by dimethylamine-promoted Dimroth rearrangement. The second method employs fluoro-diazotization followed by SNAr displacement of the 6-fluoro group by a benzylamine. The 6-N-benzylated 7-deazapurine nucleosides showed type-specific inhibition of cancer cell proliferation at micromolar concentrations and weak inhibition of human equilibrative nucleoside transport protein (hENT1).
In the fourth part of this dissertation, syntheses of C7 or C8 modified 7-deazapurine nucleosides, which might exhibit fluorescent properties, were undertaken. 8-Azidotoyocamycin was synthesized by treatment of 8-bromotoyocamycin with sodium azide. Strain promoted click chemistry of 8-azidotoyocamycin with cyclooctynes gave the corresponding 8-triazolyl derivatives. Alternatively, 7-benzotriazolyl tubercidin was synthesized by iodine catalyzed CH arylation of tubercidin with benzotriazole.
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Entezampour, Mohammad. "Quantitation of Endogenous Nucleotide Pools in Pseudomonas aeruginosa." Thesis, University of North Texas, 1988. https://digital.library.unt.edu/ark:/67531/metadc500491/.
Full textAbstract:
Nucleotide pools were extracted and quantified from Pyr^+ and Pyr^- strains of P. aerucjinosa. Strains were grown in succinate minimal medium with and without pyrimidines, and nucleotides were extracted using trichloracetic acid (TCA; 6% w/v). The pyrimidine requirement was satisfied by uracil, uridine, cytosine or cytidine. Pyr^- mutants were starved for pyrimidines for two hours before nucleotide levels were measured. This starvation depleted the nucleotide pools which were restored to wild type levels by the addition of pyrimidines to the medium. When the pyrimidine analogue, 6-azauracil, known to inhibit OMP decarboxylase, was added to cultures of the wild type strain, the uridine and cytidine nucleotides were depleted to near zero. Thus, the nucleotide pool levels of Pseudomonas strains can be manipulated.
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Visser, Daniel Finsch. "Isolation and evolution of novel nucleoside phosphorylases." Thesis, Rhodes University, 2010. http://hdl.handle.net/10962/d1004031.
Full textAbstract:
Approximately 33.4 million people are living with HIV/AIDS. Of those, 97% live in low and middle income countries, with 22.4 million in sub-Saharan Africa. Only 42% of the people who require anti-retrovirals (ARVs) in low to middle income countries are receiving anti-retroviral therapy (ART). There is a need to develop novel and cost effective methods for producing antiretroviral drugs. Stavudine and azidothymidine (AZT) were identified as potential targets because they could both be produced through a common intermediate – 5 methyluridine (5-MU). It has been established that the biocatalytic production of 5-methyluridine is possible through a reaction known as transglycosylation, in a process which has not previously been demonstrated as commercially viable. A selection of biocatalysts were expressed either in recombinant E. coli strains or in the wild type organisms, purified and then screened for their ability to produce 5-MU. A combination of Bacillus halodurans purine nucleoside phosphorylase 1 (BHPNP1) and E. coli uridine phosphorylase (EcUP) gave the highest 5-MU yield (80%). This result represents the first combination of free enzymes from different organisms, giving high yields of 5-MU under high substrate conditions. Both enzymes were purified and successfully characterised. The established pH optimum was pH 7.0 for both enzymes. Temperature optima and stability data for BHPNP1 (70 C and t1/2 at 60 C of 20.8 h) indicated that the biocatalytic step was operating within the capabilities of this enzyme and would operate well at elevated temperatures (up to 60 C). Conversely, the temperature optimum and stability data for EcUP (optimum of 40 C and t1/2 at 60 C of 9.9 h) indicated that the enzyme remained active at 40 C for the duration of a 25 h biotransformation, but at 60 C would only be operating at 20% of its optimum activity and would lose activity rapidly. BHPNP1 and EcUP were used in a bench scale (650 ml) transglycosylation for the production of 5-MU. A 5-MU yield of 79.1% was obtained at this scale with a reactor productivity of 1.37 g.l-1.h-1. Iterative saturation mutagenesis was used to rapidly evolve EcUP for improved thermostability. A moderately high throughput colorimetric method was developed for screening the mutants based on the release of p-nitrophenol upon phosphorolysis of a pyrimidine nucleoside analogue. By screening under 20 000 clones the mutant UPL8 was isolated. The mutant enzyme showed an optimum temperature of 60 C and improved stability at 60 C (t1/2 = 17.3 h). The increase in stability of UPL8 is due to only 2 mutations (Lys235Arg, Gln236Ala). These mutations may have caused an increase in stability due to interactions with other structural units in the protein, stabilization of the entrance to the binding pocket, or by decreasing the flexibility of the α-helix at the N-terminus. Transglycosylation experiments showed that the mutant enzyme UPL8 is a superior catalyst for the production of 5-MU. A 300% increase in reactor productivity was noted when free enzyme preparations of UPL8 was combined with BHPNP1 at 1.5% m.m-1 substrate loading. The high yield of 5-MU (75-80% mol.mol-1) was maintained at 9% m.m-1 substrate loading. A commercially viable productivity of 31 g.l-1.h-1 was thus realised. Further optimisation of the process could produce still higher productivities. Future work in directed evolution of nucleoside phosphorylases is envisaged for improved stability and enhanced substrate range for application to other commercially relevant transglycosylation reactions.
