Current Genetics

, Volume 24, Issue 5, pp 421–427 | Cite as

Targeted integration into the Acremonium chrysogenum genome: disruption of the pcbC gene

  • Markus Walz
  • Ulrich Kück
Original Articles

Abstract

The cephalosporin C-producing fungus Acremonium chrysogenum was transformed to hygromycin B resistance using different vector constructs. These constructs contain sequences of the pcbC gene from A. chrysogenum, encoding isopenicillin N synthetase. Detailed analysis of transformants, including pulsed-field gel electrophoresis (PFGE), suggests that integration of multiple vector copies takes place predominantly via non-homologous integration. By increasing the length of vector-DNA homologous to genomic DNA, integration occurs more frequently into chromosome VI, carrying the endogencous pcbC gene copy. In gene disruption experiments, the length of vector homology required to obtain cephalosporin C-minus transformants was investigated. Inactivation of the pcbC gene was observed only when homologous fragments of more than 3.0 kb were used on both sites of the resistance cassette. Southern analysis indicated homologous, as well as heterologous, integration of recombinant DNA. The integration of multiple vector copies leads to the appearance of truncated pcbC transcripts.

Key words

Acremonium chrysogenum pcbC gene Transformation Gene disruption Pulsed-field gel electrophoresis 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Barredo JL, Díez B, Alvarez E, Martín JF (1989) Mol Gen Genet 16:453–459Google Scholar
  2. Binninger DM, Le Chevanton L, Skrzynia C, Shubkin CD, Pukkila PJ (1991) Mol Gen Genet 227:245–251Google Scholar
  3. Buckholz RG, Gleeson MAG (1991) Bio/Technology 9:1067–1072Google Scholar
  4. Case ME (1986) Genetics 113:569–587Google Scholar
  5. Cheevadhanarak S, Renno DV, Saunders G, Holt G (1991) Gene 108:151–155Google Scholar
  6. Durand N, Reymond P, Fèvre M (1991) Curr Genet 19:149–153Google Scholar
  7. Fincham JRS (1989) Microbiol Rev 53:148–170Google Scholar
  8. Gems D, Johnstone IJ, Clutterbuck AJ (1991) Gene 98:61–67Google Scholar
  9. Giasson L, Specht CA, Milgrim C, Novotny CP, Ullrich RC (1989) Mol Gen Genet 218:72–77Google Scholar
  10. Goyon C, Faugeron G (1989) Mol Cell Biol 9:2818–2827Google Scholar
  11. Gritz L, Davies J (1983) Gene 25:179–188Google Scholar
  12. Gutiérrez S, Díez B, Montenegro E, Martín JF (1991) J Bacteriol 173:2354–2365Google Scholar
  13. Gwyn Jones I, Sealy-Lewis HM (1989) Curr Genet 15:135–142Google Scholar
  14. Hasty P, Rivera-Pérez J, Bradley A (1991) Mol Cell Biol 11:5586–5591Google Scholar
  15. Hata Y, Kitamoto K, Gomi K, Kumagai C, Tamura G (1992) Curr Genet 22:85–91Google Scholar
  16. Hoge JHC, Springer J, Zanting B, Wessles JGH (1982) Exp Mycol 6:225–232Google Scholar
  17. Hoskins JA, O'Callaghan N, Queener SW, Cantwell CA, Wood JS, Chen VJ, Skatrud PL (1990) Curr Genet 18:523–530Google Scholar
  18. Hunter GD, Bailey CR, Arst HN Jr (1992) Curr Genet 22:377–383Google Scholar
  19. Isogai T, Fukagawa M, Aramori I, Iwami M, Kojo H, Ono T, Ueda Y, Kohsaka M, Imanaka H (1991) Bio/Technology 9:188–191Google Scholar
  20. Keller NP, Bergstrom GC, Yoder OC (1991) Curr Genet 19:227–233Google Scholar
  21. Khasanov FK, Zvingila DJ, Zainullin AA, Prozorov AA, Bashkirov VI (1992) Mol Gen Genet 234:494–497Google Scholar
