权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Elucidation of DNA replication mechanism of Protobothrops flavoviridis (Habu snake)

阐明 Protobothrops flavoviridis（哈布蛇）的 DNA 复制机制

基本信息

批准号：
16580079
负责人：
ODA-UEDA Naoko
金额：
$ 2.05万
依托单位：
Sojo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

We have found that snake venom isozymes with a variety of physiological activities from Protobothrops (formally known as Trimesurus) flavoviridis had evolved in an accelerated manner, and believed that mostlikely the diversity in such venomous isozyme genes had produced by accumulation of point mutations. To understand the molecular mechanism underlying mutation in P.flavoviridis, we have cloned P.flavoviridis homolog of the Escherichia coli dinB gene which encode DNA polymerase IV and known as error-prone DNA polymerase. Total RNA from P.flavoviridis oocyte was used as template for first strand cDNA synthesis by used the SMART cDNA Library construction kit. Degenerate primers were designed based on conserved sequence in the Xenopus laevis (frog) and Gallus gallus (chicken). Multiple rounds of 5' and 3' rapid amplification of cDNA ends were used to extend the putative P.flavoviridis genes using BD SMART RACE cDNA ampification kit. Fibroblast cells from the embryo of P.flavoviridis were prepared for chromosome mapping and fluoresence in situ hybridaization. Multiple alignments were constructed with the CLUSTAL X program.The P.flavoviridis DinB sequence of cDNA 2882 bp contains an ORF of 2.5 kiobases (kb) that can encode a protein of 833 amino acids. The amino acid sequence of P.flavoviridis DinB protein contain N-terminal nucleotidyl transferase domain, two tandem HhH domain implicated of DNA binding, and nuclear locarization signal like domain and proliferating cell nuclear antigen domain as being conserved in DinB superfamily, and share significant identity (〜84%) with G.gallus (chiken). In a phylogenetic tree of DinB superfamily, P.flavoviridis DinB make a branch with G.gallus out of other eukaruote such as H.sapiens or mouse. Fluoresence in situ hybridization of chromosome represents that P.flavoviridis dinB gene localized to chromosome 6 (macrochromosome) unlike G.gallus's which localized to microchromosome.

我们发现，具有多种生理活性的黄绿原棘头蛇蛇毒同工酶的进化是以加速的方式进行的，并认为这种有毒同工酶基因的多样性很可能是通过点突变的积累而产生的。为了了解黄绿假单胞菌突变的分子机制，我们克隆了黄绿假单胞菌编码DNA聚合酶IV和易错DNA聚合酶的大肠杆菌dinB基因的同源物。利用SMART cDNA文库构建试剂盒，以黄绿青霉卵母细胞总RNA为模板合成第一链cDNA。根据非洲爪蟾（Xenopus laevis）和原鸡（Gallus gallus）的保守序列设计简并引物。使用BD SMART RACE cDNA扩增试剂盒对cDNA末端进行多轮5'和3'快速扩增以延伸推定的黄绿青霉基因。从黄绿青霉胚胎中提取成纤维细胞，进行染色体定位和荧光原位杂交。通过CLUSTAL X程序进行多重序列比对，得到黄绿青霉DinB基因cDNA全长2882 bp，开放阅读框为2.5 kb，编码833个氨基酸。黄绿脓杆菌DinB蛋白的氨基酸序列包含N-末端核苷酸转移酶结构域、两个串联的DNA结合结构域、核定位信号样结构域和增殖细胞核抗原结构域等DinB超家族中保守的结构域，与鸡球虫（G.gallus（chiken））的同源性高达84%。在DinB超家族的系统发育树中，黄绿青霉DinB与鸡球虫（G.gallus）形成一个分支，与其他真核生物如智人（H.sapiens）或小鼠形成一个分支。染色体原位杂交结果表明，黄绿青霉dinB基因定位于第6号染色体（大染色体），而鸡胚的dinB基因定位于小染色体。