The purpose of this study was to characterize recombinant plasraids carrying Drosophila melanogaster tRNA genes. The two groups of recombinant plasmids studied were those which carried tRNA₄Val genes and those with tRNA₄,₇Ser genes. pDt92 and pDt120, both tRNA₄Val gene-carrying plasmids, were characterized initially to determine the number of inserts they contained and the size of the inserts. For plasmids containing multiple inserts, the insert which carried the tRNA₄Val gene was also determined. These characteristics were studied by HindIII digestion of the plasmid DNA, agarose gel electrophoresis, Southern transfer onto nitrocellulose filters and hybridization to [¹²⁵I] tRNA₄Val. It was found that both, pDt92 and pDt120 contained two inserts each of sizes 0.5kb and 1.7kb,and 2.0kb and 5.*fkb respectively, with the 0.5kb and 2.0kb fragments carrying the tRNA₄Val genes. pDt92 and pDt120 then were recloned so as to contain only the fragments which carried the tRNA₄Val genes, namely the 0.5kb and 2.0kb fragment respectively. pDt92RC and pDt120RC plus three other tRNA₄,₇Ser gene containing plasmids, pDt16, pDt17RC and pDt27RC were further characterized by the technique of in situ hybridization to study the organization of these tRNA genes on the Drosophila genome. Four of these plasmids with the exception of pDt17RC hybridized to only one site on the Drosophila chromosome. Both, pDt92RC and pDt120RC hybridized to the 90BC site on the right arm of the third chromosome; pDt16 and pDt27RC hybridized to the 12DE site on the first or the X chromosome. pDt17RC on the other hand hybridized predominantly to the 12DE site and to a lesser extent to 2}E (2L), 56D (2R), 62D (3L) and 64D (3L) sites. These in situ hybridization results when studied together with those reported by Dunn et al. (1979b) show that genes for a single species of tRNA are located on more than one site on the Drosophila genome. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

In the present study Drosophila melanogaster was used to define the organization and expression of tRNA genes. The three major Drosophila valine tRNAs were isolated and purified by standard chromatographic techniques. Nucleoside analysis indicated that of these tRNAs only tRNA₄Val contained inosine. All three tRNAsVal contained ribothymidine, therefore they resemble yeast tRNAVal in this regard but not the mammalian tRNAsVal which lack ribothymidine. The purified tRNAs were labelled with ¹ ² ⁵I and used to determine the location of the genes for these tRNAs utilizing the technique of in situ hybridization to salivary gland chromosomes. tRNA₄Val hybridized consistently to one site on the right arm of the second chromosome, 56D, which is close to the site of 5S RNA, 56F. tRNA₃bVal hybridized to two sites, 84D and 92B, both on the right arm of the third chromosome. The labelling of site 84D was approximately twice as heavy as that of 92B. Dr. A. Delaney (unpublished) has shown that approximately 13 genes code for tRNA₃bVal per haploid genome. The in situ hybridization data suggests that the 13 genes are divided such that approximately 8 genes are at site 84D and 5 genes are at site 92B. Evidence to support this supposition is derived from measurements on the amount of tRNA₃bVal in mutant flies deficient or duplicated for site 84D on one of their two homologous third chromosomes. tRNA₃bVal amounts, measured relative to the other tRNAVal isoacceptors decrease 31% in the deficiency and increase 30% in the duplication. These results demon- strate a direct relationship of the amount of tRNA₃bVal to gene dosage because the duplication has 8 extra genes, which is a 30% increase and the deletion has 8 fewer genes, a 30% decrease. Finally, it was shown that the amount of total tRNSVal increased by 17% in the duplication but did not decrease in the deletion. This result demonstrates the amount of valine tRNA is under a type of control in which the amount of total valine tRNA is increased to compensate for the deficiency of a single isoacceptor. Also the coding properties of four tRNASer isoacceptors were determined. