The transcriptional promoter sequences for the Rhizobium meliloti nitrogen fixation genes nifA and nifB were cloned to a β-galactosidase gene fusion plasmid vector and transferred by homologous recombination to a specialized transducing phage. The promoter fusions were then transduced to a recombination deficient strain of Escherichia coli as single-copy lysogens and analysed under defined aerobic and anaerobic conditions. The lysogenic strains contained plasmids encoding either of two transcriptional activator proteins, NifA or FixJ, produced from a constitutive plasmid promoter. The expression of the nifA and the nifB promoters was found to be sensitively regulated by the carbon source used for anaerobic fermentation or anaerobic respiration, the redox potential of the terminal electron acceptor used for anaerobic respiration, and the growth phase of anaerobic cultures. The repression of nit promoter expression by oxygen respiration was specifically compared to anaerobic respiration of alternative electron acceptors. Both nifA and nifB promoter expression decreased exponentially as the reduction potential of the terminal respiration reaction increased. The repressive effect of oxygen appears to be due soley to the exponential relationship between nit promoter expression and the redox potential of oxygen respiration. In addition to separate fusions of the nifA and nifB promoters to β-galactosidase, a single-copy fusion of the entire nifA-nifB region was constructed. In this construct, plasmid-encoded FixJ protein stimulated the expression of a chromosomal nifA gene to produce the NifA protein, which then stimulated the expression of the nitB promoter. This strain produced 20-fold lower activity than a strain in which nifB promoter expression was stimulated by plasmid-encoded NifA protein. Finally, the nifA locus was found to contain a transcriptionally active element, oriented opposite to the nifA promoter. / Graduation date: 1992

