Tran, Cam Thanh Lucy; (2003) Molecular analysis of human DDAH genes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Endogenously produced asymmetrically methylated arginine residues are competitive inhibitors of all isoforms of nitric oxide synthase (NOS). Two isoforms of the enzyme dimethylarginine dimethylaminohydrolase (DDAH I and DDAH II) exist in mammals. These specifically hydrolyse asymmetrically methylated arginine residues to citrulline and methylamines. Regulation of asymmetric methylarginine concentration may provide a novel mechanism for the regulation of NOS activity in vivo. In order to carry out a molecular analysis of human DDAH genes, genomic clones were obtained by library screening and database searching. This thesis describes the mapping of the DDAH1 gene to chromosome lp22 and the DDAH2 gene to the MHC III region of 6p21.3. The MHC III region has been linked with susceptibility to diseases such as rheumatoid arthritis and insulin-dependent diabetes mellitus - diseases in which altered nitric oxide biosynthesis has been implicated in pathogenesis. These gene localisations support gene structure comparisons that suggest that the two isoforms of DDAH have arisen by gene duplication. In order to understand transcriptional regulation of the DDAH2 gene, its 5' sequence was analysed. A 1.6Kb region of DDAH2 was cloned upstream of a reporter gene. This construct was expressed when transfected into endothelial cells. Serial deletions of the promoter identified a region between -927 to -657 relative to the translational start site that directed basal expression. Site-specific deletions made within this region revealed the contribution of several response elements to basal promoter activity. The functional roles of the transcription factors that bind these elements suggested that DDAH2 might be involved in cellular differentiation and development. Transcriptional regulation of DDAH2 by retinoic acid, a potent morphogen was also demonstrated. Induction of DDAH2 by retinoic acid in endothelial cells increased nitric oxide production and this could be partially blocked by a DDAH inhibitor. This thesis has thus examined the evolutionary origins of DDAH genes and studied regulation of DDAH2 gene expression. Together, the data provide some insight into the possible physiological functions of DDAH genes.

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