An experimental microcosm system developed to investigate the community dynamics of two- and three-fungal species interactions is described. The microcosm consists of tessellated nutrient agar tiles allowing the varied prescription of: (1) the number of interacting fungal species; (2) the spatial organisation (patchiness) of the distribution of individuals; and (3) the scale of the interaction arena. The system also allows the quantification of interaction outcome in terms of species occupancy within each tile using a destructive re-isolation based plating technique. The outcome and reproducibility of small-scale, pairwise confrontations were used to predict the behaviour of larger two- and three-species interactions. The influence of experimental factors such as species patch size and spatial distribution on the community dynamics were also investigated. The spatial heterogeneity displayed during large-scale three species tessellations was represented by a novel application of principal component analysis, which showed good intuitive agreement with visual assessment of the interaction outcome patterns. The development of a non-destructive method, based on green fluorescent protein labeling of one of the organisms studied, to continuously monitor the development of the interactions is also described. Results indicated that for two species interactions of equal patch size the final outcomes of the large-scale tessellations could be extrapolated from the behaviour of relevant small-scale binary confrontations. However, further investigation revealed that species patch size influenced the temporal dynamics of the system. It was shown that larger patch sizes of species increased the time taken for one species to replace the other. Results also showed that extrapolation of the behaviour of large-scale three species confrontations were not possible from combative hierarchy information derived from the outcome of binary tile confrontations. The outcomes of the three species interactions were shown to be neither random nor fully deterministic and that a degree of stochasticity was displayed in outcome for all tessellations. These findings suggest that the initial spatial distribution of species influence outcome and reproducibility of the interactions. The model therefore demonstrates the complex and coordinated behaviour of fungal mycelia on fungal community development. The work to develop a non-destructive analysis system for the study of fungal interactions was unsuccessful. The work attempted to introduce the gene for green fluorescent protein into the genome of one of the study organisms, <i>C. marmorata</i>, via genetic transformation. Although, the introduction and expression of exogenous genetic material into the genome of <i>C. marmorata</i> was not successful, it was possible to isolate protoplasts from this species. This is the first known report of this for this species.

No description available.

In the model organism Schizosaccharomyces pombe, the Cds1 (checking DNA synthesis 1) kinase is activated at the S-phase checkpoint upon stalling of the replication fork during DNA synthesis. Under normal conditions (300C), the role of the full-length protein kinase is to activate downstream processes resulting in mitotic arrest,protection of the stalled replication fork, and prevention of continued DNA replication in an unfavourable environment. In this way, Cds1 acts to ensure the reversible arrest of DNA synthesis. However, under stress conditions such as raised temperature or specific DNA damaging drugs, shorter variants of this protein kinase are rapidly expressed. Initial experimentation revealed the origin of the predominant variant, named Cds1-B, as the internal translation initiation site Methionine 159. The expressed protein is N-terminally truncated, missing amino acids residues 1-158, and therefore lacking the regulatory SQ/TQ and FHA domains whilst retaining the kinase domain. Absence of these regulatory regions suggests that this variant is free to act outside of the chromatin environment to fulfil roles different to that of its full-length counterpart, however it is unlikely to be active as a kinase as it is unable to participate in the model of activation currently proposed in the literature. Experimentation in this project was aimed at elucidating the role of this Cds1-B variant through analysis of drug sensitivity and checkpoint control efficacy, and evaluation of kinase activity. Current literature and results discussed here suggest an interesting hypothesis in which variants are expressed in response to specific genotoxic stresses in order to self-regulate kinase activity and selectively mediate cellular response either by the DNA replication checkpoint effector Cds1 or the DNA damage checkpoint effector Chk1. Mediation of effector kinase action is a promising avenue for cancer treatment, and with a potential Cds1-B homologue for human Chk2 identified (splice variant isoform 13), gaining a greater nderstanding of these variant mechanisms in yeast will aid the development of new therapeutic interventions.

No description available.

No description available.

