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Investigations into origin and fate of uracil in the mouse genomeDingler, Felix January 2015 (has links)
No description available.
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The regioselectivity of the photoadditions of 5-substituted uracils /Dennis, Frances Grimm January 1979 (has links)
No description available.
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Biochemical characterization and mutational analysis of human uracil-DNA glycosylaseChen, Cheng-Yao 09 December 2004 (has links)
PCR-based codon-specific random mutagenesis and site-specific mutagenesis
were performed to construct a library of 18 amino acid changes at Arg276 in the
conserved leucine-loop of the core catalytic domain of human uracil-DNA glycosylase
(UNG). Each Arg276 mutant was then overproduced in E. coli cells and purified to
apparent homogeneity by conventional chromatography. All of the R276 mutant
proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein
(Ugi) in vitro, suggesting that the active site structure of the mutant enzymes was not
perturbed. The catalytic activity of all mutant proteins was reduced; the least active
mutant, R276E, exhibited 0.6% of wild-type UNG activity, whereas the most active
mutant, R276H, exhibited 43%. Equilibrium binding measurements utilizing a 2-
aminopurine-deoxypseudouridine DNA substrate showed that all mutant proteins
displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed
cross-linking of the R276 mutants to single-stranded DNA was much less
compromised. Using a concatemeric [³²P]U·A DNA polynucleotide substrate to assess
enzyme processivity, UNG was shown to use a processive search mechanism to locate
successive uracil residues, and Arg276 mutations did not alter this attribute. A
transient kinetics approach was used to study six different amino acid substitutions at
Arg276 (R276C, R276E, R276H, R276L, R276W, and R276Y). When reacted with
double-stranded uracil-DNA, these mutations resulted in a significant reduction in the
rate of base flipping and enzyme conformational change, and in catalytic activity.
However, these mutational effects were not observed when the mutant proteins were
reacted with single-stranded uracil-DNA. Thus, mutations at Arg276 effectively
transformed the enzyme into a single-strand-specific uracil-DNA glycosylase. The
nuclear form of human uracil-DNA glycosylase (LTNG2) was overproduced in E. coli
cells and purified to apparent homogeneity. While UNG2 retained ~9 % of UNG
activity, it did form a stable complex with Ugi. Paradoxically, low concentrations of
NaC1 and MgC1₂ stimulated UNG2 catalytic activity as well as the rate of rapid
fluorescence quenching; however, the rate of uracil flipping was reduced. When
UNG2 bound pseudouracil-containing DNA, conformational change was not detected. / Graduation date: 2005
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THE RELATIONSHIP OF RNA SYNTHESIS TO PROTEIN SYNTHESIS, DURING URACIL STARVATION, IN A URACIL REQUIRING STRAIN OF SACCHAROMYCES CEREVISIAEBullaro, Joseph Carava, 1936- January 1970 (has links)
No description available.
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Factors controlling orotic acid metabolism and the biosynthesis of uridine nucleotidesBlair, Donald George Ralph, January 1961 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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A neutralization-reionization mass spectrometry and computational analysis of 3-hydroxypyridine, 2-hydroxypyridine/2-(1H)pyridone, and uracil /Wolken, Jill K. January 2000 (has links)
Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 145-150).
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Part I: Mechanistic study of the base mediated fragmentation of 5-fluorouracil - alkene photoadducts to 5-substituted uracils. Part II: Mechanistic study of the base mediated fragmentation of pirimidinedione -alkene photoadducts to pyridones/Kaminski, Victor Vincent January 1984 (has links)
No description available.
