Replacement of the macronuclear ribosomal RNA genes of a mutant Tetrahymena using electroporation.
The macronucleus of the ciliate Tetrahymena contains approx. 10(4) 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 greater than wt B greater than 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 greater than 1 in 10(5), by electroporation under a wide range of electrical discharge parameters. The fraction of cells surviving electroporation varied between 2 and greater than 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-1 rDNA. These findings are also consistent with the previous conclusion that the differential replication determinants reside in the 5'-nontranscribed spacer of the rDNA.
Orias
E
E
Department of Biological Sciences, University of California, Santa Barbara 93106.
Larson
D
D
Hu
Y F
YF
Yu
G L
GL
Karttunen
J
J
Løvlie
A
A
Haller
B
B
Blackburn
E H
EH
D000818:Animals; D015139:Blotting, Southern; D002467:Cell Nucleus; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D004558:Electric Stimulation; D009154:Mutation; D012335:RNA, Ribosomal / Q000235:genetics*; D015183:Restriction Mapping; D013768:Tetrahymena / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D004275:DNA, Ribosomal; D012335:RNA, Ribosomal