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Guo, Wenyue. "A study of structure and function of two enzymes in pyrimidine biosynthesis." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2772.
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Thesis advisor: Evan R. Kantrowitz<br>Nucleotides, the building blocks for nucleic acids, are essential for cell growth and replication. In E. coli the enzyme responsible for the regulation of pyrimidine nucleotide biosynthesis is aspartate transcarbamoylase (ATCase), which catalyzes the committed step in this pathway. ATCase is allosterically inhibited by CTP and UTP in the presence of CTP, the end products of the pyrimidine pathway. ATP, the end product of the purine biosynthetic pathway, acts as an allosteric activator. ATCase undergoes the allosteric transition from the low-activity and low-affinity T state to the high-activity and high-affinity R state upon the binding of the substrates. In this work we were able to trap an intermediate ATCase along the path of the allosteric transition between the T and R states. Both the X-ray crystallography and small-angle X-ray scattering in solution clearly demonstrated that the mutant ATCase (K164E/E239K) exists in an intermediate quaternary structure shifted about one-third toward the canonical R structure from the T structure. The structure of this intermediate ATCase is helping to understand the mechanism of the allosteric transition on a molecular basis. In this work we also discovered that a metal ion, such as Mg2+, was required for the synergistic inhibition by UTP in the presence of CTP. Therefore, the metal ion also had significant influence on how other nucleotides effect the enzyme. A more physiological relevant model was proposed involving the metal ion. To better understand the allosteric transition of ATCase, time-resolved small-angle X-ray scattering was utilized to track the conformational changes of the quaternary structure of the enzyme upon reaction with the natural substrates, PALA and nucleotide effectors. The transition rate was increased with an increasing concentration of the natural substrates but became over one order of magnitude slower with addition of PALA. Addition of ATP to the substrates increased the rate of the transition whereas CTP or the combination of CTP and UTP exhibited the opposite effect. In this work we also studied E. coli dihydroorotase (DHOase), which catalyzes the following step of ATCase in the pyrimidine biosynthetic pathway. A virtual high throughput screening system was employed to screen for inhibitors of DHOase, which may become potential anti-proliferation and anti-malarial drug candidates. Upon the discovery of the different conformations of the 100's loop of DHOase when substrate or product bound at the active site, we've genetically incorporated an unnatural fluorescent amino acid to a site on this loop in the hope of obtaining a better understanding of the catalysis that may involve the movement of the 100's loop<br>Thesis (PhD) — Boston College, 2012<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
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Harris, Katharine Morse. "Studies of structure, function and mechanism in pyrimidine nucleotide biosynthesis." Thesis, Boston College, 2012. http://hdl.handle.net/2345/2594.
