Longer DNA exhibits greater potential for cell-free gene expression

Cell-free gene expression systems have been valuable tools for understanding how transcription/translation can be regulated in living cells. Many studies have investigated the determining factors that affect gene expression. Here we report the effect of the length of linearized reporter DNAs encoding the firefly luciferase gene so as to exclude the influence of supercoiling. It is found that longer DNA molecules exhibit significantly greater potency in gene expression; for example, the expression level for DNA with 25.7 kbp is 1000-times higher than that for DNA of 1.7 kbp. AFM observation of the DNA conformation indicates that longer DNA takes shrunken conformation with a higher segment density in the reaction mixture for gene expression, in contrast to the stiff conformation of shorter DNA. We propose an underlying mechanism for the favorable effect of longer DNA on gene expression in terms of the enhancement of access of RNA polymerase to the shrunken conformation. It is expected that the enhancement of gene expression efficiency with a shrunken DNA conformation would also be a rather general mechanism in living cellular environments.

Reconstitution of Cytokinin Signaling in Rice Protoplasts

The major components of the cytokinin (CK) signaling pathway have been identified from the receptors to their downstream transcription factors. However, since signaling proteins are encoded by multigene families, characterizing and quantifying the contribution of each component or their combinations to the signaling cascade have been challenging. Here, we describe a transient gene expression system in rice (Oryza sativa) protoplasts suitable to reconstitute CK signaling branches using the CK reporter construct TCSn:fLUC, consisting of a synthetic CK-responsive promoter and the firefly luciferase gene, as a sensitive readout of signaling output. We used this system to systematically test the contributions of CK signaling components, either alone or in various combinations, with or without CK treatment. The type-B response regulators (RRs) OsRR16, OsRR17, OsRR18, and OsRR19 all activated TCSn:fLUC strongly, with OsRR18 and OsRR19 showing the strongest induction by CK. Cotransfecting the reporter with OsHP01, OsHP02, OsHP05, or OsHK03 alone resulted in much weaker effects relative to those of the type-B OsRRs. When we tested combinations of OsHK03, OsHPs, and OsRRs, each combination exhibited distinct CK signaling activities. This system thus allows the rapid and high-throughput exploration of CK signaling in rice.

miR-1185-1 and miR-548q Are Biomarkers of Response to Weight Loss and Regulate the Expression of GSK3B

The aim of the present investigation was to identify putative miRNAs involved in the response to weight loss. Reverse-transcribed RNA isolated from white blood cells (WBCs) of a subpopulation from the Reduction of the Metabolic Syndrome in Navarra-Spain (RESMENA-S) study (low-responders (LR) and high-responders (HR)) was hybridized in a gene expression microarray. Moreover, miRNAs were sequenced by miRNA-Seq. It was found that miR-548q and miR-1185-1 were overexpressed in HR, both in the microarray and in the miRNA-Seq. A bioinformatic prediction of putative target genes of the selected miRNAs found that GSK3B, a putative target for miR-548q and miR-1185-1, was downregulated in HR. Particular 3′-UTR binding regions of GSK3B were cloned downstream of the firefly luciferase gene. HEK-293T cells were co-transfected with either 0.25 μg of empty pmiR-GLO or pmiR-GLO-548q-3′-UTR/pmiR-GLO-1185-1-3′-UTR, and 7.5 pmol of miR-548q/miR-1185-1 mimics, demonstrating that miR-1185-1 bound to the 3′-UTR region of GSK3B. THP-1 cells were transfected with either 20/40 nM of miR-548q/miR-1185-1 mimics, evidencing that miR-1185-1inhibited the expression of the gene when transfected at doses of 20/40 nM, whereas miR-548q inhibited GSK3B expression at a dose of 40 nM. As a conclusion, miR-548q and miR-1185-1 levels in WBCs are biomarkers of response to weight-loss diets and could be involved in the regulation of the proinflammatory gene GSK3B.

Double Recombinant Mycobacterium bovis BCG Strain for Screening of Primary and Rationale-Based Antimycobacterial Compounds

ABSTRACTConventional antimycobacterial screening involves CFU analysis, which poses a great challenge due to slow growth of mycobacteria. Recombinant strains carrying reporter genes under the influence of constitutive promoters allow rapid and wide screening of compounds but without revealing their modes of action. Reporter strains using pathway-specific promoters provide a better alternative but allow a limited screening of compounds interfering with only a particular metabolic pathway. This reduces these strains to merely a second-line screening system, as they fail to identify even the more potent compounds if they are not inhibiting the pathway of interest. In this study, we have generated a double recombinantMycobacterium bovisBCG strain carrying firefly andRenillaluciferase genes as two reporters under the control of a constitutive and an inducible mycobacterial promoter. The presence of dual reporters allows simultaneous expression and analysis of two reporter enzymes within a single system. The expression profile of the firefly luciferase gene, rendered by a constitutive mycobacterial promoter, coincides with the decline in bacterial growth in response to a wide range of antimycobacterial drugs, while the enhanced expression ofRenillaluciferase mirrors the selective induction of the reporter gene expression as a result of pathway-specific inhibition. Thus, the double recombinant strain allows the screening of both primary and rationally synthesized antimycobacterial compounds in a single assay. The inhibiting response of drugs was monitored with a dual-luciferase reporter assay which can be easily adapted in high-throughput mode.