The cytochrome P450 (CYP) superfamily of enzymes catalyses the oxidative metabolism of lipophilic xenobiotics such as drugs and environmental chemicals, and also plays an essential role in the biosynthesis and metabolism of endogenous compounds such as cholesterol and bile acids, vitamins, steroids, arachidonic acid and eicosanoids. Cytochrome P450 2J2 (CYP2J2) is a recently identified member of the human CYP protein family that is highly expressed in the heart, vasculature, liver and other tissues. CYP2J2 metabolises arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs), which have a number of potent biological activities including cytoprotective, vasodilatory and anti-inflammatory effects. Given its widespread tissue distribution and the biological actions of EETs, CYP2J2 is likely to play an important role in cellular physiology, and altered expression of CYP2J2 may have pathophysiological consequences. Indeed, recent literature studies have indicated that CYP2J2 protein levels are decreased in vascular endothelial cells exposed to hypoxia and reoxygenation, and that maintenance of CYP2J2 expression enhances cell survival. Thus, CYP2J2 expression may be impaired in diseases or conditions associated with decreased oxygen availability, such as ischaemic heart disease, stroke and atherosclerosis, and this may contribute to their pathogenic consequences. Despite its likely importance in human physiology and pathophysiology, very little is known about the regulation of CYP2J2 gene expression. The aim of this study was to investigate the molecular mechanisms that control expression of the CYP2J2 gene. In particular, this study was designed to identify factors that regulate the expression of the CYP2J2 gene in the liver-derived HepG2 cell line during normoxia and hypoxia. A 2.4 kb fragment of the 5???-flanking region of the CYP2J2 gene (corresponding to nucleotides -2341 to +98, relative to the translation start site) was isolated from a human genomic library. Automated searching of the upstream regulatory region of CYP2J2 identified several putative binding sites for the transcription factor activator protein-1 (AP-1). Because AP-1 activity is altered in hypoxia, the possibility that AP-1 may participate in the regulation of CYP2J2 expression in hypoxia was explored. Cell culture studies examined the relationship between the expression of CYP2J2, and the AP-1 genes c-fos and c-jun, in HepG2 cells cultured in normoxia and hypoxia. Down-regulation of CYP2J2 mRNA and protein in hypoxic HepG2 cells was associated with the pronounced up-regulation of c-Fos protein from an undetectable level in normoxic cells; c-Jun protein levels were readily detectable in normoxia, and were also increased in hypoxia. Transient transfection studies revealed distinct effects of Fos and Jun proteins on CYP2J2 promoter activity. While the CYP2J2 promoter was strongly activated by c-Jun, c-Fos was inactive, and also abolished gene transactivation elicited by c-Jun. These results suggest that the constitutively expressed c-Jun is important in the maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia stimulates the formation of c-Fos/c-Jun heterodimers, which do not support CYP2J2 transcription, leading to gene down-regulation. Experiments with CYP2J2 promoter deletion constructs revealed that the region between -152 to -50 bp relative to the translation start site was crucial for activation of CYP2J2 by c-Jun. Electrophoretic mobility shift assays (EMSAs) and transfection studies identified two distinct elements within this region that were involved in c-Jun-dependent transactivation: an AP-1-like element at -56 to -63 bp, and an atypical AP-1 element at -105 to -95 bp. c-Jun homodimers interacted specifically with both elements. Separate mutagenesis of either element significantly impaired c-Jun-dependent transactivation of CYP2J2, while mutagenesis of both elements eliminated c-Jun-responsiveness. EMSAs established that c-Jun, but not c-Fos, interacted with both elements in normoxic HepG2 cells. Furthermore, mutagenesis of either c-Jun-response element significantly decreased the basal transcriptional activity of the CYP2J2 promoter in HepG2 cells, while mutagenesis of both elements almost completely suppressed basal promoter activity. These findings indicate a pivotal role for c-Jun in the maintenance of CYP2J2 expression in normoxic cells. Transfection studies indicated that c-Fos suppresses c-Jun-dependent activation of CYP2J2 at both the -56/-63 bp and -105/-95 bp c-Jun-response elements. However, c-Fos-dependent inhibition appears to be mediated by distinct mechanisms at these two regulatory elements. While both elements interacted with c-Jun homodimers, only the -105/-95 bp element was able to interact with c-Fos/c-Jun heterodimers. Thus, the up-regulation of c-Fos in hypoxia, and the shift from c-Jun homodimers to c-Fos/c-Jun heterodimers, directly decreased c-Jun binding and transactivation at the -56/-63 bp element. In contrast, up-regulation of c-Fos in hypoxia altered the composition of proteins bound at the -105/-95 bp element from c-Jun to c-Fos/c-Jun. Inhibition of promoter activity occurs because c-Fos/c-Jun heterodimers can occupy, but not transactivate, the CYP2J2 promoter via the -105/-95 bp element. In summary, this thesis provides novel information on the molecular mechanisms that control the differential expression of the human CYP2J2 gene in normoxia and hypoxia. In particular, this study has established that the AP-1 proteins c-Jun and c-Fos play a crucial role in modulating the transcriptional activation of the CYP2J2 promoter in response to cellular stress. Binding of c-Jun to two distinct c-Jun-response elements within the CYP2J2 proximal promoter induces transcriptional activation of the CYP2J2 gene and is essential for maintenance of CYP2J2 expression in normoxic cells. The up-regulation of c-Fos in hypoxia promotes the formation of c-Fos/c-Jun heterodimers, which inhibit transcriptional activation of the CYP2J2 promoter by c-Jun, thus contributing to decreased CYP2J2 expression in hypoxia. Impaired expression of CYP2J2 may contribute to cellular injury in diseases such as atherosclerosis and stroke, and a greater understanding of the mechanisms responsible for mediating altered CYP2J2 expression may eventually lead to therapeutic strategies that manipulate the expression of this important human gene.

Includes bibliographical references / 183, [111] leaves, [6] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1990

Bibliography: p. 90-96 / v, 96 p. [70] leaves [26] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1984

Bibliography: leaves 133-144 / 144 p., [15] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1986

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1986. / Includes bibliographical references.

Thesis (Ph.D.)--Ohio University, March, 2007. / Includes bibliographical references (leaves 165-175)

Thesis (Ph.D) -- McMaster University, 1997 / Includes bibliographical references Also available via World Wide Web.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.