Summary
Phycomyces protoplasts transformed with a plasmid containing the bacterial gene for kanamycin resistance grow in the presence of G418, a kanamycin analogue. The plasmid also contains a Phycomyces DNA sequence that supports autonomous replication in yeast. We obtained about 250 transformants per microgram DNA or one per 5000 viable protoplasts. The transformant phenotype is retained under selective conditions and lost in the majority of the vegetative spores. Recovered plasmids and Southern analysis indicate that the plasmid probably replicates autonomously in Phycomyces.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ballance DJ, Buxton FP, Turner G (1983) Transformation of Aspergillus nidulans by the orotidine-5-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Commun 112:284–289
Bergman K, Burke P, Cerdá-Olmedo E, David CN, Delbrück M, Foster KW, Goodell EW, Heisenberg M, Meissner G, Zalokar M, Dennison DS, Shropshire W (1969) Phycomyces. Bacteriol Rev 33:99–157
Case ME, Schweizer M, Kushner SR and Giles NH (1979) Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA 76:5259–5263
Cerdá-Olmedo E (1977) Behavioral genetics of Phycomyces. Annu Rev Microbiol 31:535–547
Cerdá-Olmedo E (1985) Carotene mutants of Phycomyces. In: Law JH, Rilling HC (eds) Methods Enzymol 110:220–243
Davis RW, Botstein D, Roth J (1980) Advanced bacterial genetics laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Heisenberg M, Cerdá-Olmedo E (1968) Segregation of heterokaryons in the asexual cycle of Phycomyces. Mol Gen Genet 102:187–195
Jiménez A, Davies J (1980) Expression of a transposable antibiotic resistance element in Saccharomyces. Nature 287:869–871
Kinsey JA (1985) Neurospora plasmids. In: Bennett JW, Lasure L (eds) Gene manipulations in fungi. Academic Press, pp 254–258
Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206
Lipson ED (1983) Sensory information processing at the cellular level: the light response system in Phycomyces. In: Scott GP, Wahner HW (eds) Radiation and cellular response. Iowa State Univ Press, Ames, pp 133–152
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Murray AW, Szostak JW (1983) Pedigree analysis of plasmid segregation in yeast. Cell 34:961–970
Oka A, Sugisaki H, Takanami M (1981) Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147:217–226
Revuelta JL, Jayaram M (1986) Transformation of Phycomyces blakesleeanus to G-418 resistance by an autonomous replicating plasmid. Proc Natl Acad Sci USA 83:7344–7347
Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J Mol Biol 113: 237–251
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Specht CA, DiRusso CC, Novotny CP, Ullrich RC (1982) A method for extracting high molecular weight deoxyribonucleic acid from fungi. Anal Biochem 119:158–163
Stinchcomb DT, Thomas M, Kelly J, Selker E, Davis RW (1980) Eukaryotic DNA segments capable of autonomous replication in yeasts. Proc Natl Acad Sci USA 77:4559–4563
Stohl LL, Akins RA, Lambowitz AM (1984) Characterization of deletion derivatives of an autonomous replicating Neurospora plasmid. Nucleic Acids Res 12:6169–6178
Suárez T (1985) Obtencion de protoplastos y transformacion en Phycomyces blakesleeanus. PhD Thesis, University of Salamanca
Suárez T, Orejas M, Eslava AP (1985) Isolation, regeneration and fusion of Phycomyces blakesleeanus spheroplasts. Exp Mycol 9:203–211
Sutter RP (1975) Mutations affecting sexual development in Phycomyces blakesleeanus. Proc Natl Acad Sci USA 72:127–130
Sutter RP (1977) Regulation of the first stage of sexual development in Phycomyces blakesleeanus. In: O'Day DH, Itorgen PA (eds) Eukaryotic microbes as model as developmental systems. Marcel Dekker, New York, vol 2, pp 251–272
Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissman JH, Lockington RA, Davies RW (1983) Transformation by integration in Aspergillus nidulans. Gene 26:205–221
Turner G, Ballance DJ (1985) Cloning and transformation in Aspergillus. In: Bennet JW, Lasure L (eds) Gene manipulations in fungi. Academic Press, pp 259–278
Van Heeswijck R, Roncero MIG (1984) High frequency transformation of Mucor with recombinant plasmid DNA. Carlsberg Res Commun 49:691–702
Webster TD, Dickson RC (1983) Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903. Gene 26:243–252
Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474
Zakian VA, Scott JF (1982) Construction, replication and chromatin structure of TRP1 R1 circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA. Mol Cell Biol 2:221–232
Author information
Authors and Affiliations
Additional information
Communicated by C.P. Hollenberg
Rights and permissions
About this article
Cite this article
Suárez, T., Eslava, A.P. Transformation of Phycomyces with a bacterial gene for kanamycin resistance. Molec. Gen. Genet. 212, 120–123 (1988). https://doi.org/10.1007/BF00322453
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00322453