Full textAbstract:
Thesis advisor: Evan R. Kantrowitz<br>Thesis advisor: Mary F. Roberts<br>Living organisms depend on enzymes for the synthesis using small molecule precursors of cellular building blocks. For example, the amino acid aspartate is synthesized in one step by the amination of oxaloacetate, an intermediate compound produced in the citric acid cycle, exclusively by means of an aminotransferase enzyme. Therefore, function of this aminotransferase is critical to produce the amino acid. In the Kantrowitz Lab, we seek to understand the molecular rational for the function of enzymes that control rates for the biosynthesis of cellular building blocks. If one imagines the above aspartate-synthesis example as a single running conveyer belt, any oxaloacetate that finds its way onto that belt will be chemically transformed to give aspartate. We can extend this notion of a conveyer belt to any enzyme. Therefore, the rate at which the belt moves dictates the rate of synthesis. Now imagine many, many conveyer belts lined in a row to give analogy to a biosynthesis pathway requiring more than one enzyme for complete chemical synthesis. This is such the case for the biosynthesis of nucleotides and glucose. Nature has developed clever tricks to exquisitely control the rate of product output but means of altering the rate of one or some of the belts in the line of many, without affecting the rate of others. This type of biosynthetic rate regulation is termed allostery. Studies described in this dissertation will address questions of allosteric processes and the chemistry performed by two entirely different enzymes and biosynthetic pathways. The first enzyme of interest is fructose-1,6-bisphosphatase (FBPase) and its role in the biosynthesis of glucose. Following FBPase introduction in Chapter One, Chapter Two describes the minimal atomic scaffold necessary in a new class of allosteric type 2 diabetes drug molecules to effect catalytic inhibition of Homo sapiens FBPase. Following, is the second enzyme of interest, aspartate transcarbamoylase (ATCase) and its role in the biosynthesis of pyrimidine nucleotides. Succeeding ATCase introduction in Chapter Three, Chapter Four describes a body of work exclusively about the catalysis by ATCase. This work was inspired by the human form of the enzyme following the human genome project completion providing data that show likely Homo sapiens ATCase is not allosterically regulated. Chapter Five describes work on a allosterically-regulated, mutant ATCase and provides a biochemical model for the molecular rational for the catalytic inhibition upon cytidine triphosphate (CTP) binding to the allosteric site. The experimental techniques used for answering research questions were enzyme X-ray crystallography, in silico docking, kinetic assay experiments, genetic sub-cloning and genetic mutation<br>Thesis (PhD) — Boston College, 2012<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
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Rodriguez, Rodriguez Mauricio. "Pyrimidine nucleotide de novo biosynthesis as a model of metabolic control." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4425.
Full textAbstract:
This manuscript presents a thorough investigation and description of metabolic control
dynamics in vivo and in silico using as a model de novo pyrimidine biosynthesis.
Metabolic networks have been studied intensely for decades, helping develop a detailed
understanding of the way cells carry out their biosynthetic and catabolic functions.
Biochemical reactions have been defined, pathway structures have been proposed,
networks of genetic control have been examined, and mechanisms of enzymatic activity
and regulation have been elucidated. In parallel with these types of traditional
biochemical analysis, there has been increasing interest in engineering cellular
metabolism for commercial and medical applications. Several different mathematical
approaches have been developed to model biochemical pathways by combining
stoichiometric and/or kinetic information with probabilistic analysis, or deciphering the
comparative logic of metabolic networks using genomic-derived data. However, most of
the research performed to date has relied on theoretical analyses and non-dynamic
physiological states. The studies described in this dissertation provide a unique effort
toward combining mathematical analysis with dynamic transition experimental data.
Most importantly these studies emphasize the significance of providing a quantitative framework for understanding metabolic control. The pathway of de novo biosynthesis of
pyrimidines in Escherichia coli provides an ideal model for the study of metabolic
control, as there is extensive documentation available on each gene and enzyme involved
as well as on their corresponding mechanisms of regulation. Biochemical flux through
the pathway was analyzed under dynamic conditions using middle-exponential growth
and steady state cultures. The fluctuations of the biochemical pathway intermediates and
end products transitions were quantified in response to physiological perturbation.
Different growth rates allowed the comparison of rapid versus long-term equilibrium
shifts in metabolic adaptation. Finally, monitoring enzymatic activity levels during
metabolic transitions provided insight into the interaction of genetic and biochemical
mechanisms of regulation. Thus, it was possible to construct a robust mathematical
model that faithfully represented, with a remarkable predictability, the nature of the
metabolic response to specific environmental perturbations. These studies constitute a
significant contribution to the fields of quantitative biochemistry and metabolic control,
which can be extended to other cellular processes as well as different organisms.
