"In situ" translation: use of the cytoskeletal framework to direct cell-free protein synthesis.
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 competent 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.
Biegel
D
D
Department of Physiology, University of Connecticut Health Center, Farmington 06030.
Pachter
J S
JS
D000818:Animals; D002460:Cell Line; D046508:Culture Techniques / Q000379:methods; D003598:Cytoskeletal Proteins / Q000235:genetics*; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure; D007700:Kinetics; D051379:Mice; D008855:Microscopy, Electron, Scanning; D011132:Polyribosomes / Q000502:physiology / Q000648:ultrastructure; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics
D003598:Cytoskeletal Proteins; D012333:RNA, Messenger