Episomal and Integrated Maintenance of Foreign DNA in Giardia Lamblia

Mol Biochem Parasitol. 1998 Apr 1;92(1):59-69. doi: 10.1016/s0166-6851(97)00225-9.

Abstract

Giardia lamblia is an early diverging eukaryote which causes gastrointestinal disease throughout the world. Different subgroups of Giardia have been defined based on several biochemical and genetic criteria. We have developed a method for stably introducing DNA into the nuclei of the parasite using puromycin acetyltransferase (pac) as a dominant selectable marker. Transfected circular DNAs were maintained as episomes in the isolate WB, a representative of one Giardia subgroup. When input DNAs were linearized, integration was observed to occur by homologous recombination producing gene replacements in this isolate. In isolate GS, which represents a different subgroup, both linear and circular transfected DNAs were integrated into the genome by homologous recombination. In GS, linear DNA again produced gene replacements, while circular DNA produced duplicative integration events. The failure of GS to replicate episomes may reflect differences in the structure or recognition of DNA replication origins between these subgroups. A plasmid shuttle vector was also developed for expression of other genes in Giardia lamblia. Utilizing the green fluorescent protein as a reporter gene in the WB isolate, we show that gene expression from this vector correlated with plasmid copy number over a range of two orders of magnitude. Together these tools should greatly enhance our ability to study both the basic biology and the pathogenesis of this ubiquitous parasite.

MeSH terms

  • Acetyltransferases / genetics
  • Animals
  • Drug Resistance / genetics
  • Genetic Markers
  • Giardia lamblia / genetics*
  • Green Fluorescent Proteins
  • Luminescent Proteins / biosynthesis
  • Luminescent Proteins / genetics
  • Plasmids*
  • Recombination, Genetic*
  • Selection, Genetic
  • Transfection*
  • Triose-Phosphate Isomerase / genetics

Substances

  • Genetic Markers
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Acetyltransferases
  • puromycin N-acetyltransferase
  • Triose-Phosphate Isomerase