Functional and structural analyses of the ubiquitin conjugating enzyme UBE2I gene

UBC9(UBE2I)是一种SUMO-1结合酶，而MITF是一种对黑素细胞分化起重要作用的特异性转录因子。以前我们利用酵母双杂交系统发现了UBC9与MITF之间的特异性相互作用。为了阐明UBC9的功能，我们开发了切割小鼠UBC9 mRNA的核酶。此外，我们还对人和小鼠UBC9的基因组结构进行了研究。(1)核酶对UBC9基因的调控和功能分析：我们设计了针对小鼠UBC9基因的特异性Hummer头型核酶。我们通过体外转录的方法获得了小鼠UBC9的mRNA和核酶。通过混合实验，我们证实了这些核酶在体外对小鼠UBC9基因的切割作用。通过转染实验，我们证实了其中一些核酶能切割培养细胞中的小鼠UBC9基因。构建了MITF-GFP融合蛋白表达载体，结果表明MITF主要定位于细胞核内。我们正在利用这些UBC9核酶和MITF-GFP表达载体研究UBC9与MITF的相互作用以及UBC9对哺乳动物细胞MITF功能的影响。(2)人和小鼠UBC9的基因结构：我们分离了含有人和小鼠UBC9基因的P1噬菌体克隆。首先，我们鉴定了人类的UBC9基因。该基因全长约20kb，由7个外显子组成。人类UBC9基因的启动子区域富含GC，缺乏一个共识的TATA盒。我们还阐明了小鼠UBC9基因由7个外显子组成。

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.