Translational repression of endogenous thymidine kinase mRNA in differentiating and arresting mouse cells.
We observed that decline of thymidine kinase (TK) enzyme activity was severalfold faster than the decay of full length TK mRNA during growth arrest of 3T6 mouse fibroblasts or during differentiation of myoblasts (C2Cl12) or F9 embryonal carcinoma cells. In order to study the molecular mechanism of this disparate behavior, a polyclonal antiserum against mouse TK was raised in rabbit. High level expression of mouse TK polypeptide in Escherichia coli was achieved with a T7 RNA polymerase-directed expression system. Using the antiserum in immunoblotting, no indication for a pool of inactive enzyme was found during differentiation of F9 or growth arrest of 3T6 cells. Pulse labeling of these cells in vivo with [35S]methionine showed a more than 6-fold decrease in the rate of TK-protein synthesis of in F9 cells after 3 days of treatment with retinoic acid as well as in 3T6 cells after 16 h under low serum. This was not due to increased turnover of the protein as measured in pulse chase experiments. In addition, full length TK mRNA stayed associated with polysomes under these conditions in F9 as well as 3T6 cells. Taken together the results suggest that endogenous TK mRNA becomes translationally repressed under a variety of conditions when mouse cells cease to grow.
Knöfler
M
M
Institute of Molecular Biology, Vienna Biocenter, University of Vienna, Austria.
Waltner
C
C
Wintersberger
E
E
Müllner
E W
EW
D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D003001:Cloning, Molecular; D004794:Enzyme Repression; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013724:Teratoma; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014212:Tretinoin; D012313:RNA; D008715:Methionine; D013937:Thymidine Kinase; D013936:Thymidine