Chitin synthase 1 plays a major role in cell wall biogenesis in Neurospora crassa.
In filamentous fungi, chitin is a structural component of morphologically distinct structures assembled during various phases of growth and development. To investigate the role of chitin synthase in cell wall biogenesis in Neurospora crassa, we cloned a chitin synthase structural gene and examined the consequences of its inactivation. Using degenerate oligonucleotide mixtures designed on the basis of conserved sequences of the Saccharomyces cerevisiae CHS1 and CHS2 polypeptides, a DNA fragment encoding a similar predicted amino acid sequence was amplified from N. crassa genomic DNA. This product was used to probe N. crassa libraries for a gene homologous to one of the yeast genes. Full-length genomic and partial cDNA clones were identified, isolated, and sequenced. The amino acid sequence deduced from a cloned 3.4-kb gene [designated chitin synthase 1 (chs-1)] was very similar to that of the S. cerevisiae CHS1 and CHS2 and the Candida albicans CHS1 polypeptides. Inactivation of the N. crassa chs-1 gene by repeat-induced point mutation produced slow-growing progeny that formed hyphae with morphologic abnormalities. The chs-1RIP phenotype was correlated with a significant reduction in chitin synthase activity. Calcofluor staining of the chs-1RIP strain cross-walls, residual chitin synthase activity, and the increased sensitivity of the chs-1RIP strain to Nikkomycin Z suggest that N. crassa produces additional chitin synthase that can participate in cell wall formation.
Yarden
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Department of Biological Sciences, Stanford University, California 94305.
Yanofsky
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002473:Cell Wall / Q000201:enzymology / Q000502:physiology*; D002687:Chitin Synthase / Q000235:genetics / Q000502:physiology*; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal / Q000201:enzymology; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal / Q000502:physiology; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012689:Sequence Homology, Nucleic Acid
D005656:Fungal Proteins; D002687:Chitin Synthase