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
Ballance DJ, Buxton FP, Turner G (1983) Biochem Biophys Res Commun 112:284–289
Ballance DJ, Turner G (1985) Gene 36:321–331
Beggs JD (1978) Nature (London) 275:104–109
Clarke L, Carbon J (1980) Nature (London) 287:504–509
Clutterbuck AJ (1974) Aspergillus nidulans. In: King RC (ed) Handbook of genetics, vol 1. Plenum Press, New York London, p 447
Cove DJ (1966) Biochim Biophys Acta 113:51–56
Denhardt DT (1966) Biochem Biophys Res Commun 23:641–646
Futcher AB, Cox BS (1984) J Bacteriol 157:283–290
Grant DM, Lambowitz AM, Rambosek JA, Kinsey JA (1984) Mol Cell Biol 4:2041–2051
Hicks JB, Hinnen A, Fink GR (1979) Cold Spring Harbor Symp Quant Biol 43:1305–1313
Hsiao C-L, Carbon J (1979) Proc Natl Acad Sci USA 76:3829–3833
Ish-Horowicz D, Burke JF (1981) Nucleic Acids Res 9:2989–2998
Johnstone IL, Hughes SG, Clutterbuck AJ (1985) EMBO J 4:1307–1311
Kinnaird JH, Keighren MA, Kinsey JA, Eaton M, Fincham JRS (1982) Gene 20:387–396
Larionov V, Kouprina N, Karpova T (1984) Gene 28:229–235
Lederberg EM, Cohen SN (1974) J Bacteriol 119:1072–1074
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Murray AW, Szostak JW (1983a) Cell 34:961–970
Murray AW, Szostak JW (1983b) Nature (London) 305:189–193
Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) J Mol Biol 113:237–251
Sakaguchi J, Yamamoto M (1982) Proc Natl Acad Sci USA 79:7819–7823
Southern EM (1975) J Mol Biol 98:503–517
Stinchcomb DT, Thomas M, Kelly J, Selker E, Davis RW (1980) Proc Natl Acad Sci USA 77:4559–4563
Stohl LL, Lambowitz AM (1983a) Proc Natl Acad Sci USA 80: 1058–1062
Stohl LL, Lambowitz AM (1983b) Anal Biochem 134:82–85
Stohl LL, Akins RA, Lambowitz AM (1984) Nucleic Acids Res 12:6169–6178
Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) Proc Natl Acad Sci USA 76:1035–1039
Szostak JW, Wu R (1979) Plasmid 2:536–554
Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW (1983) Gene 26:205–221
Yelton MM, Hamer JE, Timberlake WE (1984) Proc Natl Acad Sci USA 81:1470–1474
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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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DOI: https://doi.org/10.1007/BF00405100