Cloning and characterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruption of which decreases germination frequency and hyphal growth.

We cloned a chitinase-encoding gene from Aspergillus nidulans by polymerase chain reaction using degenerated oligonucleotide primers designed from the conserved amino acid sequences among chitinases from yeasts and Rhizopus spp. The cloned gene, named chiA, encoded a polypeptide consisting of 660 amino acids. Disruption of chiA had no effect on hyphal or conidiophore morphology, but germination frequency and hyphal growth rate decreased substantially. Expression of chiA was investigated using Escherichia coli beta-galactosidase as a reporter enzyme. The beta-galactosidase activity was present during hyphal growth and increased twice as the conidiophores developed. In situ staining of beta-galactosidase activity found high expression in metulae, phialides, and conidia during conidiophore development, indicating that the expression of chiA is developmentally regulated. This is the first report to isolate a chitinase gene from A. nidulans and investigate its functions using the gene disruption technique and gene fusion methods in filamentous fungi.

[1]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[3]  C. Yanofsky,et al.  Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa. , 1991, Genes & development.

[4]  R. Waring,et al.  Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes. , 1989, Gene.

[5]  A. Ohta,et al.  Isolation and characterization of two chitin synthase genes from Aspergillus nidulans. , 1994, Bioscience, biotechnology, and biochemistry.

[6]  D. Ballance.  Sequences important for gene expression in filamentous fungi , 1986, Yeast.

[7]  P. Robbins,et al.  Molecular cloning and characterization of chitinase genes from Candida albicans. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  T. Kirkland,et al.  Isolation and characterization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccidioides immitis. , 1995, Gene.

[9]  P. Blaiseau,et al.  Primary structure of a chitinase-encoding gene (chi1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases. , 1992, Gene.

[10]  M. Van Montagu,et al.  Characterization of ech-42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism. , 1994, Proceedings of the National Academy of Sciences of the United States of America.