Construction of autonomously replicating vectors for complementation analysis of disruption mutants in a ku70 deletion background in Aspergillus niger.

Neuza Carvalho*, Mark Arentshorst, Cees van den Hondel and Arthur Ram.

Author address: 

Institute of Biology Leiden, Leiden University, Molecular Microbiology, Kluyver Centre for Genomics of Industrial Fermentation, W assenaarseweg 64, 2333 AL Leiden, The Netherlands.

Abstract: 

Mutants with a defective Non-Homologous-End-Joining (NHEJ) pathway are a very powerful tool for fungal genetic engineering. Several reports over the last few years have shown that mutants in the NHEJpathway (ku70/ku80 mutants) are very efficient recipients for gene targeting and achieve homologous targeting efficiencies up to 100%. To prove that a phenotype is associated with the deletion of a certain gene, the gene of interest is transformed back to the gene deletion strain which will ectopically integrate into the genome. However, phenotype complementation becomes difficult in a ku70 deletion background because ectopic integration frequencies are low and the gene will preferably integrate via homologous recombination, thereby replacing again the disrupted gene. One way to circumvent this problem is to clone the gene for complementation into an autonomously replicating plasmid containing a selection marker. Under selective pressure the plasmid is maintained, giving the wild type phenotype; once the selective pressure is removed, the plasmid is gradually lost and the mutant phenotype is again observed. W e have constructed autonomously replicating vectors containing either the pyrG or Hygromycine as selection markers and a unique NotI site for easy cloning of complementing genes. These vectors have been successfully used for complementation analysis of gene deletion mutants in A. niger. *Student poster
2009

abstract No: 

15

Full conference title: 

6th International Aspergillus Meeting
    • Asperfest 6 (2009)