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 lambda ZAPII-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 (cel1) 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 reesi were found upstream from the ATG start codon in cel1. Nine short intervening sequences disrupt the cel1 coding sequence, and a strong bias against codons ending with G and A was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Raguz
S
S
Division of Biosphere Sciences, King's College London, UK.
Yagüe
E
E
Wood
D A
DA
Thurston
C F
CF
D000364:Agaricus / Q000235:genetics* / Q000254:growth & development; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D003062:Codon; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D014170:Transformation, Genetic
D003062:Codon; D004271:DNA, Fungal; D005656:Fungal Proteins; D002482:Cellulose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase