Transformation as a method of increasing gene copy number and gene expression in the basidiomycete fungusCoprinus cinereuss

✍ Scribed by Fiona M. Mellon; Lorna A. Casselton

• Publisher: Springer-Verlag
• Year: 1988
• Tongue: English
• Weight: 567 KB
• Volume: 14
• Category: Article
• ISSN: 0172-8083
• DOI: 10.1007/bf00521268

✦ Synopsis

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.