A tetracycline-controlled transrepressor protein has been engineered to silence transcriptional activities of eukaryotic promoters that are stably integrated into the chromatin of human cells. By fusing the KRAB domain of human Kox1 to the Tet repressor derived from Tn10 of Escherichia coli, a tetracycline-controlled hybrid protein (TetR-KRAB) was generated and constitutively expressed in HeLa cells. The TetR-KRAB protein binds to tet operator (tetO) sequences in the absence but not in the presence of tetracycline. When TetR-KRAB bound to tetO sequences upstream of the immediate-early promoter-enhancer of human cytomegalovirus (CMV), the expression of a CMV-driven luciferase reporter construct (ptetO7-CMV-L) was repressed in transient transfection experiments. This silencing was found to operate on different promoters and from tetO sequences placed more than 3 kb from the transcriptional start site. We constructed a stable, doubly transfected cell line (TIS-10) carrying a chromosomally integrated ptetO7-CMV-L reporter construct and expressing the TetR-KRAB protein. Inducible gene expression has been a valuable tool for the study of gene function in bacteria, yeasts, and Drosophila melanogaster. In mammalian cells, eukaryotic promoter systems that respond to inducing agents such as glucocorticoid hormone (19, 23), heat shock (40), heavy metal ions (28), or interferon (32) have been used. To circumvent limitations due either to the leakiness of utilized promoters or the pleiotropic effects of inducing agents, chimeric transcription factors have been generated, for example, by fusing the strong transcriptional activator domain of VP16 to the Tn10-derived prokaryotic tetracycline repressor (TetR) protein (16). In general, chimeric transcription factors of this kind, TetR-VP16 (16), GAL4-VP16 (33), LexA-VP16 (7), and LacR-VP16 (22), are targeted to minimal promoters which are fused to multiple DNA binding sites specific for the factor in question. This results in marked transcriptional stimulation of the respective minimal promoter. In order to modulate the transcriptional activity of chimeric transcription factors, movable steroid binding domains originating from the glucocorticoid receptor (30) or the estrogen receptor (38) have been fused to sequencespecific DNA binding domains. In the case of a GAL4-VP16-estrogen receptor fusion, estrogen administration has been shown to produce a functionally active chimeric transcription factor that activates a target promoter (6).In the tetracycline system, the TetR protein fused to the transactivating domain of VP16 (called tTA) has been shown to bind to and strongly activate minimal promoter systems containing seven tetracycline operator (tetO) sequences (15, 16). The binding of tTA to the tetO sequences is blocked by tetracycline, preventing the activation of the target promoter which generates a very tight genetic switch (16).In this report, we present a novel system for tetracyclinecontrolled silencing of eukaryotic promoters. Here, the KRAB repressor domain of the human ...

The strength in vivo of 14 promoters was determined in a system which permits the quantitation of RNA synthesis with high accuracy. Up to 75‐fold differences in promoter strength were measured and the most efficient signals are promoters from coliphages T7 and T5. Their activity approaches the strength of fully induced promoters of the rRNA operons which may be close to the functional optimum of a single sequence. By contrast, a synthetic ‘consensus promoter’ belongs to the less efficient signals. Our data show that optimal promoter function can be achieved by alternate structures and strongly suggest that information outside of the ‘classical’ promoter region contributes to promoter activity.

Endothelin-converting enzyme 1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Two different isoforms (ECE-1a and ECE-1b) have previously been identified for human ECE-1. In the present study we have cloned a novel human ECE-1 isoform, termed ECE-1c, and have thus shown for the first time the existence of three distinct ECE-1 isoforms. The three isoforms differ only in their N-terminal regions and are derived from a single gene through the use of alternative promoters. Ribonuclease protection experiments revealed that, although the relative levels of the three isoform mRNA species vary between human tissues, ECE-1c mRNA is generally the predominant isoform messenger. Immunofluorescence microscopy analysis showed distinct subcellular localizations for the three isoforms: whereas ECE-1a and ECE-1c are localized at the cell surface, ECE-1b was found to be intracellular and showed significant co-localization with a marker protein for the trans-Golgi network. We determined that the three isoforms have similar kinetic rate constants (Km, kcat and Vmax) for the processing of big endothelin 1 and that the big endothelin isoforms 1, 2 and 3 are cleaved with similar relative velocities of 1.0:0.1:0.1 by the three isoenzymes.

After binding to a promoter Escherichia coli RNA polymerase is in contact with a region of about 70 bp. Around 20 bp of this sequence are transcribed. Information encoded within this transcribed region is involved in late steps of the functional program of a promoter. By changing such ‘down‐stream’ sequences promoter strength in vivo can be varied more than 10‐fold. By contrast, information for early steps of the promoter program such as recognition by the enzyme and formation of a stable complex resides in a central core region of about 35 bp. Our data show that the strength of a promoter can be limited at different levels of the overall process. Consequently promoters of identical strength can exhibit different structures due to an alternate optimization of their program.

The novel drug PX20606 activates the bile acid receptor FXR and shows beneficial effects in experimental liver cirrhosis: In the liver, it reduces scarring and inflammation, and also widens blood vessels. Thus, PX20606 leads to an improved blood flow through the liver and decreases hypertension of the portal vein. Additionally, PX20606 improves the altered intestinal barrier and decreases bacterial migration from the gut.