Functional and structural analyses of the ubiquitin conjugating enzyme UBE2I gene

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UBC9 (UBE2I) is a SUMO-1 conjugating enzyme and MITF is a specific transcription factor which is important for melanocyte differentiation. Previously we found the specific interaction between UBC9 and MITF with yeast two-hybrid system. In order to elucidate the function of UBC9,we developed the ribozymes which cleavage the mouse UBC9 mRNA. Furthermore, we characterized the genomic structures of the human and mouse UBC9.(1)Gene Regulation and Functional Analyses of UBC9 with Ribozymes:We designed the specific hummer head-type ribozymes for mouse UBC9 mRNA. We made the mouse UBC9 mRNA and ribozymes by in vitro transcription. By mixing experiment we elucidated that these ribozymes cut the mouse UBC9 mRNA in vitro. We confirmed that some of these ribozymes cut the mouse UBC9 mRNA in cultured cells by transfection experiment. Furthermore, we constructed the MITF-GFP fusion protein expression vector and showed that MITF locates in the nucleus primalily. We are now investigating the interaction between UBC9 and MITF and the effect of UBC9 for MITF function in mammalian cultured cells with these UBC9 ribozymes and MITF-GFP expression vector.(2)Gene Structures of Human and Mouse UBC9:We isolated the P1 phage clones which contain the human and mouse UBC9 genes. First, we characterized the human UBC9 gene. It spans about 20 Kb and consists of 7 exons. The promoter region of the human UBC9 gene is GC-rich and lacks a consensus TATA box. We also elucidated that the mouse UBC9 gene consists of 7 exons.