Transformation as a method of increasing gene copy number and gene expression in the basidiomycete fungus Coprinus cinereus.
Seven transformants having varying numbers of non-homologously integrated copies of the isocitrate lyase gene, acu-7, were analysed for enzyme activity. Maximum levels of activity, 3.8 times that of the wild type, were observed in a transformant with only two gene copies whereas eight gene copies in another transformant led to only 25% wild type activity. Acu+ transformants were not selected directly for expression of acu-7 but as cotransformants. Analysis of 14 transformants not expressing acu-7 showed that four contained transforming DNA sequences and significantly, two had evidence of non-homologously integrated tandem duplications of the entire acu-7 plasmid DNA. The site of integration of the gene was thus important in determining whether or not it was expressed and to what level it was expressed. A comparison of induced and uninduced levels of enzyme activity confirmed that the enzyme was still tightly regulated.
Mellon
F M
FM
School of Biological Sciences, Queen Mary College, London, UK.
Casselton
L A
LA
D000363:Agaricales / Q000235:genetics*; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003302:Coprinus / Q000201:enzymology / Q000235:genetics*; D004790:Enzyme Induction; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D007522:Isocitrate Lyase / Q000096:biosynthesis / Q000235:genetics; D015183:Restriction Mapping; D014170:Transformation, Genetic
D007522:Isocitrate Lyase