Abstract
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.
Highlights
Of full length thymidine kinase (TK) mRNA during growth arrest of 3T6 On the other hand, terminally differentiating or continumouse fibroblasts or during differentiation of myo- ously cycling cells appear to control TK activity mainly at blasts (C2C112) or F9 embryonal carcinoma cells
Full length TK mRNA stayed tions that human TK protein stabilityis drastically reduced associated with polysomes under these conditions in F9 during mitosis (Sherley andKelly, 1988b)
Takentogether the results suggest carboxyl terminus of the polypeptide chain are essential for that endogenous TK mRNA becomes translationally specific degradation
Summary
The abbreviations used are: TK, thymidine kinase; DMEM, Dulso far observed only after transfection of multiple copies of becco’s modified Eagles’ medium; FCS, fetal calf serum; IPTG, isopropyl-0-D-thiogalactopyranosideP; BS, phosphate-buffered saline. We chose to work mainly with F9 mouse teratocarcinoma cells and 3T6 mouse fibroblasts. The former cell type can be differentiated at high serum concentrations into a structure resembling parietal endoderm in vitro under the influence of the morphogen all-trans-retinoic acid (Stricklandand Mahdavi, 1978). 3T6 cells are able to undergo a reversible growth arrestunder low serum concentrations (Mullner et al, 1983). Some experiments were done with with mouse myoblasts differentiatinginto myotubes under low serum and ratmyoblasts which undergo the same process without a change in growth factor levels by reaching confluence. The results obtained indicate that translation control contributes to endogenous TK gene inactivation in all cell types tested by a mechanism that is independent of growth factor concentrations in the medium
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