Enteropathogenic Escherichia coli (EPEC) is an enteric pathogen that causes potentially fatal infantile diarrhea. A type III secretion system is employed by EPEC to inject bacterial effector proteins directly into host intestinal epithelial cells. The multivalent chaperone, CesT, interacts with nine effectors and is a significant contributor to EPEC pathogenesis. A putative transcriptional promoter region was identified directly upstream of cesT. In silico analyses identified conserved elements that suggest the cesT promoter is recognized by ?70. Using transcriptional fusions to lux reporter genes I showed that the cesT promoter region is active under conditions known to induce virulence-gene expression. I conclude that the cesT promoter is active early during an in vitro assay, and regulated by different mechanisms than those affecting the Ptir operon promoter.

In the AcMNPV genome, ~28% of the genes are arranged divergently on opposite strands with an intergenic region of <1 kbp. In this configuration, a bidirectional promoter generally drives expression of both genes. However, no baculovirus bidirectional promoters have been characterized in any detail. We chose the AcMNPV chiA/v-cath intergenic region to serve as a model to characterize transcriptional regulation of bidirectional gene pairs during AcMNPV infection. We sequentially truncated putative upstream regulatory regions of chiA and v-cath to identify sequences essential for transcriptional initiation. Forty bp of the chiA gene 5’-flanking region was sufficient to support chiA transcription at half the level of the AcΔCC+CC repair virus. Interestingly, v-cath transcription from viruses containing only 40 bp of their upstream 5’-flanking region was found to be higher by 4-fold relative to the level of native expression. Linker-scanning mutagenesis that inserted 5 bp linkers spanning the chiA/v-cath intergenic region identified nucleotides critical for the transcriptional activation of both genes. From this, nucleotides -36 to -45, of the v-cath gene were found to negatively regulate v-cath mRNA expression. Quantitative RT-PCR studies revealed a 2-4 fold higher chiA mRNA expression relative to v-cath possibly explaining why translation of CHIA can be detected 6 hours earlier than V-CATH. This study identifies upstream regions of viral chiA and v-cath required for initiation of transcription and provides the first insight into baculovirus mechanisms for transcriptional regulation of interdependent gene pairs.

No description available.

Strawberry (Fragaria ananassa) is an economically important soft fruit that is highly valued as a fresh product and flavouring. During ripening, strawberry fruits undergo a number of physiological changes affecting colour, texture and flavour. An understanding of these changes at the biochemical and molecular level will be important in developing strategies for enhancing the quality attributes of this fruit. A cDNA encoding a ripening- enhanced acyl carrier protein (RE-ACP) was previously isolated from strawberry fruit. AC? is an essential component of fatty acid synthesis in both plants and animals. The aims of this thesis were to isolate and characterise this and other members of the ACP multigene family expressed in strawberry fruit. Six closely related putative AC? cDNA isoforms were identified from strawberry. Two of these were obtained by screening a cDNA library from ripe fruit and three were obtained by a technique known as candidate fragment length polymorphism (CFLP) that utilised ACP gene-specific primers for AFLP-cDNA display. Northern analysis was not able to differentiate their expression but ACP was highly up-regulated in ripening fruit whereas low levels of expression were detected in other strawberry tissues, including achenes (seeds), expanding leaves and flowers. The RE-ACP was over-expressed in E. coli and the recombinant protein partially purified. The over-expressed protein had a M(_r) of 20kDa on SDS-PAGE and appeared to form a dimer. A genomic library was constructed from F. ananassa from which two different genomic clones closely related to RE-ACP were obtained. Promoter analysis indicated the presence of regulatory elements. The characterization of putative ACP cDNA and genomic clones, including the 5' upstream regions, is described and their possible role in strawberry fruit is discussed. Key words: Strawberry, fruit, ripening, gene expression, genomic, cDNA, fatty acid, acyl carrier protein, aroma, promoter.

