title: Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries.
abstract: 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-alpha (TNF-alpha)-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-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-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.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kawasaki
authors.forename: Reiko
authors.initials: R
authors.lastname: Onuki
authors.forename: Eigo
authors.initials: E
authors.lastname: Suyama
authors.forename: Kazunari
authors.initials: K
authors.lastname: Taira
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D016337:RNA, Catalytic; D014409:Tumor Necrosis Factor-alpha; C427204:hammerhead ribozyme