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A C Ward

Publications and source records attributed to A C Ward.

At least 91 records · Page 5Linked to original sources
PubMed1994

Characterisation of the urease-encoding gene complex of Yersinia enterocolitica.

A cosmid gene library of chromosomal DNA from Yersinia enterocolitica A2635 (serogroup O:8) was constructed in Escherichia coli. Subcloning of a urease-positive (Ure+) clone revealed a region of 6.6 kb that was sufficient for expression of Ure activity in E. coli. Sequencing of this fragment disclosed seven ORFs transcribed in the same direction. On the basis of homology to known Ure, these were designated ureA, ureB, ureC, ureE, ureF, ureG and ureD, which are predicted to encode polypeptides of 11.1, 17.9, 61.0, 29.5, 25.0, 24.1 and 36.4 kDa, respectively. The polypeptides encoded by the ure gene complex of Y. enterocolitica are significantly divergent from those encoded by the ure operons of other Enterobacteriaceae, which appear to be closely related to each other. This suggests that the ure genes were acquired by Y. enterocolitica from an unrelated organism or alternatively, that they diverged from those of other Enterobacteriaceae some considerable time ago.

Base Sequence↗
PubMed1994

Vectors for Cu(2+)-inducible production of glutathione S-transferase-fusion proteins for single-step purification from yeast.

We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.

Amino Acid Sequence↗
PubMed1994

Expression and characterisation of the influenza A virus non-structural protein NS1 in yeast.

The influenza A virus non-structural protein NS1 was produced using a copper-inducible expression system in the yeast Saccharomyces cerevisiae. The protein produced had a molecular weight of 26 kDa by SDS-PAGE and was reactive with anti-NS1 antisera. The recombinant NS1 protein was targetted to the nucleolus/nuclear envelope fraction of the yeast cell nucleus, showing that its localisation signals remain functional in yeast. In addition, immune-electron microscopy detected cytoplasmic inclusions reminiscent of those seen in cells infected with some influenza strains. The NS1 protein was shown to be capable of in vivo self-interaction which probably forms the basis of its propensity to form inclusions. Expression of the protein was found to be toxic to yeast cells expressing it, supporting a role for the protein in the shutdown of influenza virus-infected cells. Deletion mapping of NS1 pointed to 2 regions of the molecule being important for this toxicity: a basic C-terminal stretch which has been shown to act as a nuclear localisation signal, and an N-terminal region implicated in RNA binding.

Amino Acid Sequence↗
PubMed1994

Rapid identification of species within the Mycobacterium tuberculosis complex by artificial neural network analysis of pyrolysis mass spectra.

An artificial neural network (ANN) was trained to distinguish between Mycobacterium tuberculosis and M. bovis with averaged pyrolysis mass spectra from duplicate subcultures of four strains of each of these species, each pyrolysed in triplicate. Once trained, the ANN was interrogated with spectrum data from the original organisms (the "training set") and from 26 other mycobacterial isolates (the "challenge set") of the M. tuberculosis complex (MTBC). Eight strains of M. bovis and 13 of M. tuberculosis, whether sensitive or variously resistant to antituberculosis drugs, were identified in agreement with conventional identification. Four strains of "M. africanum" were identified as M. bovis. Of two atypical M. tuberculosis strains from South India, one was identified as M. tuberculosis and the other as M. bovis. Six strains of BCG proved heterogeneous; two gave equivocal identifications, three were identified as M. bovis and one was identified as M. tuberculosis.

Humans↗
PubMed1994

Bacteria isolated from nasal and tonsillar samples of clinically healthy Rocky Mountain bighorn and domestic sheep.

Nasal and tonsillar samples were collected from 14 free-ranging clinically healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis) and 10 domestic sheep (Ovis aries). We identified 194 bacterial isolates, including 101 from bighorn and 93 from domestic sheep. Of these isolates, 115 were gram-positive and 79 were gram-negative. Staphylococcus species were the most numerous gram-positive organisms and had a higher incidence in samples from domestic than from bighorn sheep. In contrast Streptococcus species were present in higher numbers in samples from bighorn sheep. Pasteurella haemolytica, the most common gram-negative bacterium, was isolated from five of five tonsillar but from none of ten nasal samples of domestic sheep, and from seven of eight tonsillar and three of ten nasal samples of bighorn sheep. Most bacteria isolated were considered opportunistic pathogens. However, of the bacteria isolated, P. haemolytica, P. multocida, and Actinomyces pyogenes are most frequently associated with respiratory disease.

