Stepwise selection of TetR variants recognizing tet operator 6C with high affinity and specificity 1 1Edited by R. Ebright

Helbl, Vera; Tiebel, Beatrix; Hillen, Wolfgang

doi:10.1006/jmbi.1997.1539

Cited by 40 publications

(17 citation statements)

References 15 publications

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“…Finally, tens of thousands of homologues are available from many host organisms and there is evidence that they exhibit sequence specific binding to disparate operator sequences 22 . Small differences in the amino acid sequence and operator nucleotides have been shown to yield high-affinity, orthogonal interactions 32,33 . The potential for orthogonality is also large; coding theory predicts that there is an upper limit of 130 helix-turn-helix repressors that could function in one cell without exhibiting crosstalk 34 .…”

Section: Introductionmentioning

confidence: 99%

Genomic mining of prokaryotic repressors for orthogonal logic gates

et al. 2013

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Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply “part mining” to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and do not interact with other promoters. Each repressor:promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOR gates, there are >1054 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.

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Section: Introductionmentioning

confidence: 99%

Genomic mining of prokaryotic repressors for orthogonal logic gates

et al. 2013

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“…Two mutant repressor͞operator combinations exhibited particularly remarkable properties. By exchanging the three amino acids, E, W, and H, at positions 37, 43, and 44 of TetR for S, R, and N, respectively, a mutant protein was obtained that specifically recognizes an operator sequence where the G at position 6 of the 19-bp-long sequence of dyad symmetry has been replaced by a C (21). Similarly, the exchange of amino acids E, P, and Y at positions 37, 39, and 42 for A, Q, and M, respectively, yielded a TetR mutant that binds with high specificity to a tetO sequence that contains a C instead of a T at position 4 (22).…”

Section: Resultsmentioning

confidence: 99%

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

Baron

Schnappinger

Helbl

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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112

108

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A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function in vitro and in vivo. In addition, the system may be of value for a variety of practical applications, including gene therapy.

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“…Residues in this helix form numerous sequence specific and nonspecific interactions with tetO DNA (45). Mutations at position 37 may alter the orientation of the recognition helix and thus also indirectly affect DNA binding (46,47). Most rtTA variants with a polar side chain at position 37 (37C, 37K, 37Q, 37R, 37S, and 37T) are equally or more active than wild-type rtTA (37E).…”

Section: Discussionmentioning

confidence: 99%

The Genetic Stability of a Conditional Live HIV-1 Variant Can Be Improved by Mutations in the Tet-On Regulatory System That Restrain Evolution

Zhou

Vink

Klaver

et al. 2006

Journal of Biological Chemistry

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Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because more quickly replicating pathogenic virus variants may evolve after vaccination. As an alternative vaccine approach, we have previously presented a doxycycline (dox)-dependent HIV-1 variant that was constructed by incorporating the tetracycline-inducible gene expression system (Tet-On system) into the viral genome. Replication of this HIVrtTA variant is driven by the dox-inducible transcriptional activator rtTA and can be switched on and off at will. A large scale evolution study was performed to test the genetic stability of this conditional live vaccine candidate. In several long term cultures, we selected for HIV-rtTA variants that no longer required dox for replication. These evolved variants acquired a typical amino acid substitution either at position 19 or 37 in the rtTA protein. Both mutations caused rtTA activity and viral replication in the absence of dox. We designed a novel rtTA variant with a higher genetic barrier toward these undesired evolutionary routes. The corresponding HIV-rtTA variant did not lose dox control in long term cultures, demonstrating its improved genetic stability.

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Stepwise selection of TetR variants recognizing tet operator 6C with high affinity and specificity 1 1Edited by R. Ebright

Cited by 40 publications

References 15 publications

Genomic mining of prokaryotic repressors for orthogonal logic gates

Genomic mining of prokaryotic repressors for orthogonal logic gates

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes

The Genetic Stability of a Conditional Live HIV-1 Variant Can Be Improved by Mutations in the Tet-On Regulatory System That Restrain Evolution

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