Isolation of micronuclei from Euplotes octocarinatus and identification of an internal eliminated sequence in the micronuclear gene encoding γ-tubulin 2

摘要: Summary A method for the isolation of micronuclear DNA from Euplotes octocarinatus has been developed. After cell lysis lysate is layered on top a Percoll gradient consisting 50% placed 80% in which had produced by high speed centrifugation. During following centrifugation at 24 000 g max 20 minutes macronuclei accumulate zone slightly above bottom centrifuge tube, while micronuclei as well some endosymbiotic bacteria and small macronuclear fragments below interface layers. The fraction can directly be used DNA. Contamination was analysed Southern hybridization using γ-tubulin 2 cDNA probe. By comparison signal intensities obtained copies genes ratio up to 1400 one macronucleus calculated. Using DNA, gene encoding isolated PCR. sequence revealed 547 bp internal eliminated (IES). It contains three repeats flanked pair hexa-nucleotide direct repeats.