Hammerhead ribozymes against gamma-glutamylcysteine synthetase mRNA down-regulate intracellular glutathione concentration of mouse islet cells.
gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione synthesis and is thought to play a significant role in intracellular detoxification systems. To specifically suppress gamma-GCS gene expression, we constructed two different hammerhead ribozymes against gamma-GCS mRNA transcripts. Two cleavage sites were targeted as follows: site 1 for anti-gamma-GCS ribozyme (H), a GUU triplet located from +348 to +350 of the gamma-GCS heavy chain, and site 2 for anti-gamma-GCS ribozyme (L), a GUU triplet located from +235 to +237 of the gamma-GCS light chain. The anti-gamma-GCS ribozymes effectively cleaved gamma-GCS mRNA in a cell-free system. When transfected into a Min-6 mouse islet cell line, these anti-gamma-GCS ribozymes not only suppressed gamma-GCS gene expression, but also reduced intracellular glutathione concentration. These results suggest that the ribozyme-mediated down-regulation of gamma-GCS gene expression may be useful for analyzing the glutathione-associated cellular defense systems of pancreatic islet cells.
Kijima
H
H
Department of Pathology, Tokai University School of Medicine, Isehara, Japan. hkijima@is.icc.u-tokai.ac.jp
Tsuchida
T
T
Kondo
H
H
Iida
T
T
Oshika
Y
Y
Nakamura
M
M
Scanlon
K J
KJ
Kondo
T
T
Tamaoki
N
N
D000818:Animals; D002460:Cell Line; D015536:Down-Regulation / Q000502:physiology*; D005721:Glutamate-Cysteine Ligase / Q000235:genetics*; D005978:Glutathione / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D051379:Mice; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection / Q000235:genetics
D016337:RNA, Catalytic; D012333:RNA, Messenger; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione