Cloning and genetic analysis of subtilases in sapstaining fungi.
In order to assess the genetic variance of a group of homologous subtilases in sapstaining fungi, molecular techniques were employed. First, PCR screening with degenerate primers and dot-blot analyses were used to screen 31 different isolates, representing nine species, of sapstaining fungi for the presence of subtilase-like sequences. Restriction fragment length polymorphism PCR and sequence analysis techniques were then used to determine the inter- and intraspecies variation of these genes. A labelled chemiluminescent probe was then used to screen an Ophiostoma floccosum 387N genomic library for subtilase genes. Over ten positive clones were found and one was subcloned and sequenced. Randomly amplified cDNA ends PCR techniques were then employed to obtain full-length subitilase gene sequence information from isolates of four different species. The obtained sequences were found to be homologous with other fungal subtilases and common structural features of the inferred proteins with proteinase K were apparent. Southern blot analysis was used to verify and determine the copy number of the subtilase genes in these fungi. In the work presented here, it was found that all of the isolates tested seemed to contain some sort of subtilase gene sequence and that there was inter- and intraspecies variation in the number and type of subtilase genes present. The data indicated that three distinct groups of subtilase genes are present in sapstaining fungi. However, individual isolates were found to contain only one or two of these gene types.
Forest Science Centre, Department of Wood Science, University of British Columbia, Vancouver V6T 1Z4, British Columbia, Canada.
Brad
B
Hoffman
Colette
C
Breuil
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004271:DNA, Fungal; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D004271:DNA, Fungal; D013381:Subtilisins