| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 90 === Citrate synthase is an almost ubiquitous enzyme. Its role is a catalyst of the entry point reaction for entry of two-carbon units into the citric acid cycle. It is an essential step in the biosynthesis of amino acids. Some organisms have a single citrate synthase, while a few, have as many as three. Peroxisomes show a remarkable metabolic plasticity. Their size, numbers, protein composition and biochemical functions vary that depending on the organism, cell type and/or environmental condition. By screening developmentally morphological mutants generated by the restriction enzyme-mediated integration (REMI) mutagenesis, we have found two mutants WTC127 and WTC180. The plaques of WTC127 cells have sparse structures, and cannot form the terminal fruiting bodies, and the sizes of plaques are strikingly smaller than wild type cells on the bacterial plate. The disrupted gene of mutant strain WTC127 encodes a citrate synthase homology protein, CshA, contained a conserved peroxisomal targeting signal PTS2 nonapeptide sequence at the N terminus. Phagocytosis of cshA- cells is slightly worse than wild type and the results of flow cytometry are the same. The growth of cshA- cells have a strikingly defect in axenic medium, but mitosis of cshA- cells is normal. Finally, mixing cshA- cells with K. aerogenes and developing on KK2 agar, the aberrant developmental phenotype of cshA- mutant can be observed at 72 hr, and the aggregation of cshA- cells delayed about 20 hours. The presence of bacteria interferes the multicellular development of cshA- cells, but it is still unclear why cshA- cells cannot form mature fruiting bodies. Because the developmental program of WTC127 is normal on KK2 agar, we are interested in the role of another citrate synthase gene, gltA (glutamate auxotroph). We determined the existence of other citrate synthase homology protein, and GltA was considered the potential another citrate synthase in Dictyostelium. Analyzing the 3’-terminal of cDNA fragment, there is not any signal peptide including in C-terminal of GltA. RT-PCR analysis indicated that gltA mRNA is expressed throughout the development. We think that gltA is a substitute for cshA- cell during development. The WTC180 cells formed distinctly smaller structures than wild type on a bacterial plate. The disrupted gene of mutant strain WTC180 is a novel gene. Mutant cells were able to form normal aggregation streams upon starvation, but the formation of tip mound delayed about 6 hours. The terminal structures, very small size of fruiting bodies, completed at 36 hr.
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