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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.