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 thermoph ila, the rDNA is excised from its germ-line chromosome, rearranged int o a palindrome, and amplified to 10(4) copies. We have identified a ci s-acting germ-line mutation, rm11/6, that prevents amplification of th e rDNA in all but approximately 1 in 10(5) cells when it is the only r DNA allele in the developing macronucleus. The rmm11/6 mutation reside s in a conserved element required for excision, the chromosome breakag e sequence (Cbs) flanking the 3' end of the rDNA. Surprisingly, the rm m11/6 mutation only weakly affects excision of the rDNA from its germ- line location; at least 25% of cells heterozygous for this mutation co rrectly excise the affected rDNA allele. In heterozygotes, when this r DNA allele is excised, it is also poorly amplified. The rDNA amplifica tion defect caused by this mutation is not overcome by delaying amplif ication 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 th e rDNA is restricted in the developing macronucleus. We propose that t he rmm11/6 mutation delays excision of the rDNA and that the developme ntal progression of the macronucleus past a restricted window for ampl ification is responsible for the severe amplification defect caused by this weak rDNA excision mutation.