| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 89 === The Pia protein plays an essential role in the development of Dictyostelium discoidium. Pia participates in the cAMP signal transduction and has been shown to function in between the heterotrimeric G protein and the adenylate cyclase enzyme. The action mechanism of Pia, however, remains unclear. By searching the sequence databases, a Saccharomyces cerevisiae gene , TSC11, has been found to be homologus to the PiaA gene. The open reading frame of TSC11 predicts a protein of 164.4 kD and no known functional motifs can be found in the TSC11 sequence. When the entire TSC11 gene is deleted in S. cerevisiae, the yeast cells are not viable. Therefore, TSC11 is an essential gene in S. cerevisiae.
Little is known about the function or action mechanism of Tsc11p. Aiming at identifying the localization of Tsc11p in the S. cerevisiae cells, I prepared and purified two different fusion proteins of Tsc11p(1160~1430aa), used them as the antigens for immunizing rabbits, obtained anti-Tsc11p antibodies, and used the antibodies to do Western analysis on cellular fractions. The results demonstrate that Tsc11p was mainly localized to the nuclear fractions. I have also expressed a tri-HA tagged full-length Tsc11p in S. cerevisiae and demonstrated the ability of tagged Tsc11p to function as the wild type Tsc11p in a plasmid shuffling experiment. When anti-HA antibodies were used to detect the tri-HA tagged Tsc11p, the results also showed the nuclear localization.
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