MOUSE THYMIDINE KINASE STABILITY IN-VIVO AND AFTER IN-VITRO TRANSLATION

Using a combination of centrifugal elutriation and recultivation of sy nchronised cell populations we could show that murine thymidine kinase (TK) is rapidly degraded during mitosis in polyoma virus-transformed mouse fibroblasts, in parallel to the time-course for loss of cyclin A . Transformation is no prerequisite for the instability phenotype sinc e artificial overexpression of TK under the control of a constitutive promoter in normal mouse fibroblasts also resulted in rapid turnover o f TK during mitosis. The decay of TK protein could be partially mimick ed in vitro with enzymatically active protein translated in a rabbit r eticulocyte lysate: full length polypeptide was lost slightly more rap idly in the presence of G2/M cytosolic extracts than with G1/S prepara tions. In addition, an enzymatically active C-terminal truncation of 3 7 amino acids at Gln-196 was completely stable under the conditions te sted, confining the instability domain between residues 196 to 233. Th ese experiments also indicated the border for intact TK since translat ion products up to Tyr-189 or less were completely inactive. This was also confirmed by a mutant TK protein from mouse F9tk(-) teratocarcino ma cells which harboured a similar deletion. (C) 1997 Elsevier Science B.V.