Characterization of the Class I alpha-mannosidase gene family in the filamentous fungus Aspergillus nidulans

We describe the cloning and sequence characterization of three Class I alph a-1,2-mannosidase genes from the filamentous fungus Aspergillus nidulans. W e used degenerate PCR primers to amplify a portion of the alpha-1,2-mannosi dase IA gene and used the PCR fragment to isolate the 2495 nt genomic gene plus several hundred bases of flanking region. Putative introns were confir med by RT-PCR. Coding regions of the genomic sequence were used to identify two additional members of the gene family by BLAST search of the A. nidula ns EST sequencing database. Specific PCF: primers were designed to amplify portions of these genes which were used to isolate the genomic sequences. T he 1619 nt coding region of the alpha-1,2-mannosidase IB gene and the 1759 nt coding region of the alpha-1,2-mannosidase IC gene, plus flanking region s, were fully sequenced. All three genes appeared to encode type-II transme mbrane proteins that are typical of Class I alpha-1,2-mannosidases. The ded uced protein sequences were aligned with 11 published Class I alpha-1,2-man nosidases to determine sequence relationships. All three genes exhibited hi gh similarity to other fungal alpha-1,2-mannosidases. The alpha-1,2-mannosi dase IB exhibited very high similarity to the Aspergillus satoi and Penicil lium citrinum alpha-1,2-mannosidases and likely represents an orthologue of these genes. Phylogenetic analysis suggests that the three A. nidulans Cla ss I alpha-1,2-mannosidases arose from duplication events that occurred aft er the divergence of fungi from animals and insects. This is the first repo rt of the existence of multiple Class I mannosidases in a single fungal spe cies. (C) 2000 Elsevier Science B.V. All rights reserved.