Programmed -1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift -1 nt and continue ...translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins.
Clostridium difficile infection (CDI) is a serious problem within the healthcare environment where the bacterium causes symptoms ranging from mild diarrhoea to life-threatening colitis. In addition ...to its principal virulence factors, Toxin A and Toxin B, some
C. difficile strains produce a binary toxin (CDT) composed of two sub-units namely CDTa and CDTb that are produced and secreted from the cell as two separate polypeptides. Once in the gut these fragments have the potential to combine to form a potent cytotoxin whose role in the pathogenesis of CDI is presently unclear. Here, we describe expression and purification methods for recombinant CDTa and CDTb produced in
Escherichia coli. We show that purified CDTa and CDTb can combine to form an active CDT which is cytotoxic to Vero cells. In addition, the purification processes described will allow milligram quantities of binary toxin fragments to be produced for further functional and structural studies.
Isolates of hepatitis E virus (HEV) have recently been described from China that are distinct from Burmese, Mexican and US viruses and constitute a novel genotype (genotype 4). Here, the complete ...genomic sequence of a representative isolate of genotype 4 HEV, amplified directly from the stool of an acutely infected patient, is presented. Analysis of the entire sequence confirms our previous conclusion, based upon partial sequence data, that these Chinese isolates belong to a novel genotype. Typical of genetic variation in HEV, most nucleotide substitutions occur in the third base of the codon and do not affect the amino acid sequence. The genotype 4 virus is unusual in that a single nucleotide insertion in the ORF 3 region changes the initiation of ORF 3, and perhaps also ORF 2. The consequences of these changes are discussed.
Reviews "Mosaics of faith : floors of pagans, Jews, Samaritans, Christians, and Muslims," by Rina Talgam (Yad Ben-Zvi Press and Pennsylvania State University Press, 2014).
Programmed -1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift -1 nt and continue translating in the ...new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3'-adjacent to a 'slippery' heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of -1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3'-adjacent stem-loop structure, but is not appreciably affected by the 'StopGo' sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46-76 % efficient.
To manage extensive walking track (trail) systems effectively, managers need information about the condition, stability and likely rates of deterioration of tracks. This information may be ...impractical to obtain from ground inspections, particularly if the track systems of interest encompass hundreds or even thousands of kilometres of tracks. Two trials were undertaken in Tasmania, Australia to assess the practicality of using a GIS-based methodology to predict track ‘types’, types being classes of environmental and track-orientation variables that are associated with characteristic rates of widening and erosion as tracks develop. In the first trial, type values previously measured at 500 18 m long monitoring sites located across a wide range of environments were compared with those predicted for 50–75 m long track segments that included or overlapped the sites. In the second trial, the type values of 300 75 m track segments distributed across five tracks were measured in the field and predicted using a refined version of the methodology. The reliability of the methodology was slightly improved in the second trial, in which 50% of the predictions were accurate and 38% were out by one category. Predictions of the statistical distribution of types were prone to bias due to local conditions on individual tracks, but agreed closely with the measured distribution across the entire data set. The methodology was used to assess track types across the 1700 km track system managed by the Tasmanian Parks and Wildlife Service, as a basis for identifying and prioritising management responses including track stabilisation works. It is likely that with further refinement and with better GIS information, the methodology could reliably predict the stability of individual tracks.
► A GIS-based methodology is proposed for predicting walking track stability ‘types’. ► The reliability of the methodology was tested across a wide range of environments. ► The statistical distribution of types is predicted reliably for extensive track systems. ► The methodology is useful in planning management responses such as track ‘hardening’. ► The methodology was used to assess types across a 1700 km track system in Tasmania.
Clostridium botulinum neurotoxin type A is a potently toxic protein of 150 kDa with specific endopeptidase activity for the SNARE protein SNAP-25. Proteolytic cleavage of BoNT/A with trypsin leads to ...removal of the C-terminal domain responsible for neuronal cell binding. Removal of this domain result in a catalytically active, non-cell-binding derivative termed LH
N/A. We have developed a purification scheme to prepare LH
N/A essentially free of contaminating BoNT/A. LH
N/A prepared by this scheme retains full enzymatic activity, is stable in solution, and is of low toxicity as demonstrated in a mouse toxicity assay. In addition, LH
N/A has minimal effect on release of neurotransmitter from a primary cell culture model. Both the mouse bioassay and in vitro release assay suggest BoNT/A is present at less than 1 in 10
6 molecules of LH
N/A. This represents a significant improvement on previously reported figures for LH
N/A, and also the light chain domain, previously purified from BoNT/A. To complement the preparation of LH
N/A from holotoxin, DNA encoding LH
N/A has been introduced into
Escherichia coli to facilitate expression of recombinant product. Expression and purification parameters have been developed to enable isolation of soluble, stable endopeptidase with a toxicity profile enhanced on that of LH
N/A purified from BoNT/A. The recombinant-derived material has been used to prepare antisera that neutralise a BoNT/A challenge. The production of essentially BoNT/A-free LH
N/A by two different methods and the possibilities for exploitation are discussed.
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