Summary
Aspergillus niger tryptophan auxotrophic mutants have been isolated after UV irradiation of conidiospores. The mutants belong to two different complementation groups, trpA and trpB, which complement each other in heterokaryons. Neither of the mutations could be complemented with the cloned A. niger trpC gene. To obtain A. niger trpC mutants in a direct way, gene inactivation by cotransformation was performed. For this purpose an in-frame gene fusion between the A. niger trpC and Escherichia coli lacZ genes was constructed and shown to be functionally expressed after introduction into A. niger by cotransformation with the pyrA gene as selective marker. Among the β-galactosidase expressing cotransformants, obtained with either circular or linearized vectors, no trpC mutants were detected, even after enrichment. Such mutants, however, could be obtained by cotransformation of A. niger with specific fragments of the fusion gene. Biochemical analysis of the cotransformants indicated that in nearly all cases the fusion gene had replaced the wild-type trpC gene. Genetic analysis showed that the trpC mutation is not linked to any of the A. niger loci described so far. The trpC mutants can be complemented by the cloned A. niger trpC gene as well as by the A. nidulans trpC gene.
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References
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles II. A multipurpose cloning system. Gene 2:95–113
Bos CJ (1985) Induced mutation and somatic recombination as tools for genetic analysis and breeding of imperfect fungi. PhD thesis, Agricultural University Wageningen, Wageningen, The Netherlands
Bos CJ, Debets AJM, Swart K, Huybers A, Kobus G, Slakhorst SM (1988) Genetic analysis and the construction of master strains for the assignment of genes to six linkage groups in Aspergillus niger. Curr Genet 14:437–443
Buxton FP, Gwynne DI, Garven S, Sibley S, Davies RW (1987) Cloning and molecular analysis of the ornithine carbamoyl transferase gene of Aspergillus niger. Gene 60:255–266
Casadaban MJ, Martinez-Arias A, Shapira SK, Chou J (1983) Beta-Galactosidase gene fusions for analyzing gene expression in E. coli and yeast. Methods Enzymol 100:293–308
Crawford IP (1980) Gene fusions in the tryptophan pathway: tryptophan synthetase and phosphoribosylanthranilate isomerase: indole glycerolphosphate synthase. In: H Bisswanger and E Schmincke-Ott (Ed.) Multifunctional proteins. John Wiley & Sons, New York, pp 151–173
Davis MA, Cobbett CS, Hynes MJ (1988) An amdS-lacZ fusion for studying gene regulation in Aspergillus. Gene 63:199–212
Fries N (1947) Experiments with different methods of isolating physiological mutations of filamentous fungi. Nature 159:199
Goosen T, Bloemheuvel G, Gysler C, Bie DA de, Broek HWJ van den, Swart K (1987) Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene. Curr Genet 11:499–503
Hütter R, De Moss JA (1967) Enzyme analysis of the tryptophan pathway in Aspergillus nidulans. Genetics 55:241–247
Hynes MJ (1979) Fine structure mapping of the amdS structural gene and its controlling region in Aspergillus nidulans. Genet Res 91:381–392
Kos T, Kuyvenhoven J, Wernars K, Bos CJ, Broek HWJ van den, Pouwels PH, Hondel CAMJJ van den (1985) Isolation and characterization of the Aspergillus niger trpC gene. Gene 39:231–238
Kos T, Kuyvenhoven A, Hessing HGM, Pouwels PH, Hondel CAMJJ van den (1988) Nucleotide sequence of the Aspergillus niger trpC gene: structural relationship with analogous genes of other organisms. Curr Genet 13:137–144
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Miller BL, Miller KY, Timberlake WE (1985) Direct and indirect gene replacements in Aspergillus nidulans. Mol Cell Biol 5:1714–1721
Paietta JV, Marzluf GA (1985) Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa. Curr Genet 9:383–388
Perkins DD, Radford A, Newmeyer D, Björkman M (1982) Chromosomal loci of Neurospora crassa. Microbiol Rev 46:426–570
Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, Hondel CAMJJ van den (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117–124
Roberts CF (1967) Complementation analysis of the tryptophan pathway in Aspergillus nidulans. Genetics 55:233–239
Struhl K, Stinchcomb DT, Scherer S, Davis RW (1979) High frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci USA 76:1035–1039
Timberlake WE, Boylan MT, Cooley MB, Mirabito PM, O'Hara EB, Willett CE (1985) Rapid identification of mutation-complementing restriction fragments from Aspergillus nidulans cosmids. Exp Mycol 9:351–355
Van Gorcom RFM, Pouwels PH, Goosen T, Visser J, Broek HWJ van den, Hamer JE, Timberlake WE, Hondel CAMJJ van den (1985) Expression of an Escherichia coli β-galactosidase fusion gene in Aspergillus nidulans. Gene 40:99–106
Van Gorcom RFM, Punt PJ, Pouwels PH, Hondel CAMJJ van den (1986) A system for the analysis of expression signals in Aspergillus. Gene 48:211–217
Van Hartingsveldt W, Mattern IE, Zeijl CMJ van, Pouwels PH, Hondel CAMJJ van den (1987) Development of a homologous transformation system for Aspergillus niger based on the pyrG gene. Mol Gen Genet 206:71–75
Wernars K, Goosen T, Wennekes LMJ, Visser J, Bos CJ, Broek HWJ van den, Gorcom RFM van, Hondel CAMJJ van den, Pouwels PH (1985) Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene. Curr Genet 9:361–368
Wernars K, Goosen T, Wennekes LMJ, Swart K, Hondel CAMJJ van den, Broek HWJ van den (1987) Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes. Mol Gen Genet 209:71–77
Yelton MM, Hamer JE, Souza ER de, Mullaney EJ, Timberlake WE (1983) Developmental regulation of the Aspergillus nidulans trpC gene. Proc Natl Acad Sci USA 80:7576–7580
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Communicated by C.P. Hollenberg
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Goosen, T., van Engelenburg, F., Debets, F. et al. Tryptophan auxotrophic mutants in Aspergillus niger: Inactivation of the trpC gene by cotransformation mutagenesis. Molec. Gen. Genet. 219, 282–288 (1989). https://doi.org/10.1007/BF00261189
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DOI: https://doi.org/10.1007/BF00261189