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Zhu, Anting [Verfasser]. "Identification and characterization of pyrimidine 5'-nucleotidases in Arabidopsis thaliana / Anting Zhu." Hannover : Technische Informationsbibliothek (TIB), 2019. http://d-nb.info/1183701845/34.
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Berthod, Thomas. "Synthèse d'oligonucléotides comportant des lésions radio- et photo-induites des bases pyrimidiques." Université Joseph Fourier (Grenoble ; 1971-2015), 1996. http://www.theses.fr/1996GRE10224.
Full textAbstract:
De nombreuses modifications des bases de l'adn peuvent etre generees par divers facteurs comme les agents oxydants ou cancerigenes, les rayonnements afin d'evaluer les consequences biologiques et physico-chimiques de ces dommages, il est necessaire de posseder des modeles plus complexes de ceux-ci qui peuvent etre obtenus par leur incorporation dans des oligonucleotides par voie chimique. Ce travail est consacre a la preparation de fragments d'adn contenant des derives de la 2'-desoxyuridine. Le premier volet de ce travail a consiste a preparer un synthon phosphoramidite de la 5-formyl-2'-desoxyuridine et a l'incorporer dans des oligonucleotides de synthese. La suite de ce travail concerne la mise en evidence et la caracterisation d'une nouvelle lesion, resultant de l'oxydation de la thymidine: la 5-carboxy-2'-desoxyuridine. Par ailleurs, la preparation d'oligonucleotides comportant cette lesion a ete realisee. La troisieme partie de ce travail correspond a l'incorporation d'un troisieme defaut dans des oligonucleotides: la 5,6-dihydro-2'-desoxyuridine (dhdu). Une etude plus complete sur les produits de degradation de cette lesion, susceptibles de se former en milieu alcalin, a ete ensuite menee avec le monomere et avec un trinucleotide, d(gdhdut). Dans une derniere partie, la recherche de conditions analytiques de separation de produits d'oxydation de la thymidine par electrophorese capillaire est presentee
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Cockrell, Gregory Mercer. "New Insights into Catalysis and Regulation of the Allosteric Enzyme Aspartate Transcarbamoylase." Thesis, Boston College, 2013. http://hdl.handle.net/2345/3156.
Full textAbstract:
Thesis advisor: Evan R. Kantrowitz<br>The enzyme aspartate transcarbamoylase (ATCase) is an enzyme in the pyrimidine nucleotide biosynthetic pathway. It was once an attractive target for anti-proliferation drugs but has since become a teaching model due to kinetic properties such as cooperativity and allostery exhibited by the Escherichia coli form of the enzyme. ATCase from E. coli has been extensively studied over that last 60 years and is the textbook example of allosteric enzymes. Through this past research it is understood that ATCase is allosterically inhibited by CTP, the end product of pyrimidine biosynthesis, and allosterically activated by ATP, the end product of the parallel purine biosynthetic pathway. Part of the work discussed in this dissertation involves further understanding the catalytic properties of ATCase by examining an unregulated trimeric form from Bacillus subtilis, a bacterial ATCase that more closely resembles the mammalian form than E. coli ATCase. Through X-ray crystallography and molecular modeling, the complete catalytic cycle of B. subtilis ATCase was visualized, which provided new insights into the manifestation of properties such as cooperativity and allostery in forms of ATCase that are regulated. Most of the work described in the following chapters involves understanding allostery in E. coli ATCase. The work here progressively builds a new model of allostery through new X-ray structures of ATCase*NTP complexes. Throughout these studies it has been determined that the allosteric site is bigger than previously thought and that metal ions play a significant role in the kinetic response of the enzyme to nucleotide effectors. This work proves that what is known about ATCase regulation is inaccurate and that currently accepted, and taught, models of allostery are wrong. This new model of allostery for E. coli ATCase unifies all old and current data for ATCase regulation, and has clarified many previously unexplainable results<br>Thesis (PhD) — Boston College, 2013<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
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Romieu, Anthony. "Synthèse d'oligonucléotides modifiés comportant des lésions radio-induites des bases puriques et pyrimidiques." Université Joseph Fourier (Grenoble), 1999. http://www.theses.fr/1999GRE10140.
