LambdaN-GFP: an RNA reporter system for live-cell imaging

We describe a GFP-based RNA reporter system (lambdaN-GFP) to visualize RNA molecules in live mammalian cells. It consists of GFP fused to an arginine-rich peptide derived from the phage lambda N protein, lambdaN22, which binds a unique minimal RNA motif and can be used to tag any RNA molecule. LambdaN-GFP uses a small and easy to engineer RNA tag, reducing the likelihood of perturbing the function of the tagged RNA molecule.     

New in vitro reporter gene bioassays for screening of hormonal active compounds in the environment

Identification of chemicals with endocrine-disrupting activities in the past two decades has led to the need for sensitive assays for detection and monitoring of these activities in the environment. In vitro reporter gene assays represent a relatively fast and easy-to-perform method for detection of compounds that are able to bind to hormonal receptors and stimulate or silence their transactivation activity, thus interfering with the hormone signaling pathways. This paper reviews upgrades on reporter gene assays performed during the last decade. The utilization of new reporter genes (luciferase and green fluorescent protein coding genes) significantly improved the sensitivity of the tests and made them faster. Reporter gene assays now represent a high-throughput system for screening chemicals for hormonal activity. Finally, modification of test set-ups for testing anti-hormonal activities also enabled measurements of endocrine-disrupting activities in complex environmental samples such as sediments and wastewater treatment plant effluents.     

Structure of an amplifiable DNA sequence in Streptomyces lividans 66

Summary Spontaneous chloramphenicol-sensitive mutants of Streptomyces lividans 66 had previously been shown to be very unstable and to yield arginine auxotrophic mutants at a frequency of 25% of spores; the Arg^- mutants had amplified a particular 5.7 kb DNA sequence to over one hundred tandem copies per genome. In this paper we report the cloning of the amplifiable region from amplified and wild-type strains. This showed that the amplifiable fragment is already present as a duplication in wild type cells. Hybridisation experiments also demonstrated that in the amplified strains there was a deletion of neighbouring DNA sequences to one side of the amplifiable element; sequences to the other side remain intact.     

Synthesis of probes for RNA using Qbeta-replicase.

Qβ replicase was used to make labeled RNA complementary to 5S, 18S and 28S ribosomal RNA and 9S histone mRNA. These copies appear to be the same size as the parental RNA strand. The kinetics of annealing of these probes shows a proportionality between the rate and the complexity of the RNA molecule used as template.     

Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter

By insertion of a DNA fragment, containing the phage lambda pR promoter and the pM-promoted cI857 allele of the lambda repressor gene, in plasmid R1 upstream of the replication control genes, cloning vectors have been constructed which are present in one copy per chromosome at temperatures below 37 degrees C, and which display uncontrolled replication at 42 degrees C. Derivatives have been made which carry the R1 par region, stabilizing the plasmid at low temperature when grown in the absence of selection pressure. Cells harbouring these plasmids stop growing after 1-2 h incubation at 42 degrees C, and at this time 50% of the total DNA in the cells is plasmid DNA corresponding to more than 1000 plasmid molecules per cell. Concomitant with plasmid amplification at the high temperature, synthesis of plasmid-coded gene products is amplified, and these vectors can therefore be utilized for obtaining greatly enhanced yields of gene products that may be detrimental to the host cell when present in large amounts.     

Boundaries for an RNA world.

Before large, specific peptides could be made, in an RNA world our ancestors may have used an RNA-like molecule for both catalysis and information storage. Taken together, RNA activities suggest RNA could have performed most of the essential informational functions of an ancient organism (i.e. those functions most similar to RNA's role today). In contrast, though compellingly reasoned optimism about RNA-catalyzed small-molecule metabolism is plentiful, fundamental problems remain to be solved.     

A fluorescence-based reporter substrate for monitoring RNA editing in trypanosomatid pathogens

RNA editing regulates mitochondrial gene expression in trypanosomatid pathogens by creating functional mRNAs. It is catalyzed by a multi-protein complex (the editosome), and is found to be essential in both insect stage and mammalian blood stream form of Trypanosoma brucei. This particular form of RNA editing is unique to trypanosomatids, and thus provides a suitable drug target in trypanosomatid pathogens. Here, we demonstrate the feasibility of a rapid and sensitive fluorescence-based reporter assay to monitor RNA editing based on ribozyme activity. We could validate our new assay using previously identified inhibitors against the essential RNA editing ligase. The principle advantages of this assay are: (i) the use of non-radioactively labeled materials, (ii) sensitivity afforded by fluorescence instrumentation applicable to high-throughput screening of chemical inhibitors against the essential editosome and (iii) a rapid and convenient ‘mix and measure’ type of assay in low volume with a high signal to noise ratio. This assay should enhance rapid identification and characterization of the editosome inhibitors primarily based on the overall composition of the editosomes from T. brucei. These inhibitors could also be tested against the editosomes from the closely related pathogens including T. cruzi and Leishmania species.     

