Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph.D.)--Bowling Green State University, 2006. / Document formatted into pages; contains xii, 145 p. : ill. (some col.) Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 4 uppsatser.

All guanosine residues in tRNA react with glyoxal to form adducts. No breakage of phosphodiester bonds of the tRNA occurs during the reaction. These adducts contain vicinal hydroxyl functions which can complex with borate or be oxidized by periodate to give N-formyl guanosine residues. The latter in tRNA afford little if any protection against attack by RNase T₁. The rate of cleavage at guanosine by this enzyme is slowed by the glyoxal adduct. Cleavage at guanosine by RNase T₂ occurs at less than 10% of the residues under conditions where the enzyme splits all other phosphodiester bonds. When borate is complexed with the adduct, cleavage by RNase T₁ is practically eliminated. The protection of tRNA with this borate glyoxal adduct followed by digestion with RNase T₁ offers a method to selectively cleave tRNA at inosine and 1-methyl guanosine residues The sequential removal of nucleotides from tRNA by snake venom phosphodiesterase (exonuclease) is completely stopped by a guanosine residue possessing a glyoxal-borate complex. Protection of pG with this adduct prevents removal of the phosphate by snake venom 5'-nucleotidase. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The structural features involved in recognition of transfer ribonucleic acids (tRNAs) by aminoacyl-tRNA synthetases have been investigated using an inhibition assay. It was expected that any oligo- or polynucleotide possessing any of the structural features required of a tRNA for recognition by its aminoacyl-tRNA synthetase would cause specific competitive inhibition of that enzyme. Oligonucleotides from a pancreatic r ibonuc lease digest of RNA were separated according to chain length, then certain isoplithic fractions were subfractionated on the basis of composition. A number of the separated oligonucleotides caused inhibition of valine, lysine and arginine estexification, but in no case was this specific for a given amino acid or kinetically competitive . Oligonucleotides from a ribonuclease T₁ digest of yeast tRNA were separated into isopLithic fractions , but none of these, were significantly inhibitory toward valine, lysine, aspartic acid or phenylalanine esterification to tRNA at the levels tested. Oligonucleotides from a pancreatic ribonuclease digest of poly A and poly U, when separated into isoplithic fractions, did not competitively or specifically inhibit lysine or phenylalanine esterification to tRNA. A number of studies were conducted to determine whether or not periodate-oxidized tRNA (tRNAox) could be used as a starting point for recognition studies. Attempts were made to find conditions under which inhibitory samples of tRNAox could be partially digested with nucleases but still retain the capacity to inhibit aminoacylation. These experiments gave no indication of success and so were abandoned. Cyanoethylated tRNA was oxidized with periodate to determine whether or not it had the capacity to inhibit, but the results were inconclusive. Samples of tRNA were subjected to stepwise degradation and a collection of degraded species was obtained. These were found to vary in their capacity to inhibit valine, lysine and phenylalanine esterification to tRNA. Thus, none of the degraded species inhibited phenylalanine esterification, whereas all of those prepared inhibited valine esterification. Lysine esterification had an intermediate response to these tRNAs. The necessary control experiments were carried out to show that the reagents used in the degradations had no effect on acceptor activities. The response of valine and phenylalanine esterification to variations in magnesium ion concentrations was measured and it was shown to differ significantly for each amino acid. Phenylalanine esterification was shown to be strongly inhibited by excessive quantities of magnesium ion while valine esterification was not. A need for a quantity of magnesium ion in excess of that amount chelated by adenosine triphosphate was demonstrated. Attempts were made to measure the extent of binding of magnesium ion to tRNA under the conditions of aminoacylation assays by the equilibration dialysis technique. However, the errors of the method used for magnesium ion determination were too great to allow any firm conclusions to be drawn. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

A number of structures had been previously proposed for prokaryotic 5S RNA, eukaryotic 5S RNA and eukaryotic 5.8S RNA based on a minimal amount of experimental evidence. None of the models could be adapted to all 5S RNA and 5.8S RNA species, although experimental evidence suggested similar structures. Therefore, a cloverleaf structure was proposed, based on laser Raman evidence (G.A. Luoma, M. Sc. Thesis). This structure could be adapted to E. coli 5S RNA, S. cerevisiae 5S RNA and S. cerevisiae 5.8S RNA while satisfying the limited amount of experimental evidence. To increase the experimental structural data, ultraviolet spectroscopy, circular dichroism spectroscopy, low field proton nuclear magnetic resonance spectroscopy and electron spin resonance spectroscopy were performed on S. cerevisiae 5S RNA, E. coli 5S RNA and wheat germ 5S RNA. Also, 5-fluorouracil was incorporated into S. cerevisiae 5S RNA to attempt to obtain further structural evidence. Finally, the presence of multiple conformations in the 5S RNA species was determined. This substantial physical characterisation is in total support of the cloverleaf model for these 5S RNA species. The cloverleaf model has also been adapted to all known sequences of 5S RNA and 5.8S RNA. For eukaryotic 5S RNA, the cloverleaf can explain the evolution of the various species. For prokaryotic 5S RNA, the cloverleaf can explain the inter-conversion of the native and B-form conformers. Finally, for 5.8S RNA, the cloverleaf model can explain the ability of yeast 5.8S RNA and E. coli 5S RNA to bind the same ribosomal proteins. / Science, Faculty of / Chemistry, Department of / Graduate

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Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Cheng Tat Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 106-114). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of content --- p.iii / Abbreviations --- p.iv / List of figures --- p.v / List of tables --- p.vi / Chapter Chapter One --- Introduction / Chapter 1.1 --- Ribozyme --- p.1 / Chapter 1.1.1 --- RNA world hypothesis --- p.2 / Chapter 1.1.2 --- Hammerhead ribozyme --- p.3 / Chapter 1.1.3 --- Applications of hammerhead ribozymes --- p.4 / Chapter 1.1.4 --- Allosteric ribozyme --- p.4 / Chapter 1.1.5 --- Ribozyme screening system --- p.5 / Chapter 1.2 --- Other RNA as gene silencing agents --- p.7 / Chapter 1.2.1 --- RNAi --- p.7 / Chapter 1.2.2 --- Antisense RNA --- p.10 / Chapter 1.3 --- Project Overview --- p.11 / Chapter 1.3.1 --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies --- p.11 / Chapter 1.3.2 --- Construction of anti-Elfin ribozyme and its application on gene silencing study --- p.11 / Chapter Chapter Two --- Materials and Methods / Chapter 2.1 --- Cloning techniques --- p.13 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.13 / Chapter 2.1.2 --- Restriction digestion of DNA --- p.13 / Chapter 2.1.3 --- Ligation of DNA fragments --- p.14 / Chapter 2.1.4 --- Preparation of competent cells --- p.15 / Chapter 2.1.5 --- Transformation of competent cells --- p.16 / Chapter 2.1.6 --- Gel extraction --- p.16 / Chapter 2.1.7 --- Plasmid preparation --- p.17 / Chapter 2.1.7.1 --- Mini scale plasmid preparation --- p.17 / Chapter 2.1.7.2 --- Medium scale