Full textAbstract:
Divers facteurs comme des agents oxydants ou cancerigenes, les rayonnement ultraviolets et ionisants, peuvent engendrer des modifications des bases de l'adn. Afin d'evaluer les consequences biologiques et physico-chimiques de ces dommages, l'obtention de courts fragments d'adn (oligonucleotides), de sequence definie (20 a 50 bases de long) et comportant une ou plusieurs modifications en des sites bien precis est primordiale. La synthese oligonucleotidique est aujourd'hui la methode de choix pour preparer de tels composes modeles. Ce travail est consacre a la preparation de fragments d'adn synthetiques contenant des nucleosides modifies formes lors de la radiolyse ou de la photosensibilisation des acides nucleiques. La premiere partie de cette these concerne la preparation d'un synthon phosphoramidite de la 5-hydroxy-2-desoxycytidine et l'incorporation de ce dernier dans des oligonucleotides de synthese de 14 a 33 bases de long. La deuxieme partie (chapitres iii et iv) concerne la synthese et l'incorporation de lesions radio-induites originales : les cyclonucleosides. Les deux diastereoisomeres (5r)- et (5s)- des 5,8-cyclopurine-2-desoxyribonucleosides ont ete inseres separement dans differents oligonucleotides (3 a 22 bases de long) en utilisant la chimie phosphoramidite classique. L'incorporation de la (5s, 6s)-5,6-cyclo-5,6-dihydrothymidine a egalement ete effectuee. La structure particuliere de ce cyclonucleosides (perte du caractere aromatique de l'heterocycle azote) ainsi que sa faible reactivite (determinee au cours de nos experiences) nous ont contraint au developpement d'une strategie de synthese radicalement differente de celle utilisee pour les 5,8-cyclopurine-2-desoxyribonucleosides. La troisieme partie de ce travail est consacre a la preparation d'un synthon phosphoramidite pour la 4-hydroxy-8-oxo-7,8-dihydro-2-desoxyguanosine. Au cours de l'une des etapes de synthese de ce precurseur, nous avons pu facilement separer les deux diastereoisomeres (4r)- et (4s)- de ce nucleoside modifie. Ils ont ete incorpores separemment dans les fragments d'adn synthetiques; l'epimerisation de la position c-4 n'etant pas observee au cours de la synthese sur support solide et lors de l'etape de deprotection ammoniacale. La derniere partie de ce manuscrit concerne la preparation d'oligonucleotides (2 a 9 bases de long) contenant un nucleoside modifie precurseur du radical 5-(2-desoxyuridilyl)methyle : la 5-(phenylthiomethyl)-2-desoxyuridine. Ces substrats ont ete utilises dans des etudes mecanistiques ayant pour but de preciser la reactivite de cet intermediaire radicalaire. Pour chacun des dommages incorpores une attention toute particuliere a ete portee a l'integrite des oligonucleotides synthetises. L'utilisation de differentes methodes analytiques (spectrometrie de masse et analyses clhp et maldi-tof des digestions enzymatiques) a permis de demontrer la purete des produits obtenus.
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Santos, Barbara Helen Cortat. "Papel biológico dos dímeros de pirimidina em células humanas irradiadas com radiação UVA." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-07122010-101150/.
