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

Abstract

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