Summary
Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences. After optimizing the transformation procedure, transformation efficiencies of 15–21 transformants/μ plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the β-tubuline gene of a benomyl-resistant Neurospora crassa mutant, the cotransformation rate was determined. Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies. In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions. The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies. Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus. Fractination of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus.
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References
Barrett V, Derjon RK, Lemke PA (1990) Genetic transformation of a mycorrhizal fungus. Appl Microbiol Biotechnol 33:313–316
Begueret J, Razanamparany V, Perrot M, Barreau C (1984) Cloning gene ura5 for the orotidylic acid pyrophosphorylase of the filamentous funugs Podospora anserina: transformation of ptotoplasts. Gene 32:487–492
Berges T, Barreau C (1989) Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina. J Gen Microbiol 135:601–604
Brygoo Y, Debuchy R (1985) Transformation by integration in Podospora anserina. I. methodology and phenomenology. Mol Gen Genet 200:128–131
Churchill ACL, Ciuffetti LM, Hansen DR, Van Etten HD (1990) Transformation of the fungal pathogen Cyrphonectria parasitica with a variety of heterologous plasmids. Curr Genet 17:25–31
Cooley RN, Shaw RK, Franklin FCH, Caten CA (1988) Transformation of the phytopathogenic fungus Septoria nodorum to hygromycin B resistance. Curr Genet 13:383–389
Esser K (1974) Podospora anserina. In: King RC (ed) Handbook of Genetics, Vol I. Plenum Press, New York, pp 531–551
Esser K, Kuenen R (1967) Genetics of Fungi, Springer, Berlin Heidelberg New York
Esser K (1985) Genetic control of aging: the mobile intron model. In: Bergener M, Ermini M, Stähelin HB (eds) The 1984 Sandoz Lectures in Gerontology. Thresholds in Aging. Academic Press, London, pp 3–20
Esser K, Kück U, Lang-Hinrichs C, Lemke P, Osiewacs HD, Stahl U, Tudzynski P (1986) Plasmids of Eukaryotes, Springer, Berlin Heidelberg New York Tokyo
Ferman ML, Oliver RP (1988) The transformation of protoplasts of Leptosphaera maculans to hygromycin B resistance. Curr Genet 13:327–330
Fernandez-Larrea J, Stahl U (1989) Transformation of Podospora anserina with a dominant resistance gene. Curr Genet 16:57–60
Kelly JM, Hynes MJ (1987) Multiple copies of the amdS gene of Aspergillus nidulans cause titration of trans acting regulatory proteins. Curr Genet 12:21–31
Kistler HC, Benny UK (1988) Genetic transformation of the fungal plant with pathogen, Fusarium oxysporum. Curr Genet 13:145–149
Kück U (1989) Mitochondria DNA rearrangements in Podospora anserina. Exp Mycol 13:111–120
Kück U, Osiewacz HD, Schmidt U, Kappelhoff B, Schulte E, Esser K (1985) The onset of senescence is affected by DNA rearrangements of a discontinuous mitochondrial gene in Podospora anserina. Curr Genet 9:373–382
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Marcou D, Picard-Bennoun M, Simonet JM (1984) Genetic map of Podospora anserina. In: O'Brien (ed) Genetic Maps, Vol 3, Cold Spring Harbor Laboratory, pp 253–261
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 (acu7). Mol Gen Genet 210:352–357
Miller BL, Miller KY, Timberlake WE (1985) Direct and indirect gene replacement in Aspergillus nidulans. Mol Cell Biol 5:1714–1721
Mohr G (1988) Grundlagen, Anwendungen und Möglichkeiten der Stammverbesserung biotechnologisch relevanter Hyphenpilze durch die Gentechnik: Integrative Transformation von Aspergillus niger. Dissertationes Botanicae 131
Mullaney EJ, Punt PJ, Hondel CAMJJ van Den (1988) DNA mediated transformation of Aspergillus ficuum. Appl Microbiol Biotechnol 28:451–454
Oliver RP, Roberts IN, Harling R, Kenyon L, Punt PJ, Dingenmanse MA, Hondel CAMJJ van den (1987) Transformation of Fulvia fulva, a fungal pathogen of tomato, to hygromycin B resistance. Curr Genet 12:231–233
Orbach MJ, Porro EB, Yanofsky C (1986) An electrophoretic karyotype of Neurospora crassa. Mol Cell Biol 6:2452–2461
Osiewacz HD, Weber A (1989) DNA mediated transformation of the filamentous fungus Curvularia lunata using a dominant selectable marker. Appl Microbiol Biotechnol 30:375–380
Osiewacz HD, Hermanns J, Marcou D, Triffi M, Esser K (1989) Mitochomdrial DNA rearrangements are correlated with a delayed amplification of the mobile intron (plDNA) in a long-lived mutant of Podospora anserina. Mutation Res 219:9–15
Osiewacz HD (1990) Molecular analysis of aging processes in fungi. Mutation Res 237:1–8
Osiewacz HD, Clairmont C, Huth M (1990) Electrophoretic karyotype of the ascomycete Podospora anserina. Curr Genet 18:481–483
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, Hondel CAMJJ van den (1987) Transformation of Aspergillus based on the hygromycin B resistance marker form Escherichia coli. Gene 56:117–124
Razanamparany V, Begueret J (1986) Positive screening and transformation of ura5 mutants in the fungus Podospora anserina: characterization of the transformant. Curr Genet 10:811–817
Sainsard-Chanet A, Begel O (1986) Transformation of yeast and Podospora anserina: innocuity of senescence-specific DNAs. Mol Gen Genet 204:443–451
Selker EU, Cambareri EB, Jensen BC, Haack KR (1987) Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51:741–752
Timberlake W, Boylan MT, Coolee MB, Mirabito PM, O'Hara EB, Willet CE (1985) Rapid identification of mutation-complementating restriction fragments from Aspergillus nidulans cosmid. Exp Mycol 9:351–355
Vieira J, Messing J (1982) The pUC plasmid, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Wernars K, Goosen T, Weenekes BMJ, Swart K, Hondel CAMJJ van den, Brook HWJ van den (1987) Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes. Mol Genet 209:71–77
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13 mp18 and pUC19 vectors. Gene 33:103–119
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Osiewacz, H.D., Skaletz, A. & Esser, K. Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes. Appl Microbiol Biotechnol 35, 38–45 (1991). https://doi.org/10.1007/BF00180633
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DOI: https://doi.org/10.1007/BF00180633