Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRN

Monitoring mis‐acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site‐specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run‐off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser‐scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl‐tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell type

A Modular One-Generation Reproduction Study as a Flexible Testing System for Regulatory Safety Assessment

The European Union’s Registration, Evaluation and Authorisation of Chemicals (REACH) legislation mandates testing and evaluation of approximately 30,000 existing substances within a short period of time, beginning with the most widely used “high production volume” (HPV) chemicals. REACH testing requirements for the roughly 3000 HPV chemicals specify three separate tests for reproductive toxicity: two developmental toxicity studies on different animal species (OECD Test Guideline 414) and a two-generation reproduction toxicity study (OECD TG 416). These studies are highly costly in both economic and animal welfare terms. OECD TG 416 is a fertility study intended to evaluate reproductive performance of animals in the P and F1-generations following repeated exposure to a test substance. It can also be used to detect adverse effects on structural and functional development. Thus, it has conventionally been preferred to the one-generation study (OECD TG 415). Recently, the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) proposed that routine two-generation studies could in most cases be replaced with an “enhanced” one-generation study (Reuter et al. [1]). The flexible design proposed by HESI-ACSA allows for the addition of one or more specialised modules, if triggered (e.g. production of a second generation or the investigation of classical developmental toxicity or developmental neuro- or immunotoxicity). Significantly, however, the HESI-ACSA proposal was designed for use in the safety assessment of pesticidal, as opposed to industrial, chemicals. Thus for the purposes of REACH, a streamlined one-generation study that also examines structural development would be the most efficient means of addressing current information requirements for HPV chemicals. This study represents a flexible testing system that can be modified to meet regulatory needs in a variety of sectors

The reaction centre of the photounit of Rhodospirillum rubrum is anchored to the light-harvesting complex with four-fold rotational disorder

The minimal photounit of the photosynthetic membranes of the purple non-sulphur bacterium Rhodospirillum rubrum, comprising the reaction centre and the light-harvesting complex has been purified and crystallised in two dimensions in the presence of added phospholipids, and subsequently visualised by electron microscopy after negatively-staining. The position of the reaction centres within the light-harvesting ring has been determined at low resolution by the application of a new analysis for rotationally disordered identical units (here the reaction centres) within a two-dimensional crystalline lattice comprised of perfectly aligned unit cells (here the light-harvesting complexes). The reaction centre was found to preferentially occupy one of four orientations within the light-harvesting complex. The light-harvesting complex appears to be distorted to C4 symmetry, thus assuming a squarish shape when visualised by negative staining. A tentative structural model of the reaction centre-light-harvesting complex photounit which fits the experimental data is propose

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12′ position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12′ lysines in the α, γ and δ subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK a values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in β or ε subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK a values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12′ lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of α subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channe

Micropositioning and microscopic observation of individual picoliter-sized containers within SU-8 microchannels

We describe the fabrication and application of a bioanalytical chip, made of SU-8 photoresist, comprising integrated, high aspect-ratio microfluidic channels, suitable to manipulate and investigate vesicles, cell fragments and biological cells. A central micrometer-sized aperture allows electrical particle counting and planar membrane experiments, microfluids allow (sub)micrometer-sized objects to be transported and addressed with different chemicals. Here we show how lipid vesicles are positioned with micrometer precision within the micro-channels by means of pressure and electrophoretic movement. Our approach is suited for controlling and investigating (bio)chemical synthesis and cellular signalling processes in ultrasmall individual vesicles by electro-optical technique

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chem. aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amts. of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately det. suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control expts. showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential exptl. parameters for expanding the scope of the suppressor tRNA technol. to different cell types. [on SciFinder (R)