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Chlamydia is a clinically significant organism that exhibits a unique stage-specific developmental cycle, involving the interconversion between two metabolically distinct forms. The completion of eight chlamydial genome sequences identified three different RNA polymerase sigma factor (ó) genes. Temporal gene expression analysis has predicted that each ó may play an integral role in controlling the development cycle. This thesis examines the role of the chlamydial alternate sigma factor, ó54 (rpoN) and the potential mechanism for the control the developmental cycle and disease pathogenesis. To achieve this, we searched the genome for putative ó54 promoters, validated the findings by DNA-binding assays, and examined the roles of the genes predicted to be regulated by ó54. This study applied a bioinformatics approach to search for additional ó54 regulated genes in C. trachomatis L2. A reduced consensus sequence (TGGCACnnnnnTTGC) identified two previously published ó54 promoter sequences upstream of CT652.1 and CT683. A modified consensus sequence (TGG-N9-TGC) was applied to the C. trachomatis D genome in Findpatterns yielded 512 potential targets of which 20 by virtue of sequence orientation and distance upstream of the predicted ORF start codon Primer extension analysis of total RNA isolated at 24 hours post-infection mapped the 5' RNA end upstream for acpS (CT100), yhf0_1 (CT258), SAM (CT404), lpxA (CT531), hypothetical proteins CT652.1 and CT683, and htrA (CT823) to the predicted ó54 promoters. Three candidates (CT291, CT404, and CT847) were mapped to putative ó70-like ó66 promoters. No transcript start sites were detected for the remaining ó54 promoter candidates. Two transcripts were detected from predicted ó66 and ó54 tandem promoters upstream of CT404. Primer extension analysis of the CT404 transcripts from RNA isolated at 4, 8, 12, 24 and 32 hours post-infection showed a decrease between 12 hours and 24 hours post-infection in transcripts thought to be generated from the predicted ó66 promoter. Transcripts from the predicted ó54 promoter were identified throughout development. Temporal gene expression profiles of the candidate genes with predicted ó54 promoters (CT652.1, CT683, CT100, CT258, CT531 and CT823) were resolved throughout the C. trachomatis L2 developmental cycle using real-time PCR. Transcripts for CT608 and CT609 were detected early in the cycle, while strong transcript levels were detected for CT258, CT531 and CT823 after the appearance of CT609 (rpoN). Low levels of CT652.1 and CT683 were measured, in the mid to late phase of the cycle, and transcripts for CT100 appeared at lower levels during the middle phase of the cycle. The functional assay of the predicted ó54 promoters required the generation of recombinant C. trachomatis L2 ó54 (rRpoN). The C. trachomatis rpoN was cloned into a bacterial expression system (pQE70) and the recombinant proteins purified for subsequent DNA mobility shift assays. Expression of rRpoN was hampered by low copy numbers, and unusual physical characteristics. DNA binding and mobility shift assays using rRpoN extracts against the chlamydial CT652.1 ó54 promoter, plus two characterised E. coli ó54 promoters (hypA and hycA), were successful if E. coli core RNA polymerase was added to the assay. All 20 candidates with predicted ó54 promoters were analysed with EMSA using rRpoN extract. The promoters upstream of CT100, CT223, CT258, CT322, CT652.1 and CT683 showed affinity towards the recombinant rRpoN-E. coli core RNA polymerase holoenzyme complex. Searches for potential chlamydial ó54 transcription initiation activators were made using the Multiple Em for Motif Elucidation (MEME) software, looking to identify the DNA binding motifs. The upstream promoter regions of CT100, CT223, CT258, CT322, CT531, CT652.1, CT683 and CT823 in C. trachomatis L2 and orthologs found in other species of Chlamydia were analysed. The software identified a near palindromic sequence upstream of CT100 orthologs in C. trachomatis D and C. trachomatis MoPn (CAACCCAAC and CACCACAAC) where as a CT531- and CT823-specific motif was also discovered (CCGTTGTAGAATCTC). It is beginning to emerge that ó54 may regulate the expression of proteins required for the formation of the cell wall. Since the expression of the ó54 transcript, rpoN, coincides with the morphological change from the non-infectious RB to the infectious EB, predictions could be made concerning which genes are potentially regulated by ó54.

Gliomas are the commonest primary tumours of the brain and glioblastoma multiforme (GBM) represents more than 50% of this group. GBM remains a neurosurgical conundrum since patients often succumb to the disease within one year. Surgery followed by radiation and medical regimens over the years have had minimal impact on the prognosis of patients with this cancer and hence, alternative and novel therapeutic modalities are required if the survival of patients with this disease is to be significantly improved. The ATM gene, which is mutated in the disease ataxia-telangiectasia (A-T), is implicated in response to radiation-induced DNA damage, leading to profound radiosensitivity. By reducing the levels of ATM in the radioresistant GBM cells through antisense or RNA interference (RNAi) technology delivered by lentiviruses, malignant GBM tumour cells were successfully sensitised to radiation treatment. In conjunction with surgery, this strategy will provide an enhanced therapeutic intervention especially in the case of GBM where the tumour is untreatable. In this thesis, analysis of the D-3-Phosphoglycerate dehydrogenase promoter in a GBM cell line as well as the development of a novel rat model for GBM using a bioluminescent F98 cell line will also be presented.

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Thesis (M.S.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on July 31, 2008). Research advisor: Martha M Howe, Ph.D. Document formatted into pages (vii, 127 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 103-116).

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p.215-229