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Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes.

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PMC
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  • Research Article

Abstract

The ribosomal RNA genes (rDNA) of Tetrahymena thermophila macronucleus exist as free linear 21-kilobase molecules that contain replication origins and telomeres. A mutation in this gene confers resistance to the antibiotic paromomycin. We have isolated rDNA from such a mutant (strain p2f), microinjected it into the macronucleus of a sensitive strain, and obtained drug-resistant cells at a frequency of 1-3%. The transformed cells have a distinct and stable phenotype. The rDNA of the transformants contains the expected sequences of the mutant rDNA as determined by oligonucleotide hybridization. rDNA from a different inbred line (C3-368), which contains heteromorphic restriction sites, has also been used for injection, and the results confirm the fact that the injected rDNA is indeed present in the transformants. Injection of rDNA from the C3 strains also increases the transformation frequency 5- to 10-fold and leads to the total replacement of the resident rDNA of the B-inbred strains. This is presumably due to the replication dominance of rDNA from the C3 strains over that of the B strains. Using this method, we have also been able to transform developing cells, at similar frequencies, by microinjecting into the macronuclear anlagen.

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