“In situ” translation: Use of the cytoskeletal framework to direct cell-free protein synthesis |
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Authors: | Diane Biegel Joel S Pachter |
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Institution: | (1) Department of Physiology, University of Connecticut Health Center, 06030 Farmington, Connecticut |
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Abstract: | Summary We have developed a novel, “in situ” translation system derived from cultured cells that are subject to mild detergent extraction.
By using a low concentration of nonionic detergent to gently permeabilize cells while they remain adherent to a substrate,
cytoskeletal frameworks are obtained that are devoid of membraneous barriers yet retain much the same topological arrangement
of mRNA, ribosomes and cytostructure that exists “in vivo”. Data indicate that when these cytoskeletal frameworks are supported
by a ribosome-depleted, nuclease-treated, reticulocyte lysate supernatant, they are capable of resuming translation of their
attached polysomes for at least 40 minutes. Emulsion autoradiography of ongoing protein synthesis demonstrates that protein
synthetic activity is ubiquitous throughout the population of extracted cells, and not confined to a less well-extracted subset.
Computer-assisted, two-dimensional gel analysis reveals that the pattern of proteins produced by such extracted cells is approximately
70% coincident with that produced by unextracted cells, including proteins of molecular weight as great as 200 kilodaltons.
Furthermore, a continued increase in intensity of almost all proteins during the first 40 minutes of translation suggests
that translational re-initiation, in addition to polysome run-off, is also taking place. Collectively, these findings indicate
that much of the translational machinery remains both intact and competant in this cytoskeletal-based translation system.
As such, this system should prove extremely useful in identifying molecular factors operant during certain types of translation
control and in further examining the role played by the cytoskeleton in regulating gene expression.
This work was supported by grants from the American Cancer Society (#NP-683) and from the University of Connecticut Health
Center Research Foundation. |
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Keywords: | cytoskeleton ribosomes messenger RNA translation |
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