Functional and structural analyses of the ubiquitin conjugating enzyme UBE2I gene

UBC 9（UBE 2 I）是SUMO-1结合酶，MITF是黑素细胞分化的特异性转录因子。我们在酵母双杂交系统中发现了UBC 9与MITF之间的特异性相互作用。为了阐明UBC 9的功能，我们开发了切割小鼠UBC 9 mRNA的核酶。此外，我们表征了人类和小鼠UBC 9的基因组结构。（1）核酶对UBC 9基因的调控及功能分析：设计了针对小鼠UBC 9 mRNA的hummer头型核酶。我们通过体外转录的方法制备了小鼠UBC 9 mRNA和核酶。通过混合实验，证实了这些核酶在体外对小鼠UBC 9 mRNA具有切割作用。我们通过转染实验证实了其中一些核酶在培养的细胞中切割小鼠UBC 9 mRNA。进一步构建了MITF-GFP融合蛋白表达载体，并初步证实MITF的表达定位于细胞核内。我们现在正在研究UBC 9和MITF之间的相互作用和UBC 9对MITF功能的影响，在哺乳动物培养细胞中使用这些UBC 9核酶和MITF-GFP表达载体。（2）人和小鼠UBC 9的基因结构：我们分离了含有人和小鼠UBC 9基因的P1噬菌体克隆。首先，我们鉴定了人类UBC 9基因。它跨越约20 Kb，由7个外显子组成。人UBC 9基因的启动子区富含GC，缺乏共有TATA盒。我们还阐明了小鼠UBC 9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.