Preparation of a cell-free translation system from PC12 cell.
The postmitochondrial fraction (S10) contains the cellular components essential for translation, and a high-salt wash (HSW) of the ribosomes is enriched in eukaryotic initiation factors. This report describes the preparation of a cell-free translation system utilizing an S10 extract from PC12 cells. The products synthesized from either firefly luciferase mRNA or PC12 cell poly(A) RNAs in the PC12-S10 extract were increased by the addition of the HSW from PC12 cells. Increases in the translation of luciferase mRNA by the addition of PC12-HSW were dose-dependent and also dependent on the time of incubation. The translation of human epidermal growth factor receptor (hEGFR) mRNA could also be detected in the PC12-S10 extract translation system by immuno-precipitation. N-linked glycosylation of the translation products also was observed. The efficiency of translation was altered by the addition of Mg2- or K+, and optimization of the concentrations of these ions was necessary for each mRNA. The translation system made from PC12 cells, then, is capable of the synthesis of proteins of relatively high molecular weight and should be useful for analyzing mechanisms of translational control during proliferation and differentiation of cells from a neuronal lineage.
Shibutani
M
M
Section of Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kim
E
E
Lazarovici
P
P
Oshima
M
M
Guroff
G
G
D000818:Animals; D002474:Cell-Free System; D004815:Epidermal Growth Factor / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007163:Immunosorbent Techniques; D008156:Luciferases / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D016716:PC12 Cells / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D008156:Luciferases; D008274:Magnesium; D011188:Potassium