Generation and characterization of hammerhead ribozymes targeting rodent metallothionein-I and -II ribonucleic acid

Toxicol Appl Pharmacol. 1999 Dec 15;161(3):294-301. doi: 10.1006/taap.1999.8821.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Cadmium / toxicity
  • Cell Line, Transformed
  • Cell Survival / drug effects
  • Clone Cells
  • DNA Primers / chemistry
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism*
  • Male
  • Metallothionein / biosynthesis
  • Metallothionein / genetics*
  • Mice
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Prostate / cytology
  • Prostate / drug effects
  • Prostate / metabolism
  • RNA, Catalytic / chemistry
  • RNA, Catalytic / metabolism*
  • RNA, Messenger / metabolism*
  • Rats
  • Transcription, Genetic
  • Transfection

Substances

  • DNA Primers
  • RNA, Catalytic
  • RNA, Messenger
  • Cadmium
  • Metallothionein