Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations.
Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations.
复制标题
通过抑制连接子插入诱变构建莫洛尼鼠白血病病毒突变体：可行插入突变的位置。
DOI：
10.1073/pnas.81.13.4149
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发表时间：
1984
影响因子：
11.1
通讯作者：
Goff,SP
中科院分区：
文献类型：
--
作者：
Lobel,LI;Goff,SP
描述了一种高效的插入突变生成方法。该过程涉及使用带有SuIII+抑制tRNA基因的220碱基对（bp） EcoRI片段作为插入诱变原。将待诱变的质粒DNA通过各种方法线性化，将抑制片段连接到裂解位点。在MacConkey乳糖板上携带lac- amber突变的大肠杆菌中可以很容易地检测到成功插入突变体；几乎100%的红色菌落都含有片段的插入。随后去除SuIII+基因并进行再循环，如果原始切割是钝端，则留下一个12bp的插入；如果原始切割产生3bp的内聚末端，则留下一个9bp的插入。该技术，以及传统的十体和十二体连接体连接体突变技术，被用于在Moloney小鼠白血病病毒基因组的克隆DNA拷贝中产生大量插入突变文库。许多有活力的突变体被分离出来，在基因组的不同区域有9-，10-和12-bp的插入。活突变的图谱位置表明，病毒的长末端重复序列和gag和env基因的部分对改变相当不敏感。虽然大多数突变在许多传代中都是稳定的，但一些突变丢失了插入的DNA；我们推测，插入在这些突变体中是有害的，并且病毒的继续传递被逆转录者选择为过度生长。
A highly efficient method for the generation of insertion mutations is described. The procedure involves the use of a 220-base-pair (bp) EcoRI fragment bearing the SuIII+ suppressor tRNA gene as an insertional mutagen. The plasmid DNA to be mutagenized is linearized by a variety of means, and the suppressor fragment is ligated into the site of cleavage. Successful insertion mutants can be readily detected in Escherichia coli carrying lac- amber mutations on MacConkey lactose plates; virtually 100% of the red colonies contain insertions of the fragment. Subsequent removal of the SuIII+ gene and recyclization leaves a 12-bp insertion if the original cleavage was blunt-ended and a 9-bp insertion if the original cleavage generated 3-bp cohesive termini. This technique, as well as conventional linker mutagenesis with decamer and dodecamer linkers, was used to generate a large library of insertion mutations in cloned DNA copies of the genome of Moloney murine leukemia virus. A number of viable mutants were isolated bearing 9-, 10-, and 12-bp insertions in various domains of the genome. The map positions of the viable mutations suggest that the viral long terminal repeats and portions of the gag and env genes are quite insensitive to alteration. Although most of the mutations were stable for many passages, some of the mutants lost the inserted DNA; we presume that the insertion was somewhat deleterious in these mutants and that continued passage of the virus selected for overgrowth by a revertant.
促性腺激素释放激素及其激动剂抑制未成熟和成年垂体切除大鼠的睾丸黄体生成激素受体和类固醇生成。
影响因子：
4.8
作者：
Bambino,TH;Schreiber,JR;Hsueh,AJ
通讯作者：
Hsueh,AJ
来自支持细胞富集培养物的废培养基中的一种因子，可刺激 Leydig 细胞中的类固醇生成
影响因子：
4.1
作者：
G. Verhoeven;J. Ćailleau
通讯作者：
J. Ćailleau