Summary
The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5′-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr+ transformants at a frequency of 40 transformants per μg of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per μg of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr+. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.
Similar content being viewed by others
References
Ballance DJ, Turner G (1985) Development of a high-frequency transforming vector for Aspergillus nidulans. Gene 36:321–331
Ballance DJ, Turner G (1986) Gene cloning in Aspergillus nidulans: isolation of the isocitrate lyase gene (acuD). Mol Gen Genet 202:271–275
Barbesgaard P (1977) Industrial enzymes produced by members of the genus Aspergillus. In: Smith JE, Pateman JA (eds) Genetics and physiology of Aspergillus. Br Myc Soc Symp Series no. 1. Academic Press, London, pp 391–404
Bennet JW (1985) Molds, manufacturing and molecular genetics. In: Timberlake WE (ed) Molecular genetics of filamentous fungi. Alan R. Liss Inc., New York, pp 345–366
Berry DR, Chmiel A, Al Obaidi Z (1977) Citric acid production by Aspergillus niger. In: Smith JE, Pateman JA (eds) Genetics and physiology of Aspergillus. Br Myc Soc Symp Series. no. 1. Academic Press, London, pp 405–426
Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197:345–346
Boel E, Hjort I, Svensson B, Norris F, Fiil NP (1984) Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely rated mRNAs. EMBO J 3:1097–1102
Bos CJ (1986) Induced mutation and somatic recombination as tools for genetic analysis and breeding of imperfect fungi. Thesis, Agricultural University, Wageningen, The Netherlands
Buxton FP, Radford A (1983) Cloning of the structural gene for orotidine-5′-phosphate decarboxylase of Neurospora crassa by expression in Escherichia coli. Mol Gen Genet 190:403–405
Buxton FP, Gwynne DI, Davies RW (1985) Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans. Gene 37:207–214
Clutterbuck AJ (1974) Aspergillus nidulans. In: King RC (ed) Handbook of genetics I. Bacteria, bacteriophages and fungi. Plenum Press, New York, pp 447–510
Frischauf AM, Lehrach H, Poustka A, Murray N (1983) Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842
Johnstone IL, Hughes SG, Clutterbuck AJ (1985) Cloning an Aspergillus nidulans developmentally regulated gene by transformation. EMBO J 4:1307–1311
Kelly MK, Hynes MJ, (1985) Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J 4:475–479
Lhoas P, (1967) Genetic analysis by means of the parasexual cycle in Aspergillus niger. Genet Res 10:45–61
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning — A laboratory manual. Cold Spring Harbor Laboratory Cold Spring Harbor, New York
Newbury SF, Glazebrook JA, Radford A (1986) Sequence analysis of the pyr-4 (orotidine 5′-P decarboxylase) gene of Neurospora crassa. Gene 43:51–58
Pontecorvo G, Roper JA, Hemmons LM, MacDonald KD, Bufton AWJ (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238
Struhl K, Stinchcomb DT, Scherer S, Davies RW (1979) High frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039
Smith JE, Pateman JA (eds) (1977) Genetics and physiology of Aspergillus Br Myc Soc Symp Series no. 1. Academic Press, London
Upshall A (1986) Genetic and molecular characterization of argB+ transformants of A. nidulans. Curr Genet 10:593–599
Van Gorcom RFM, Pouwels PH, Goosen T, Visser J, Van den Broek HWJ, Hammer JE, Timberlake WE, van den Hondel CAMJJ (1985) Expression of an Escherichia coli β-galactosidase fusion gene in Aspergillus nidulans. Gene 40:99–106
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp 18 and pUC19 vectors. Gene 33:103–119
Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474
Yelton MM, Timberlake WE, van den Hondel CAMJJ (1985) A cosmid for selecting genes by complementation in Aspergillus nidulans: selection of developmentally regulated yA locus. Proc Natl Acad Sci USA 82:834–838
Author information
Authors and Affiliations
Additional information
Communicated by C.P. Hollenberg
Rights and permissions
About this article
Cite this article
van Hartingsveldt, W., Mattern, I.E., van Zeijl, C.M.J. et al. Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206, 71–75 (1987). https://doi.org/10.1007/BF00326538
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00326538