Current Genetics

, Volume 50, Issue 3, pp 183–191 | Cite as

Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments

  • Kostyantyn V. Dmytruk
  • Andriy Y. Voronovsky
  • Andriy A. Sibirny
Research Article

Abstract

The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata.

Keywords

Insertional mutagenesis Gene tagging Yeast Riboflavin Candida famata 

Notes

Acknowledgments

We are grateful to Dr. Meg Woolfit (Trinity College, University of Dublin, Ireland) for critical reading of the manuscript.

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Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Kostyantyn V. Dmytruk
    • 1
  • Andriy Y. Voronovsky
    • 1
  • Andriy A. Sibirny
    • 1
    • 2
  1. 1.Institute of Cell BiologyNAS of UkraineLvivUkraine
  2. 2.Department of Metabolic EngineeringRzeszow UniversityRzeszowPoland

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