An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

Background: Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. Results: We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange (TM) site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Conclusion: Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange (TM). Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange T) but increased the overall success rates.Publisher PDFPeer reviewe

Investigation of taste tainting in salmon flesh in the Ribble catchment

This report presents the findings of the first phase of an investigation into the cause(s) of taints in salmonid fish in the River Ribble, commissioned by the North West Region of the Environment Agency. There have been reports of tainting in fish taken from both the estuary and the freshwater river for many years, but the contaminants involved and their source and transport pathway are unknown. Tainting by phenols has been of specific concern in the past. The work programme comprised: examination of tainting reports; collection of salmonids; their submission for taste testing; literature review; analysis of fish flesh using gas chromatography-mass spectrometry (GCMS) and analysis of river bed sediments. From enquiries, three common descriptors of the 'taint' were identified: disinfectanty; diesely; and muddy. The incidence of taints appears transient/irregular and may therefore relate to the incidence of discharges and specific threshold concentrations of pollutants. The literature review showed that a wide range of organic compounds including many industrial chemicals, and others which are naturally occurring, can taint fish flesh. Taste testing confirmed the presence of tainted salmon and trout in the Ribbie Catchment. It identified a low incidence of 'untainted' fish but demonstrated the 'taint' to be not specific to one tainting substance. Differences were found both between the species and fish from different parts of the catchment. Overall, most fish exhibited an unpleasant flavour, though this may have been influenced to some extent by the fact that most were sexually mature. The worst tainting was found in trout from the river Calder: a soapy/chemical aftertaste. An unpleasant earthy/musty flavour distinguished the salmon from the trout. Phenol was shown to have been a minor issue during the present study, whilst no hydrocarbon taints were identified. Examination of tissue from the eight salmon exhibiting the worst taints revealed the presence of aromatic hydrocarbons, but no phenolic compounds. Other notable substances present in the fish were siioxanes and benzophenone. Data from sediment analysis is presented which shows the main compounds present to be aromatic and polyaromatic hydrocarbons, that concentrations at two locations R. Darwen and R. Calder were significantly higher than at other sites, and that some phenolic compounds were detected at low levels. A paucity of fish flesh taste descriptors linked to specific compounds prevented an obvious correlation to be made between the tastes observed and the organic compounds detected. Descriptors frequently used by the taste testing panel (e.g. earthy, musty, astringency, chemical) cannot be linked to any of the compounds identified in the tissue analyses. No taste information was available from the literature on siioxanes. Aromatic hydrocarbons though present in tissue and sediments were not identified as tainting

Factors Influencing the Prognosis in Tuberculous Meningitis Treated With Streptomycin

From a study of 102 cases of tuberculous meningitis admitted to Ayrshire Central Hospital, Irvine, and Belvidere Hospital, Glasgow, between June, 1948, and December, 1951, endeavour has been made to analyse the factors that may have had a bearing on the ultimate outcome, expressed in terms of both quantity and quality of survivors. The patients were all treated with streptomycin, with or without adjuvants, in the pre-isoniazid era. Diagnosis was confirmed bacteriologically in 82% of the cases and was not in doubt in any of the others. 66 (64.7%) the patients survived for a minimum of 5 years. In order to determine the inter-relation of all the factors that have been taken into consideration and ascertain the individual effects of each on the ultimate outcome and on the incidence of complications and sequelae, it is clear that a much vaster experience would be required than can be gained by one person - for example, an investigation of the dimensions of the trials conducted by the Medical Research Council using many centres. In a comparatively small series such as this, in which the figures tabulated were obtained in the experience of one person, the results may admittedly be vitiated by the interplay of different factors. An attempt has, however, been made to assess the major factors, and the following are the main conclusions reached. The study was divided into six sections. A. Factors prior to the institution of treatment. Prognosis was significantly better in patients (a) between the ages of 3 and 30 years, (b) with less advanced disease, (c) fully conscious, (d) well nourished, (e) without radiological evidence of miliary or adult pulmonary tuberculosis, (f) with high initial CSF cell count and chloride content. B. Treatment Regime: 7 different regimes of treatment were employed. All patients were given intramuscular and intrathecal streptomycin (or dihydro streptomycin), and in all hut 6 the duration of the initial course of treatment was not less than 6 months. Allowing for the limited numbers in each treatment group, it seemed justifiable to make the following deductions from the results; (a) less than 6 months' treatment was inadequate; (b) prolonged intrathecal therapy did not have any special advantage over shorter courses; (c) the use of dihydrostreptomycin was not associated with a significantly higher mortality rate compared with streptomycin; (d) neither streptokinase nor PPD enhanced the results; the latter, indeed, seemed to be detrimental. C. Type of response to treatment: A good initial response was not necessarily an indication of a favourable outcome. Nor could an accurate prognosis be made on the basis of early disappearance or long persistence of any or all of the main symptoms or signs. The CSF sugar level was not found to have the same prognostic significance as other observers have attached to it. The CSF protein level seemed of greater value in the assessment of prognosis than that of any other CSF constituent; no patient died whose CSF protein became normal - but 7 such patients relapsed. D. Incidence and influence of spinal block; A significantly higher mortality was noted in the 22 (21.6%) patients who developed obstruction to the flow of CSF It seemed likely that patients with initial CSF protein over 200 mg. per 100 ml. may have had incipient block as a significantly high proportion of such patients developed it. Neither streptokinase nor PPD proved to be of any value in relieving the obstruction; indeed it appeared that both, especially the latter, may have been responsible for its development in some cases. E. Incidence and influence of relapse; 20 relapses occurred in 16 patients who had completed a previous course of treatment; 1 of these patients succumbed after 3 relapses. The incidence of relapse did not appear to be related to the initial treatment regime provided this was of 6 months' duration, but a very high proportion of patients relapsed after an initial course lasting 3 1/2 months. All relapses occurred within 1 year, and most within 3 months, of the completion of the previous course of treatment. Most were detected by the recurrence of symptoms and signs and confirmed by CSF findings. A full course of treatment was instituted as soon as relapse was diagnosed, and the prognosis proved to be as good as for the initial illness

Spectator no more, the role of the membrane in regulating ion channel function

CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government. Research funds for this study were also partly covered by a Tenovus Scotland grant (Tenovus Grant Application T15/41), awarded to CP. JHN is supported by the Chinese National Thousand Talents Program, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award.A pressure gradient across a curved lipid bilayer leads to a lateral force within the bilayer. Following ground breaking work on eukaryotic ion channels, it is now known that many proteins sense this change in the lateral tension and alter their functions in response. It has been proposed that responding to pressure differentials may be one of the oldest signaling mechanisms in biology. The most well characterized mechanosensing ion channels are the bacterial ones which open when the pressure differential hits a threshold. Recent studies on one of these channels, MscS, have developed a simple molecular model for how they sense and adapt to pressure. Biochemical and structural studies on mechanosensitive channels from eukaryotes have disclosed pressure sensing mechanisms. In this review, we highlight these findings and discuss the potential for a general model for pressure sensing.PostprintPeer reviewe

Synthesis of Hybrid Cyclopeptides through Enzymatic Macrocyclization

Acknowledgements We thank Dr. G. Mann and Dr. A. Bent for supplying the enzymes and the useful discussions, and Dr. T. Lebl for the useful NMR discussions. This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1), the Wellcome Trust [TripleTOF 5600 mass spectrometer (094476), the MALDI TOF-TOF Analyzer (079272AIA), 700 NMR], and the EPSRC UK National Mass Spectrometry Facility at Swansea University. J.H.N., M.A.J., and N.J.W. are named on patents that have been filed by St. Andrews and Aberdeen Universities to commercialize the enzymes (PatG) and (LynD) used in the study. No income derives from the patent.Peer reviewedPublisher PD

Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase

We thank Dr. Rafael Guimaraes da Silva for helpful discussions on enzyme kinetics. We also thank Professor David Lilley, Dr. Alasdair Freeman and Dr. Anne-Cecile Declais at the University of Dundee for training and usage of their QFM-4000 quenched-flow apparatus.Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising drug-like molecules due to their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we put forward a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial-rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is amongst the fastest macrocyclases described to date, and this work is a necessary step towards designing broad specificity efficient macrocyclases.Publisher PDFPeer reviewe

Bacterial polysaccharide synthesis and export

The work is supported by the Chinese National Thousand Talents Program-, Wellcome Trust Senior Investigator Award (WT100209MA) and Royal Society Wolfson Merit Award.All domains of life make carbohydrate polymers and by anchoring them to lipid molecules they can decorate the outside of the cell. Polysaccharides are linked to proteins by glycosylation, a process found in both bacteria and in higher organisms. Bacteria do have other distinct uses for carbohydrate polymers; in gram-negative bacteria glycolipids form the outer leaflet of the outer membrane and in many pathogens (both gram-positive and gram-negative) sugar polymers are used to build a capsule or are secreted into the environment. There are parallels, but of course differences, in the biosynthesis of glycolipids between prokaryotes and eukaryotes, which occur at the membrane. The translocation of large sugar polymers across the outer membrane is unique to gram-negative bacteria. Recent progress in the molecular understanding of both the biosynthesis at the inner membrane and the translocation across the outer membrane are reviewed here.Publisher PDFPeer reviewe

Mechanisms of cyanobactin biosynthesis

This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1).Cyanobactins are a diverse collection of natural products that originate from short peptides made on a ribosome. The amino acids are modified in a series of transformations catalyzed by multiple enzymes. The patellamide pathway is the most well studied and characterized example. Here we review the structures and mechanisms of the enzymes that cleave peptide bonds, macrocyclise peptides, heterocyclise cysteine (as well as threonine and serine) residues, oxidize five-membered heterocycles and attach prenyl groups. Some enzymes operate by novel mechanisms which is of interest and in addition the enzymes uncouple recognition from catalysis. The normally tight relationship between these factors hinders biotechnology. The cyanobactin pathway may be particularly suitable for exploitation, with progress observed with in vivo and in vitro approaches.PostprintPeer reviewe

Crystallization and preliminary crystallographic analysis of the bacterial capsule assembly-regulating tyrosine phosphatases Wzb of Escherichia coli and Cps4B of Streptococcus pneumoniae

The crystallization is reported of two bacterial tyrosine phosphatases which belong to different enzyme families despite their ability to catalyse identical reactions

MtsslWizard: In Silico Spin-Labeling and Generation of Distance Distributions in PyMOL

MtsslWizard is a computer program, which operates as a plugin for the PyMOL molecular graphics system. MtsslWizard estimates distances between spin labels on proteins quickly with user-configurable options through a simple graphical interface. In default mode, the program searches for ensembles of possible MTSSL conformations that do not clash with a static model of the protein. Once conformations are assigned, distance distributions between two or more ensembles are calculated, displayed, and can be exported to other software. The program’s use is evaluated in a number of challenging test cases and its strengths and weaknesses evaluated. The benefits of the program are its accuracy and simplicity