| Summary: | 碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 91 === The nucleolus is the site of rDNA transcription, rRNA processing and ribosome biogenesis. Human Nopp140 (hNopp140) is a hyperphosphorylated protein of the nucleolus. At interphase, hNopp140 localizes in nucleolus as a dotted manner. When cells enter mitosis, hNopp140 is further phosphorylated and disperses throughout the entire cytoplasm. JY3 has been identified by a monoclonal antibody raised against hNopp140-immunoprecipitates. Based on immunoprecipitation, I demonstrated that JY3, a 100 kD protein, existed in the immunocomplex of hNopp140. JY3 was able to be co-immunoprecipitated with different truncated forms of hNopp140, indicating that there are multiple interacting domains. In asynchronized HeLa cells, JY3 appeared in nucleus as a dynamic pattern. In a subset of interphase cells, JY3 existed in nucleolus as a granular pattern, but in others, it exhibited as nuclear speckles, a pattern similar to that of slicing factors. When cells entered mitosis, JY3 were accumulated on the surface of chromosomes. At telophase and early G1 phase, JY3 colocalized with hNopp140 and apparently clustered in nucleolar organizer regions. In order to find out the identity of JY3 protein, I performed immunoscreening of a λgt11 library. Two candidates, ANT1 and TCOF1, were obtained. Both proteins ectopically expressed in HeLa cells and 293T cells were recognized by JY3. ANT1 behaved as a dynamic protein either forming granules in nucleolus or like the splicing factors spreading in nucleoplasm. ANT1 was proven to present in the hNopp140-immunocomplex with a molecular weight of 100 kD. It strongly suggests that JY3 protein is equivalent to ANT1. Only partial TCOF1 cDNA was obtained so far. This truncated TCOF1, missing the C-terminal end, was able to target to nucleolus and appeared as a dotted manner. TCOF1 (140~200 kD), structurally similar to hNopp140, is related to a human autosomal dominant genetic disease, named Treacher Collins Syndrome. Links between TCOF1 and JY3 protein require further investigation.
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