A novel promoter sequence is involved in the oxidative stress-induced expression of the adult T-cell leukemia-derived factor (ADF)/human thioredoxin (Trx) gene.

Adult T cell leukemia-derived factor (ADF) is a human thioredoxin (Trx) and is a disulfide reducing protein with various biological functions. We found that expression of the ADF/Trx gene was increased by oxidative agents such as hydrogen peroxide, diamide and menadione in Jurkat cells. Analysis using a CAT expression vector plasmid under the control of the ADF/Trx gene promoter revealed that CAT gene expression in Jurkat cells was increased after exposure to oxidative agents. A series of deletion analyses showed that a region from -976 to -890 of the 5' flanking sequence was required for enhancement of ADF/Trx promoter activity against the oxidative agents. Gel mobility shift assay revealed the specific DNA binding activities to the sequences from -953 to -930 in the nuclear extracts from the Jurkat cells. The sequences in this region showed no homology with any known consensus sequences for DNA binding factors. It is suggested that ADF/Trx gene expression is enhanced through a novel cis-acting regulatory element responsive for the oxidative stress and a new factor(s) is involved in this oxidative stress responsive element.     

Light regulatory sequences are located within the 5'' portion of the Fed-1 message sequence.

We have previously shown that element(s) mediating a light-induced increase in the abundance of Fed-1 mRNA in the leaves of transgenic tobacco plants are located within the transcribed portion of the gene. As part of an effort to define the mechanism of this effect, we report here that cis-acting elements capable of mediating a 5-fold light-induced increase in the abundance of this mRNA are located within a region comprising the 5' leader and first third of the Fed-1 coding sequence. No activity was detected in the 3' untranslated region of the gene. In a gain-of-function assay, the 5' region was found to be capable of conferring light responsiveness on three different reporter sequences, although experiments with the gusA reporter were complicated by an apparent negative light effect on the stability of this mRNA. Deletion experiments show that at least one essential light regulatory element is located in the 5' untranslated region of Fed-1 between nucleotides +19 and +57. Additional Fed-1 sequences, including a portion of the protein coding region, are required to confer positive responsiveness on the gusA reporter. These additional sequences may include specific light regulatory elements or simply provide an environment in which the leader element can function normally.     

Cellular proteins from human monocytes bind to dengue 4 virus minus-strand 3' untranslated region RNA

The synthesis of plus and minus RNA strands of several RNA viruses requires as a first step the interaction of some viral regulatory sequences with cellular and viral proteins. The dengue 4 virus genome, a single-stranded, positive-polarity RNA, is flanked by two untranslated regions (UTR) located in the 5' and 3' ends. The 3'UTR in the minus-strand RNA [3'UTR (-)] has been thought to function as a promoter for the synthesis of plus-strand RNA. To study the initial interaction between this 3'UTR and cellular and viral proteins, mobility shift assays were performed, and four ribonucleoprotein complexes (I through IV) were formed when uninfected and infected U937 cells (human monocyte cell line) interacted with the 3'UTR (-) of dengue 4 virus. Cross-linking assays with RNAs containing the complete 3'UTR (-) (nucleotides [nt] 101 to 1) or a partial sequence from nt 101 to 45 and nt 44 to 1 resulted in specific binding of some cellular proteins. Supermobility shift and immunoprecipitation assays demonstrated that the La protein forms part of these complexes. To determine the region in the 3' UTR that interacted with the La protein, two deletion mutants were generated. The mutant (del-96), with a deletion of nt 96 to 101, was unable to interact with the La protein, suggesting that La interacted with the 5' portion of the 3'UTR (-). Complex I, which was the main ribonucleoprotein complex formed with the 3'UTR (-) and which had the fastest electrophoretic migration, contained proteins such as calreticulin and protein disulfide isomerase, which constitute important components of the endoplasmic reticulum.     

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization

Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5' untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation. Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5' UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5' UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wild-type levels of D1 protein, confirming that the psbA 5' UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5' UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA- background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5' UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.