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A radiação ultravioleta (UV) pode ser absorvida por diferentes moléculas celulares, incluindo o DNA no qual provoca distorções estruturais. As lesões mais comuns induzidas pela radiação UV são o ciclobutano de pirimidina (CPD) e o fotoproduto (6-4)-pirimidina-pirimidona [(6-4)PPs]. Estas lesões podem ser reparadas pela fotorreativação, caracterizada por ter uma única proteína (fotoliase) que remove lesões empregando luz visível (320-500 nm) como fonte de energia. Foram identificados dois tipos de fotoliases que diferem por sua especificidade ao substrato: CPD-fotoliase e (6-4)-fotoliase. Um outro mecanismo de reparo é o reparo por excisão de nucleotídeos (NER), um mecanismo que envolve múltiplos passos e proteínas. Enquanto os efeitos genotóxicos da UVC e UVB já estão relativamente esclarecidos e bem aceitos, ainda existem controvérsias sobre a genotoxicidade da radiação UVA, devido ao fato de ser fracamente absorvida pelo DNA. Alguns autores acreditam que os seus principais efeitos são gerados de forma indireta pela produção de espécies reativas de oxigênio enquanto outros acreditam que a UVA pode gerar danos ao DNA de forma direta, provocando a formação de dímeros de pirimidina. O objetivo deste trabalho foi verificar os efeitos genotóxicos da radiação UVA em fibroblastos humanos deficientes e proficientes em NER utilizando adenovírus recombinantes contendo uma ou outra fotoliase para verificar se as lesões CPD e (6-4)PP são geradas pela UVA e se elas teriam alguma importância nas respostas verificadas após irradiação. Foi verificado que as células deficientes no gene XPA são mais sensíveis à radiação UVA quando comparadas às células selvagens. Por meio da detecção imunológica, confirmamos a geração das lesões CPD, (6-4)PP e Dewar, fotoisômero da lesão (6-4)PP, após irradiação com UVA no genoma de células humanas. Empregando vetores adenovirais para transdução de fotoliase específica para lesões tipo CPD ou (6-4)PP, confirmamos que de fato essas lesões são formadas em células humanas deficientes em reparo de DNA após irradiação com UVA. Além disso, esses vírus permitiram verificar a relevância biológica dessas lesões na indução de morte celular em células XP-A irradiadas. De fato, os dados indicam que para doses baixas de radiação UVA essas lesões desempenham um importante papel na indução de morte. Não podemos descartar, porém, que lesões indiretas (provavelmente geradas por estresse oxidativo) também tenham papel na indução de morte pela radiação UVA, o que parece ser mais importante a doses médias e altas dessa radiação.<br>Ultraviolet radiation (UV) is absorbed by different cellular molecules, including DNA in which induces structural distortions. The most common lesions induced by UV radiation are the cyclobutane pyrimidine (CPD) and the photoproduct (6-4)-pyrimidine-pyrimidone [(6-4)PP]. These lesions can be repaired by the photoreactivation, characterized by a single protein (photolyase) that removes lesions using visible light (320-500 nm) as energy source. Two types of photolyases had been identified that differ by their substrate specificity: CPD-photolyase and (6-4)-photolyase. Another repair mechanism is the nucleotide excision repair (NER), a mechanism that involves multiple steps and proteins. While the genotoxic effects of UVB and UVC are already relatively well-understood and accepted, there is still controversy about the genotoxicity of UVA radiation, due to its low absorption by DNA. Some authors believe that the major effects are generated indirectly by the production of reactive oxygen species, while others believe that UVA can cause damage to DNA directly, inducing the formation of pyrimidine dimers. The aim of this study was to assess the genotoxic effects of UVA radiation in human fibroblasts deficient and proficient in NER, using recombinant adenovirus expressing the photolyases to verify if CPDs and (6-4)PPs are generated by UVA and whether they had any importance in the responses observed after irradiation. It was found that cells deficient in the XPA gene are more sensitive to UV radiation compared to wild type cells. By immunological detection, we confirm the generation of CPD, (6-4)PP and Dewar, photoisomer of the (6-4)PP lesion, in the genome of human cells after irradiation with UVA. Using adenoviral vectors for the transduction of photolyases specific for CPD or (6-4)PP lesions, we confirm that in fact these lesions are generated in human cells deficient in DNA repair after irradiation with UVA. Moreover, these viruses allowed us to verify the biological relevance of these lesions in the induction of cell death in irradiated XP-A cells. In fact, our data indicates that for low doses of UVA radiation, these lesions play important roles in the induction of death. We cannot rule out, however, that indirect lesions (probably caused by oxidative stress) could also have a role in the induction of death by UVA radiation, which seems to be more important in intermediate and high doses of this radiation.
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Cunha, Silveira Sandra. "Voies de sauvetage du pool des pyrimidines et du NAD : des alliés inattendus dans le maintien de la stabilité du génome." Thesis, Paris Sciences et Lettres (ComUE), 2018. http://www.theses.fr/2018PSLET011.