Saccharomyces cerevisiae (bakers'/brewers' yeast) is a ubiquitous dietary constituent in the developed world. Previous studies, using semi-quantitative ELISA techniques, suggested that patients with Crohn's disease have higher titres of IgG and IgA isotypespecific antibodies to this yeast than are found in normal control subjects or patients with ulcerative colitis. For this study, in order to allow more stringent assay standardisation and more meaningful numerical comparison of the relative antigen-binding capacities of different sera, a quantitative ELISA was developed for measurement of anti-yeast antibodies, using a soluble extract of yeast (sacc) as the antigen. The finding of raised levels of yeast antibodies in Crohn's disease was confirmed, and the data suggest that this may be related to the presence of disease in the small bowel, although this latter observation did not reach statistical significance. Patients with chronic liver disease also had higher antibody levels than controls, but less markedly so than in Crohn's disease. When sera were tested in a similar assay for antibodies to bovine casein, no difference was found between controls and the Crohn's or liver disease group. The response of peripheral blood mononuclear cells (PBMC) to sacc was examined using a proliferation assay measuring uptake of tritiated thymidine. Cells from normal controls demonstrated dose-dependent proliferation, the time-course of which resembled that obtained with known recall antigens. Following separation of cell populations by rosetting with sheep erythrocytes, the responding cells were shown to be T-lymphocytes and the magnitude of the response was sensitive to the number of antigen-presenting cells present in the culture. When positive selection with immunomagnetic beads was used to further separate T-cells into highly purified CD4+ and CD8+ populations, responsiveness to yeast co-separated with the CD4+ subset. Following negative selection of cells expressing CD45RO or CD45RA, responsiveness was largely, but not exclusively, confined to the CD45RO+ population. Limiting dilution analysis of peripheral blood T-cells gave estimates of the sacc-specific precursor cell frequency in keeping with values previously reported for recall antigens, although the experimental data could not be shown to conform to single-hit kinetics. By sequential stimulation in long term culture, it was possible to obtain populations of cells which were uniquely responsive to sacc but unresponsive to other recall antigens. At some concentrations of sacc, proliferation responses of PBMC from Crohn's disease patients were higher than those in normal subjects, but the difference was not convincing overall. Digestion of sacc with pronase abolished the T-cell response but left specific antibody-binding intact, supporting the suggestion that antibody recognition is dependent on carbohydrate epitopes. Yeast cell wall mannan is implicated as the likely site of B-cell epitopes; evidence pertaining to T-cell epitopes is less conclusive. Thus, this study provides evidence that immune sensitisation to a common dietary constituent frequently occurs in the normal population, leading to detectable humoral and cellular immune responses. The T-cell response appears to be genuinely antigen-specific, and not due to non-specific lymphocyte activation. The gastrointestinal lymphoid system may be the site at which primary sensitisation occurs. In patients with Crohn's disease, the humoral response is enhanced, possibly as a consequence of inflammatory processes in the small bowel.

Feedings of Trichoderma viride with ring-tritiated aromatic amino-acids have shown that m-tyrosine is not an obligatory precursor of gliotoxin and that during conversion of phenylalanine into gliotoxin, neither loss nor migration of the ring protons occurs. A phenylalanine epoxide intermediate is proposed.

Eukaryotic cells transcribe a vast array of non-coding RNAs, most of which have not been assigned a functional role. The work presented here reveals a novel mechanism of transcriptional repression that is mediated by the non-coding RNA prt (pho1-repressing transcript). The prt transcript is shown to recruit a histone deacetylase, Clr3, to repress pho1. This gene encodes a secreted acid phosphatase essential for phosphate acquisition in fission yeast. In the presence of phosphate, prt is produced from an upstream promoter and leads to silencing of pho1. Thus far, this has been explained by prt transcription leading to deposition of repressive methylation over the locus. However, this explanation is known to be incomplete since deletion of the only known histone methyltransferase does not lead to pho1 induction comparable to deletion of the prt promoter. This suggests that another mechanism must be involved in mediating transcriptional interference via non-coding transcription. In the present study the putative ncRNA-binding protein Seb1, together with the chromatin modifying complex SHREC, is demonstrated to associate with prt to elicit silencing of pho1 by a mechanism that is independent of H3K9 methylation and instead relies on deacetylase activity provided by the Clr3 component of SHREC. These data reveal a previously uncharacterised layer of ncRNA-mediated gene regulation and provide important conceptual advances in understanding the mechanisms governing the phenomenon known as transcriptional interference.

No description available.

No description available.