Functional artificial luciferases as an optical readout for bioassays

This study elucidates functional artificial luciferases (ALucs) wholly synthesized for bioassays and molecular imaging. The ALucs bearing epitopes were newly created by amending the sequences of our previously reported ALucs in light of a multi-sequence alignment and hydrophobicity search. The synthesized ALucs are survived in live cells and stable in culture media for 25 days after secretion. The epitopes in ALucs are exposed during the secretion process and indeed valid for column purification and immunological assays. The ALucs exerted a 9400-times stronger optical intensity with a coelenterazine derivative (CTZ i), when compared with Renilla reniformis luciferase 8.6–535. A supersecondary structure of ALuc30 was predicted with respect to the X-ray crystallographic information of the coelenterazine-binding protein (CBP). The structure revealed that ALuc30 has a room for accommodating the iodide of CTZ i. This study guides on how to create functional artificial luciferases and predicts the structural details with the current bioinformatics technologies.     

Establishment of an efficient RNA silencing system in Trichoderma koningii using DsRed as a reporter

We aimed to establish an efficient RNA interference (RNAi) system in the industrially important filamentous fungus Trichoderma koningii using the DsRed protein as a reporter of the silencing process. To accomplish this, a DsRed expression cassette was transformed into T. koningii, and a recombinant strain that stably expressed DsRed was obtained. Next, a vector-directing expression of a DsRed hairpin RNA was constructed and transformed into the T. koningii recipient strain. Approximately 79 % of transformants displayed a decrease in DsRed fluorescence, and expression of DsRed in some transformants appeared to be fully suppressed. Characterization of randomly selected transformants by genomic DNA PCR analysis, real-time PCR quantification, and western blot confirmed downregulation of gene expression at different levels. The RNA silencing approach described here for T. koningii is effective, and the DsRed reporter gene provides a convenient tool for identification of silenced fungal transformants by their DsRed fluorescence compared to the control strain. The results of this study demonstrate the power of RNAi in T. koningii, which supports the use of this technology for strain development programs and functional genomics studies in industrial fungal strains.     

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.     

Live-cell imaging of viral RNA genomes using a Pumilio-based reporter

We describe a method for localizing plant viral RNAs in vivo using Pumilio, an RNA-binding protein, coupled to bimolecular fluorescence complementation (BiFC). Two Pumilio homology domain (PUMHD) polypeptides, fused to either the N- or C-terminal halves of split mCitrine, were engineered to recognize two closely adjacent eight-nucleotide sequences in the genomic RNA of tobacco mosaic virus (TMV). Binding of the PUMHDs to their target sites brought the split mCitrine halves into close proximity, allowing BiFC to occur and revealing the localization of viral RNA within infected cells. The bulk of the RNA was sequestered in characteristic inclusion bodies known as viral replication complexes (VRCs), with a second population of RNA localized in discrete particles distributed throughout the peripheral cytoplasm. Transfer of the TMV Pumilio recognition sequences into the genome of potato virus X (PVX) allowed the PVX RNA to be localized. Unlike TMV, the PVX RNA was concentrated in distinctive 'whorls' within the VRC. Optical sectioning of the PVX VRCs revealed that one of the viral movement proteins was localized to the centres of the RNA whorls, demonstrating significant partitioning of viral RNA and proteins within the VRC. The utility of Pumilio as a fluorescence-based reporter for viral RNA is discussed.     

Synthesis of RNA having cap structure.

RNA consisting 43 nucleotides bearing cap structure was synthesized (Figure). In the first place, 9 mer of a leader sequence with the cap structure (F-1) was synthesized by the phosphotriester method and followed by the capping reaction. Next, 32 mer of a cistron was divided into two fragments and each was synthesized by the phosphoramidite method. The 3'-end nucleotide of the RNA, a modified guanosine 5'-phosphate, was introduced to F-3 by use of P1-2',3'-O-methoxymethylene guanosine-5'-yl P2-adenosine-5'-yl diphosphate (A5' ppGmM) with T4 RNA ligase. The chemically synthesized RNA fragments were ligated with T4 RNA ligase to afford the desired RNA.     

New antifungal peptides. Synthesis,bioassays and initial structure prediction by CD spectroscopy

The synthesis, in vitro evaluation and conformational study of KKWKMRRNQFWIKIQR-NH₂, HFRWRQIKIWFQNRRMKWKK-NH₂ and RQPKIWFPNRRKPWKK-NH₂ acting as antifungal agents are reported. These peptides displayed a moderate but significant antifungal effect against both pathogenic fungi Candida albicans and Cryptococcus neoformans. The conformational analysis of these peptides was carried out using both theoretical and experimental methods.