Michael P. Whitehead

Investigation of a PCR-based method for the routine identification of British Armillaria species

Some Armillaria species cause major disease problems in both forestry and gardens. In order to determine routinely and rapidly the identity of an isolate, a PCR-based method was investigated. A fragment, approx. 900 bp, of the IGS region of the ribosomal DNA was amplified from 96 different isolates of six species of Armillaria collected from the U.K. and mainland Europe. Armillaria mellea, A. gallica and A. tabescens produced unique digestion patterns when the PCR fragment was restricted with Alu I. A. ostoyae could be distinguished from A. borealis and A. cepistipes when the PCR fragment was restricted with Bsm I. Previously unreported RFLP patterns were determined for A. mellea and A. gallica . The existence of A. gallica isolates which are putative heterozygotes for the rDNA cluster is also reported. Based on differences in banding patterns. A. mellea, A. gallica, A. cepistipes and A. borealis were subdivided further into groups which correlated with differences in cultural morphology.

Survival of Escherichia coli O157:H7 in traditional African yoghurt fermentation.

Growth and survival of a nontoxigenic strain of Escherichia coli O157:H7 (ATCC 43888) was determined in traditionally fermented pasteurized milk. Preheated milk was inoculated with 1% (v/v) of a mixed culture of Lactobacillus delbrueckii ssp. bulgaricus (NCIMB 11778) and Streptococcus salivarius ssp. thermophilus (NCIMB 110368) and incubated at 25, 30, 37 or 43 degrees C for 24 h. E. coli O157:H7 (10(5) CFU/ml) were introduced into the milk pre- and post-fermentation. Fermented milk samples were subsequently stored at either 4 degrees C (refrigerator temperature) or 25 degrees C (to mimic African ambient temperature) for 5 days. After 24 h of fermentation, the pH of the samples fermented at the higher temperatures of 37-43 degrees C decreased from 6.8 to 4.4-4.0 ( +/- 0.2) whereas at the lower temperature of 25 degrees C, the pH decreased to pH 5.0 +/- 0.1. During this period, viable counts for E. coli O157:H7 increased from 10(5) to 10(8) - 10(9) CFU/ml except in milk fermented at 43 degrees C wherein viability declined to 10(4) CFU/ml. In fermented (25-30 degrees C) milk stored at 4 degrees C for 5 days, E. coli O157:H7 viability decreased from 10(8-9) to 10(6-7) CFU/ml whereas milk fermented at 43 degrees C resulted in loss of detectable cells. In contrast, storage of fermented milk samples at 25 degrees C for 5 days eventually resulted in complete loss of viability irrespective of fermentation temperature. Stationary phase E. coli O157:H7 inoculated post-fermentation (25 and 43 degrees C) survived during 4 degrees C storage, but not 25 degrees C storage. Fermentation temperature and subsequent storage temperature are critical to the growth and survival of E. coli O157:H7 in traditional fermented products involving yoghurt starter cultures.

Cloning of a novel gene encoding a C2H2 zinc finger protein that alleviates sensitivity to abiotic stresses in Aspergillus nidulans

We report the cloning and sequencing of a DNA fragment encoding a putative C 2 H 2 zinc finger protein from Aspergillus nidulans. The gene was isolated by complementation cloning of a salt sensitive phenotype of the A. nidulans sltAl mutant. A 3.8 kb Pst I fragment that restored wild type salt tolerance contained one large open reading frame of 2202 bp. The predicted protein (StzA) from this reading frame comprises 698 amino acids and has three Zinc fingers along with a putative transcriptional activation domain rich in acidic amino acids. The corresponding sequence from a sltAl mutant contains a premature STOP codon resulting in loss of the putative transcriptional activator in the C-terminal region. The Zinc fingers show conserved motifs with a number of transcription factors including CreA from A. nidulans and the human Wilms tumour susceptibility protein WT-1.

Horizontal transfer of bacterial polyphosphate kinases to eukaryotes: implications for the ice age and land colonisation

