This thesis concerns the atomistic investigation of the extra-cellular protein angiogenin. The main aims of this project are to propose new potential inhibitors to the angiogenin, and to investigate how they might bind to the protein. Two main families of inhibitors have been investigated; firstly, anthracycline antineoplastic antibiotics of which adriamycin is the parent drug; and secondly, uridine and cytidine nucleotide derivatives. The project has been divided into four research areas; conformational analysis of adriamycin; conformational analysis of nucleotide derivatives; a comparative study of the published crystal structure of angiogenin and a published in-house homology model of the protein; finally a series of docking studies to explore the similarity of the active site of angiogenin to ribonuclease A. Angiogenin has been shown to be 68% similar to ribonuclease A on an atom-by-atom basis, and a new sequence alignment has been produced following the release of the crystal structure of angiogenin.

Tuberculosis is one of the leading causes of mortality globally. Although this disease has been around for many generations, treatment and management of the disease remains a daunting challenge. M. tb, is one of the most famous tuberculosis causing organisms, however there are many other mycobacterial strains and species that are also responsible for human mortality, globally. Not all mycobacterial species, however, are disease causing. It is only a few strains such as M. tb H37Rv, M. tb CDC1551, M. tb F11 and M. bovis which are responsible for causing disease. The rest are relatively harmless. What are the genetic differences between these virulent and avirulent strains that dictate a strain's behavior? The answers to these and many other questions lie hidden within the genomes of these organisms. Due to the great advances in DNA sequencing techniques, it is now now possible to more quickly and cheaply, sequence whole bacterial genomes in a single experimental run (High-throughput sequencing). Comparative genomics is therefore extremely relevant and important to be able to handle the dubious amounts of genomic data being poured into our public databases. Several comparative genomics environments already exist on the web today, however the goal of this project is to produce a web-based, comparative genomics environment which not only incorporates basic comparative genomics functions but also, novel tools such as the Seqword Genome Browser (SWGB) and the Mycobacterial Comparison Project (MCP). Using these tools, some interesting comparative genomics findings regarding certain strains of Mycobacteria are made. We reveal several genomic islands within M. avium and M. tb H37Rv. It is shown that certain genes which are usually found to be conserved among other bacteria, tend to be rather divergent among the mycobacteria. 'Mutational hotspots' containing many DNA replication genes are observed to have higher mutation rates relative to the rest of the genome which perhaps accounts for the slow-growth rate of these bacteria. By looking at the genetic profile of PE-PGRS genes in mycobacteria it was shown that M. tb H37Rv and M. tb F11 were actually closer for several genes than when compared to strain H37Ra. The finding was unexpected as H37Ra is known to be derived from H37Rv. These findings are extremely important in the area of TB research as it is of extreme importance to be able to trace areas of greater or lower selection within mycobacteria. Automated sequence comparison such as this is also important for tracking drug resistance markers and other features within mycobacteria so that more focused research can be carried out. The built system was tested and validated with mycobacteria, however, the system is flexible and designed with the intent of inclusion of any prokaryotic organism. It is hoped that systems such as these, and other advances in sequence comparison technology in the future, will provide the understanding needed to better control and cure diseases in the future. / Thesis (PhD)--University of Pretoria, 2010. / Biochemistry / unrestricted

A 1.57 kb XhoI restriction fragment derived from the TOL plasmid pDKI was subcloned into the E. Coli plasmid pUC19. The complete nucleotide sequence of this XhoI fragment was determined using both the chemical cleavage and chain termination DNA sequencing methods.

Previous research has shown varying degrees of renal damage on exposure to equal amounts of cadmium in occupationally exposed mining and factory workers. Further work has shown that in vitro exposure of human peripheral lymphocytes to the same cadmium levels resulted in significant variation in Metallothionein (MT) mRNA transcriptional induction over basal MT mRNA expression in a series of individuals. This variation could account for the differences in renal Cd toxicities identified previously. In this study, the human MT-IIₐ gene was cloned from 12 individuals, and the 5'promoter region was sequenced for each to determine the extent of promoter nucleotide variation. This is of interest since such an analysis has not been done in the past. No study has been done to look at the degree of polymorphism in a particular promoter region. Thus, there are no data on the degree of nucleotide drift or change which can occur in promoter regulatory elements. Such a study could provide insight into whether promoter changes could result in the type of variation described above. It could also give some insight into the degree of variation in sequences in the literature. The results obtained indicated that the human MT-IIₐ promoter region is highly conserved, with only one polymorphic site identified at position 557, between the glucocorticoid responsive element and the fourth metal regulatory element sequences. This suggests that promoter variation is not likely a significant yfactor in MT mRNA induction variability, although further analysis would be needed to show this since only 12 people were analyzed. The results were compared against a study of nucleotide polymorphism in Drosophila melanogaster, which is the only other data on nucleotide variation specifically (Kreitman, 1983; Kreitman and Hudson, 1991). As well, a number of discrepancies were noted from the original published sequences in the literature, suggesting that errors are likely published in genomic sequence which are never identified, except through trial and error. This has potential repercussions when considering the use of such sequence in cloning and sequencing projects, like the sequencing of the human genome, since this would depend on the accuracy of previously published data. / Thesis / Master of Science (MS)

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Computer Science / Doctoral / Doctor of Philosophy

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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.