The cytosolic chitinase from Neurospora crassa
Chitinases have been implicated in aspects of growth and morphology of chitin containing fungi such as hyphal branching, anastamosis and, possibly, apical growth itself. Cloning of Neurospora crassa chitinase genes would allow a molecular genetic analysis of the involvement of chitinases in such phenomena through the study of regulation, subcellular location and the use of specific mutagenesis. The cytosolic chitinase was partially purified to a high specific activity and characterised. Allosamidin, the first specific inhibitor of chitinase to be described, was shown to be a potent inhibitor of the cytosolic chitinase. Direct sequencing of the chitinase protein was not possible due to the inability to purify the protein to homogeneity. However, protein sequence information was obtained from protein electroblotted onto Immobilon polyvinylide difluoride membrane, after first identifying the chitinase protein band in situ after polyacrylamide gel electrophoresis using a specific activity stain. Protein sequence data was used to design an oligonucleotide probe for the screening of a genomic library in the cosmid vector pSV50. Screening of the library resulted in the identification of a single positive cosmid. The region of cosmid 23D6 that showed hybridisation to the oligonucleotide was subcloned and sequenced. The oligonucleotide was shown to hybridise to a sequence that had a single mis-match. However, when the sequence surrounding the oligonucleotide binding site was converted to an amino acid sequence, using the reading frame for the chitinase protein sequence, no homology with the remaining chitinase protein sequence was seen.