BackgroundStudies of online database(s) showed that convincing examples of eukaryote PPKs derived from bacteria type PPK1 and PPK2 enzymes are rare and currently confined to a few simple eukaryotes. These enzymes probably represent several separate horizontal transfer events. Retention of such sequences may be an advantage for tolerance to stresses such as desiccation or nutrient depletion for simple eukaryotes that lack more sophisticated adaptations available to multicellular organisms. We propose that the acquisition of encoding sequences for these enzymes by horizontal transfer enhanced the ability of early plants to colonise the land. The improved ability to sequester and release inorganic phosphate for carbon fixation by photosynthetic algae in the ocean may have accelerated or even triggered global glaciation events. There is some evidence for DNA sequences encoding PPKs in a wider range of eukaryotes, notably some invertebrates, though it is unclear that these represent functional genes.Polyphosphate (poly P) is found in all cells, carrying out a wide range of essential roles. Studied mainly in prokaryotes, the enzymes responsible for synthesis of poly P in eukaryotes (polyphosphate kinases PPKs) are not well understood. The best characterised enzyme from bacteria known to catalyse the formation of high molecular weight polyphosphate from ATP is PPK1 which shows some structural similarity to phospholipase D. A second bacterial PPK (PPK2) resembles thymidylate kinase. Recent reports have suggested a widespread distribution of these bacteria type enzymes in eukaryotes.ResultsOn – line databases show evidence for the presence of genes encoding PPK1 in only a limited number of eukaryotes. These include the photosynthetic eukaryotes Ostreococcus tauri, O. lucimarinus, Porphyra yezoensis, Cyanidioschyzon merolae and the moss Physcomitrella patens, as well as the amoeboid symbiont Capsaspora owczarzaki and the non-photosynthetic eukaryotes Dictyostelium (3 species), Polysphondylium pallidum and Thecamonas trahens. A second bacterial PPK (PPK2) is found in just two eukaryotes (O. tauri and the sea anemone Nematostella vectensis). There is some evidence for PPK1 and PPK2 encoding sequences in other eukaryotes but some of these may be artefacts of bacterial contamination of gene libraries.ConclusionsEvidence for the possible origins of these eukaryote PPK1s and PPK2s and potential prokaryote donors via horizontal gene transfer is presented. The selective advantage of acquiring and maintaining a prokaryote PPK in a eukaryote is proposed to enhance stress tolerance in a changing environment related to the capture and metabolism of inorganic phosphate compounds. Bacterial PPKs may also have enhanced the abilities of marine phytoplankton to sequester phosphate, hence accelerating global carbon fixation.

Postmortem degradation of porcine articular cartilage.

Postmortem decompositional changes to articular cartilage were analysed to help establish a new methodology in determining the postmortem interval. The cartilage was collected from porcine trotters buried in simulated shallow graves for different time periods. The trotters were dissected to expose the cartilage located at the metatarsal joint. Numerous macroscopic changes including a colour change, gradual degradation of cartilage and adjacent soft tissues and a loss of cartilage covering articular facets were observed. Further analysis was conducted using light microscopy (LM) and scanning electron microscopy (SEM) to assess microstructural changes. Both LM and SEM showed gradual morphological and structural changes to the tissue over time, along with loss of nuclear material. Tissue surface analysis with SEM highlighted orthorhombic shaped crystals that appear at approximately three weeks postmortem and persist until six weeks postmortem. Both microscopic and macroscopic characteristics followed a recognisable succession over the burial times observed. These results indicate that postmortem degradation of articular cartilage may be useful for estimating a presumptive postmortem interval.

Identification of crystals forming on porcine articular cartilage: a new method for the estimation of the postmortem interval.

Articular cartilage was examined to determine its decomposition sequence and its potential for assessing the postmortem interval. Scanning electron microscopy of articular cartilage from buried porcine trotters showed the presence of microcrystals on the synovial surface. These orthorhombic pyramidal or “coffin”‐shaped crystals, appeared at 3 weeks (22 days) after interment and disappeared after 6 weeks. The disappearance of these crystals was linked to decompositional changes to the integrity of the synovial joint. The formation and disappearance of these crystals was associated with a pH change at the cartilage surface. Scanning electron microscopy–energy‐dispersive X‐ray (SEM‐EDX) analysis showed that the five main elements contained within these crystals were carbon, nitrogen, oxygen, magnesium, and phosphorous. Such elemental analysis suggested the crystals may be struvite (MgNH4PO46(H2O)). Bacteria cultured from the cartilage synovial surface produced struvite crystals when grown in suitable media and were identified by DNA analysis to be Comamonas sp.

Fungal gene sequences make excellent models for teaching data mining

A brief introductory exercise in the use of on-line databases to examine fungal genes and their products is described. Fungal genes make particularly good teaching models owing to their relatively simple eukaryotic structure and wide range of homologues in higher organisms including humans. An evaluation of students’ reactions to the exercise is included.

Assigning Level in Data-Mining Exercises.

Abstract There is currently much interest in ascribing outcomes to Masters (M) level programmes. It is particularly difficult to define M level outcomes in bioinformatics for students on non-specialist programmes. An approach is described that attempts to discriminate undergraduate from M level in a data-mining exercise. Differentiation of level is based upon the taxonomic origin of a DNA sequence, the relative increase in gene complexity from lower to higher eukaryote and the initiative required to use a wider range of databases and analytical tools.

Investigating the Postmortem Molecular Biology of Cartilage and its Potential Forensic Applications

This study investigated the postmortem molecular changes that articular cartilage undergoes following burial. Fresh pig trotters were interred in 30‐cm‐deep graves at two distinct locations exhibiting dissimilar soil environments for up to 42 days. Extracts of the metacarpophalangeal (MCP) and metatarsophalangeal (MTP) joint cartilage from trotters disinterred weekly over 6 weeks were analyzed by Western blot against the monoclonal antibody 2‐B‐6 to assess aggrecan degradation. In both soil conditions, aggrecan degradation by‐products of decreasing molecular size and complexity were observed up to 21 days postmortem. Degradation products were undetected after this time and coincided with MCP/MTP joint exposure to the soil environment. These results show that cartilage proteoglycans undergo an ordered molecular breakdown, the analysis of which may have forensic applications. This model may prove useful for use as a human model and for forensic investigations concerning crimes against animals and the mortality of endangered species.