Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location

The internal ribosomal entry site (IRES)from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Compared head-to-head with dual-luciferase reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a similarly configured pIRES vector. It also produced nearly twice as much protein as pCITE-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. The preferred IRES (viral bases 273-845) and the minimum IRES (viral bases 400-836) for optimum activity are illustrated.     

Leishmania chagasi: a tetracycline-inducible cell line driven by T7 RNA polymerase

Trypanosomatid protozoa lack consensus promoters for RNA polymerase (RNAP) II. However, the artificial insertion of the T7 promoter (P(T7)) and the tetracycline repressor into Trypanosoma brucei cell lines expressing T7RNAP allows P(T7)-driven gene expression to be tetracycline-inducible. These cell lines provide a molecular tool to address protein function by several recombinant approaches. We describe here the development of an analogous Leishmania chagasi cell line bearing the genes for exogenous T7RNAP and the tetracycline repressor inserted in the multi-gene alpha-tubulin locus. A plasmid construct with P(T7) and the tetracycline operator upstream of a reporter gene, when introduced into this cell line as episomal plasmids or chromosomal insertion into the non-coding strand of an 18SrRNA gene, resulted in tetracycline-inducible expression of the reporter as much as 16- and 150-fold, respectively. The reporter was under a much tighter control when chromosomally inserted than extra-chromosomally born. Furthermore, P(T7) augmented the reporter's expression 2-fold more in comparison to P(T7)-less constructs. This cell line is the first Leishmania spp. that allows the exogenous T7RNAP-driven gene expression to be tetracycline-inducible; and may provide a useful tool for addressing protein function by manipulating expression levels of Leishmania endogenous genes.     

DNA sequence for the T7 RNA polymerase promoter for T7 RNA species II

The DNA sequence for the T7 late region class III promoter for T7 RNA species II has been determined. I have found that the DNA sequence for this promoter presented in an earlier report (Oakley et al., 1979) is incorrect and that this class III promoter contains a 23 base-pair sequence identical to those present in all other T7 class III promoters (Rosa, 1979). The T7 RNA species II promoter has been located at 68% on the T7 genome.     

A novel T7 system utilizing mRNA coding for T7 RNA polymerase

The T7 system dose not require the relocation of a reporter gene to the nucleus for its gene expression in the cytoplasm, but relies on the co-localization of T7 RNA polymerase (T7 RNAP) enzyme and reporter gene DNA that is controlled by the T7 promoter. In the present study, we developed a new T7 system in that gene expression can occur at a higher level than those using conventional systems. Insertion of 5'- and 3'-untranslated regions (UTR) of beta-globin gene into a reporter gene enhanced the reporter gene expression, presumably due to the stability and efficient translation of the mRNA. Instead of the T7 RNAP protein used in conventional methods, moreover, transfection of cells with T7 RNAP mRNA, which has been modified by inserting beta-globin 5'- and 3'-UTR sequences as well as the cap and poly(A) tail structures, further enhanced the reporter gene expression. Thus, this novel T7 system using T7 RNAP mRNA may be powerful for the efficient gene expression of DNA exogenously provided in the cytoplasm.     

Strong suppression of chloramphenicol acetyltransferase expression by inducing T7 RNA polymerase

Summary Suppression of gene expression can be used as a way to study gene regulation. Expression of chloramphenicol acetyltransferase (CAT) underE. coli promoter was inhibited by 92 % by induction of T7 RNA polymerase which transcribes from a T7 promoter opposingE. coli promoter.     

A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei. This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei, including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.