Generation and characterization of hammerhead ribozymes targeting rodent metallothionein-I and -II ribonucleic acid
Graduate School of Biomedical Sciences; Department of Surgery
Medical Subject Headings
Animals; Base Sequence; Cadmium; Cell Line, Transformed; Cell Survival; Clone Cells; DNA Primers; Epithelial Cells; Male; Metallothionein; Mice; Molecular Sequence Data; Nucleic Acid Conformation; Prostate; RNA, Catalytic; RNA, Messenger; Rats; Transcription, Genetic; Transfection
Life Sciences | Medicine and Health Sciences
Two trans-acting hammerhead ribozymes (Rzs), Rz1-2 and Rz4-9, were designed and shown to be effective in cleaving rat or mouse metallothenein (MT)-I and MT-II mRNA, respectively, at position +147/148, in a sequence-specific manner. The catalytic efficiency for Rz1-2 on rat MT-I was found to be 678 M(-1)s(-1) and that for Rz4-9 on rat MT-II cRNA was found to be 372 M(-1)s(-1). An expression vector, pRz(4-9/1-2), containing both Rzs linked in tandem, was constructed and used to transfect NbE-1 cells, an immortalized ventral prostate epithelial cell line. Two stable-transfected lines, NbE-1(Rz2) and NbE-1(Rz3), expressing substantial levels of (Rz1-2/Rz4-9) and minimal levels of MT-I and MT-II transcripts were found to exhibit increased sensitivity to cadmium (Cd)-induced cytotoxicity compared to the parent line. This article is the first to report successful degradation of rodent MT mRNA in vitro and in cellulo by hammerhead Rzs.
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Citation: Toxicol Appl Pharmacol. 1999 Dec 15;161(3):294-301. Link to article on publisher's site
Toxicology and applied pharmacology
Lee, Kai-Fai; Lau, Kin-Mang; and Ho, Shuk-Mei, "Generation and characterization of hammerhead ribozymes targeting rodent metallothionein-I and -II ribonucleic acid" (2000). GSBS Student Publications. 653.