ISOLATION AND CHARACTERIZATION OF A CELLULOSE-GROWTH-SPECIFIC GENE FROM AGARICUS-BISPORUS

The edible basidiomycete, Agaricus bisporus, produces extracellular endoglucanase. Endoglucanase production is induced by cellulose and repressed by fructose in A. bisporus grown on minimal medium, and is regulated in activity during fruiting body development. An anti-endoglucanase antibody was used to isolate cellulase-related genes. Three main polypeptides of 38, 58, and 60 kDa were immunoprecipitated by the antibody from products of in vitro cell-free translation of mRNAs isolated from cellulose-grown mycelium. No cross-reaction was detected with the translated products from fructose-grown mycelium. This antibody was used to immunoscreen a lambdaZAPII-cDNA expression library made from mRNA isolated from cellulose-grown mycelium. Two cDNA cross-reacting clones, pSRc110 and pSRc200, were isolated. Clones pSRc110 and pSRc200 cross-hybridized and had the same restriction map. Clone pSRc200 hybrid selected an mRNA that on cell-free translation produced a 38-kDa polypeptide. The cDNA fragment from pSRc200 hybridized to a 1.3-kb mRNA from cellulose-grown mycelium. No hybridization was observed when using fructose-grown mycelium mRNA. Thus, the gene (cell) expressing the 1.3-kb mRNA, is differentially regulated by the carbon source of the culture medium. The cell gene was isolated in a 8.9-kb EcoRI genomic fragment after hybridization to pSRc200. Sequences similar to those in the egl1 and cbh2 genes from Trichoderma reesei were found upstream from the ATG start codon in cell. Nine short intervening sequences disrupt the cell coding sequence, and a strong bias against codons ending with G and A was observed. CEL1 (protein encoded by cell) showed a primary structure in which four putative functional domains were recognized: a predicted 29-amino-acid (aa) signal peptide, a core of 233 aa, a Pro-Ser-Thr-rich domain of 22 aa, and a C-terminal 36-aa cellulose-binding domain similar to those found in other fungal cellulolytic enzymes. No homology was observed between the CEL1 core and any beta-glycanase sequences described to date.