Identification of genes that function in the TNF-α-mediated apoptotic pathway using randomized hybrid ribozyme libraries
Identification of genes that function in the TNF-α-mediated apoptotic pathway using randomized hybrid ribozyme libraries
复制标题
使用随机混合核酶文库鉴定在 TNF-α 介导的细胞凋亡途径中起作用的基因
作者：
H. Kawasaki;R. Onuki;E. Suyama;K. Taira
既然许多基因组的序列都是可用的，就需要快速鉴定功能基因的方法。我们在这里描述了一个简单的系统，用于分离在肿瘤坏死因子-α（TNF-α）介导的细胞凋亡途径中起作用的基因，使用随机底物结合臂的RNA解旋酶相关核酶文库。由于靶位点的可接近性大大限制了胞内核酶的有效使用，因此常规核酶文库的有效性一直很低。为了克服这一障碍，我们将能够与内源性RNA解旋酶相互作用的RNA基序（poly（A）-tail）连接到核酶上，使得所得的解旋酶连接的杂合核酶可以更容易地攻击靶位点，而不管它们的二级或三级结构。当引入核酶文库后细胞表型发生变化时，可以通过对活性核酶克隆进行测序来鉴定导致这些变化的基因。在TNF-α介导的细胞凋亡中，当将核酶文库导入MCF-7细胞时，存活的克隆完全或部分抵抗TNF-α诱导的细胞凋亡。我们使用这种方法鉴定了许多促凋亡基因和以前未表征的基因的部分序列。我们的基因发现系统应该普遍适用于各种系统中功能基因的鉴定。
Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-α (TNF-α)–mediated pathway of apoptosis, using RNA helicase–associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-α-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-α-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.
影响因子：
56.9
作者：
H. Tang;G. Gaietta;W. Fischer;Mark Ellisman;F. Wong-Staal
通讯作者：
H. Tang;G. Gaietta;W. Fischer;Mark Ellisman;F. Wong-Staal