Generation and characterization of hammerhead ribozymes targeting rodent metallothionein-I and -II ribonucleic acid.
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.
Lee
K F
KF
Department of Surgery, University of Massachusetts, 55 Lake Avenue North, Worcester, Massachusetts, 01655, USA.
Lau
K M
KM
Ho
S M
SM
D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000633:toxicity; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D002999:Clone Cells; D017931:DNA Primers / Q000737:chemistry; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011467:Prostate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection
D017931:DNA Primers; D016337:RNA, Catalytic; D012333:RNA, Messenger; D002104:Cadmium; D008668:Metallothionein