Studies on the Human Glutathione Peroxidase Gene and Related DNA Sequences.

Glutathione peroxidase (GPX) is a selenoenzyme consisting of four identical subunits. Each subunit is approximately 21KD in size and contains selenium in the form of selenocysteine in the active site. Mammalian glutathione peroxidase activity is highest in erythrocytes with high levels also found in liver and kidney. The enzyme catalyses the reduction of hydrogen peroxide and organic hydroperoxides using glutathione as the reducing agent.The enzyme plays a central role in antioxidative defence and hence has been implicated in a variety of clinical conditions where free radicals and hydroperoxides are thought to be involved, e. g. cancer, rheumatoid arthritis, and cardiovascular disease. It has also been implicated in selenium-deficiency diseases, and diseases where abnormalities in levels of selenium are seen, since severe nutritional selenium deficiency has been shown to cause a deficiency in glutathione peroxidase.The functional GPX gene in humans is located on chromosome 3, and consists of two exons and one intron. Published sequences of human and animal GPX genes reveal that the selenocysteine residue is encoded by the "termination" codon UGA. Recent evidence suggests that selenocysteine is incorporated co-translationally, the UGA codon being suppressed by a selenocyteyl-tRNA and novel translation factors.The aims of this investigation were to isolate and characterise the human GPX gene and related sequences. These sequences would then be used as hybridisation probes in the analysis of mRNA levels in patients with diseases where modulation of glutathione peroxidase levels is thought to be implicated.In this thesis I present results to demonstrate:-i) The isolation and characterisation of the human genomic sequence coding for the GPX gene and identification of this with the sequence of the human GPX gene recently published by other authors.ii) The isolation of a cDNA apparently coding for a novel GPX-related selenopeptide, GPRP, and a comparison of this with the human GPX gene to determine the relationship between the two.iii) The use of subcloned DNA fragments and synthetic oligonucleotides to distinguish between the two sequences in hybridisation experiments with human genomic DNA and mRNA.iv) Analysis of mRNA levels for GPX and GPRP in white blood cells from patients with rheumatoid arthritis and hyperlipidemia.Results are discussed with particular reference to the relationship between the two genes. No significant differences in GPX or GPRP mRNA levels were seen in patients with rheumatoid arthritis or hyperlipidemia compared to controls. Therefore if GPX or GPRP levels are altered in these conditions it is not as a result of changes in gene expression at the transcriptional level.After completion of this thesis, it was demonstrated that the GPRP sequence was identical to the 5' end of the mouse gene. The results concerning the relationship between the human GPX gene and GPRP will therefore have to be viewed in the light of this information. A full discussion of these results has been included in this thesis