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S Biebinger

Publications and source records attributed to S Biebinger.

4 recordsLinked to original sources
PubMed1997

Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei.

We previously described a system for exogenous control of gene expression in procyclic trypanosomes which depends upon the binding of a tetracycline-inducible repressor to operators situated at the transcriptional start site of the PARP promoter. The recombinant constructs are introduced into non-transcribed spacers of the ribosomal RNA repeat, in an orientation opposite to that of rRNA transcription. Using this system, gene expression could be regulated over four orders of magnitude, but it was not possible to express toxic gene products because selection of recombinant trypanosomes depended on the activity of the inducible promoter. We describe here the characteristics of vectors that include two promoters: a tetracycline-inducible one to drive expression of the toxic products, and a constitutive one to drive transcription of the selectable marker. Relatively high levels of non-induced (non-tetracycline-dependent) expression were seen in some trypanosome clones; this was not usually due to read-through of multiple tandemly-integrated plasmids or tet operator mutations. A variety of constructs differing in resistance marker, 3'-untranslated region (3'-UTR) and the nature of the constitutive promoter was tested. Vectors allowing the successful expression of toxic and other genes in both life cycle stages with regulation factors of up to 700 fold were obtained.

Animals↗
PubMed1996

The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context.

African trypanosomes are extracellular protozoan parasites that are transmitted from one mammalian host to the next by tsetse flies. Bloodstream forms express variant surface glycoprotein (VSG); the tsetse fly (procyclic) forms express instead the procyclic acidic repetitive protein (PARP). PARP mRNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP, VSG and rRNA promoters in permanently-transformed blood-stream and procyclic form trypanosomes, using reporter gene constructs that showed no post-transcriptional regulation. When the constructs were integrated in the rRNA non-transcribed spacer, the ribosomal RNA and VSG promoters were not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid vector sequences was also reduced.

Animals↗