Replacement of the macronuclear ribosomal RNA genes of a mutant Tetrahymena using electroporation

The macronucleus of the ciliate Tetrahymena contains approx. 10⁴ ribosomal RNA gene molecules (rDNA) in the form of linear, autonomously replicating palindromes. Previous studies have shown that macronuclear rDNA molecules derived from wild-type (wt) inbred strain C3 out-replicate those derived from wt inbred strain B, in macronuclei initially heterozygous for both, leading to the complete loss of the B rDNA. However, rmm-1, a cis-acting laboratory-induced mutation obtained previously by mutagenesis of inbred strain C3, causes the mutant rmm-1 rDNA to be completely out-replicated by B rDNA. These findings suggest the following hierarchy of replication potential: wt C3 > wt B > C3-rmm-1. We used electroporation to test whether cells containing only rmm-1 macronuclear rDNA are favorable recipients for transformation with either wt B or C3 donor rDNA molecules. The donor rDNA molecules carried the selectable marker Pmr (paromomycin resistance) located in the coding region of the 17S rRNA. Transformants were obtained, at a frequency > 1 in 10⁵, by electroporation under a wide range of electrical discharge parameters. The fraction of cells surviving electroporation varied between 2 and > 95% in successful experiments. Replacement (‘transplacement’) of the recipient rDNA was observed, consistent with the prediction that B and C3 rDNA should out-replicate rmm-l rDNA. These findings are also consistent with the previous conclusion that the differential replication determinants reside in the 5'-nontranscribed spacer of the rDNA.