Electroporation-Mediated Replacement of a Positively and negatively Selectable β-Tubulin Gene in Tetrahymena thermophila

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-05, Vol.91 (10), p.4549-4553
• Main Authors: Gaertig, Jacek, Thatcher, Thomas H., Gu, Long, Gorovsky, Martin A.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Blotting, Southern; Chlamydomonas - genetics; DNA; DNA - analysis; DNA - genetics; DNA Primers; DNA probes; Electroporation; Gene Transfer Techniques; Genes; Genetic mutation; Genetic vectors; Microtubules; Molecular Sequence Data; Mutagenesis, Site-Directed; Paclitaxel - toxicity; Phenotypes; Plasmids; Point Mutation; Polymerase Chain Reaction; RNA, Messenger - analysis; RNA, Messenger - biosynthesis; Tetrahymena thermophila; Tetrahymena thermophila - drug effects; Tetrahymena thermophila - genetics; Tubulin - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.10.4549

Replacement of lysine-350 by methionine in the β-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed β-tubulin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxolsensitive transformants could be retransformed with a wild-type gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tubulin gene function and show that electroporation allows facile gene replacement in Tetrahymena.