compensate for the defective levels of Pth protein thus reducing the thermosensitivity of the mutant cells, although the mechanism of this suppression is presently unknown. In the present study, we have quantiﬁed the cellular levels of Pth(Ts) protein and of a number of tRNA species in transformed pth(Ts) cell lines in order to gain an insight into the suppression of thermosensitivity of pth(Ts) mutants through tRNALys overproduction. MATERIALS AND METHODS Strains and plasmids E.coli strain AA7852, a pth(Ts) derivative of strain CP-78 [F , arg , leu , thr , his , thiamine , T1s] (1) was employed in the study. The plasmid constructs harbouring tRNA genes were (i) pVH119, bearing valUa, valUb, valUg and lysV genes; (ii) pVH125, a derivative of pVH119 carrying Nucleic Acids Research, 2006, Vol. 34, No. 5 1565 valUa, valUb, valUg and partly deleted lysV genes; (iii) pVH124, a derivative in which all of these tRNA genes had been deleted (7); (iv) ptRNAHisCCA, carrying argX, hisR, leuT and proM genes; and (v) pTH2, containing the thrW gene (7). Growth assays Isolated colonies of E.coli strain AA7852, that had been independently transformed with different plasmid constructs, were streaked onto plates of Luria–Bertani (LB) medium containing 100 mg/ml ampicillin (LB-Ap) and incubated over- night at 32, 41 or 43 C (Figure 1A). For the determination of bacterial growth in liquid culture (Figure 2A), an aliquot of a 32 C overnight culture in LB-Ap was diluted 1:100 with fresh medium and further incubated at 32 C to an OD600 of 0.2. Equal portions of the resulting culture were incubated at 32 or 41 C, and cellular growth followed by determination of OD600 with respect to time. Figure 1. Suppression of the Pth(Ts) phenotype mediated by the overproduction of tRNALys maintains moderate levels of the Pth(Ts) protein. (A) Depicts the cellular growth of the pth(Ts) mutant strain AA7852 separately transformed with pVH124 (DvalU, DlysV), pVH125 (valU, DlysV) or pVH119 (valU, lysV) incubated at different temperatures. Isolated colonies of the independent transformants were streaked onto LB-Ap plates and incubated overnight at the indicated temperatures. (B) Presents the immunodetection of Pth(Ts) in the pth(Ts) mutant strain AA7852 separately transformed with pVH124, pVH125, pVH119, ptRNAHisCCA (argX, hisR, leuT, proM) or pTH2 (thrW) and grown at 32 C prior to transfer at time ¼ 0 min at 41 or 43 C. The concentration of Pth(Ts) protein was estimated by immunoblot analysis. The left lane shows purified wild-type Pth protein, which migrates slightly faster in SDS–PAGE than the Pth(Ts) variant (arrowed) (3).