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High efficient homologous integration via tandem exo-beta-1,3-glucanase genes in the common mushroom, Agaricus bisporus

Rhee, M.D. van de, Mendes, O., Werten, M.W.T., Huizing, H.J., Mooibroek, H.
Current genetics 1996 v.30 no.2 pp. 166-173
genetic transformation, glycosidases, messenger RNA, restriction mapping, structural genes, Agaricus bisporus, open reading frames, genetic code, plasmid vectors, amino acid sequences, homologous recombination, gene expression, fruiting bodies, introns, mycelium
Homologous integration was studied in the common mushroom, Agaricus bisporus, using a plasmid (pHAG3-1) carrying the hygromycin-resistance gene and a 3.2-kb genomic fragment from A. bisporus. Homologous integration was found in 30-60% of the transformants obtained with pHAG3-1 linearized at three different positions within the homologous sequence, generating either blunt, 5'- or 3'-protruding ends. The genomic fragment was found to contain two homologous open reading frames in tandem, which showed 60% similarity to exo-beta-1,3-glucanases from Saccharomyces cerevisiae and Candida albicans. The level of the corresponding mRNA is low in the vegetative mycelium and relatively high in fruiting bodies. In the vegetative mycelium of a transformant with tandemly integrated pHAG3-1 plasmids at the homologous position, exoglucanase mRNA was strongly increased without any apparent effect on growth rate or morphology.