Karyotype analysis of Neoparamoeba pemaquidensis CCAP1560/4 and its IRO. Ethidium bromide-stained pulsed-field gels are shown alongside Southern blots using probes designed against the N. pemaquidensis internal transcribed spacer (ITS) sequence and telomeric repeat (TTTAGGG), as well as the IRO spliced-leader (SL) RNA gene. Southern hybridizations were performed using the Roche DIG System (Roche Diagnostics, Mannheim, Germany). Hansenula wingei and Saccharomyces cerevisiae were used as DNA size markers. Cell densities of DNA plugs of Neoparamoeba pemaquidensis CCAP1560/4 are shown, as are the chromosome sizes of the markers. (A) Pulsed-field gel run with 1% agarose, 0.5× Tris-borate-EDTA (TBE), 14°C, a run time of 24 h at 6 V/cm, 120° angle, and a 60- to 120-s pulse time. (B) A 1% agarose pulsed-field gel, 0.5× TBE, 14°C, 48-h running time, 3 V/cm, 106° angle, and 500-s pulse time.