No description available.

I have measured the metabolism of tRNA$ sp{ rm Asn}$ in Friend cells undergoing erythroid differentiation, using an assay exploiting the tRNA$ sp{ rm Asn}$ gene's ability to specifically hybridize tRNA$ sp{ rm Asn}$. The concentrations of tRNA$ sp{ rm Asn}$ measured as a proportion of the total tRNA population (relative concentration) decreased until day 3 post-induction, and then increased on day 4. The relative concentrations of tRNA$ sp{ rm Asn}$ are influenced by tRNA$ sp{ rm Asn}$ having lower turnover rate in comparison to the total tRNA population, and by the relative rate that labelled, newly synthesized tRNA$ sp{ rm Asn}$ accumulates in vivo. The rate of tRNA$ sp{ rm Asn}$ synthesis in nuclei isolated from Friend cells at different times during differentiation also fluctuates. This fluctuation may reflect changes in RNA polymerase III activity in these isolated nuclei.

Thesis (Ph. D.)--University of Oregon, 2005. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 102 - 112). Also available for download via the World Wide Web; free to University of Oregon users.

No description available.

Two different cloned human DNA segments encompassing transfer RNA gene and pseudogene clusters have been isolated from a human gene library harbored in bacteriophage lambda Charon 4-A. One clone (designated as λhVal7) encompassing a 20.5-kilobase (Kb) human DNA insert was found to contain a valine transfer RNA_AAC gene and several Alu-like elements by Southern blot hybridization analysis and DNA sequencing with the dideoxyribonucleotide chain-termination method in the bacteriophage M13mp19 vector. Another lambda clone (designated as λhLeu8) encompassing a 14.3-Kb segment of human DNA was found to contain a methionine elongator transfer RNA_CAT pseudogene and other as yet unidentified transfer RNA pseudogenes.

Two phage lambda clones encompassing human tRNA genes have been isolated from a human gene library harbored in bacteriophage lambda Charon-UA. One of the clones (designated as hLeuU) containing a 20-kb human DNA fragment was isolated and found to contain a cluster of four tRNA genes. An 8.2-kb Hindlll fragment encompassing the four tRNA genes was isolated from the 20-kb fragment and subcloned into pBR322 for restriction mapping and DNA sequence analysis. The four tRNA genes are arranged as two tandem pairs with the first pair containing a proline tRNAAGQ gene and a leucine tRNAAAQ gene and the second pair containing another proline tRNAAGG gene and a threonine tRNAuQU gene. The two pairs are separated about 3 kb from each other, and the leucine tRNAAAG gene is of opposite polarity from the other three tRNA genes. The tRNA transcription units were sequenced by a unidirectional deletion dideoxyribonucleotide chain-termination method in the M13mpl8 and 19 vectors. The coding regions of the four tRNA genes contain characteristic internal split promoter sequences and do not encode intervening sequences nor the CCA trinucleotide found in mature tRNAs. The proline t R N A A G G gene is separated from the leucine t R N A A A Q gene by a 725-bp intergenic region and the second proline t R N A A G Q is 315 bp downstream of the threonine t R N A U G U gene. The coding sequences of the two proline tRNA genes are identical. The 3'-flanking regions near the 3*-ends of these four tRNA genes have typical RNA polymerase III termination sites of at least four c o n s e c u t i v e T nt. There is no homology between the 5'-flanking regions of these genes. All four tRNA genes are potentially functional, since they are transcribed by RNA polymerase III in an in vitro HeLa cell extract. Another phage lambda clone (designated as XhLeu8) was also found to contain four tRNA genes. One of the tRNA genes was characterized by DNA sequencing. The tRNA gene has an anticodon for leucine, but has three base substitutions from the leucine tRNA^A G and the leucine tRNAAAQ gene of AhLPT, occurring in the D-stem, D-loop and T-loop regions. The substitution in the T-loop is a C to T transition at an otherwise invariant position of the consensus sequence, 5'-GTTC-3'» within the B-block of the internal split promotor. Thus, this gene may more properly be classified as a leucine tRNA pseudogene.

This dissertation covers two projects linked by their involvement in the modification of tRNA bases. The first project focused on an investigation of a role for the modified base Archaeosine, the ubiquitous modification in tRNA in the archaeal domain. Initial work was performed on a set of in vitro prepared tRNA modified to feature either the canonical guanine base at position 15, preQ0 (TGT product) or Archaeosine (ArcS product). There was very little difference in the thermal stability of tRNAs containing these modifications in the halophilic H. volcanii tRNASer or E. coli tRNAGln. In tRNAGln taken from M. thermautotrophicum however, there was a 2°C increase in melting point in 50 mM MgCl2 upon modification to archaeosoine. Benefitting from the development of genetic tools for the generation of specific deletion mutants of the thermophile Thermococcus kodakarensis, it was possible to start investigation of tRNAs that have been hypomodified in vivo due to the lack of arcTGT (TK0760) and ArcS (TK2156). In vitro modified equivalents of the GlnCUG isoacceptor were also prepared. Thermal stability of these tRNAs show virtually identical melting transitions, with a biphasic denaturation occurring at all magnesium concentrations tested. Isolation of the CUG isoacceptor from the in vivo maturated total tRNA pool allowed melts of specifically hypomodified tRNAs. Those containg Archaeosine (WT) and genetically encoded guanine (∆tgt) showed identical melting profiles with Tm beyond the 98°C limit of the experiment. In the preQ0 containing in vivo RNA the shows a lag in its magnesium response, and a more persistent biphasic melting profile. At 10mM Mg2+ concentration the preQ0 containing tRNA is approaching a Tm of 98°C though the turn over point in the melt is not well defined. The second project was to investigate the product of base treatment of the oxidized cofactor NAD(P)+. This cofactor is involved in the biosynthesis of preQ1 from preQ0 in bacterial systems and at low concentrations it can be difficult to quantify enzyme activity based on direct quantitation. Under these conditions a fluorescence based method where by the production of NAD(P)+ is measured rather than the consumption of NAD(P)H. Base treatment of the oxidized cofactor generates a fluorescent species with an efficiency of 95%. The assay has been used extensively by our group to track activity of various enzymes including QueF, however the identity of the fluorophore had not been established. Purification of the fluorescent product was achieved by isocratic HPLC in water using a reverse phase column. It was found that the assay conditions previously used (7.5M NaOH for 2 hours) were actually counterproductive for maximizing fluorescence yield. Incubation at 2M NaOH gave a 35% increase in product yield. The isolated product was determined to have molecular weight of 123.0318 (3.6 ppm by accurate mass ESI MS). 1H and 13C NMR were used to confirm the structure to be that of 2-hydroxynicotinaldeyde. It was also possible to determine the quantum yield for the molecule to 0.11. Work carried out previously on pyridinium based NADP analogs is consistent with the identity of the fluorophore presented here.

Thesis (Ph.D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xvi, 147 p.; also includes graphics (some col.). Includes abstract and vita. Advisor: Joseph A. Krzycki, Dept. of Microbiology. Includes bibliographical references (p. 122-147).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 176-188).

Thesis (Ph. D.)--Montana State University--Bozeman, 2005. / Typescript. Chairperson, Graduate Committee: Michele Hardy. Includes bibliographical references (leaves 107-133).