In eukaryotes, regulation of translation and decay of messenger RNA are critical for fine-tuned control of gene expression. An important point of control is the key transition where mRNAs exit translation and assemble into a non-translating mRNP state that can accumulate in cytoplasmic granules such as P bodies and/or Stress granules. In the budding yeast Saccharomyces cerevisiae , the activators of decapping Dhh1 and Pat1 appear to promote the exit of mRNAs from translation. In my work, summarized below, I describe a new regulator of translation repression and mRNA degradation, Stm1, and its novel mode of action. First, I identified Stm1 as a novel regulator of translation repression and mRNA decay. Stm1 shows several genetic interactions with Pat1 and Dhh1, in a manner consistent with Stm1 promoting the function of Dhh1. This suggests that Stm1 has a role to play in translation repression and/or activation of mRNA decay. stm1 δ strains are defective in the degradation of a subset of mRNAs that include EDC1 and COX17 . These results strongly argue that Stm1 is a novel addition to the mRNA degradation machinery. Second, I have shown that Stm1, a known ribosome-associated protein, can bind and stall 80S ribosomes to repress translation and promote decay. Stm1 is able to repress translation and stall an 80S complex in vitro . Several mutations were identified in the protein, which link the in vitrophenotype to its biological functionin vivo. The analysis of different steps in translation reveals Stm1 functions in a novel manner to inhibit translation after the formation of an 80S complex. Since most of the regulation of translation is thought to happen at the stage of initiation, this study reveals a novel mode of translation regulation. These results also provide a direct and mechanistic link between ribosome function, inhibition of translation and the degradation of messenger RNAs.

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.