Characterization and measurement of metallothionein messenger RNA of C57BL mouse liver.
Liver poly(A)+RNA of Cd2+-treated C57BL mouse was characterized by cell-free translation, particularly intending to establish a procedure to measure the levels of messenger RNA coding for metallothioneins (MT-mRNA). Intact polysomes were obtained by Mg2+ precipitation from the liver cytoplasm of mice injected with 1 mg Cd2+/kg body wt. Poly(A)+RNA isolated from the polysomes was translated by a wheat germ cell-free system and the [35S]cysteine-labeled translation products were analyzed by sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis and fluorography. MTs were identified in the translation products directed by the RNA from the Cd2+-treated mice, but not in the translation products directed by the RNA from untreated mice. Relative incorporation of [35S]cysteine into MTs was determined by densitometrical quantification of the MT bands, and was found to be linear up to a RNA concentration of 150 micrograms/ml in the translation reaction mixture, showing that this system is suitable for the measurement of translatable MT-mRNA levels. Cd2+ stimulated the total levels of cell-free translation (1.4-fold at 20-60 micrograms/ml), not specifically to MT-mRNA. MT-mRNA sedimented at 9S in a sucrose gradient, and its size was comparable with rat and human MT-mRNAs.
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005470:Fluorometry; D008099:Liver / Q000032:analysis*; D008297:Male; D008668:Metallothionein / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D011132:Polyribosomes / Q000187:drug effects; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis*
D012333:RNA, Messenger; D002104:Cadmium; D008668:Metallothionein