CHS2, a chitin synthase gene from the oomycete Saprolegnia monoica

Abstract

PCR was used to amplify fragments corresponding to the chitin synthase (CHS) genes from the Oomycetes Saprolegnia monoica, Phytophthora capsicum and Achlya ambisexualis, utilizing as primers, oligonucleotides designed from the conserved region of CHS genes of chitinous fungi. Chitin synthase homologues were found in the three cellulosic fungi. The chitin synthase 2 gene (CHS2) from S. monoica was cloned, sequenced and characterized. The amino acid sequence deduced from the CHS2 genomic DNA revealed several domains, corresponding to the catalytic domains and polypeptide signatures, of high identity with CHS genes from chitinous fungi. Existence of a CHS gene family in S. monoica was supported by the identification of two CHS sequences among the PCR products, the localization of CHS homologues on two chromosomes, and the detection of two transcripts in mycelia and protoplasts. Polyclonal anti-chitin synthase antibodies raised against the N-terminal and the neutral fragments of the CHS2 products revealed, respectively, two and four proteins in membrane fractions and a truncated active form in entrapped product. The overall comparison of the structure and organization of CHS genes indicates that in spite of their divergent evolution, Oomycetes and chitinous fungi have evolved with conserved chitin synthase systems.