Gareth J. Murtagh

In vitro stability and immunoreactivity of the native and recombinant plant food 2S albumins Ber e 1 and SFA‐8

Background The ability of an intact protein to reach the circulatory system may be a prerequisite to allergenicity and many allergens, particularly those from plant foods, have been found to be consistently more resistant to digestion by pepsin than other proteins.

Reproductive systems: Sex and the single lichen

Most lichenized fungi produce abundant sexual structures, and in many species sexual spores seem to provide the only means of dispersal. For example, 90% of lichens found in Great Britain and Ireland produce ascomata (fruit bodies) containing sexually derived ascospores, whereas only 29% form symbiotic vegetative propagules. Sex in lichenized fungi has been assumed to equate with outcrossing, but failure to induce sexuality in vitro has prevented experimental investigation of their breeding systems.

Sex and the single lichen.

Most lichenized fungi produce abundant sexual structures, and in many species sexual spores seem to provide the only means of dispersal. For example, 90% of lichens found in Great Britain and Ireland produce ascomata (fruit bodies) containing sexually derived ascospores, whereas only 29% form symbiotic vegetative propagules. Sex in lichenized fungi has been assumed to equate with outcrossing, but failure to induce sexuality in vitro has prevented experimental investigation of their breeding systems.

The Disulphide Mapping, Folding and Characterisation of Recombinant Ber e 1, an Allergenic Protein, and SFA8, Two Sulphur-rich 2 S Plant Albumins

We have cloned and expressed genes encoding the allergenic brazil nut 2S albumin (Ber e 1) and the sunflower albumin 8 (SFA8) in the methylotrophic yeast Pichia pastoris. We show that both proteins were secreted at high levels and that the purified proteins were properly folded. We also showed that Ber e 1 is glycosylated during secretion and that the glycan does not interfere with the folding or immunoreactivity. The disulphide map of the Ber e 1 protein was experimentally established and is in agreement with the conserved disulphide structure of other members of the 2S albumin family. A model three-dimensional structure of the allergen was generated. During the expression studies and through mutation we have also shown that alteration of the sequences around the Kex2 endoproteolytic processing site in the expressed fusion protein can compromise the secretion by targeting part of the protein for possible degradation. The secreted production of these properly folded sulphur-rich plant albumins presents an opportunity to delineate the attributes that make an allergen and to facilitate the diagnosis and therapy of type I allergy.

Molecular and physiological diversity in the bipolar lichen-forming fungus Xanthoria elegans

Genetic and physiological diversity was assessed in isolates of the lichen-forming fungus Xanthoria elegans originating from a range of geographic localities and climatic regimes including polar and temperate regions. In vitro cultures were established and isolates examined by production of multi-locus RAPD markers and by sequence comparison of the ITS region of the rDNA tandem repeat unit. Both molecular techniques revealed significant variability. Phylogenetic analysis of RAPD profiles (226 markers) clustered isolates from particular locations together and Mantels test showed a correlation between genetic divergence and extent of geographic separation. Phylogenies based on ITS sequence divergence were not well supported and did not correlate with the geographic origin of samples, suggesting a single locus approach to be less informative than RAPD data in a study of this nature. Physiological investigations also revealed significant variability between isolates with different geographic origins. Temperature had a significant effect on relative growth rate (RGR) such that X. elegans originating from sites with lower mean annual temperatures had significantly higher RGRs at all test temperatures between 2° and 18°C. Enhanced metabolic activity might be an adaptation for growth in colder climates. The results demonstrate high genetic diversity within this morphologically variable species in response to geographic and environmental factors, and are discussed in relation to data obtained for non-lichenised fungal species and the possibility of cryptic speciation.

Stability of recombinant 2 S albumin allergens in vitro.

Two well known 2 S albumins, Ber e 1 from brazil nut and sunflower 2 S albumin 8 (SFA-8), have been expressed in a eukaryotic system and purified. Analysis of recombinant versions of Ber e 1 and SFA-8 revealed them to be significantly more resistant to digestion by pepsin than BSA, and to be stable for up to 30 min in simulated gastric fluid. Unfolding monitored by CD indicated that both proteins were also very resistant to denaturation induced by heat and low pH. These results suggest that, although the ability of 2 S albumins to reach the circulatory system may be a prerequisite for the allergenicity of this group of proteins, stability is just one of a number of characteristics that provoke a selective immune response.

A new species of Tetramitus in the benthos of a saline antarctic lake

A new amoeboflagellate, Tetramitus vestfoldii n. sp. was discovered in the microbial mat of Pendent Lake, Vestfold Hills, Eastern Antarctica. Isolates were examined using light microscopy, electron microscopy and molecular analysis of the small subunit ribosomal DNA (SSU rDNA). Microscopical observations revealed a markedly eruptive, limax amoeboid stage with a clear hyaline zone, whilst flagellates were tapered posteriorly with a cytostome and anterior rostrum from which 4 flagella of equal length arose. The SSU rDNA was sequenced. Alignment of the sequence with published sequences of some other amoeboflagellates revealed that this species is best placed within the Vahlkampfiidae. The T. vestfoldii SSU rDNA was found to show greatest similarity with that of T. rostratus , a species with which it also shares several structural characteristics. Inorganic nutrient measurements indicate that the benthos of Pendent Lake could be a favourable environment for a bacterivorous predator such as Tetramitus . Ice cover on Pendent Lake was seen throughout the study period and may have reduced light intensity in the microbial mat; a low light intensity would appear to be a requirement for this species. The new species is defined on the basis of a pronounced taper towards the posterior end in the flagellate stage, an intolerance to salt concentrations greater than 30 and phylogenetic resolution using SSU rDNA sequence comparisons.

Sex and the single lichen: Reproductive systems

Most lichenized fungi produce abundant sexual structures, and in many species sexual spores seem to provide the only means of dispersal. For example, 90% of lichens found in Great Britain and Ireland produce ascomata (fruit bodies) containing sexually derived ascospores, whereas only 29% form symbiotic vegetative propagules. Sex in lichenized fungi has been assumed to equate with outcrossing, but failure to induce sexuality in vitro has prevented experimental investigation of their breeding systems.