Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene.
Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene.
复制标题
含有 FMR-1 基因 CGG 三联体重复序列的质粒的克隆、表征和特性。
DOI：
10.1006/jmbi.1996.0273
复制
发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Wells,RD
中科院分区：
文献类型：
--
作者：
Shimizu,M;Gellibolian,R;Oostra,BA;Wells,RD
人类脆性x综合征（一种非孟德尔遗传的智力低下疾病）的FMR-1基因在5 '非翻译区含有一个遗传不稳定的CGG区域。疾病的严重程度与CGG束的长度有关。在大肠杆菌中，通过三种策略克隆了28个含有（CGG）ninserts的稳定质粒（≤6 ~ 240），这些质粒具有不同程度和类型的序列中断（多态性），并且在不同的方向上。一些较短的片段是通过直接克隆合成的寡核苷酸制备的，较长的片段是通过从脆性x患者的cDNA或从这些序列的扩增或缺失中克隆（CGG）81、（CGG）11AGG(CGG)60CAG(CGG)8的多片段克隆而成的。杆菌。插入物的遗传稳定性，特别是对于较长的片段，取决于序列长度、多态性的存在、宿主细胞基因型、插入物在载体中的方向以及在载体中克隆的位置。对完全甲基化和非甲基化质粒的二维琼脂糖凝胶电泳研究以及化学探针研究显示缺乏下缠绕结构或可接近的碱基对。这些dna能够对脆性x综合征的分子基础进行一系列遗传和生化研究。
The FMR-1 gene for the human fragile-X syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5′ non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG)ninserts (wheren=6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies inEscherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG)81, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-X patient or from expansions or deletions of these sequences inE. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-X syndrome.