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chs-4, a class IV chitin synthase gene fromNeurospora crassa

Abstract

InSaccharomyces cerevisiae, most of the cellular chitin is produced by chitin synthase III, which requires the product encoded by theCSD2/CAL1/DIT101/KT12 gene. We have identified, isolated and structurally characterized aCSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomyceteNeurospora crassa and have used a “reverse genetics” approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of aN. crassa gene (designatedchs-4) encoding a polypeptide belonging to a class of chitin synthases which we have designated class IV. The predicted polypeptide encoded by this gene is highly similar to those ofS. cerevisiae andCandida albicans. N. crassa strains in whichchs-4 had been inactivated by the Repeat-Induced Point mutation (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in thechs-4 RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme inN. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.

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References

  1. Beth Din A, Yarden O (1994) TheNeurospora crassa chs-2 gene encodes a non-essential chitin synthase. Microbiology 140:2189–2197

  2. Bowen AR, Chen-Wu JL, Momany M, Young R, Szaniszlo PJ, Robbins PW (1992) Classification of fungal chitin synthases. Proc Natl Acad Sci USA 89:519–523

  3. Bulawa CE (1992)CSD2, CSD3, andCSD4, genes required for chitin synthesis inSaccharomyces cerevisiae: theCSD2 gene product is related to chitin synthases and to developmentally regulated proteins inRhizobium species andXenopus laevis. Mol Cell Biol 12:1764–1776

  4. Bulawa CE (1993) Genetics and molecular biology of chitin synthesis in fungi. Annu Rev Microbiol 47:505–534

  5. Bulawa CE, Slater M, Cabib E, Au-Young J, Sburlati A, Adair WL, Robbins PW (1986) TheS. cerevisiae structural gene for chitin synthase in not required for chitin synthesis in vivo. Cell 46:213–225

  6. Burnett JH (1979) Aspects of the structure and growth of hyphal walls. In: Burnett JH, Trinci APJ (eds) Fungal walls and hyphal growth. Cambridge University Press, Cambridge, pp 1–25

  7. Cabib E, Mol PC, Shaw JA, Choi WJ (1993) Biosynthesis of cell wall and septum during yeast growth. Arch Med Res 24:301–303

  8. Chen-Wu JL, Zwicker J, Bowen AR, Robbins PW (1992) Expression of chitin synthase genes during yeast and hyphal growth phases ofCandida albicans. Mol Microbiol 6:497–502

  9. Choi WJ, Santos B, Duran A, Cabib E (1994) Are yeast chitin synthases regulated at the transcriptional or posttranslational level? Mol Cell Biol 14:7685–7694

  10. Davis RH, de Serres FJ (1970) Genetic and microbiological research techniques forNeurospora crassa. Methods Enzymol 17A:79–143

  11. Gold SE, Kronstad JW (1994) Disruption of two genes for chitin synthase in the phytopathogenic fungusUstilago maydis. Mol Microbiol 11:897–902

  12. Hunsley D, Gooday GW (1974) The structure and development of septa inNeurospora crassa. Protoplasma 82:125–146

  13. Kollar R, Petrakova E, Ashwell G, Robbins PW, Cabib E (1995) Architecture of the yeast cell wall — the linkage between chitin and beta (1 → 3)-glucan. J Biol Chem 270:1170–1178

  14. Mahadevan PR, Tatum EL (1965) Relationship of the major constituents of theNeurospora crassa cell wall to wild-type and colonial morphology. J Bacteriol 90:1073–1081

  15. Mehmann B, Brunner I, Braus GH (1994) Nucleotide sequence variation of chitin synthase genes among ectomycorrhizal fungi and its potential use in taxonomy. Appl Environ Microbiol 60:3105–3111

  16. Mellado E, Aufauvre-Brown A, Specht CA, Robbins PW, Holden DW (1995) A multigene family related to chitin synthase genes of yeast in the opportunistic pathogenAspergillus fumigatus. Mol Gen Genet 246:353–359

  17. Metzenberg RL, Stevens JN, Selker EU, Morzycka-Wroblewska E (1985) Identification and chromosomal distribution of 5S rRNA genes inNeurospora crassa. Proc Natl Acad Sci USA 82:2067–2071

