| Budget Amount *help |
¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Research Abstract |
Human autoantibodies have proven to be useful reagents for identifying novel nuclear antigens. Several autoantibodies are known to recognize proteins which are highly conserved in evolution and which are involved in essential intracellular functions such as DNA replication, transcription, and splicing of pre-mRNA, In this study, we isolated cDNA clones encoding transcripton factor ZF5, which is present in speckle structures, a novel MAP kinase kinase (MKK6) library, and a novel nuclear speckle-type POZ protein designated SPOP were isolated from a HeLa cDNA by this immunoscreening approach with autoimmune patient sera. Moreover, we have cloned human dymple, a novel dynamin-family member. The full-length cDNA sequence encodes a protein composed of 736 amino acids with a molecular mass of 80 kDa. Amino acid sequence most resembles yeast Dnmlp and Vps1p. Dymple lacks a proline-rich C-terminal domain, through which dynamin binds to SH3 domains to be activated. Northern blot analysis revealed two transcript sizes of 2.5kb and 4.2kb with alternative polyadenylation at highest levels in brain, skeletal muscle and testis. It was further established that there are three patterns of alternative splicing producing in-frame deletions in the coding sequence of dymple in a tissue-specific manner. When overexpressed, wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern, while an N-terminal deletion mutant formed large aggregates bounded by a TGN marker. Since dynamin participates in clathrin-mediated endocytosis through a well-characterized mechanism, the existence of a dynamin-like molecules in each specific vesicle transport pathway has been predicted. Our findings suggest that dyniple may be the first example of such a subfamily in mammalian cells other than dynamin-itself, though its precise role and membrane localization remain to be resolved.
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