In this article we report the in vivo and in vitro characterization of single chain tetracycline repressor (scTetR) variants in Escherichia coli. ScTetR is genetically and proteolytically stable and exhibits the same regulatory properties as dimeric TetR in E.coli. Urea-dependent denaturation of scTetR is independent of the protein concentration and follows the two-state model with a monophasic transition. Contrary to dimeric TetR, scTetR allows the construction of scTetR mutants, in which one subunit contains a defective inducer binding site while the other is functional. We have used this approach to establish that scTetR needs occupation of both inducer binding sites for in vivo and in vitro induction. Single mutations causing loss of induction in dimeric TetR lead to non-inducible scTetR when inserted into one half-side. The construction of scTetR H64K S135L S138I (scTetRi2) in which one half-side is specific for 4-dedimethylamino-anhydrotetracycline (4-ddma-atc) and the other for tetracycline (tc) leads to a protein which is only inducible by the mixture of tc and 4-ddma-atc. Fluorescence titration of scTetRi2 with both inducers revealed distinct occupancy with each of these inducers yielding roughly a 1:1 stoichiometry of each inducer per scTetRi2. The properties of this gain of function mutant clearly demonstrate that scTetR requires the binding of two inducers for induction of transcription.

(CK-K) S U M M A R Y Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy. (J Histochem Cytochem 56:477-485, 2008)