The Luc2 gene enhances reliability of bicistronic assays

Luciferases are prominent reporters in molecular and cellular biology investigations including miRNA target studies and the determination of Internal Ribosome Entry Site (IRES) activities in bicistronic assays. A majority of the current bicistronic vectors contain a firefly luciferase reporter as the second cistron. One reason for this is the presence of cryptic transcription start sites inside the luciferase gene. We present here an experimental evaluation of the cryptic transcription within the latest version of the firefly luciferase gene, luc2. Using flow cytometric analysis, we observed a negligible amount of cryptic transcriptional activity that was only slightly above the background of untransfected cells. Nevertheless, quantitative reverse transcription PCR experiments revealed a six-to-nine-fold gradual increase of transcription along the coding region of the gene. The level of cryptic transcription from the coding region of the improved luc2 firefly luciferase gene is significantly lower when compared to the luc+ gene. In summary, the luc2 better fulfills the requirements of bicistronic assays than the previous luc+ version. The observed low cryptic transcription activity in luc2 could be limiting only in cases where weak IRESs are studied.

Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening

The firefly luciferase gene is commonly used in cell-based reporter assays. Convenient luciferase assay reagents for use in high-throughput screening (HTS) are commercially available. However, the high cost of these reagents is not within the means of some academic laboratories. Therefore, we set out to develop an affordable luciferase assay reagent applicable in an HTS format using simple liquid-handling steps. The reagent was homemade from individual chemical components and optimized for luminescence intensity and stability. We determined the minimal concentrations of the most expensive components, dithiothreitol (DTT) and D-luciferin, resulting in a total assay reagent cost of less than 1 cent per sample. Signal stability was maximized by omission of coenzyme A and reduction of DTT concentration. The assay was validated in a high-throughput setting using two cancer cell lines carrying a p53-dependent luciferase reporter construct and siRNAs modulating p53 transcriptional activity. Induction of p53 activity by silencing PPM1D or SYVN1 and reduction of p53 activity by silencing p53 remained constant over a 2-h measurement period, with good assay quality (Z′ factors mostly above 0.5). Hence, the luciferase assay described herein can be used for affordable reporter readout in cell-based HTS.

A Polymorphism In the 3'UTR Region of ABCB1 Is Associated with Increased Allele Activity with Corresponding Increases In P-Glycoprotein Expression and Function

Abstract 3990 Multidrug resistance is one of the major prognostic factors in acute myeloid leukemia (AML). Expression of p-glycoprotein (p-gp), a member of the ABC transporter family and encoded by the ABCB1 gene, is associated with failure to respond to chemotherapy. Control of MDR1 expression is multifactorial and to-date emphasis has been on the 5'-promoter region of the gene. Evidence is now accumulating regarding a role for the 3'-untranslated region (3' UTR) of genes in controlling expression. We therefore studied a single nucleotide polymorphism (SNP) in the 3'UTR of ABCB1 (rs3842) and determined its effect on p-gp expression. Samples were available on 450 AML patients who had been entered into the UK LRF AML14 and NCRN AML15 trials. The distribution of the rs3482 A/G SNP was determined using the SNaPshot method; 10% of samples had their genotype confirmed by sequencing. P-gp protein expression and function were analysed by flow cytometry of CD45-gated cells; expression using MRK-16 and function by assessing the modulation of R123 accumulation by PSC833. To determine the activity of the two polymorphic alleles the 3'UTR of ABCB1 was cloned into a modified pGL3 luciferase vector and the normalised luciferase activity was assessed for both the A and G allele. The distribution of the rs3482 SNP in our AML patient cohort was 71% AA, 27% AG and 1.5% GG. These results are comparable with the published prevalence of the SNP in European populations. Approximately 60% of AML patients do not express p-gp so we therefore focused upon the 40% of patients who expressed p-gp. We found that in the p-gp-expressing patients a higher p-gp protein and function was associated with patients with the wild–type homozygous AA genotype when compared to the GA+GG genotypes. Furthermore when the ABCB1 3'UTR was cloned downstream of the firefly luciferase gene and transfected into HL-60 cells the ‘A’ construct demonstrated significantly increased activity when compared to the G construct (normalized ratio (firefly luciferase/renilla luciferase): A 7.7, G 1.9; n=4, p<0.05). Outcome data was available on 414 of the intensively treated trial patients. Whilst the ABCB1 3'UTR polymorphism had no impact on overall survival or relapse risk there was a trend towards an increased relapse free survival in patients with the wildtype AA genotype (RFS AA 37%; AG+GG 28%, p=0.08). This may correspond to higher p-gp expression in immunocompetent cells post-remission. In conclusion this work demonstrates that the ABCB1 rs3482 3'UTR SNP is functionally significant with the A allele associated with higher activity and a concomitant increase in p-gp expression and function in p-gp expressing patient samples. Disclosures: No relevant conflicts of interest to declare.

Regulation of Translational Efficiency by Disparate5′-UTRs of PPARγSplice Variants

The PPAR-γgene encodes for at least 7 unique transcripts due to alternative splicing of five exons in the5′-untranslated region (UTR). The translated region is encoded by exons 1–6, which are identical in all isoforms. This study investigated the role of the5′-UTR in regulating the efficiency with which the message is translated to protein. A coupledin vitrotranscription-translation assay demonstrated that PPAR-γ1, -γ2, and -γ5 are efficiently translated, whereas PPAR-γ4 and -γ7 are poorly translated. Anin vivoreporter gene assay using each5′-UTR upstream of the firefly luciferase gene showed that the5′-UTRs for PPAR-γ1, -γ2, and -γ4 enhanced translation, whereas the5′-UTRs for PPAR-γ5 and -γ7 inhibited translation. Models of RNA secondary structure, obtained by the mfold software, were used to explain the mechanism of regulation by each5′-UTR. In general, it was found that the translational efficiency was inversely correlated with the stability of the mRNA secondary structure, the presence of base-pairing in the consensus Kozak sequence, the number of start codons in the5′-UTR, and the length of the5′-UTR. A better understanding of posttranscriptional regulation of translation will allow modulation of protein levels without altering transcription.