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Behaviour of recombinant plasmids in Aspergillus nidulans: structure and stability

Summary

A pyrG Aspergillus strain was transformed with plasmid pDJB-1, derived from pBR325 by insertion of the Neurospora crassa pyr4 gene (orotidine 5′-phosphate carboxylase), giving mitotically unstable transformants. Aspergillus DNA which acted as an “autonomously replicating sequence” (ARS) in yeast was inserted into pDJB-1 and the resulting construct, pDJB12.1, gave mitotically stable transformants when introduced into Aspergillus. Transformants obtained with pDJB-1 and pDJB12.1 gave few pyr progeny in crosses to a pyrG + strain. Southern hybridisation analysis of pyr + transformants obtained with pDJB-1 revealed restriction fragments expected for integrated plasmid but transformants obtained with pDJB12-1 showed only bands derived from free plasmid. pDJB-1 and derivatives of pDJB12.1 could be recovered from transformants. These derivatives could not be explained by straightforward excision of integrated pDJB12.1 sequences but could result from recombination between plasmid molecules. Hybridisation of undigested transformant DNAs showed that the transforming DNA was present in a high molecular weight form. These results suggest: (1) pDJB12.1 derivatives and possibly pDJB-1 can replicate autonomously in Aspergillus; (2) A. nidulans DNA acting as an ARS in yeast enhances replication and/or segregation of transforming plasmids in Aspergillus; and (3) recombinant plasmids may undergo rearrangements when introduced into Aspergillus.

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Abbreviations

PABA:

para-amino benzoic acid

EDTA:

disodium salt of ethylene diamine tetra-acetic acid

SDS:

sodium dodecyl sulphate

DTT:

dithiothreitol

UV:

ultra violet

SSC:

standard saline citrate; 0.15 M sodium chloride, 0.015 M trisodium citrate pH 7.

ARS('s):

autonomously replicating sequence(s)

kb:

kilobase pairs

References

  1. Ballance DJ, Buxton FP, Turner G (1983) Biochem Biophys Res Commun 112:284–289

  2. Ballance DJ, Turner G (1985) Gene 36:321–331

  3. Beggs JD (1978) Nature (London) 275:104–109

  4. Clarke L, Carbon J (1980) Nature (London) 287:504–509

  5. Clutterbuck AJ (1974) Aspergillus nidulans. In: King RC (ed) Handbook of genetics, vol 1. Plenum Press, New York London, p 447

  6. Cove DJ (1966) Biochim Biophys Acta 113:51–56

  7. Denhardt DT (1966) Biochem Biophys Res Commun 23:641–646

  8. Futcher AB, Cox BS (1984) J Bacteriol 157:283–290

  9. Grant DM, Lambowitz AM, Rambosek JA, Kinsey JA (1984) Mol Cell Biol 4:2041–2051

  10. Hicks JB, Hinnen A, Fink GR (1979) Cold Spring Harbor Symp Quant Biol 43:1305–1313

  11. Hsiao C-L, Carbon J (1979) Proc Natl Acad Sci USA 76:3829–3833

  12. Ish-Horowicz D, Burke JF (1981) Nucleic Acids Res 9:2989–2998

  13. Johnstone IL, Hughes SG, Clutterbuck AJ (1985) EMBO J 4:1307–1311

  14. Kinnaird JH, Keighren MA, Kinsey JA, Eaton M, Fincham JRS (1982) Gene 20:387–396

  15. Larionov V, Kouprina N, Karpova T (1984) Gene 28:229–235

  16. Lederberg EM, Cohen SN (1974) J Bacteriol 119:1072–1074

  17. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

  18. Murray AW, Szostak JW (1983a) Cell 34:961–970

  19. Murray AW, Szostak JW (1983b) Nature (London) 305:189–193

  20. Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) J Mol Biol 113:237–251

  21. Sakaguchi J, Yamamoto M (1982) Proc Natl Acad Sci USA 79:7819–7823

  22. Southern EM (1975) J Mol Biol 98:503–517

  23. Stinchcomb DT, Thomas M, Kelly J, Selker E, Davis RW (1980) Proc Natl Acad Sci USA 77:4559–4563

  24. Stohl LL, Lambowitz AM (1983a) Proc Natl Acad Sci USA 80: 1058–1062

  25. Stohl LL, Lambowitz AM (1983b) Anal Biochem 134:82–85

  26. Stohl LL, Akins RA, Lambowitz AM (1984) Nucleic Acids Res 12:6169–6178

  27. Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035–1039

  28. Szostak JW, Wu R (1979) Plasmid 2:536–554

  29. Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW (1983) Gene 26:205–221

  30. Yelton MM, Hamer JE, Timberlake WE (1984) Proc Natl Acad Sci USA 81:1470–1474

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Correspondence to D. W. MacDonald.

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Barnes, D.E., MacDonald, D.W. Behaviour of recombinant plasmids in Aspergillus nidulans: structure and stability. Curr Genet 10, 767–775 (1986). https://doi.org/10.1007/BF00405100

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Key words

  • Aspergillus
  • Transformation
  • Recombinant plasmids
  • Autonomously replicating sequences