Summary
A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per μg DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus.
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Arnau J, Murillo FJ, Torres-Martinez S (1988) Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus. Mol Gen Genet 212:375–377
Arnau J, Jepsen LP, Stroman P (1991) Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides. Mol Gen Genet 225:193–198
Ballance DJ, Turner G (1985) Development of a high frequency transforming vector for Aspergillus nidulans. Gene 36:321–331
Beri RJ, Lewis EL, Turner G (1988) Behaviour of a replicating mitochondrial DNA sequence from Aspergillus amstelodomi in Saccharomyces cerevisiae and Aspergillus nidulans. Curr Genet 13:479–486
Daboussi MJ, Ajeballi A, Gerlinger C, Blaiseau PL, Bouvier I, Cassan M, Lebrun MHL, Parisot D, Brigoo Y (1989) Transformation of seven species of filamentous fungi using the nitrate reductase gene of Aspergillus nidulans. Curr Genet 15:453–456
Graaf L, Van den Broek H, Visser J (1988) Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans. Curr Genet 13:315–321
Hanahan D (1986) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning, vol 1. A practical approach. IRL Press, Oxford, Washington DC, pp 109–135
Holmes PS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
Iimura Y, Gomi K, Vzu H, Hara S (1987) Transformation of Aspergillus oryzae through plasmid mediated complementation of a methionine auxotrophic mutation. Agric Biol Chem 51:323–328
Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Mattern IF, Unkles S, Kinghorn JR, Pouwels PH, Van den Hondel CAMJJ (1987) Transformation of Aspergillus oryzae using the A. niger pyrG gene. Mol Gen Genet 210:460–461
Mellon FM, Little PFR, Casselton LA (1987) Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: isolation and expression of the isocitrate lyase gene (acu-7). Mol Gen Genet 210:352–357
Oakley BR, Rinehart JE, Mitchel BL, Oakley CE, Carmona C, Gray GL, May GS (1987a) Cloning, mapping and molecular analysis of the pyrG (orotidine-5′-phosphate decarboxylase) gene of Aspergillus nidulans. Gene 61:385–399
Oakley CE, Weil CF, Kretz PL, Oakley BR (1987b) Cloning of the riboB locus of Aspergillus nidulans. Gene 53:293–298
Revuelta JL, Jayaram M (1986) Transformation of Phycomyces blakesleeanus to G-418 resistance by an autonomously replicating plasmid. Proc Natl Acad Sci USA 83:7344–7347
Roncero MIG (1984) Enrichment method for the isolation of auxotrophic mutants of Mucor using the polyene antibiotic N-glycosyl-polifungin. Carlsberg Res Commun 49:685–690
Roncero MIG, Jepsen LP, Stroman P, Van Heeswijck R (1989) Characterization of a leuA gene and an ARS element from Mucor circinelloides. Gene 84:335–343
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Schipper MAA (1976) On Mucor racemosus and related species. Studies in Mycology 12:1–40
Sealey PG, Southern EM (1987) Electrophoresis of DNA. In: Rickwood D, Hames BD (eds) Gel electrophoresis of nucleic acids. A practical approach. IRL Press, Oxford, Washington DC, pp 39–75
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Suarez T, Eslava AP (1988) Transformation of Phycomyces with a bacterial gene for kanamycin resistance. Mol Gen Genet 212:120–123
Unkles SE, Campbell EI, Carrez D, Grieve C, Contreras S, Fiers W, Van den Hondel CAMJJ, Kinghorn JR (1989) Transformation of Aspergillus niger with the homologous nitrate reductase gene. Gene 78:157–166
Van Hartingsveldt W, Mattern IE, Van Zeijl CMJ, Pouwels PH, Van den Hondel CAMJJ (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75
Van Heeswijck R (1986) Autonomous replication of plasmids in Mucor transformants. Carlsberg Res Commun 51:433–443
Van Heeswijck R, Roncero MIG (1984) High frequency transformation of Mucor with recombinant plasmid DNA. Carlsberg Res Commun 49:691–702
Wnendt S, Jacobs M, Stahl U (1990) Transformation of Aspergillus giganteus to hygromycin B resistance. Curr Genet 17:21–24
Wöstemeyer J, Burmester A, Wiegel C (1987) Neomycin resistance as a dominantly selectable marker for transformation of the zygomycete Absidia glauca. Curr Genet 12:625–627
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Communicated by C. van den Hondel
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Anaya, N., Roncero, M.I.G. Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene. Molec. Gen. Genet. 230, 449–455 (1991). https://doi.org/10.1007/BF00280302
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DOI: https://doi.org/10.1007/BF00280302