  18. Motoyama T, Sudoh M, Horiuchi H, Ohta A, Takagi M (1994a) Isolation and characterization of two chitin synthase gene ofRhizopus oligosporus. Biosci Biotech Biochem 58:1685–1693

  19. Motoyama T, Kojima N, Horiuchi H, Ohta A, Takagi M (1994b) Isolation of a chitin synthase gene (chsC) ofAspergillus nidulens. Biosci Biotech Biochem 58:2254–2257

  20. Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene forβ-tubulin from a benomyl-resistant mutant ofNeurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461

  21. Orbach MJ, Sachs MS, Yanofsky C (1990) TheNeurospora crassa arg-2 locus: structure and expression of the gene encoding the small subunit of the arginine-specific carbamoyl phosphate synthethase. J Biol Chem 265:10981–10987

  22. Pammer M, Briza P, Ellinger A, Schuster T, Stucka R, Feldman H, Breitenbach M (1992)DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls. Yeast 9:1089–1099

  23. Reissig JL, Stringer JL, Leloir LF (1955) A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem 217:959–966

  24. Robbins PW, Albright C, Benfield B (1988) Cloning and expression of aStreptomyces plicatus chitinase (chitinase-63) inEscherichia coli. J Biol Chem 263:443–447

  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  26. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

  27. Schmidt JC, Edson CM, Brody S (1975) Changes in glucosamine and galactoseamine levels during conidial germination inNeurospora crassa. J Bacteriol 122:1062–1070

  28. Sedmak JJ, Grossbery SE (1977) A rapid sensitive and versatile assay for protein using Coomassie brilliant blue (G-250). Anal Biochem 79:544–552

  29. Selker EU (1990) Premeiotic instability of repeated sequences inNeurospora crassa. Annu Rev Genet 24:579–613

  30. Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation inNeurospora crassa. Proc Natl Acad Sci USA 85:6870–6874

  31. Shaw JA, Mol PC, Bowers B, Silverman SJ, Valdivieso MH, Duran A, Cabib E (1991) The function of chitin synthases 2 and 3 in theSaccharomyces cerevisiae cell cycle. J Cell Biol 114:111–123

  32. Staben C, Jensen B, Singer M, Pollock J, Schectman M, Kinsey J, Selker E (1989) Use of a bacterial hygromycin B resistance gene as a dominant selectable marker inNeurospora crassa transformation. Fungal Genet Newslett 36:79–81

  33. Sudoh M, Nagahasi S, Doi M, Ohta A, Takagi M, Arisawa M (1993) Cloning of the chitin synthase 3 gene fromCandida albicans and its expression during the yeast-hyphal transition. Mol Gen Genet 241:351–358

  34. Valdivieso MH, Mol PC, Shaw JA, Cabib E, Duran A (1991)CAL1, a gene required for activity of chitin synthase 3 inSaccharomyces cerevisiae. J Cell Biol 114:101–109

  35. Vogel HJ (1956) A convenient growth medium forNeurospora crassa (medium N). Microbiol Genet Bull 13:42–43

  36. Yanai K, Kojima N, Takaya N, Horiuchi H, Ohta A, Takagi M (1994) Isolation and characterization of two chitin synthase genes fromAspergillus nidulans. Biosci Biotech Biochem 58:1828–1835

  37. Yarden O, Yanofsky C (1991) Chitin synthase 1 plays a major role in cell wall biogenesis ofNeurospora crassa. Genes Dev 5:2420–2430

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Author information

Correspondence to Oded Yarden.

Additional information

A. Beth Din and C. A. Specht contributed equally to this work

Communicated by C. A. M. J. J. van den Hondel

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Din, A.B., Specht, C.A., Robbins, P.W. et al. chs-4, a class IV chitin synthase gene fromNeurospora crassa . Molec. Gen. Genet. 250, 214–222 (1996). https://doi.org/10.1007/BF02174181

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Key words

  • Neurospora
  • Ascomycete
  • Fungal cell wall