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Le syndrome de Bloom (SB) est une maladie humaine autosomique récessive rare résultant d’une mutation sur les deux copies du gène BLM, qui code pour la protéine BLM, une 3’-5’ ADN hélicase de la sous-famille recQ. Les cellules SB présentent une forte instabilité génétique et les patients atteints du SB sont prédisposés au développement de tous les types de cancers affectant la population générale. La déplétion en BLM conduit à une chute drastique de l’expression de la cytidine désaminase (CDA), une enzyme de la voie de sauvetage des pyrimidines qui catalyse la désamination hydrolytique de la cytidine (C) et de la désoxycytidine (dC) en uridine (U) et désoxyuridine (dU). La déficience en CDA conduit à un excès de dC et de dCTP (désoxycytidine triphosphate) dans les cellules SB, mais également dans les cellules qui expriment BLM, ce qui entraîne une instabilité génétique. Effectivement, ce déséquilibre du pool de pyrimidines conduit à une diminution significative de l’activité basale de la poly (ADP-ribose) polymérase 1 (PARP-1) qui, elle-même, entraîne une réduction de l’activation de Chk1. Ceci affaiblit l‘efficacité des points de contrôle du cycle cellulaire en aval, favorisant l’accumulation en mitose de séquences d’ADN non répliquées qui conduisent à une formation excessive de ponts anaphasiques ultrafins (UFB). Ces séquences concernent essentiellement des régions du génome « difficiles à répliquer », comme les centromères et les sites fragiles. L’objectif de mon projet thèse était de décrypter le mécanisme conduisant à la réduction de l’activité basale de PARP-1 dans les cellules déficientes en CDA. Nous avons effectué une étude comparative des métabolomes de deux couples de lignées isogéniques exprimant ou non CDA qui a révélé une augmentation du niveau de nicotinamide (NAM), substrat de la nicotinamide phosphoribosyltransférase (NAMPT), et une diminution du niveau de nicotinamide mononucléotide (NMN), produit de la NAMPT, dans les cellules déficientes en CDA. Nous avons confirmé la réduction de l’activité de la NAMPT nucléaire dans les cellules déficientes en CDA. Nous avons également montré que la déplétion en NAMPT par ARN interférence ou l’inhibition chimique de l’activité de la NAMPT reproduit la réduction de l’activité basale de PARP-1 dans les cellules exprimant CDA, et pas dans les cellules déficientes en CDA. De plus, l’expression exogène de la NAMPT sauvage, mais pas celle de la NAMPT mutée dans son site catalytique, restaure complètement l’activité basale de PARP-1 dans les cellules déficientes en CDA, entraînant de fait une normalisation de la fréquence des UFBs dans ces cellules. Ces résultats indiquent que la réduction de l’activité basale de PARP-1 dans les cellules déficientes en CDA résulte de la diminution de l’activité de la NAMPT. Nous proposons un modèle dans lequel l’accumulation intracellulaire de dC /dCTP résultant de la déficience en CDA pourrait entraver l’activité de la NAMPT nucléaire, provoquant une accumulation intracellulaire de NAM, un inhibiteur naturel connu de PARP-1, qui par conséquent réduirait l'activité basale de PARP-1. Nos résultats révèlent pour la première fois, un lien entre la déficience en CDA et le métabolisme du nicotinamide, voie métabolique essentielle pour le maintien de l’intégrité de la cellule<br>Bloom syndrome (BS) is a rare human autosomal recessive disorder resulting from mutations in both copies of the BLM gene, encoding BLM, a 3’-5’ RecQ DNA helicase. BS cells present a strong genetic instability and BS patients are predisposed to a wide range of cancers that commonly affect the general population. BLM depletion leads to the downregulation of cytidine deaminase (CDA), an enzyme of the pyrimidine salvage pathway that catalyzes the hydrolytic deamination of cytidine (C) and deoxycytidine (dC) to uridine (U) and deoxyuridine (U), respectively. CDA defect leads to an excess of cellular dC and deoxycytidine triphosphate (dCTP) in either BS cells or BLM-expressing cells, that jeopardizes genome stability. Indeed, this nucleotide pool disequilibrium leads to a significant reduction of basal Poly (ADP-ribose) polymerase 1 (PARP-1) activity. The resulting low levels of PARP-1 activity disturb Chk1 