A weak germ-line excision mutation blocks developmentally controlled amplification of the rDNA minichromosome of Tetrahymena thermophila.
During development of the somatic macronucleus of Tetrahymena thermophila, the rDNA is excised from its germ-line chromosome, rearranged into a palindrome, and amplified to 10(4) copies. We have identified a cis-acting germ-line mutation, rmm11/6, that prevents amplification of the rDNA in all but approximately 1 in 10(5) cells when it is the only rDNA allele in the developing macronucleus. The rmm11/6 mutation resides in a conserved element required for excision, the chromosome breakage sequence (Cbs) flanking the 3' end of the rDNA. Surprisingly, the rmm11/6 mutation only weakly affects excision of the rDNA from its germ-line location; at least 25% of cells heterozygous for this mutation correctly excise the affected rDNA allele. In heterozygotes, when this rDNA allele is excised, it is also poorly amplified. The rDNA amplification defect caused by this mutation is not overcome by delaying amplification with the DNA synthesis inhibitor aphidicolin, indicating that rDNA excision and amplification are not experimentally separable. Our experiments provide the first evidence that the capacity to amplify the rDNA is restricted in the developing macronucleus. We propose that the rmm11/6 mutation delays excision of the rDNA and that the developmental progression of the macronucleus past a restricted window for amplification is responsible for the severe amplification defect caused by this weak rDNA excision mutation.
Kapler
G M
GM
Department of Microbiology and Immunology, University of California, San Francisco 94143.
Blackburn
E H
EH
D000818:Animals; D015139:Blotting, Southern; D002875:Chromosomes; D004261:DNA Replication; D004275:DNA, Ribosomal / Q000235:genetics*; D005784:Gene Amplification; D005854:Germ Cells; D009154:Mutation; D010641:Phenotype; D016053:RNA, Protozoan / Q000235:genetics; D016808:Tetrahymena thermophila / Q000235:genetics* / Q000254:growth & development
D004275:DNA, Ribosomal; D016053:RNA, Protozoan