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A New LC Column for the Separation and the Quantitation of Nucleotides

Description: A new column, Dionex AS4A, (polystyrenedivinylbenzene matrix) used for the separation of ribonucleotides and deoxyribonucleotides for the first time, and previously used for ion analysis was found superior to conventional silica columns because it separates ribonucleotides and deoxyribonucleotides. Resolution of dGTP was not possible with the Dionex column and CTP and GDP often co-eluted. Using conventional silica columns, monophosphates separated from diphosphates and diphosphates from triphosphates. Using the new Dionex column resolves all three simultaneously. The Dionex column resolved nucleotides with sharper peaks than silica columns, and the longer its retention time the better was the resolution. This Dionex column is stable, with 80 runs possible without cleaning while resolving ribonucleotides and deoxyribonucleotides to the picomole level.
Date: December 1987
Creator: Brock, Patricia C. (Patricia Charlene)
Partner: UNT Libraries

Prediction of Post Mortem Interval from Degradation of Endogenous Nucleotides in Human Subjects

Description: High Performance Liguid Chromatography was used to measure degradation of nucleotides in human cadavers for the purpose of prediction of post mortem interval. Endogenous nucleotides were extracted from integumentary tissue of six(6) human cadavers using six percent(6%) tricholoacetic acid. Linear regression statistical techniques were used to determine linearity of degradation of various nucleotide pools.
Date: April 1993
Creator: Williams, John Burgess
Partner: UNT Libraries

Quantitation of Endogenous Nucleotide Pools in Pseudomonas aeruginosa

Description: 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.
Date: August 1988
Creator: Entezampour, Mohammad
Partner: UNT Libraries

Characterization of aspartate transcarbamoylase in the archaebacterium Methanococcus jannaschii

Description: The ATCase characterized in this study is from the extreme thermophilinc Archaebacterium, Methanococcus jannaschii. The enzyme was very stable at elevated temperatures and possessed activity from 20ºC to 90ºC. M. jannaschii ATCase retained 75 percent of its activity after incubation at 100ºC for a period of 90 minutes.
Date: December 1996
Creator: Stewart, John E. B. (John Edward Bakos)
Partner: UNT Libraries

Endogenous Nucleotide Pools in Growing Cells of Azotobacter Vinelandii

Description: The objective of this investigation was to examine the changes in the nucleotide pools of Azotobacter vinelandii during the growth cycle. Endogenous ribonucleotides were extracted from A. vinelandii using trichloroacetic acid (TCA; 12% w/v). The 5' mono-, di- and triphosphates of adenine, guanine, uracil and cytosine were separated and quantified by anion-exchange high performance liquid chromatography. Results indicated that the adenylate energy charge of A. vinelandii paralleled the growth rate during exponential phase and that it declined rapidly as the stationary phase was reached. In addition, the amount of each nucleotide in A. vinelandii tended to increase in the logarithmic phase and decrease in the stationary phase in a similar manner to the energy charge.
Date: August 1987
Creator: Lee, Yick-Shun
Partner: UNT Libraries

Genome Sequence Databases (Overview): Sequencing and Assembly

Description: From the date its role in heredity was discovered, DNA has been generating interest among scientists from different fields of knowledge: physicists have studied the three dimensional structure of the DNA molecule, biologists tried to decode the secrets of life hidden within these long molecules, and technologists invent and improve methods of DNA analysis. The analysis of the nucleotide sequence of DNA occupies a special place among the methods developed. Thanks to the variety of sequencing technologies available, the process of decoding the sequence of genomic DNA (or whole genome sequencing) has become robust and inexpensive. Meanwhile the assembly of whole genome sequences remains a challenging task. In addition to the need to assemble millions of DNA fragments of different length (from 35 bp (Solexa) to 800 bp (Sanger)), great interest in analysis of microbial communities (metagenomes) of different complexities raises new problems and pushes some new requirements for sequence assembly tools to the forefront. The genome assembly process can be divided into two steps: draft assembly and assembly improvement (finishing). Despite the fact that automatically performed assembly (or draft assembly) is capable of covering up to 98% of the genome, in most cases, it still contains incorrectly assembled reads. The error rate of the consensus sequence produced at this stage is about 1/2000 bp. A finished genome represents the genome assembly of much higher accuracy (with no gaps or incorrectly assembled areas) and quality ({approx}1 error/10,000 bp), validated through a number of computer and laboratory experiments.
Date: January 1, 2009
Creator: Lapidus, Alla L.
Partner: UNT Libraries Government Documents Department

Pyrimidine nucleotide metabolism in Rhizobium meliloti: purification of aspartate transcarbamoylase from a pyrimidine auxotroph

Description: Rhizobium aspartate transcarbamoylase (ATCase; EC 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.
Date: December 1990
Creator: Eguae, Samuel Iyamu
Partner: UNT Libraries

Creation and characterization of an Escherichia coli and Pseudomonas putida hybrid aspartate transcarbamoylase

Description: 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.
Date: December 1992
Creator: Ruley, Jill R. (Jill Rosanne)
Partner: UNT Libraries

Radial Compression High Performance Liquid Chromatography as a Tool for The Measurement of Endogenous Nucleotides in Bacteria