activation and decrease the efficiency of downstream checkpoints, leading to the accumulation, during mitosis, of unreplicated DNA at some “difficult-to-replicate” loci in the genome, such as centromeres, fragile sites, leading to excess ultrafine anaphase bridge (UFB) formation. The objective of my PhD project was to decipher the mechanism leading to the reduction of basal PARP-1 activity in the absence of CDA. We performed a metabolomic study that revealed an increase in nicotinamide (NAM) levels, the substrate of nicotinamide phosphoribosyltransferase (NAMPT), and a decrease in nicotinamide mononucleotide (NMN) levels, the product of NAMPT. We confirmed the reduction of the nuclear NAMPT activity in CDA-deficient cells. We found that the siRNA-mediated NAMPT knockdown or the chemical NAMPT inhibition reproduce the reduction of basal PARP-1 activity in CDA-proficient cells, but not in CDA-deficient cells. Moreover, expression of exogenous wild type NAMPT, but not of the NAMPT catalytic mutant, fully rescued the reduction of basal PARP1 activity, and the subsequent increase in UFB frequency in CDA-deficient cells. These results indicate that the reduced basal PARP-1 activity in CDA-deficient cells is due to a reduced NAMPT activity. We propose a model in which the intracellular accumulation of dC/dCTP resulting from CDA deficiency might impair the nuclear NAMPT activity, resulting in an intracellular accumulation of NAM, a known natural inhibitor of PARP-1, that consequently reduces PARP-1 activity. Our results highlight for the first time a link between cytidine deaminase deficiency and nicotinamide metabolism, a pathway essential for the maintenance of cell integrity
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Girault, Isabelle. "Identification et dosage immunologique de lésions de l'ADN." Grenoble 1, 1995. http://www.theses.fr/1995GRE10123.
Full textAbstract:
Divers facteurs (agents oxydants, rayonnements) peuvent engendrer des modifications de bases de l'adn. Le premier volet de ce travail de these a consiste a identifier les produits de degradation des bases pyrimidiques par l'ozone. L'identification de ces composes a permis de mettre en evidence deux nouveaux derives et de conclure a un mecanisme d'action specifique. La suite du travail concerne la preparation d'anticorps diriges contre des modifications de bases de l'adn. Nous avons mis au point une nouvelle methode de preparation des antigenes destines a la production d'anticorps utilises pour la detection immunologique des lesions de l'adn. Ainsi, des anticorps polyclonaux diriges contre la 8-oxo-7,8-dihydroguanine (8-oxogua) ont ete obtenus. L'utilisation de ces anticorps a permis de preparer des colonnes d'immunoaffinite, en vue de prepurifier la 8-oxogua a partir de fluides biologiques. D'autres applications ont ete realisees avec des anticorps monoclonaux. Il s'agit du dosage de 8-oxogua et de dimeres de thymine (thy<>thy) dans de l'adn isole et cellulaire. Cependant, la specificite de l'anticorps dirige contre 8-oxogua s'est averee insuffisante pour doser cette lesion par immunologie. Par contre, l'anticorps dirige contre les thy<>thy a permis de mettre en evidence, par un dosage elisa, la formation de ce defaut dans un adn isole ayant subi l'action de l'uv du vide lors du vol spatial biopan. Par ailleurs, un dosage par cytometrie en flux a permis de detecter les dimeres de thymine directement dans l'adn cellulaire irradie par le rayonnement uv-b. Dans une derniere partie, nous avons recherche des anticorps specifiques de la 8-oxogua dans le serum de diabetiques de type i. Ce travail a permis de mettre en evidence des auto-anticorps diriges, non pas contre la lesion de l'adn recherchee, mais contre l'albumine humaine traitee par naio#4 et nabh#4
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Saïag, Bernard. "Modulation de l'etat contractile du muscle lisse vasculaire par les purines, les pyrimidines et leurs recepteurs." Rennes 1, 1989. http://www.theses.fr/1989REN1A052.
Full textAbstract:
Etude de la modulation de l'etat contractile des muscles lisses arteriels (arteres caudale et femorale du rat) et veineux (veines saphene et maxillaire interne du chien) par des molecules de nature purique (adenosine. . . Atp) ou pyrimidique (utp)