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Reverse transcription in HIV-1 (human immunodeficiency virus type 1) is initiated from a tRNA, tRNALys3, that is annealed to the primer binding site (PBS) in the 5' region of viral RNA. This tRNA, along with the other major tRNALys isoacceptors, tRNALys1,2 , is selectively packaged into HIV-1 during its assembly. The formation of a tRNALys packaging/annealing complex is believed to involve the interaction between a Gag/GagPol/viral complex with a lysyl-tRNA synthetase (LysRS)/tRNALys complex, with Gag interacting specifically with LysRS, and GagPol interacting with both Gag and tRNALys. In fact, Gag particles alone will package LysRS, but GagPol, which binds tRNA Lys, is also required for incorporation of the tRNALys. / The model we propose for the tRNALys packaging/annealing complex predicts a possible interaction between LysRS and Pol sequences in GagPol, which might facilitate transfer of tRNALys3 from LysRS to the reverse transcriptase (RT) thumb domain where tRNALys3 binds. In this work, we demonstrate that, in addition to its interaction with Gag, LysRS also interacts with sequences within the connection/RNaseH domains in RT. Since these RT domains are not required for tRNALys packaging into HIV-1, the LysRS/Pol interaction is probably not involved in the transfer of tRNALys3 to RT. The LysRS/Pol interaction may instead be involved in tRNALys3 annealing since the connection domain in RT has been found to be required for this process. Also, since an interaction has been reported between Gag and Pol sequences in GagPol, we also investigated whether the Gag/LysRS/Pol interaction played an important role in stabilizing the Gag/Pol interaction, and found, using siRNA to LysRS, that it did not. / tRNALys3 annealing to viral RNA is promoted by nucleocapsid sequences in Gag and by mature NCp7, and we have examined the roles of Gag and NCp7 in this process. Gag- and NC-facilitated tRNALys3 annealing to HIV-1 RNA were measured both in vivo and in vitro, indirectly by the ability of annealed tRNALys3 to prime reverse transcription, and directly by measuring the occupancy of the PBS by tRNALys3. While tRNALys3 annealing can be carried out by both Gag and NCp7, exposure (in vivo or in vitro) of the tRNALys3/viral RNA complex to NCp7 is required for optimum placement of the tRNALys3. This is indicated by 1) tRNALys3's reduced ability to incorporate the first dNTP, dCTP, and 2) its more ready displacement from the PBS by DNA synthesized from a downstream primer. / It has been previously demonstrated that APOBEC3G (A3G) can inhibit tRNA Lys3 annealing to viral RNA, and we have used A3G to further dissect the roles of Gag and NCp7 in annealing, both in vitro and in vivo. Experiments studying how APOBEC3G (A3G) inhibits tRNA Lys3 annealing indicate that in protease-positive viruses, Gag-facilitated tRNALys3 annealing may only playa minor role. In vivo and in vitro, A3G only inhibits NCp7-facilitated annealing, and not Gag-facilitated annealing. Nevertheless, while Gag is able to show 70-80% of the annealing efficiency of NCp7 in a protease-negative virus, A3G can reduce annealing efficiency in protease-positive viruses to 40%. This appears to be due to the fact that, in vitro, the presence of NCp7 makes prior Gag-facilitated annealing susceptible to A3G. This suggests that in wild type viruses, any Gag-facilitated annealing of tRNALys3 to viral RNA that does occur is altered through an A3G-susceptible re-annealing by NCp7.

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Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Title from first page of PDF file (viewed Feb. 14, 2008). Includes bibliographical references.

