新基因人MOB cDNA的克隆与功能分析

Cloning and Function Analysis of A Novel Gene: Human MOB

人MOB基因被认为是在脑组织中高表达的 5次跨膜而功能未知的膜蛋白 .从胎儿和成人肾脏消减杂交cDNA文库中获得了一条新的表达序列标签 (expressedsequencetag ,EST)序列 ,该序列对应于功能未知的MOB基因 .利用生物信息学分析工具和分子生物学技术 ,从胎儿肝脏中成功地克隆了人MOB基因cDNA序列 ,同时也获得了含有完整开放读码框的大鼠和鸡MOB的电子全长序列 .人MOB基因定位于 10q11 1～ 11 2之间 ,含有一个 12 4 2bp的开放阅读框 ,第 4 15位开始的ATG可能是翻译起始位点 .核酸序列相似性搜索发现 ,其他种属中存在与之高度相似的EST序列 ,如爪蟾 (79% )、羊 (87% )、猪 (94 % )、牛 (93% ) .蛋白质序列相似性搜索发现 ,人MOB与大鼠、小鼠和鸡MOB有 97%、 97%、 91%的相似性 ,与其他一些不同种属来源的假想蛋白有 4 5 %～ 73%的相似性 .不同物种之间MOB基因核酸和蛋白质序列的高度相似说明 ,MOB是进化上高度保守的蛋白质 .保守结构域分析发现 ,人、小鼠、大鼠和鸡MOB结构域组成完全一致 ,N端均与不育α基序(sterilealphamotif,SAM )结构域显著匹配 ,随后出现匹配显著性稍差的几个结构域 .核酸和蛋白质序列的保守性提示 ,除N端约 70个氨基酸组成SAM结构域外 。

Human MOB is a novel gene first cloned in lung tissue and lymphoma tissue. It is proposed to express predominately in brain, and be a five-pass transmembrane protein. A new expressed sequence tag (EST) (GenBank accession number: BI740300) matched completely with human MOB gene was obtained. The 2 230 bp sequence of human MOB cDNA, which contains a 1 242 bp (nt 415～1 656) open reading frame, encoding a protein of 413 amino acid residues, was successfully cloned. In addition, rat and chicken MOB cDNA sequences, which also contain a 1 242 bp open reading frame, were cloned in silico by aligning dozens of overlapping rat and chicken ESTs and cDNA sequences identified from GenBank. Human MOB was mapped on chromosome 10q11.1～11.2. Homology searches with the deduced 413 amino acid residues revealed human MOB shares 97% similarity with murine MOB, 97% with rat MOB, 91% with chicken MOB and 45%～73% with lots of hypothetical proteins of various origin. The predicted protein contains SAM domain, which has been suggested to be an evolutionarily conserved protein-binding domain that is involved in the regulation of numerous developmental processes in diverse eukaryotes and can potentially function as a protein interaction module through its ability to homo- and heterooligomerise with other SAM domains. Homology searches and domain query indicate that human MOB is a member of potential phylogenetically conserved MOB family. RT-PCR revealed that human MOB was almost expressed in all kinds of tissues and cells, which is completely consistent with the in silico expression pattern identified by bioinformatics analysis. In contrast to bioinformatics analysis and previous study, however, human MOB has widespread subcelluar expression, primarily in nuclei. Further study in HeLa cells shows that overexpression of human MOB seems not to influence cell cycle and apoptosis. Taken together, human MOB is a novel phylogenetically conserved gene, which is proposed to express in almost all tissues and cells, and has widespread subcelluar expression, primarily in nuclei. It may play a role in signal transduction and thus be involved in development regulation, but seems not to influence cell cycle and apoptosis. Further investigation remains needed.