  22. Kistler HC, Benny U (1992) Gene 117:81–89Google Scholar
  23. Kück U, Walz M, Mohr G, Mracek M (1989) Appl Microbiol Biotechnol 31:358–365Google Scholar
  24. Le Chevanton L, Leblon G, Lebilcot S (1989) Mol Gen Genet 218:390–396Google Scholar
  25. Lopes TS, Hakkaart G-JAJ, Koerts BL, Raué HA, Planta RJ (1991) Gene 105:83–90Google Scholar
  26. Malmberg L-H, Hu W-S (1992) Appl Microbiol Biotechnol 38:122–128Google Scholar
  27. Mansour SL, Thomas KR, Capecchi MR (1988) Nature 336:348–352Google Scholar
  28. Mathison L, Soliday S, Stepan T, Aldrich T, Rambosek J (1993) Curr Genet 23:33–41Google Scholar
  29. Mézard C, Pompon D, Nicolas A (1992) Cell 70:659–670Google Scholar
  30. Osiewacz HD, Skaletz A, Esser K (1991) Appl Microbiol Biotechnol 35:38–45Google Scholar
  31. Picard M, Debuchy R, Julien J, Brygoo Y (1987) Mol Gen Genet 210:129–134Google Scholar
  32. Punt PJ, Greaves PA, Kuyvenhoven A, van Deutekom JCT, Kinghorn JR, Pouwels PH, van den Hondel CAMJJ (1991) Gene 104:119–122Google Scholar
  33. Queener SW, Ingolia TD, Skatrud PL, Chapman JL, Kaster KR (1985) A system for genetic transformation of Cephalosporium acremonium. In: Leive L (ed) Microbiology-1985. American Society for Microbiology, Washington D.C., pp 468–472Google Scholar
  34. Razanamparany V, Bégueret J (1988) Gene 74:399–409Google Scholar
  35. Sakai A, Shimizu Y, Hishinuma F (1990) Appl Microbiol Biotechnol 33:302–306Google Scholar
  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  37. Schiestl RH, Petes TD (1991) Proc Natl Acad Sci USA 88:7585–7589Google Scholar
  38. Selker EU, Cambareri EB, Jensen BC, Haack KR (1987) Cell 51:741–752Google Scholar
  39. Shen P, Huang HV (1986) Genetics 112:441–457Google Scholar
  40. Skatrud PL, Queener SW (1989) Gene 78:331–338Google Scholar
  41. Skatrud PL, Queener SW, Carr LG, Fisher DL (1987) Curr Genet 12:337–348Google Scholar
  42. Skatrud PL, Tietz AJ, Ingolia TD, Cantwell CA, Fisher DL, Chapman JL, Queener SW (1989) Bio/Technology 7:477–485Google Scholar
  43. Smith AW, Ramsden M, Peberdy JF (1992) Gene 114:211–216Google Scholar
  44. Smith DJ, Bull JH, Edwards J, Turner G (1989) Mol Gen Genet 216:492–497Google Scholar
  45. Stauffer JF, Schwartz LJ, Brady CW (1966) Dev Ind Microbiol 7:104–113Google Scholar
  46. Steele PE, Carle GF, Kobayshi GS, Medoff G (1989) Mol Cell Biol 9:983–987Google Scholar
  47. Struhl K (1983) Nature 305:391–397Google Scholar
  48. Struhl K, Cameron JR, Davies RW (1976) Proc Natl Acad Sci USA 73:1471–1475Google Scholar
  49. Tsukuda T, Carleton S, Fotheringham S, Holloman WK (1988) Mol Cell Biol 8:3703–3709Google Scholar
  50. Waldron C, Murphy EB, Roberts JL, Gustafson GD, Armur SL, Malcom SK (1985) Plant Mol Biol 5:103–108Google Scholar
  51. Walz M (1992) Bibl Mycol 147Google Scholar
  52. Walz M, Kück U (1991) Curr Genet 19:73–76Google Scholar
  53. Ward M, Wilson LJ, Kodama KH, Rey MW, Berka RM (1990) Bio/Technology 8:435–440Google Scholar
  54. Whitehead MP, Gurr SJ, Grieve C, Unkles SE, Spence D, Ramsden M, Kinghorn JR (1990) Gene 90:193–198Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Markus Walz
    • 1
  • Ulrich Kück
    • 1
  1. 1.Lehrstuhl für Allgemeine BotanikRuhr-Universität BochumBochumGermany

Personalised recommendations