Degree awarded with distinction on 6 December l995. A dissertation submitted to the Faculty of Science, University the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science. March. 1995 / Hepatitis B virus (HBV) infection resulting in integration of the viral DNA into host liver cell DNA is associated with the development of hepatocellular carcinoma (HCC). This is indicated by epidemiological trends, molecular studies and studies of animal models infected with viruses closely related to HBv. However, little is known about the mechanism by which the integrated HBV DNA includes HCC despite continuing analysis of the integrated HBV DNA and its surrounding cellular sequences. [Abbreviated Abstract. Open document to view full version]

TOL plasmids encode enzymes responsible for utilization of toluene and related aromatic compounds by Pseudomonas putida, ultimately converting them to central metabolic intermediates. The nucleotide sequence for the 5.6 kb xylGFJQK region of the pDK1 TOL meta operon was determined. DNA sequence analysis revealed the presence of five open reading frames corresponding to xylG (1458 bp), xylF (846 bp), xylJ (783 bp), xylQ (936 bp) and xylK (1047 bp), encoding predicted protein products of 51.6, 31.3, 27.8, 32.8, and 36.6 kDa in size, respectively. The average G+C content of the xylLTEGFJQK region was 65.7%, somewhat higher than the 58.9% seen in the immediately upstream xylXYZ region and substantially more than the 50% G+C content reported for the upper TOL operon of this plasmid. Homology comparisons were made with genes and proteins of related catabolic plasmids. The dmpCDEFG and pWWO xylGFJQK regions exhibit consistently high levels of nucleotide and amino acid homology to pDK1 xylGFJQK throughout the entire region. In contrast, although the nucleotide sequence homology of the Acinetobacter atdCDE region to xylGFJ is high, the homology of atdFG to xylQK is markedly less. Such radical changes in homology between corresponding regions of different operons, combined with variable base and codon usage patterns within and between operons, provides additional support for the idea that the upper and lower operons encoding enzymes of aromatic pathways have evolved independently of one another and that these operons have continued to exchange genetic material with homologous expression units through a series of recombination events. Recombinant plasmids were constructed for individual expression of each of the xylGFJQK genes. HMSD (XylG) and HMSH (XylF) were partially purified and characterized with respect to substrate specificity and kinetic mechanism. Evidence was obtained suggesting that the HMSD reaction occurs via a steady state ordered mechanism or a random mechanism where binding of the first substrate effects the enzyme's affinity for the second substrate.

Prodigiosins and tambjamines are natural compounds from bacterial and marine sources belonging to a family containing a common 4-methoxy-2,2'-bipyrrole core. These compounds have received a lot of interest due to their promising biological activities. Studies have suggested DNA as a potential therapeutic target for the natural prodigiosin and tambjamine due to their ability to facilitate oxidative DNA cleavage in the presence of Cu2+. Based on this we sought to study the metal binding activity of new prodigiosin and tambjamine analogues. A new prodigiosin analogue was synthesized and complexed with Cu2+. This revealed 1:1 complex formation between the tripyrrole and Cu2+ that was confirmed by mass spectra and NMR spectra analysis. In addition in situ studies also revealed that our new analogues of prodigiosin cannot bind Zn2+ when the methoxy group on ring B is replaced by an alkyl group or when one of the ring nitrogens is methylated. Our UV-Vis experiments with calf thymus DNA showed that prodigiosins and tambjamines bind DNA mainly through an external mode, suggesting that hydrogen bonding between the pyrrole ring nitrogens and the bases of DNA takes precedence over stacking interactions. For the new Cu2+ complex synthesized however, we observed spectral changes that suggest intercalation into DNA. DNA cleavage experiments revealed that the prodigiosin-Cu complex is able to convert supercoiled DNA into its linear form. The data from the gel shift assays fit well to a first-order consecutive reaction model. In addition to preformed metal complexes, we showed DNA cleavage by in situ complexation of the ligands in the presence of Cu2+. However, although we showed Zn2+ complex formation with prodigiosin analogues, in situ studies did not show induction of DNA cleavage by Zn2+ complexes under our experimental conditions.

The ability of a genetically-engineered <i>Erwirzia carotovora</i> subsp. <i>carotovora</I> (Ecc) strain to transfer recombinant chromosomal DNA or plasmids to wildtype Ecc or <i>Pseudomonas fluorescens</i> was tested on filters, within soil microcosms, and <i>in planta</i>. Ecc was engineered by chromosomal insertion of a disarmed <i>endo</i>-pectate lyase gene marked with a 1.4kb DNA fragment conferring kanamycin resistance. Plasmids RPI and pBR322 were introduced separately into engineered Ecc clones. These strains served as donors in genetic transfer experiments. No transfer of the inserted kan marker or of pBR322 was observed under any experimental condition. In filter matings, RPI was transferred to wildtype Ecc at a frequency of 3.6 X 10⁻² transconjugants per donor (TPD) and to P. <i>fluorescens</i> at a frequency of 2.4 X 10⁻⁵ TPD. In matings conducted in potato tubers inoculated using sewing needles, the respective frequencies were 4.0 X 10⁻³ and 2.0 X 10⁻³, while matings on potato slices yielded frequencies of 4.7 X 10⁻² and 2.3 X 10⁻². In soil microcosms, the maximum transfer frequencies observed were 2.3 X 10³ and 8.4 X 10⁻⁵ TPD. / Master of Science

The unfolding of recombinant DNA, from research technique to political issue, is described. As a research technique, recombinant DNA (abbreviated rDNA) has opened up new vistas in biological and other fields of research. But its potential yet unproven hazard has created uneasy feelings toward the technique. The controversial nature of the issue finally launched rDNA into the political sphere, involving scientists, the public at large, and Congress in efforts to control the development of the field. The first group to regulate rDNA was the scientists. The scientific community called for a voluntary moratorium on experiments perceived as potentially dangerous at the time. It was an unprecedented act. The National Institutes of Health subsequently issued guidelines for a safe execution of rDNA experiments to minimize potential dangers to public health and well-being. Efforts of the scientific community to control rDNA was seen, however, as a politics of expertise. Challenges to this "technocratic" approach soon emerged. Vocal members of the public suspected expert decision makers as being biased toward scientific interests, reducing rDNA to a technical issue. They rejected the experts’ tunnel vision and demanded a say in decisions. Public participation in the decision-making process precipitated community debates at locations where rDNA research was ongoing. A democratic approach to decision-making proved to be a viable policy-making mode. The ensuing local and state laws, however, seemed inadequate to cover global consequences of rDNA. In an effort to unify regulations of the field, Congress attempted to legislate on the subject. Resistance from the scientific community, which regard legislative control as rigid and unnecessary, was one of the causes of diminishing congressional interest in the matter. None of the introduced bills was enacted. For complex policy areas with uncertain yet far-reaching scientific and societal consequences -- like rDNA -- this dissertation recommends a policy-making process where scientists, interested lay persons, politicians, public administrators, and other relevant parties participate in structured communications prior to an emerging controversy. To facilitate the process, establishment of National Science Fora is recommended. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.