Wan Tai Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 102-111). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Chinese Abstract --- p.v / Acknowledgements --- p.vii / General Abbreviations --- p.ix / Abbreviations of Chemicals --- p.xi / Table of Contents --- p.xii / List of Figures --- p.xvi / List of Tables --- p.xvi / Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Soybeans --- p.1 / Chapter 1.1.1 --- Nutrient composition of soybean --- p.1 / Chapter 1.1.2 --- Nitrogen fixation and assimilation in soybean --- p.3 / Chapter 1.1.3 --- The role in nitrogen allocation and controlling the nitrogen sink-source relationship of asparagine --- p.3 / Chapter 1.1.4 --- Characterization of asparagine synthetase --- p.8 / Chapter 1.1.4.1 --- Biochemistry and molecular background of plant asparagine synthetase --- p.8 / Chapter 1.1.4.2 --- Asparagine synthetase in Arabadopsis thaliana --- p.9 / Chapter 1.1.4.3 --- "Asparagine synthesis in soybean, Glycine max" --- p.10 / Chapter 1.1.4.4 --- "Asparagine synthetase in rice, Oryza sativa" --- p.11 / Chapter 1.2 --- Seed protein quality and quantity improvement --- p.13 / Chapter 1.2.1 --- Nutrition composition of rice --- p.13 / Chapter 1.2.2 --- Molecular approaches for improving seed storage protein quality --- p.14 / Chapter 1.2.2.1 --- Protein sequence modification --- p.14 / Chapter 1.2.2.2 --- Synthetic genes --- p.16 / Chapter 1.2.2.3 --- Overexpression of homologous genes --- p.17 / Chapter 1.2.2.4 --- Transfer and expression of heterologous genes --- p.18 / Chapter 1.2.2.5 --- "Manipulation of pathway synthesizing essential amino acids, aspartate family amino acid" --- p.19 / Chapter 1.2.3 --- Research in improving rice seed protein quality and quantity --- p.22 / Chapter 1.3 --- Hypothesis and objective of this study --- p.23 / Chapter 2 --- Materials and Methods --- p.25 / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Plant materials --- p.25 / Chapter 2.1.2 --- Bacterial strains and vectors --- p.26 / Chapter 2.1.3 --- Growth conditions for soybean --- p.26 / Chapter 2.1.4 --- Chemicals and reagents --- p.26 / Chapter 2.1.5 --- "Buffer, solution and gel" --- p.26 / Chapter 2.1.6 --- Commercial kits --- p.27 / Chapter 2.1.7 --- Equipments and facilities used --- p.27 / Chapter 2.1.8 --- Primers --- p.27 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Growth condition for plant materials --- p.28 / Chapter 2.2.1.1 --- General conditions for planting soybean --- p.28 / Chapter 2.2.1.2 --- Soybean seedlings for gene expression profile analysis --- p.28 / Chapter 2.2.1.3 --- Mature soybean for gene expression profile analysis --- p.29 / Chapter 2.2.1.4 --- Mature soybean for cloning of AS I and AS2 full length cDNA --- p.30 / Chapter 2.2.1.5 --- Mature soybean seed for amino acid profile analysis --- p.30 / Chapter 2.2.1.6 --- General conditions for planting transgenic rice in CUHK --- p.30 / Chapter 2.2.1.7 --- Transgenic rice seedling for PCR screening --- p.31 / Chapter 2.2.1.8 --- Transgenic rice for functional test and seed for biochemical analysis --- p.31 / Chapter 2.2.2 --- Molecular techniques --- p.32 / Chapter 2.2.2.1 --- Total RNA extraction --- p.32 / Chapter 2.2.2.2 --- Denaturing gel electrophoresis for RNA --- p.33 / Chapter 2.2.2.3 --- Northern blot analysis --- p.33 / Chapter 2.2.2.3.1 --- Chemiluminescent detection --- p.33 / Chapter 2.2.2.3.2 --- Film development --- p.34 / Chapter 2.2.2.4 --- Preparation of single-stranded DIG-labeled PCR probes --- p.34 / Chapter 2.2.2.4.1 --- Primer design for the PCR probes of --- p.34 / Chapter 2.2.2.4.2 --- Amplification of AS1 and AS2 internal PCR fragments --- p.34 / Chapter 2.2.2.4.3 --- Quantitation of purified AS1 and AS2 PCR fragments --- p.35 / Chapter 2.2.2.4.4 --- Biased PCR to make single-stranded DNA probes --- p.35 / Chapter 2.2.2.4.5 --- Probe quantitation --- p.36 / Chapter 2.2.2.5 --- Probe specificity test --- p.37 / Chapter 2.2.2.6 --- Cloning of full length cDNA --- p.37 / Chapter 2.2.2.6.1 --- First strand cDNA synthesis from RNA of high protein content soybean leaf --- p.37 / Chapter 2.2.2.6.2 --- PCR for amplification of AS1 and AS2 full length cDNA --- p.38 / Chapter 2.2.2.6.3 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.38 / Chapter 2.2.2.6.4 --- Ligation of DNA inserts into pBluescript II KS(+) T-vector --- p.39 / Chapter 2.2.2.6.5 --- Preparation of E. coli DH5α CaCl2-mediaed competent cells --- p.39 / Chapter 2.2.2.6.6 --- Transformation of E. coli DH5α competent cell --- p.40 / Chapter 2.2.2.7 --- Screening of recombinant plasmids --- p.40 / Chapter 2.2.2.7.1 --- Isolation of recombinant plasimid DNA from bacterial cells --- p.41 / Chapter 