The human c-myc gene is a cellular proto-oncogene composed of three exons and two introns. Transcription of c-myc is controlled by two promoters, Pl and P2. The activity of these promoters is regulated by many factors, such as cellular transcription factors E2F, YYl, and HSV-1 immediate-early proteins, ICPO, ICP4. Many regulatory elements located both upstream of and between P 1 and P2 have been identified, and some of these are required for optimum expression of c-myc. In this thesis research, a region downstream from P2 in the c-myc exon 1 was identified by its response to transactivation by HSV-1 immediate-early proteins, ICPO and ICP4. The purpose of this research was to examine this region for regulatory sites that respond to HSV-1 infection. I hypothesized that after HSV-1 infection, ICPO and ICP4 activate c-myc expression, in part, through regulatory sequences present in exon 1. To test for this hypothesis, reporter plasmids containing (I) the c-myc promoter (from - 101 bp relative to Pl) and exon 1 coupled to the bacterial CAT gene were constructed. (ii) The c-myc exon sequences used were either intact (wild-type) or they were constructed with various deletions. The activities of these plasmids were examined in transient expression assays. To analyze protein binding, electrophoretic mobility shift assay (EMSA) and completion EMSAs were carried out. The results from these experiments lead to the following conclusions: (i) ICP4 and ICPO serve as activators, whereas ICP27 inhibits c-myc gene expression. (ii) The region from +332 to +513 within the c-myc exon 1 contains an important element required for transactivation of the c-myc gene by HSV-1 proteins. (iii) Cellular proteins, including factor YYl, bind to the region from +332 to +513 in the c-myc exon 1. Although the exact mechanism by which HSV-1 immediate-early proteins regulate cmyc gene expression is still not clear, it gives rise to a possibility that this regulation is caused by turning on or activation of the cellular regulatory proteins by ICP4 and ICPO. The cellular proteins in turn activate the c-myc gene expression by interacting with the ciselement downstream from P2.

The herpes simplex virus 1 (HSVl) VP5 gene codes for the major viral capsid protein. Understanding of the mechanism of how the VP5 gene is regulated in host cells will help us to understand the molecular action of the HSV 1 life cycle and its interplay with the host cell gene expression machinery (transcription and translation). This may ultimately provide scientific bases for both better prevention and cure of HSV 1 caused diseases. Previous work from Dr. Robert L. Millette' s laboratory has indicated that a 164 base pair region of the VP5 promoter gene could activate the transcription of an attached reporter gene (bacteria CAT gene). Furthermore, a 12 bp (GGCCATCTTGAA) cis-acting element situated within the 164 bp promoter region was required for the promoter activity. To understand the function of this cis-element in the regulation of VP5 transcription and to identify the trans-acting factors interacting with this element, gel mobility shift assays were first carried out using the fragment containing the 12 bp site as the probe. A cellular factor, YY 1, was found to bind to this site in a sequence specific manner. Based on the oligonucleotide competition assays, partial protease digestions, and antibody supershift assays, it became clear that two cellular factors bound to the VP5 promoter. These were related, if not identical, to the previously identified Yin-Yang- 1 factor (YY 1), and transcription factor the SPl. Site-directed mutagenesis studies indicated that these two factors bind to distinct sites on the 164 bp fragment. Point mutations studies on the 12 bp YYl binding site demonstrated that seven of the 12 bp were required for YY 1-DNA complex formation and the first four bp in the 12 bp were very important for VP5 gene regulation. Also, it was found that YY 1 performs both positive and negative regulator function in VP5 gene regulation. In conclusion, two cellular transcription factors, YY 1 and SPl, play a major role in VP5 gene expression.

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No description available.

In this thesis we present a new model for identifying dependencies within a gene regulatory cycle. The model incorporates both probabilistic and temporal aspects, but is kept deliberately simple to make it amenable for learning from the gene expression data of microarray experiments. A key simplifying feature in our model is the use of a compression function for collapsing multiple causes of gene expression into a single cause. This allows us to introduce a learning algorithm which avoids the over-fitting tendencies of models with many parameters. We have validated the learning algorithm on simulated data, and carried out experiments on real microarray data. In doing so, we have discovered novel, yet plausible, biological relationships.

Abstract not available

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- T cells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell???mediated inflammatory responses and showed reduced levels of the Th1 cell???promoting cytokine interleukin 12 and reduced interferon ?? production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicated that this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system.

The nucleopolyhedrovirus of Epiphyas postvittana (EppoMNPV) is being studied as a potential biological control agent for leafroller insects in New Zealand. The aims of this project were for the identification of putative genes that are unique to, variant in or missing from, the EppoMNPV genome and the subsequent analysis of at least one of these genes. The purpose of this was to identify and characterise genes potentially involved in the specific host range and virulence of EppoMNPV. This was achieved in two steps. Three genome regions lacking linearity between EppoMNPV and the closely related OpMNPV were previously identified and the targeted sequencing of these three regions was the first aim of this project. The collation of the entire genome sequence of EppoMNPV and comparison to the genome sequences of 22 other baculoviruses completed the identification of genes that are unique to, variant in or missing from EppoMNPV. The EppoMNPV genome was found to be 118,584 bp in size encoding 136 putative proteins. A total of 29 genes were found to be common to all baculoviruses, while the lepidopteran baculoviruses share a total of 62 genes. The genome of EppoMNPV encodes four putative unique genes, the sequence of which offers no clues as to possible function. EppoMNPV lacks a homologue of the superoxide dismutase gene common to all other lepidopteran baculoviruses The EppoMNPV IE2 homologue was identified as a 311 amino acid protein with a truncation in the N-terminal region. We hypothesised that this truncation would lead to a loss of function, which could contribute to the virulence and/or host range of EppoMNPV For this reason, the characterisation of the EppoMNPV IE2 was taken up as the second part of this project. A comparative study between the AcMNPV and EppoMNPV IE2 proteins identified no differences in function between these two proteins in Sf21 cells. The EppoMNPV IE2 was capable of trans-activating three constitutive promoters and localised to discrete nuclear bodies. Cell cycle arrest was not achieved by either IE2 protein in our cell culture system. The role of four sequence motifs common to all IE2 proteins was studied with the aid of mutational analysis. Mutation of arginine and acidic rich sequences of EppoMNPV IE2 showed only a slight decrease in trans-activation activity while mutation of the RING-finger and coiled-coil motifs reduced trans-activation to less than half that of wild type IE2. Mutation of the coiled-coil motif resulted in reduced amounts of protein localising to discrete nuclear regions. A series of deletion mutants from the N- and C-termini of EppoMNPV IE2 identified that the C-terminal 111 amino acids of EppoMNPV IE2 was sufficient for nuclear targeting. Deletion of the C-terminal 19 amino acids resulted in an IE2 mutant completely defective in both localisation and transactivation. This demonstrates that localisation to discrete nuclear regions is essential for EppoMNPV IE2 to act as a transactivator.

Includes bibliography / 100 leaves, [8] 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, 1987

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.