Biomedical subjects

Christian Berens

Publications and source records attributed to Christian Berens.

4 recordsLinked to original sources

PubMed2003

Single-chain Tet transregulators.

We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse 'tet-on' phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.

Cell Line↗

PubMed2003

Conditional gene expression by controlling translation with tetracycline-binding aptamers.

We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA-metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5'-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We defined nucleotides which influence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.

5' Untranslated Regions↗

PubMed2003

Conditional cell suicide using dox-dependent caspase-2 expression.

BACKGROUND: Adoptive immune transfer is used as an efficient treatment modality to achieve a graft-versus-leukemia effect in persisting or relapsing residual leukemic disease. Safety considerations dictate the need for equipping the transferred cells with a conditional suicide mechanism to eliminate donor T cells when graft-versus-host disease occurs. We have examined in a model system using HeLa cells whether doxycycline (dox)-dependent expression of pro-apoptotic proteins could be used as a potential new strategy for conditional cell elimination. METHODS: Four constructs encoding pro-apoptotic proteins were tested in transient transfections to identify suitable cell death inducers. Murine caspase-2 placed under Tet-control was chosen for stable transfection into cell lines carrying different dox-dependent transregulators. The efficiency of cell death induction and the expression patterns of caspase-2 were analyzed in the respective clones. RESULTS: Different levels of induced cell death were obtained depending on the properties of the transregulators used to control target gene expression. High expression levels of caspase-2 in the presence of dox were required to achieve efficient induction of cell death, while tight repression in the absence of inducer was not necessary for cell survival. Dox treatment for 48 h resulted in 94% cell death indicating a very efficient conditional suicide mechanism. CONCLUSIONS: We propose that the principle of using pro-apoptotic cellular proteins placed under appropriate dox-dependent regulation may represent an alternative conditional suicide mechanism to the frequently used herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir-system, which harbors immunological and toxicological risks.

Animals↗

PubMed2003

Gene regulation by tetracyclines. Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes.

The Tet repressor protein (TetR) regulates transcription of a family of tetracycline (tc) resistance determinants in Gram-negative bacteria. The resistance protein TetA, a membrane-spanning H+-[tc.M]+ antiporter, must be sensitively regulated because its expression is harmful in the absence of tc, yet it has to be expressed before the drugs' concentration reaches cytoplasmic levels inhibitory for protein synthesis. Consequently, TetR shows highly specific tetO binding to reduce basal expression and high affinity to tc to ensure sensitive induction. Tc can cross biological membranes by diffusion enabling this inducer to penetrate the majority of cells. These regulatory and pharmacological properties are the basis for application of TetR to selectively control the expression of single genes in lower and higher eukaryotes. TetR can be used for that purpose in some organisms without further modifications. In mammals and in a large variety of other organisms, however, eukaryotic transcriptional activator or repressor domains are fused to TetR to turn it into an efficient regulator. Mechanistic understanding and the ability to engineer and screen for mutants with specific properties allow tailoring of the DNA recognition specificity, the response to inducer tc and the dimerization specificity of TetR-based eukaryotic regulators. This review provides an overview of the TetR properties as they evolved in bacteria, the functional modifications necessary to transform it into a convenient, specific and efficient regulator for use in eukaryotes and how the interplay between structure--function studies in bacteria and specific requirements of particular applications in eukaryotes have made it a versatile and highly adaptable regulatory system.

Animals↗