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Deamination or the hydrolytic removal of one hydroxyl group from a base in DNA or RNA can lead to changes in the transcript and protein produced. Examples of this are the deamination of cytosine residues in DNA by activation induced deaminase (AID) during antibody diversification, or deamination of adenosine at the Q/R site in the GluR-B transcript by adenosine deaminase acting on RNA 2 (ADAR2), which regulates calcium permeability in neurons. The initial focus of my thesis was to characterise a putative novel deaminase APOBEC4. APOBEC4 was identified in a bioinformatic search for proteins containing the core catalytic residues common to the whole family of Cytidine Deaminase enzymes. The aim of the project was to express and purify recombinant APOBEC4 for in vitro characterization, however despite using different expression systems and purification conditions the majority of the recombinant protein was inherently insoluble and I could not isolate sufficient amounts of protein for further studies. Recombinant protein with a GST-tag was used to generate polyclonal antibodies which recognised recombinant protein but were unable to detect endogenous APOBEC4. The focus of my thesis then changed to the process of adenosine to inosine editing in RNA, which is a post-transcriptional mechanism for generating protein diversity. The enzyme family responsible for catalysing this reaction is known as ADAR, and Drosophila melanogaster has only one Adar gene. Flies lacking the Adar gene show locomotion defects and age-dependent neurodegeneration, however little is known about the molecular mechanism underlying these defects. To investigate this phenotype I performed microarray analysis on RNA isolated from heads of 5 day old flies lacking the Adar gene to characterize gene expression changes in the fly heads before neurodegeneration caused secondary effects. Analysis was also performed on Adar-null flies expressing either an active Adar gene or a catalytically-inactive Adar gene in cholinergic neurons to determine which transcripts could be directly regulated by Adar. I confirmed the microarray results by real-time PCR, and demonstrated that the changes in transcript level could be reversed by expression of either active or catalytically-inactive Adar. Expression of edited transcripts did not change dramatically. Filter-binding analysis and electrophoretic mobility shift assay revealed that recombinant ADAR could bind to all RNA transcripts analysed with similar affinity; both known substrates and potential new substrates for Adar, as well as transcripts that were chosen as negative controls due to their expression not altering in the expression microarray. Recombinant ADAR bound to dsRNA with a very high affinity; other transcripts investigated bound with considerably lower affinity, yet all transcripts investigated were bound by ADAR. Further analysis of transcript changes in Adar-null flies was investigated by performing microarray analysis with a custom-made splicing-sensitive microarray. Analysis revealed that a subset of transcripts were differentially spliced in Adar-null flies; however this group of transcripts was distinct from the group identified as being altered on the expression microarray, indicating that the splicing changes are independent of changes in expression. Analysis of exon-specific probes on the splicing array confirmed the transcript changes identified in the expression array. Real-time PCR confirmed the changes in splicing, and these transcripts were further examined by sequence analysis. This revealed several transcripts identified as altered by the AS array showed use of alternative polyadenylation sites indicating ADAR may have a role in detemining polyadenylation site selection.

Thesis (MSc (Med)(Microbiology)) -- University of Limpopo, 2012. / Mycobacterium tuberculosis is the most common cause of death world-wide and its incidence has been steadily increasing, which is more evident when comparing the global tuberculosis (T8) incidence of 9.24 million in 2006 to 9.27 million cases in 2007. African countries are the second most affected by the epidemic and South Africa is among the 22 highest burden countries most affected by T8 with a very high number of cases relative to the total population. The early diagnosis of tuberculosis and screening of contacts is the cornerstone for controlling spread of active T8 infection. T8 diagnosis becomes even more challenging in patients with immunosuppression (for example in human immunodeficiency virus (HIV) infected), in the case of latent infection and extra pulmonary T8 such as pleural T8. The definitive diagnosis of pleural T8 depends on the demonstration of M. tuberculosis in sputum, pleural fluid and pleural biopsy. Although acid fast bacilli (AF8) microscopy is a rapid, inexpensive and relatively simple method, it has low sensitivity. The culture method is more sensitive than AF8 microscopy, detecting 25-37% of all pleural tuberculosis cases however it takes 4 to 8 weeks for a visible growth on a solid medium. Therefore it is important to find a rapid and reliable test for the diagnosis of pleural T8 particularly in developing countries such as South Africa where there is a high T8 incidence and HIV infection rate.

Two molecular forms of adenosine deaminase have been isolated from bovine livers. These two forms were found to be interconvertible. Adenosine deaminase extracted from human tissues exhibited similar properties as well. Previous studies have shown the presence of a conversion factor which formed an aggregate with the small form (the C-form) of the enzymes, and the resulting enzyme complex was identified to be the large form (the A-form) of adenosine deaminase. Studies have been made with rat tissues. The C-form of the adenosine deaminase is widely distributed in the various tissues, while the large form of the enzyme is absent in those tissues examined.The outline of this study is summarized below: (1) The isolation of the conversion factor from human liver.(2) The isolation of the adenosine deaminase from the various tissues.(3) The coversion of human adenosine deaminase C-form to the A-form.(4) Quantization of the conversion of the C-form to the A-form.(5) Testing the effect of conversion on nonhuman enzymes.(6) The optimum temperature and time to yield greatest amount of conversion.The results from this study would verify any differences that might exist between the small forms of the enzyme present in different species.

Thesis (Ph. D. in Pharmacology)--Vanderbilt University, Dec. 2005. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 133-160).

A Thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg, for the Degree of Master of Science JOHANNESBURG 1986 / The adenosine deaminase enzyme from a Xhosa tribesman has been characterized. Red blood cell activity levels were 6-9% of normal whereas his white cell ADA levels were about 30% of normal. The enzyme's stability at 57°C was shown to be greatly reduced suggesting a mutation resulting in an enzyme with reduced stability in vivo. It was concluded that the discrepancy in ADA activity levels between red and white blood cells was due to the red cells being anucleate. The proband's residual ADA was found to have a Michaelis Constant (K ) for adenosine m of 47.9 ♦ IS.BuM, a value which is not significantly different from that of normal ADA (51.7 ± 11.4ufl). Red cell deoxy-ATP levels were measured and found to be elevated two-to-three times over normal levels. Red cells from ADA-deficient patients with severe combined immunodeficiency (SCID) have been reported with deoxy-ATP levels elevated about 1 000 times. It was concluded that the slight elevation of deoxy-ATP levels in the proband were too low to have any noticeable effect on functions of his immune system. Starch gel electrophoresis of red cell ADA from members of the proband's family in conjunction with red cell ADA activity levels suggested that both parents carried a gei e for 'partial' ADA deficiency, both of which had been inherited by the proband as well as one of his sibs. Isoelectric focusing studies suggested that the two, parental AUA partial deficiency genes were different from one another. It was also found that another rare allele of ADA, possibly ADA ',was segregating within the same family although this event appaars to be unconnected with the ADA partial deficiency.

The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN.

Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.

Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.