A long stringent sequence signal for programmed chromosome breakage in Tetrahymena thermophila

Programmed chromosome breakage occurs at 50-200 specific sites in the genom e of Tetrahymena thermophila during somatic nuclear (macronuclear) differen tiation, Previous studies have identified a 15 bp sequence, the Cbs (for ch romosome breakage sequence), that is necessary and sufficient to specify th ese sites. In this study we determined the effects of mutations in the Cbs on its ability to specify the chromosome breakage site and promote new telo mere formation in conjugating cells. Twenty-one constructs: with single nuc leotide substitutions covering all 15 positions of the Cbs were made and te sted. Fourteen of them (covering 11 positions) abolished breakage entirely, six (covering six positions, including the remaining four) caused partial loss of breakage function and one showed no detectable effect. This result indicates that the Cbs has an exceptionally long and stringent sequence req uirement. It offers no evidence that the Cbs contains a separate domain for promoting new telomere formation. In addition, we found that a partially f unctional Cbs retained in the macronucleus does not induce chromosome break age during vegetative growth and that excess copies of this germline-specif ic sequence in the somatic nucleus have little deleterious effect on cell g rowth.