Efficient isolation of genes differentially expressed on cellulose by suppression subtractive hybridization in Agaricus bisporus.
The production of cellulases on minimal medium in the edible mushroom Agaricus bisporus is regulated by the carbon source: induced by cellulose and repressed by glucose. In order to isolate cellulose-growth specific sequences, a cDNA library from A. bisporus using suppression subtractive hybridization (SSH) was constructed. Northern blot analysis indicated that a high level of enrichment was achieved; 183 clones were isolated. A preliminary screen with cellulose-specific genes of A. bisporus (cel1, cel2, cel3 and cel4) using Southern hybridization resulted in 28 clones to be cel3, and 5 clones were cel2. The remaining 144 clones were sequenced. Partial sequences of the following genes were found: a beta-glucosidase homologue of the blvk gene of Kluyveromyces marxianus; a cellulase homologue of an endoglucanase (avicellase III) of Aspergillus aculeatus, four different xylanases homologue of the xyn genes of different fungi, and one hexose transporter homologue to the hxtA gene of Aspergillus parasiticus. The apparent full-length of two hydrophobins homologue to the abh3 gene of A. bisporus and one histone homologue to the h2a gene of Aspergillus niger were also found. The remaining sequences did not have homology to any known genes.
Morales
Porfirio
P
Mushroom Biotechnology, College of Postgraduates, Campus Puebla, Apartado Postal 701, Puebla, Puebla 72001, Mexico.
Thurston
Chris F
CF
D000364:Agaricus / Q000235:genetics* / Q000378:metabolism; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D018076:DNA, Complementary; D005656:Fungal Proteins; D002482:Cellulose; D006026:Glycoside Hydrolases; D002480:Cellulase; C104579:CEL2 protein, Agaricus bisporus; D043366:Cellulose 1,4-beta-Cellobiosidase