Animals↗
PubMed1993

Rapid identification of streptomycetes by artificial neural network analysis of pyrolysis mass spectra.

An artificial neural network was trained to distinguish between three putatively novel species of Streptomyces using normalised, scaled prolysis mass spectra from three representative strains of each of the taxa, each sampled in triplicate. Once trained, the artificial neural network was challenged with spectral data from the original organisms, the 'training set', from additional members of the putative novel taxa and from over a hundred strains representing six other actinomycete genera. All of the streptomycetes were correctly identified but many of the other actinomycetes were mis-identified. A modified network topology was developed to recognise the mass spectral patterns of the non-streptomycete strains. The resultant neural network correctly identified the streptomycetes, whereas all of the remaining actinomycetes were recognised as unknown organisms. The improved artificial neural network provides a rapid, reliable and cost-effective method of identifying members of the three target streptomycete taxa.

Actinomycetales↗
PubMed1993

Artificial neural network analysis of pyrolysis mass spectrometric data in the identification of Streptomyces strains.

Sixteen representatives of three morphologically distinct groups of streptomycetes were recovered from soil using selective isolation procedures. Duplicated batches of the test strains were examined by Curie-point pyrolysis mass spectrometry and the first data set used for conventional multivariate statistical analyses and as a training set for an artificial neural network. The second set of data was used for 'operational fingerprinting' and for testing the artificial neural network. All of the test strains were correctly identified using the artificial neural network whereas only fifteen of the sixteen strains were assigned to the correct group using the conventional operational fingerprinting procedure. Artificial neural network analysis of pyrolysis mass spectrometric data provides a rapid, cost-effective and reproducible way of identifying and typing large numbers of microorganisms.

Bacterial Typing Techniques↗
PubMed1993

Expression of HIV-1 nef in yeast: the 27 kDa Nef protein is myristylated and fractionates with the nucleus.

The nef gene of human immunodeficiency virus type 1 (HIV-1) has been expressed in the yeast Saccharomyces cerevisiae to produce native Nef proteins. The proteins of M(r) 27 kDa and 25 kDa, produced by translation from the first and second start codons of the nef gene react with human HIV-1 antisera. Under low-level steady-state expression conditions, Nef27 undergoes myristylation and is targeted to the nuclear fraction while Nef25 is not myristylated and not nuclear localized. When produced rapidly and to high levels, Nef27 is initially present in the cytoplasm as a soluble myristylated protein that later fractionates with the nucleus.

Base Sequence↗
PubMed1993

Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis.

Pulsed-field agarose gel electrophoresis of SmaI digests of genomic DNA was used to examine lactose plasmid copy number and stability in Lactococcus lactis. In L. lactis strain DRC1, the plasmid was found to exist as a single-copy plasmid. Transconjugants of strain HID113 carrying this plasmid were unstable. Variants were isolated with improved phenotypic stability resulting from improved maintenance of the lactose plasmid or from integration of part of the plasmid into the lactococcal chromosome.

Conjugation, Genetic↗
PubMed1993

Use of DNA analysis of Pasteurella haemolytica biotype T isolates to monitor transmission in bighorn sheep (Ovis canadensis canadensis).

Pneumonia has been identified as a major cause of poor lamb survival in indigenous herds of Rocky Mountain bighorn sheep (Ovis canadensis canadensis) in central Idaho. Pasteurella haemolytica was isolated from five adult Rocky Mountain bighorn ewes captured from a free-ranging herd in central Idaho. The lambs from two of these ewes delivered by cesarean section were free of P. haemolytica until 40 days of age and after repeated contact with their dams. The lambs subsequently developed signs of pneumonia, and P. haemolytica was isolated from nasal, pharyngeal, and transtracheal wash samples from each lamb. All P. haemolytica biotype T isolates from the ewes and lambs, as well as those from a 9-month-old lamb of the same herd from which samples for culture were obtained 2 years earlier, were subjected to HaeIII restriction enzyme analysis (REA) and ribotyping. Two ribotypes and seven REA patterns were visually distinguishable by these procedures. Similarity coefficients (SAB) of 0.09 to 0.95 were calculated for the seven REA patterns. The REA patterns of the isolates from the lambs were identical (SAB = 1.0). The isolates from the lambs also had SAB values of 1.0, which was indicative of identity with one of the seven isolates cultured from the ewes at the time of capture and with the organism isolated from the 9-month-old lamb. These procedures have the discriminatory capabilities necessary to monitor the transmission of specific strains of bacteria within and between animal populations.