plasmid preparation --- p.19 / Chapter 2.1.8 --- DNA agarose gel electrophoresis --- p.20 / Chapter 2.1.9 --- Buffer and reagents --- p.21 / Chapter 2.2 --- In vitro cleavage of target RNA by ribozymes --- p.22 / Chapter 2.2.1 --- In vitro transcription of target RNA and ribozymes --- p.22 / Chapter 2.2.2 --- Purification of transcription products --- p.22 / Chapter 2.2.3 --- Ribozymatic cleavage reaction --- p.24 / Chapter 2.2.4 --- Preparation of RNA size marker templates --- p.24 / Chapter 2.2.5 --- Urea-acrylamide gel electrophoresis --- p.25 / Chapter 2.2.6 --- Autoradiography --- p.26 / Chapter 2.2.7 --- Buffer and reagents --- p.26 / Chapter 2.3 --- Detection of cellular RNA expression RT-PCR detection --- p.28 / Chapter 2.3.1 --- RNA extraction --- p.28 / Chapter 2.3.2 --- DNase I digestion --- p.29 / Chapter 2.3.3 --- Reverse transcription --- p.29 / Chapter 2.4 --- Mammalian cell culture techniques --- p.31 / Chapter 2.4.1 --- Transfection into mammalian cells --- p.31 / Chapter 2.4.2 --- Counting the number of cells --- p.32 / Chapter 2.4.2 --- Buffer and reagents --- p.32 / Chapter Chapter Three --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.1.1 --- Objectives --- p.34 / Chapter 3.1.2 --- Why anti-GFP ribozymes? --- p.34 / Chapter 3.2 --- Construction of anti-GFP ribozymes that are active in vitro --- p.36 / Chapter 3.2.1 --- Design of the anti-GFP ribozymes --- p.36 / Chapter 3.2.2 --- Construction of DNA templates for in vitro transcription --- p.40 / Chapter 3.2.3 --- GFP RNA was successfully cleaved by ribozymes --- p.45 / Chapter 3.3 --- In vivo activities of anti-GFP ribozymes --- p.48 / Chapter 3.3.1 --- Design of systems that detected anti-GFP ribozyme activities in --- p.48 / Chapter 3.4 --- The first trial - system α --- p.50 / Chapter 3.4.1 --- Cloning of ribozymes into pACYC184 --- p.51 / Chapter 3.4.2 --- IPTG interfered with GFP expression --- p.54 / Chapter 3.5 --- The second trial - system β --- p.57 / Chapter 3.5.1 --- Insertion of new multiple cloning sites into pET3d --- p.58 / Chapter 3.5.2 --- Cloning of GFP into pET-neu --- p.60 / Chapter 3.5.3 --- GFP expression was interfered by the additional T7 promoter --- p.62 / Chapter 3.6 --- The third trial - system δ --- p.65 / Chapter 3.6.1 --- Insertion of a new cloning site into pET-neu --- p.66 / Chapter 3.6.2 --- Cloning of GFP and ribozymes into pET-fn --- p.68 / Chapter 3.6.3 --- Ribozymes sequence upstream of GFP interfered with GFP expression / Chapter 3.7 --- The fourth trial - system co --- p.73 / Chapter 3.7.1 --- Insertion of new cloning sites into pET-neu --- p.74 / Chapter 3.7.2 --- Cloning of GFP and ribozymes into pET-nr --- p.76 / Chapter 3.7.3 --- Anti-GFP ribozymes did not turn off green fluorescence of GFP --- p.79 / Chapter 3.7.4 --- No in vivo cleavage of GFP was detected --- p.81 / Chapter 3.8 --- Summary --- p.83 / Chapter Chapter Four --- Construction of an anti-Elfin ribozyme and its application on gene silencing study / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Objectives --- p.84 / Chapter 4.1.2 --- Elfin --- p.84 / Chapter 4.1.3 --- Experimental plan --- p.85 / Chapter 4.2 --- In vitro cleavage of Elfin RNA by ribozyme --- p.86 / Chapter 4.2.1 --- Design of anti-Elfin ribozyme --- p.86 / Chapter 4.2.2 --- Preparation of DNA template for in vitro transcription --- p.88 / Chapter 4.2.3 --- Successful in vitro cleavage of Elfin RNA by ribozyme --- p.88 / Chapter 4.3 --- In vivo gene silencing studies of RNA tools --- p.90 / Chapter 4.3.1 --- Design of antisense RNA --- p.90 / Chapter 4.3.2 --- Design of siRNA --- p.92 / Chapter 4.3.3 --- Cloning of RNA tools into pSilencer --- p.94 / Chapter 4.3.4 --- Cloning of a neomycin resistance gene into pSilencer-R --- p.94 / Chapter 4.3.5 --- Elfin RNA was not down-regulated --- p.97 / Chapter 4.4 --- Summary --- p.100 / Chapter Chapter 5 --- Discussions --- p.101 / Reference / Appendix