Uba1 in tsTM3 cells during incubation at 39uC were examined by immunoblotting. To analyze ubiquitination activity in the nucleus, we isolated cells expressing Fucci and investigated changes of Fucci with live-cell imaging and Western blotting. Endogenous proteins related to licensing of DNA replication were also examined by Western blotting and by indirect immunolabeling. Finally, we discuss the role of Uba1 in the nucleus. The official completion of the human genome project in 2003 was perceived as a landmark event in biological science. However, to make the most of the vast amount of sequence data, several questions have to be addressed, i.e. what fraction of the genome is functional or which of these genes are of therapeutical interest. One powerful and indispensable means to analyze gene function is conditional regulation of gene expression and, consequently, many systems have been developed for this purpose. A widely used approach is based on tetracycline-dependent gene regulation which originated from the bacterial transcription factor Tet Repressor in combination with a TetRresponsive promoter. Tetracycline derivatives are then used as small molecule effectors to efficiently regulate the expression of the cloned gene of interest. These TetR-based regulatory systems have been frequently used in a variety of different organism ranging from bacteria to mammals. For a successful application in eukaryotic organisms, TetR has to be modified by adding regulatory domains like the VP16 activation domain derived from Herpes Simplex virus as a fusion to the C-terminus of TetR. The reverse tc-dependent transactivator rtTA2S-M2, which is composed of a reverse TetR variant and a VP16-derived minimal activation domain, is a highly efficient representative of these so-called tc-dependent transregulators. Addition of the tc derivative doxycycline leads to binding of this rtTA variant to the TetR-responsive promoter and to subsequent activation of gene expression. This “Tet-On” system is used efficiently to switch on gene expression and analyze the resulting effects. However, due to the long half-life of dox, switching off target gene expression can only be achieved by replacing the dox-containing medium with dox-free medium, or, in animal studies, to supply dox-free drinking water or food. In both cases, this leads only to a moderate and slow decrease in target gene expression. If a controlled and rapid shut down of target gene expression is necessary, small molecule effectors that act as dox antagonists would be of great benefit. To date, only a single small molecule has been isolated that acted as an antagonist of the tc-dependent transactivator tTA in bacteria. However, this publication was never followed-up by any additional studies, despite strong interest in such a molecule to rapidly switch target gene expression on and off in vivo. Besides tc derivatives, peptides have recently been isolated as novel effectors for TetR.