Biomedical subjects

S Jaenecke

Publications and source records attributed to S Jaenecke.

4 recordsLinked to original sources

PubMed2000

Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination.

The synthesis of eukaryotic selenoproteins involves the recoding of an internal UGA codon as a site for selenocysteine incorporation. This recoding event is directed by a selenocysteine insertion sequence in the 3'-untranslated region. Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have investigated how the rates of translational termination and selenocysteine incorporation relate to cis-acting elements in the mRNA as well as to trans-acting factors in the cytoplasm. We used cis-elements from the phospholipid glutathione peroxidase gene as the basis for this work because of its relatively high efficiency of selenocysteine incorporation. The last two codons preceding the UGA were found to exert a far greater influence on selenocysteine incorporation than nucleotides downstream of it. The efficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The combination of two or three UGA codons in one reading frame led to a dramatic reduction in the yield of full-length protein. It is therefore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of selenoprotein synthesis and its ability to compete with termination at UGA codons.

3' Untranslated Regions↗

PubMed2000

A gene containment strategy based on a restriction-modification system.

Engineering barriers to the spread of specific genes are of great interest both to increase the predictability of recombinant microorganisms used for environmental applications and to study the role of gene transfer in the adaptation of microbial communities to changing environments. We report here a new gene containment circuit based on a toxin-antidote pair that targets the cell DNA, i.e. the type II EcoRI restriction-modification system. The set-up involved linkage of the ecoRIR lethal gene encoding the EcoRI endonuclease (toxin) to the contained character in a plasmid and chromosomal insertion of the ecoRIM gene encoding the cognate EcoRI methylase (antidote) that protects the target DNA from restriction. Transfer of the contained character to a recipient cell lacking the antidote caused EcoRI-mediated chromosomal breaks, leading to cell death, thereby preventing gene spread. Using transformation and conjugation as mechanisms of DNA transfer and different environmentally relevant bacteria as recipients, we have shown that the potentially universal EcoRI-based containment system decreases gene transfer frequencies by more than four orders of magnitude. Analyses of the survivors escaping killing revealed a number of possible inactivation mechanisms.

Base Sequence↗

PubMed1999

Construction of plasmid vectors bearing a NotI-expression cassette based on the lac promoter.

We have constructed two plasmid vectors for cloning and expression of DNA fragments controlled by the lac promoter as a NotI-expression cassette. Whereas plasmid pSJ33 allows mobilization of the expression cassette into a wide variety of Gram-negative bacteria by RP4-mediated conjugation, the low-copy-number plasmid pSJP18Not facilitates cloning and expression in Escherichia coli when high gene dosage may be detrimental. In addition to their suitable cloning features (e.g. multiple cloning site, lacZ alpha fragment, compatible with ColE1-derived vectors), these plasmids are particularly useful as auxiliary vectors for cloning of the expression cassettes at the NotI site of mini-transposon elements [1, 2] and their eventual stable insertion into the host chromosome.

Cloning, Molecular↗

PubMed1996

A stringently controlled expression system for analysing lateral gene transfer between bacteria.

The lateral transfer of genetic information among microorganisms is a major force driving the outstanding adaptability of microbial communities to environmental changes. Until now little information has been obtained on gene transfer in natural ecosystems. We present here a genetic circuit for detecting and quantifying horizontal gene transfer from a defined donor microorganism to recipient organisms in the absence of selection for a recipient-specific phenotype. The system consists of an engineered lacZ (encoding beta-galactosidase) reporter gene whose expression is controlled by a synthetic regulatory element based on a fusion between the Pr promoter-operator from lambda bacteriophage and the 5' non-coding leader region of the inp gene encoding the IS 10 transposase function. Expression of this reporter cassette in the recombinant microorganism is completely shut down by two chromosomally encoded trans-acting repressors working at the level of transcription (the Cl-EK117 protein from the lambda phage), and at the level of translation (the antisense RNA-OUT of the IS 10 element). When the reporter element is transferred to a different host by any mechanism, it escapes repression and becomes expressed. The system was validated with Pseudo-monas putida, and conjugational transfer frequencies of the reporter element as low as 10(-6) were detected. The modular design and broad host range of the genetic circuit, in combination with biomarkers which permit real-time in situ detection, will facilitate the monitor-ing of gene flow in a non-disruptive manner within the environment.

Bacteria↗