Preparation of a cell-free translation system from PC12 cells |
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Authors: | Makoto Shibutani Eugene Kim Philip Lazarovici Mari Oshima Dr Gordon Guroff |
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Institution: | (1) Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, Building 49, Room 5A64, 20892 Bethesda, Maryland |
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Abstract: | 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
immunoprecipitation.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.
Special issue dedicated to Dr. Hans Thoenen. |
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Keywords: | PC12 translation S10 epidermal growth factor receptor luciferase |
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