Rat selenoprotein W (SeW) promoter activity was investigated using different concentrations of cadmium, copper, and zinc. Two fragments (404bp and 1265bp) of the SeW promoter, containing a single metal response element (MRE), were ligated into the multiple cloning site of a pGL3-Basic reporter plasmid. The constructs were transfected into cultured rat C6 (glial) and L8 (myoblast) cells and promoter activity measured by means of luciferase reporter gene fused to the SeW promoter fragments in the reporter plasmid. With post-transfection exposure of these cell lines to these metals, copper and zinc, but not cadmium, significantly increased promoter activity of the unmutated 1265bp (not 404bp) construct (p<0.05) only in the C6 cells. Mutation of the MRE sequence abolished promoter response to metal exposure but did not eliminate promoter activity. The results suggest that SeW expression in glial cells can be increased on exposure to copper and zinc and that this response is dependent on the MRE sequence present in the SeW promoter. To understand transcriptional regulation of the SeW gene, we used in vitro binding assays to identify transcription factors that may be involved in the transcriptional regulation of the SeW gene. Using protein from rat C6 (glial) cell nuclear extracts, oligonucleotides containing putative regulatory elements in the SeW promoter, and antibodies, we were able to show that the specificity protein 1(Sp1) transcription factor binds to the Sp1 consensus sequence in the SeW promoter as well as the MRE. However, the MRE, GRE, AP-1 and LF-A1 did not yield any specific binding. Although, competition analysis showed specific binding at the TFII-1 site, super-shift analysis using anti-TFII-1 antibody did not yield any super-shifted band. Therefore the SeW gene may be a target for Sp1 whose interaction with the SeW promoter may activate or repress the transcription of SeW. / Graduation date: 2003

Three experiments were conducted to evaluate the impacts of removing antibiotic growth promoters (AGP) on broiler performance and yield and to evaluate alternative products as potential replacements. In experiment one, approximately 552,000 broilers were reared in four solid-wall, tunnel ventilated houses that were divided into two paired-house facilities, each assigned one of two dietary treatments. The treated group received basal diets containing salinomycin (SAL), roxarsone (ROX) and AGP, while the control group received the same diets without ROX and AGP. Removal of ROX and AGP had no affect on average body weight and feed efficiency, while livability was significantly affected negatively by the removal of ROX and AGP. Tender, wing, drum and percentage of total white meat showed significant improvements in yield during the study, while all other parts were not affected by removal of ROX and AGP. In experiment two, an investigation was conducted to evaluate the effects on performance from feeding Bacillus subtilis spores (Gallipro®, Chr Hansen A/S, Denmark), as a direct-fed microbial additive, to commercial broiler chickens. Birds were divided among two paired-house facilities. The treatment group received basal diets supplemented with B. subtilis spores, while the control group was fed the same basal diets containing an AGP. Feed conversion ratio was significantly lower for the treatment group, while average body weight, coccidiosis lesion scores, and footpad scores were not affected by the treatments. In experiment three, 6,000 broiler chickens were equally divided among four treatment groups and reared to 49 d to determine the effectiveness mannan oligosaccharides (MOS, Bio-Mos®, Alltech, Nicholasville, Kentucky, USA) as an alternative for an AGP program and MOS plus Natustat™ (NAT, Alltech, Nicholasville, Kentucky, USA) as an alternative to an enteric health program (AGP+anticoccidial drug). Average body weight for the control (CON) and antibiotic (ANT) groups was significantly different from the MOS+NAT group, but not the MOS group. Carcass front half, carcass hind half, frame and skin yields were improved for all treatments when compared to the MOS+NAT group. Conversely, percent total white meat yield was improved with the inclusion of MOS when compared to the ANT group. The findings of this research suggest that the removal of AGP from the diets of commercial broiler chickens does not affect the performance and yield of the birds over a one year production period. Furthermore, B. subtilis spores and mannan oligosaccharides provide acceptable alternatives to an AGP program.

No description available.

For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

Cyanobacteria are solar-powered cell factories that can be engineered to supply us with renewable fuels and chemicals. To do so robust and well-working biological parts and tools are necessary. Parts for controlling gene expression are of special importance in living systems, and specifically promoters are needed for enabling and simplifying rational design. Synthetic biology is an engineering science that incorporates principles such as decoupling, standardization and modularity to enable the design and construction of more advanced systems from simpler parts and the re-use of parts in new contexts. For these principles to work, cross-talk must be avoided and therefore orthogonal parts and systems are important as they are decoupled by definition. This work concerns the design and development of biological parts and tools that can enable synthetic biology in cyanobacteria. This encompasses parts necessary for the development of other systems, such as vectors and translational elements, but with a focus on transcriptional regulation. First, to enable the development and characterization of promoters in different cyanobacterial chassis, a broad-host-range BioBrick plasmid, pPMQAK1, was constructed and confirmed to function in several cyanobacterial strains. Then, ribosome binding sites, protease degradation tags and constitutive, orthogonal promoters were characterized in the model strain Synechocystis PCC 6803. These tools were then used to design LacI-regulated promoter libraries for studying DNA-looping and the behaviour of LacI-mediated loops in Synechocystis. Ultimately, this lead to the design of completely repressed LacI-regulated promoters that could be used for e.g. cyanobacterial genetic switches, and was used to design a destabilized version of the repressed promoter that could be induced to higher levels. Further, this promoter was used to implement an orthogonal transcriptional system based on T7 RNAP that was shown to drive different levels of T7 promoter transcription depending on regulation. Also, Gal4-repressed promoters for bacteria were engineered and examined in Escherichia coli as an initial step towards transferring them to cyanobacteria. Attempts were also made to implement a light-regulated one-component transcription factor based on Gal4. This work provides a background for engineering transcription and provides suggestions for how to develop the parts further.

Thesis (J.C.L.)--Catholic University of America, 1988. / Vita. Includes bibliographical references (68-73).

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 1999. / Includes bibliographical references.

Thesis (J.C.L.)--Catholic University of America, 1988. / Vita. This is an electronic reproduction of TREN, #029-0125. Includes bibliographical references (68-73).

Thesis (M.Sc.)--York University, 2007. Graduate Programme in Chemistry. / Typescript. Includes bibliographical references (leaves 129-130). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR32034

Thesis (Ph. D.)--University of British Columbia, 2003. / Includes bibliographical references (leaves 299-308).