Animals↗
PubMed1991

Polyphasic taxonomic study of the genera Gordona and Tsukamurella including the description of Tsukamurella wratislaviensis sp. nov.

Representatives of the genera Gordona and Tsukamurella together with related actinomycetes were the subject of chemotaxonomic, numerical taxonomic and DNA homology studies. In the numerical analysis the organisms were examined for 116 unit characters and the data sorted using the simple matching, Jaccard and pattern coefficients; clustering was achieved using the unweighted pair group method with arithmetic averages algorithm. The numerical groupings were not affected by the statistics used or by test error, estimated at 1.9%. The numerical taxonomic data supported the integrity of the four validly described species of Gordona but indicated that the genus Tsukamurella might encompass a species in addition to Tsukamurella paurometabola. The putative new strains had chemical, enzymic, nutritional and tolerance properties consistent with their assignment to the genus Tsukamurella and formed a DNA homology group corresponding to the one formed by the Tsukamurella paurometabola strains. It is proposed that the new taxon be assigned to the genus Tsukamurella as Tsukamurella wratislaviensis.

Actinomycetales↗
PubMed1991

Rapid inter-strain comparison by pyrolysis mass spectrometry of coagulase-negative staphylococci from persistent CAPD peritonitis.

Pyrolysis mass spectrometry (PyMS) was used as a method of rapid inter-strain comparison of 19 isolates of coagulase-negative staphylococci from episodes of CAPD peritonitis. Thirteen isolates were from multiple, but distinct, episodes of peritonitis in 6 patients and the remaining 6 isolates were from 6 patients with single episodes. The results, expressed in terms of identity/non-identity of strains, were compared with those obtained using an established typing system comprising an extended antibiogram, determination of biotype and plasmid profile analysis. The PyMS results for inter-strain comparison were in agreement with the reference typing scheme results. PyMS can be used in this setting to rapidly obtain evidence that persistent infection is/is not likely to be due to the same organism, although it cannot be used for formal typing. The results by both methods showed that serial, apparently distinct, episodes of peritonitis over periods as long as 120 days may be due to the same strain of coagulase-negative staphylococcus. Clinically based distinctions between recurrence of infection (same strain) and re-infection (different strains) may not always be supported by the microbiological evidence.

Coagulase↗
PubMed1991

Epidemiological typing of coagulase-negative staphylococci by pyrolysis mass spectrometry.

Pyrolysis mass spectrometry (PMS) was evaluated for the epidemiological typing of coagulase-negative staphylococci (CNS) in situations in which it was necessary to distinguish between repeated isolation of the same strain from a single patient (genuine infection) and coincidental isolation of unrelated strains (contamination). Thirteen CNS isolates were examined, consisting of five pairs, each pair isolated from a single patient, and three unrelated strains. PMS analysis gave results equivalent to a conventional typing system comprising antibiogram, biotype and plasmid profile analysis. Both methods facilitate differentiation between genuine infection with CNS and the isolation of contaminants. The speed, reproducibility, versatility and relatively low cost of PMS suggest that it may be a valuable new technique for the epidemiological typing of CNS in routine clinical settings.

Coagulase↗
PubMed1990

Bacterial flora of the vagina and uterus of healthy cats.

Bacterial culturing was conducted on samples from the reproductive tracts of 53 clinically healthy female cats. Aerobic bacteria were isolated from 52 of 53 vaginal swab samples and from 2 of 29 uterine swab samples. Anaerobic bacteria were detected in 4 of 30 vaginal and 1 of 29 uterine cultures. The aerobic bacteria included species of Acinetobacter, Actinomyces, Corynebacterium, Escherichia, Haemophilus, Klebsiella, Lactobacillus, Staphylococcus, and Streptococcus. Coagulase-negative Staphylococcus, Streptococcus canis, and E coli were the most common organisms and were isolated from 56%, 52%, and 44% of the vaginal samples, respectively. Anaerobes isolated from vaginal samples included 3 species of Bacteroides and 2 isolates of Peptococcus. The single uterine anaerobe isolate was a Lactobacillus sp. The number of bacterial species isolated from each vaginal culture ranged from 1 to 8 (mean, 3). The number of colony forming units tended to vary inversely with the number of bacterial species detected in each sample.

Animals↗