VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI)
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Browsing VI Meeting on Genetics and Cellular Biology of Basidiomycetes (GCBB-VI) by Author "Burton, K.S."
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Publication Open Access Biochemistry of volatile compounds synthesis in Agaricus bisporus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Combet, E.; Henderson, J.; Eastwood, D.C.; Griffiths, G.; Burton, K.S.Agaricus bisporus unique flavour is due to the release of a set of eight-carbon volatile compounds, which biosynthetic pathway has not been elucidated yet, despite of the numerous implications of those volatile compounds. Beside their influence on crop quality, they are also important for insect perception and play a part in triggering the switch from vegetative to reproductive growth in mushrooms. 8-carbon volatiles are derived from the oxygenation and the cleavage of the polyunsaturated fatty acid linoleic acid. This reaction has similarities to the plant system, but also major differences. Examination of the enzymic mechanisms and the fatty acid chemistry suggested that the enzyme involved in the oxygenation step could be a lipoxygenase (as found in plants) or a heme-dioxygenase, similar to the recently isolated linoleate diol synthase from Gaeumannomyces graminis. In order to characterise the biochemical pathway leading to eight-carbon volatile production, we investigated fatty acid and lipids distribution in Agaricus bisporus, as well as hydroperoxide and volatile compounds levels. In parallel, we searched for candidate genes susceptible to encode the enzyme responsible for this novel oxidation route in fungi. The combination of analytical methods, such as GC-MS, with a molecular approach based on degenerate PCR and library screening provided us with a broad range of results. These results establish the relation between fatty acids and volatile compounds and enabled us to gain a better understanding of mushroom volatiles biosynthesis and lipid metabolism.Publication Open Access Expression profiling of natural antisense transcripts in Agaricus bisporus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eastwood, D.C.; Sergeant, M.; Mead, A.; Burton, K.S.Our view of the role of RNA has changed from being a passive intermediary of genetic information to acting as a regulator of gene expression in the form of short-interfering RNA, micro RNA, riboswitches and natural antisense. Long length natural antisense transcripts (NATs) have been identified for genes which are up-regulated after harvest in the fruitbody of the mushroom Agaricus bisporus. These NATs therefore are likely to be involved in the regulation of postharvest events such as development and senescence. A novel quantitative reverse transcriptase PCR technique has been developed. The data have been statistically analysed to produce expression profiles of NATs for six postharvest genes. The average antisense/sense ratios varied by three orders of magnitude, from 8.0 for shs13 to 6 x 10-3 for cruciform DNA binding protein. The expression profiles were found to be highly specific for individual genes, to be dynamic over time and highly variable between neighbouring tissues. This latter characteristic has led to the speculation that NATs may be involved in tissue differentiation. Sequence information of natural antisense transcripts form A. bisporus suggests that they are synthesized from messenger (sense) RNA by RNA-dependent RNA-polymerase. Evidence will be presented that to support the hypothesis that the level of antisense may be controlled by the 3’ processing of sense RNA.Publication Open Access Molecular characterisation and expression analysis of developmentally regulated genes in Agaricus bisporus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Sreenivasaprasad, S.; Molloy, S.; Fleming Archibald, C.; West, D.; Herman, B.; Eastwood, D.C.; Burton, K.S.; Henderson, J.Analysis of cDNA transcripts, PCR based methods and genomic library screening have been used to clone and characterise developmentally regulated genes in the cultivated white button mushroom Agaricus bisporus. Up-regulated genes identified during the rapid expansion phase of the sporophore include sugar transporter gene sut1, putative riboflavin-aldehyde-formingenzyme gene (raf) and three novel morphogenes mag2-mag4. Further, a hexose transporter gene sut2 and lectin genes abl1 and abl2, among others have been cloned from A. bisporus using PCR based strategies. Northern analysis indicated their up-regulation during sporophore differentiation and development. Sugar transporter gene sut1 transcripts increased abundantly during sporophore development and although sut1 showed varying levels of homology to other sugar transporters, its substrate preference could not be identified based on homology. Interestingly, analysis of basidiomycete genome sequences revealed the presence of a putative sut1 homolog in the white rot fungus Phanerocheate chrysosporium. On the other hand, Ab sut2 showed strong homology to fungal glucose/hexose transporters and its homologs also appear to be present in A. bitorquis and Coprinus cinereus suggesting a generic role. Analysis of the genomic cosmid clones revealed that the lectin genes abl1 and abl2 are present in close proximity to each other and further characterisation is on-going.Publication Open Access Molecular toolkit development for gene expression and gene silencing technologies in the homobasidiomycete Fungi Agaricus bisporus and Coprinus cinereus(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Heneghan, M.N.; Burns, C.; Burton, K.S.; Challen, M.P.; Bailey, A.; Foster, G.D.We have developed a “Molecular Toolkit” comprising interchangeable promoters and marker genes to facilitate transformation of homobasidiomycete mushrooms and subsequent analysis of gene expression. We will describe the testing of a wide range of promoters in both Agaricus bisporus and Coprinus cinereus when linked to a range of selectable and visual marker genes, along with the parameters required to successfully achieve foreign gene expression within these organisms. It has been previously demonstrated that a prerequisite for GFP expression in A. bisporus and C. cinereus is an intron. We describe the construction of an expression vector containing a multiple cloning site linked to an intron thus allowing different genes to be easily expressed in A. bisporus and C. cinereus. We report on the development of gene silencing technologies within A. bisporus and C. cinereus. In particular the serine protease has been targeted for gene silencing in A. bisporus. Serine protease has been implicated in post-harvest and age-related senescence of sporophores. On harvesting, mushrooms degenerate rapidly to give browned caps and loss of texture in the fruit body, and such problems can dramatically reduce sale ability of the mushrooms. Suppression of genes involved in these pathways could increase mushroom shelf-life and profitability for mushroom growers, or help to further elucidate the complex biochemical pathways involved in post-harvest degradation. Progress will also be reported on gene silencing in C. cinereus.