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A molecular analysis of dihydropyrimidine dehydrogenaseJohnston, Stephen J. January 2000 (has links)
Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the reductive catabolism of the pyrimidine bases uracil and thymine. The clinical relevance of this enzyme is illustrated in individuals presenting with the inherited metabolic disorder thymine uraciluria. This syndrome is characterised by high plasma concentrations of thymine and uracil, and may result in clinical features including mental retardation and dysmorphia. DPD is also clinically relevant in the metabolism and subsequent inactivation of the chemotherapeutic agent 5- fluououracil (5FU). DPD activity has been shown to be highly variable in populations of healthy volunteers and cancer patients, but the mechanisms of regulation of DPD activity are as yet poorly understood. The extent of this variation may determine the efficacy or the severity of the side effects of this treatment. The aim of this research was to evaluate DPD in terms of mRNA expression, protein expression, and activity in a variety of normal and tumour tissues in an attempt to gain an insight into the regulation of DPD. Protein expression and catalytic activity were measured using the well-characterised techniques of Western blotting, and the HPLC separation of 5FU metabolites respectively. However, the method evaluating DPD mRNA expression needed to be developed and validated. After the appraisal of various mRNA detection and quantitation methodologies, competitive polymerase chain reaction (cPCR) was selected as the most suitable method for evaluating DPD transcription in these studies. The RNA samples are reverse transcribed into cDNA which then undergoes PCR amplification in the presence of known amounts of a synthetic template ('competitor') and competes for PCR primers with the target of interest. In each PCR reaction different quantities of target and competitor PCR product will be of both PCR products the concentration of the target template in the cDNA sample can be determined. Competitive PCR was demonstrated to be a highly sensitive and specific method for quantitating DPD mRNA expression, and could be used for tissues with both high and low levels of DPD (liver colon respectively). The technique was also found to be highly reproducible and reliable and was deemed to be suitable for use in further studies. To gain an understanding of the regulation of DPD in colorectal tumour, and the effect it may have upon the activity of 5FU in a specific location, the expression/activity profile of DPD was assessed in colorectal tumour, matched normal colorectal tissue, colorectal metastases to liver, and matched normnal liver. DPD activity, mRNA, and protein levels were all significantly higher in the normal liver than colon, and in the normal liver compared to liver metastases. In the colorectal tissues, mRNA levels were significantly lower in the colorectal tumour than normal colonic mucosa, however no significant difference could be determined between tissues for DPD protein and activity. A good relationship was determined between DPD activity and protein expression in colorectal tumour tissue (rs=0.61, p=0.01), whereas a weaker relationship was determined between DPD mRNA and activity for all colorectal tumour, metastases, and normal tissues (0.43, p 0.1). DPD activity has been detected in most tissues tested to date but appears to be tissue specific with higher levels observed in liver and peripheral blood mononuclear cells than other tissues. In these studies, DPD mRNA, protein, and activity were all found to be higher in the human liver tissue than normal colon.
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Characterization of Escherichia coli double-strand uracil-DNA glycosylase and analysis of uracil-initiated base excision DNA repairSung, Jung-Suk 04 June 2002 (has links)
Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified
to apparent homogeneity from bacteria that were defective in uracil-DNA
glycosylase (Ung). After cloning the dug gene, recombinant Dug was
overexpressed, purified, and characterized with respect to activity, substrate
specificity, product DNA binding, and mechanism of action. Purified Dug
excised both uracil and ethenocytosine specifically from double-stranded
DNA substrates. One distinctive characteristic of Dug was that the purified
enzyme removed a near stoichiometric amount of uracil from DNA containing
U/G mispairs. The observed lack of turnover was attributed to tight binding
of Dug to the apyrimidinic-site (AP) contained in the DNA reaction product.
Catalytic activity was stimulated in the presence of E. coli endonuclease IV
that caused AP-site incision and dissociation of Dug. By using enzyme
complementation experiments, Dug was shown to initiate uracil-initiated base
excision repair (BER) in E. coli (ung) cell-free extracts. The relative rate of
repair of uracil- and ethenocytosine-containing DNA in isogenic E. coli cells
that were proficient or deficient in Ung and/or Dug was measured using a
novel competition assay. Complete ethenocytosine-initiated BER displayed an
absolute requirement for Dug and occurred at the same rate as uracil-initiated
BER in the presence of both Ung and Dug. However, the rate of Dug-mediated ethenocytosine-DNA repair was 8-fold faster than that of uracil-DNA mediated by Dug. The distribution of BER patch sizes associated with
both uracil- and ethenocytosine-containing DNA showed similar results. In
both cases, DNA repair synthesis utilized predominantly a long patch BER
mechanism involving the incorporation of 2-20 nucleotides. A previously
unidentified "very long patch" mechanism of BER involving the incorporation
of more than 200 nucleotides was identified and shown to be mediated by
DNA polymerase I. The rate-limiting step associated with uracil-initiated BER
was found to involve DNA ligase and the distribution of BER patch size was
modulated by the ratio of DNA polymerase I and DNA ligase. The fidelity of
DNA repair synthesis associated with complete uracil-DNA BER was
measured using E. coli cell-free extracts that were proficient or deficient in
Ung activity and determined to be 5.5 x 10������ and 19.7 x 10������, respectively. / Graduation date: 2003
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Mechanism of action of Escherichia coli uracil-DNA glycosylase and interaction with the bacteriophage PBS-2 uracil-DNA glycosylase inhibitor proteinLundquist, Amy J. 21 October 1999 (has links)
Graduation date: 2000
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