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ubiquitin-huntingtin nuclear inclusion body formation

泛素狩猎蛋白核包涵体形成

基本信息

批准号：
10680589
负责人：
SAITOH Yasushi
金额：
$ 2.11万
依托单位：
IWATE UNIVERSITY (1999)Tokai University (1998)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

The nuclear inclusion body formation of ubiquitin-huntingtin conjugate must be controlled by ubiquitin conjugating enzymes and deubiquitinating enzymes. One ubiquitin-conjugating enzyme was reported to bind huntingtin. In contrast, the function of deubiqitinating enzyme is not clear yet.We found a novel deubiquitinating enzyme gene named RS447-dUb in the RS447 megasatellite repeat. We examined the expression of the RS447-dUb in human tissues. Northern blot analysis using an antisense ORF probe identified 2.4 Kb transcript in heart, brain, liver and skeletal muscle. To confirm the presence of poly(A) containing RS447 RNA, cDNA clones that encodes a sense transcript of the RS447 were isolated by using a PCR-based extension method in conjunction with 5' and 3'-RACE. We also identified a functional promoter activity in upstream sequence of the RS447 ORF.We also transfected a cosmid clone containing 7 tandem copies of RS447 into mouse A9 cells and confirmed the expression of RS447 deubiquitinating enzyme.These results indicate that the RS447-repeating unit on this megasatellite repeat codes and actively expresses a functional deubiquitinating enzyme.Although it is expressed ubiquitously in human tissues, RS447-dUb exhibited a unique expression pattern in that its complementary strand is transcribed as an antisense transcript predominantly in brain. This could modulate the level of RS447-dUb expression in the human brain. Further characterizations of the expression of sense and antisense transcripts of the RS447, and relationship with ubiquitin-huntingtin nuclear inclusion body formation are currently underway.

泛素-亨廷顿偶联物核包涵体的形成必须受泛素偶联酶和去泛素化酶的控制。据报道，一种泛素结合酶与亨廷顿蛋白结合。相比之下，去遍在蛋白酶的功能尚不清楚，我们在RS 447巨卫星重复序列中发现了一个新的去遍在蛋白酶基因，命名为RS 447-dUb。我们检测了RS 447-dUb在人体组织中的表达。使用反义ORF探针的北方印迹分析鉴定了心脏、脑、肝和骨骼肌中的2.4Kb转录物。为了确认含有RS 447 RNA的poly（A）的存在，通过使用基于PCR的延伸方法结合5'和3'-RACE分离编码RS 447的有义转录物的cDNA克隆。我们还在RS 447 ORF的上游序列中鉴定了一个功能性启动子活性。我们还将含有7个串联拷贝的RS 447的粘粒克隆转染到小鼠A9细胞中，并证实了RS 447去泛素化酶的表达。这些结果表明，RS 447-这个巨卫星重复序列上的重复单元编码并主动表达一种功能性去泛素化酶。组织中，RS 447-dUb表现出独特的表达模式，因为其互补链主要在脑中转录为反义转录物。这可以调节RS 447-dUb在人脑中的表达水平。目前正在进一步研究RS 447的正义和反义转录本的表达，以及与泛素-亨廷顿蛋白核包涵体形成的关系。