Description: High performance liquid chromatography was used to measure ribonucleoside triphosphates in microbial samples. Anion exchange columns in a radial compression module were used to separate and quantify purine and pyrimidine ribonucleotides. Endogenous ribonucleoside triphosphates were extracted from Escherichia coli and pseudomonas aeruginosa using three different solvents, namely trifluorocetic acid (TFA; 0.5M), trichloroacetic acid (TCA; 6 per cent w/v) and formic acid (1.0M) Extracts were assayed for uridine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP) by using anion exchange radial compression high performance (pressure) liquid chromatography. The three extraction produres were compared for yield of triphosphates. E. coli, the TFA extraction procedure was more sensitive and reliable than TCA and formic acid extraction procedures, but , in P. aeruginosa, the best yields of ATP and GTP were obrained following extraction with TFA. Yields of UTP and CTP increased when extraction was performed in TCA. These data illustrate that different extraction produres produce different measures for different triphosphates, a point often overlooked.
Date: August 1986
Creator: Dutta, Probir Kumar
Partner: UNT Libraries

Isolation and Characterization of the Operon Containing Aspartate Transcarbamoylase and Dihydroorotase from Pseudomonas aeruginosa

Description: 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.
Date: May 1993
Creator: Vickrey, John F. (John Fredrick), 1959-
Partner: UNT Libraries

Characterization of Aspartate Transcarbamoylase and Dihydroorotase in Moraxella Catarrhalis

Description: 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.
Date: May 1998
Creator: Fowler, Michael A. (Michael Allen), 1961-
Partner: UNT Libraries

Molecular and Kinetic Characterization of the Aspartate Transcarbamoylase Dihydroorotase Complex in Pseudomonas putida

Description: 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.
Date: May 1992
Creator: Schurr, Michael J. (Michael John)
Partner: UNT Libraries

Comparative Biochemistry and Evolution of Aspartate Transcarbamoylase from Diverse Bacteria

Description: 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 ...
Date: May 1999
Creator: Hooshdaran, Massoumeh Ziba
Partner: UNT Libraries

Comparison of Aspartate Transcarbamoylase and Pyrimidine Salvage in Sporosarcina urea, Sprolactobacillus inulinus, Lactobacillus fermentum, and Micrococcus luteus

Description: 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.
Date: August 1994
Creator: Barron, Vincent N. (Vincent Neal)
Partner: UNT Libraries

Regulatory Divergence of Aspartate Transcarbamoylase from the Pseudomonads

Description: 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 ...
Date: December 1996
Creator: Linscott, Andrea J. (Andrea Jane)
Partner: UNT Libraries


Description: The Eco R1 restriction enzyme can be shown to be inhibited by nucleotides which correspond to any part of its known site of phosphodiesterase activity. A series of di-, tetra-, and hexa-nucleotide fragments were synthesized and their effect on the activity of the enzyme upon superhelical Co1 E1 DNA studied. The inhibition caused by the individual mononucleotides were also studied. In general all the nucleotide fragments showed some form of interaction with the enzyme system. Tetranucleotides were stronger inhibitors than dinucleotides, which in turn were stronger inhibitors than the mononucleotides. Within each category of inhibitors, those containing the phosphodiester bond which is acted upon by the enzyme were the strongest inhibitors. Only those fragments which were consistent with the enzymes site of activity showed competitive inhibition kinetics. Nucleotides which do not fit within the site of phosphodiesterase activity show non-competitive inhibition kinetics.
Date: November 1, 1979
Creator: Hollis, Donald F.
Partner: UNT Libraries Government Documents Department

Terminator Detection by Support Vector Machine Utilizing aStochastic Context-Free Grammar

Description: A 2-stage detector was designed to find rho-independent transcription terminators in the Escherichia coli genome. The detector includes a Stochastic Context Free Grammar (SCFG) component and a Support Vector Machine (SVM) component. To find terminators, the SCFG searches the intergenic regions of nucleotide sequence for local matches to a terminator grammar that was designed and trained utilizing examples of known terminators. The grammar selects sequences that are the best candidates for terminators and assigns them a prefix, stem-loop, suffix structure using the Cocke-Younger-Kasaami (CYK) algorithm, modified to incorporate energy affects of base pairing. The parameters from this inferred structure are passed to the SVM classifier, which distinguishes terminators from non-terminators that score high according to the terminator grammar. The SVM was trained with negative examples drawn from intergenic sequences that include both featureless and RNA gene regions (which were assigned prefix, stem-loop, suffix structure by the SCFG), so that it successfully distinguishes terminators from either of these. The classifier was found to be 96.4% successful during testing.
Date: December 30, 2006
Creator: Francis-Lyon, Patricia; Cristianini, Nello & Holbrook, Stephen
Partner: UNT Libraries Government Documents Department

Comparative chloroplast genomics: Analyses including new sequencesfrom the angiosperms Nuphar advena and Ranunculus macranthus

Description: The number of completely sequenced plastid genomes available is growing rapidly. This new array of sequences presents new opportunities to perform comparative analyses. In comparative studies, it is most useful to compare across wide phylogenetic spans and, within angiosperms, to include representatives from basally diverging lineages such as the new genomes reported here: Nuphar advena (from a basal-most lineage) and Ranunculus macranthus (from the basal group of eudicots). We report these two new plastid genome sequences and make comparisons (within angiosperms, seed plants, or all photosynthetic lineages) to evaluate features such as the status of ycf15 and ycf68 as protein coding genes, the distribution of simple sequence repeats (SSRs) and longer dispersed repeats (SDR), and patterns of nucleotide composition.
Date: March 1, 2007
Creator: Raubeso, Linda A.; Peery, Rhiannon; Chumley, Timothy W.; Dziubek,Chris; Fourcade, H. Matthew; Boore, Jeffrey L. et al.
Partner: UNT Libraries Government Documents Department

Aspartate Transcarbamoylase of Aeromonas Hydrophila

Description: 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.
Date: December 2000
Creator: Higginbotham, Leah
Partner: UNT Libraries

BioInformatics, Phylogenetics, and Aspartate Transcarbamoylase

Description: 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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Date: August 2000
Creator: Cooke, Patrick Alan
Partner: UNT Libraries