2.2.2.7.2 --- PCR screening on recombinant plasmids --- p.41 / Chapter 2.2.2.7.3 --- DNA gel electrophoresis --- p.41 / Chapter 2.2.2.8 --- Sequencing and homology search --- p.42 / Chapter 2.2.2.9 --- Functional test using transgenic plant --- p.43 / Chapter 2.2.2.9.1 --- Preparation of chimeric gene constructs and recombinant plasmids --- p.43 / Chapter 2.2.2.9.2 --- Agrobacterium mediated transformation into rice calli to regenerate transgenic AS1/ AS2 rice --- p.44 / Chapter 2.2.2.10 --- PCR Screenig of homozygous and heterozygous transgenic plants --- p.44 / Chapter 2.2.2.10.1 --- Isolation of genomic DNA from transgenic plants --- p.45 / Chapter 2.2.2.10.2 --- PCR screening using genomic DNA --- p.46 / Chapter 2.2.2.11 --- Quantitative PCR analysis on transgenic plants --- p.48 / Chapter 2.2.3 --- Biochemical Analysis --- p.49 / Chapter 2.2.3.1 --- Quantitative amino acid analysis in mature soybean seeds --- p.49 / Chapter 2.2.3.2 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.49 / Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Amino acid analysis on mature soybean seeds --- p.50 / Chapter 3.2 --- Expression pattern analysis of AS genes by Northern Blot analysis --- p.54 / Chapter 3.2.1 --- Making of single strand digoxigenin (DIG)-labeled probe --- p.54 / Chapter 3.2.2 --- Probe specificity --- p.57 / Chapter 3.2.3 --- AS expression level under light/dark treatments by Northern Blot analysis --- p.58 / Chapter 3.2.4 --- AS expression level in young seedlings by Northern Blot analysis --- p.62 / Chapter 3.2.5 --- AS expression level in podding soybean by Northern Blot analysis --- p.64 / Chapter 3.3 --- Cloning of AS genes from high protein content soybeans --- p.66 / Chapter 3.3.1 --- "PCR amplification of AS1 and AS2 full length cDNA from the first-strand cDNA of high portein content cultivar soybean, YuDoul2" --- p.66 / Chapter 3.3.2 --- Nucleotide sequences analysis of AS1 and AS2 full-length cDNA clones --- p.68 / Chapter 3.4 --- Construction of AS1 and AS2 transgenic rice --- p.75 / Chapter 3.4.1 --- Construction of AS1 and AS2 constructs --- p.75 / Chapter 3.4.2 --- Transformation of chimeric gene constructs into Agrobacterium tumefaciens --- p.75 / Chapter 3.4.3 --- Agrobacterium mediated transformation into Oryza sativa calli to regenerate transgenic rice --- p.76 / Chapter 3.4.4 --- PCR screening of transgene from transgenic AS1 and AS2 rice --- p.76 / Chapter 3.4.5 --- Quantitative PCR analysis of the transgene expression --- p.81 / Chapter 3.4.6 --- Quantitative amino acid analysis in mature transgenic rice grain --- p.83 / Chapter 4 --- Discussion --- p.89 / Chapter 4.1 --- The role of asparagine and asparagine synthetase in nitrogen assimilation and sink-source relationship in soybean --- p.89 / Chapter 4.2 --- Comparative study of AS between different high seed protein content crops --- p.92 / Chapter 4.3 --- The attempt to find out the reason for the strong AS1 expression detected in high protein soybean cultivars --- p.92 / Chapter 4.4 --- Other factors affecting seed protein contents --- p.93 / Chapter 4.5 --- Rice seed quality improvement by nitrogen assimilation enhancement --- p.94 / Chapter 4.6 --- Comparative study of amino acid profile and seed total protein in other transgenic rice --- p.95 / Chapter 4.7 --- Possible reason of higher seed protein content in AS2 transgenic rice --- p.96 / Chapter 4.8 --- Selectable marker --- p.97 / Chapter 5 --- Conclusion and Prespectives --- p.99 / Chapter 6 --- References --- p.102 / Chapter 7 --- Appendix --- p.112 / Appendix I: Major chemicals and reagents used in this research --- p.112 / "Appendix II: Major buffer, solution and gel used in this research" --- p.114 / Appendix III: Commercial kits used in this research --- p.117 / Appendix IV: Major equipments and facilities used in this research --- p.118 / Appendix V: Primer list --- p.119

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 15, 2008). Lys,3 is superscript in the title. Includes bibliographical references.

Thesis (Ph.D. in Biochemistry) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 191-200). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.