Improvements to Gene Deletion in the Fungal Pathogen Cryptococcus Neoformans: Absence of Ku Proteins Increases Homologous Recombination, and Co-Transformation of Independent DNA Molecules Allows Rapid Complementation of Deletion Phenotypes

Fungal Genet Biol. 2006 Aug;43(8):531-44. doi: 10.1016/j.fgb.2006.02.007. Epub 2006 May 22.

Abstract

Cryptococcus neoformans is a pathogenic fungus that is relatively amenable to molecular genetic analysis, including gene deletion. However, rates of homologous recombination can be low, so obtaining specific gene deletion transformants is challenging. We have utilized two new technologies, cku deletion strains to improve the efficiency of gene deletions in this organism, and co-transformations. The Ku70-Ku80 heterodimer is predicted to be an essential part of the non-homologous end-joining process in C. neoformans. Here we show that a deletion in one or both of these proteins results in an increase in the rates of homologous recombination. Importantly, we demonstrate that after generation of a strain with a particular deletion of interest, the cku deletion can be removed by mating and segregation. We also utilize co-transformation of wild-type genes and selectable markers on separate linear DNA molecules to complement a deletion event. We show that co-transformation results in the successful restoration of wild-type phenotype, though variations in this phenotype often occur.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cryptococcus neoformans / genetics*
  • Cryptococcus neoformans / physiology
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / physiology
  • Female
  • Fungal Proteins / genetics
  • Fungal Proteins / physiology
  • Gene Deletion*
  • Genes, Fungal
  • Genetic Complementation Test
  • Mice
  • Mice, Inbred BALB C
  • Phenotype
  • Recombination, Genetic*
  • Sequence Homology
  • Transformation, Genetic*

Substances

  • DNA-Binding Proteins
  • Fungal Proteins