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

Abstract

Programmed chromosome breakage occurs at 50-200 specific sites in the genome of Tetrahymena thermo-phila during somatic nuclear (macronuclear) differentiation. Previous studies have identified a 15 bp sequence, the Cbs (for chromosome breakage sequence), that is necessary and sufficient to specify these 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 telomere formation in conjugating cells. Twenty-one constructs with single nucleotide substitutions covering all 15 positions of the Cbs were made and tested. 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 requirement. It offers no evidence that the Cbs contains a separate domain for promoting new telomere formation. In addition, we found that a partially functional Cbs retained in the macronucleus does not induce chromosome breakage during vegetative growth and that excess copies of this germline-specific sequence in the somatic nucleus have little deleterious effect on cell growth.

Figure 1

rDNA vector with mutated Cbs inserts. The micronuclear rDNA clone FANA3 contains a 72 bp synthetic polylinker in the 3′-spacer region (at position 10144) of the rDNA which serves as a site for insertion. It also contains the Cbs at its native locations at both ends of the rDNA and all sequences needed for rDNA processing and expression. A series of clones containing a mutated version of the Cbs were constructed by inserting a 27 bp oligonucleotide containing the mutated sequence into the polylinker at the SmaI site or between the SmaI and NotI sites.

Figure 2

A PCR assay for detecting rDNA termini. (A) The strategy of the assay. PCR was carried out using rdn10026 and Tel as primers and the transformant DNA as template. If breakage and telomere addition occur at the inserted Cbs, the rDNA should produce a short (0.13 kb) fragment after PCR. Otherwise a 0.41 kb DNA fragment should be produced due to breakage at the native 3′ Cbs of the rDNA. PCR products from the endogenous rDNA is shorter (0.31 kb) due to absence of the linker and the insert. (B) The results of an experiment to determine the sensitivity of the PCR assay. In this experiment total DNAs extracted from FA142 and FANA3 transformants were mixed in different ratios (1:100 and 1:1000) and used as templates for PCR. The results show that the 0.13 kb fragment is detectable when its template is only 1/100 of the total (lane 1), but not when it is 1/1000 (lane 2).

Figure 3

Effects of base substitutions on Cbs function. To analyze each construct listed in Table 1, total DNA from several independent transformants generated (numbers shown in parentheses in Table 1) were mixed and analyzed by the PCR assay described in Figure 2. These constructs (or controls) are: lane 1, control endogenous DNA from strain CU427; lane 2, FANA3 (without Cbs insertion); lane 3, FA142 (wild-type Cbs); lane 4, 1:100 mixture of transformants from FA142 and FANA3; lanes 5–21, FA4G, FA8T, FA6A, FA2T, FA12G, FA6G, FA6T, FA5G, FA12T, FA15C, FA9A, FA9T, FA7G, FA15A, FA10T, FA10A and FA3T, respectively. The 0.41 kb fragment is detected in FANA3 and Cbs mutant transformants. The 0.13 kb fragment is detected in transformants of FA142, FA6A, FA7G, FA15A and FA3T. The 0.31 kb fragment is generated from endogenous rDNA and found in varying amounts in some transformants.

Figure 4

Analysis of individual transformants from clones with breakage activities. DNA from individual transformed lines derived from clones displaying some breakage activity was analyzed by the same method as described in Figure 2. Transformants from three clones are shown here as examples. Each lane contains the PCR product from an individual line. Clones used to generate these transformants were: lanes 1–6, FA6A; lanes 7–9, FA1G; lanes 10–14, FA3T.

Figure 5

Fate of the Cbs in macronuclei of vegetative cells. Two of the FA1G transformants (see Fig. 4, lanes 8 and 9) were used to analyze the fate of rDNA with a mutated, partially functional Cbs in vegetatively growing cells. Four subclones were isolated from one and three from the other transformant and propagated for 20 and 120 generations before harvesting for the PCR analysis described in Figure 2. Lanes 1–7 are PCR products of these subclones after 20 generations of growth and lanes 8–14 are products of the same subclones (in the same order) after 120 generations of growth. Subclones analyzed in lanes 1–4 and 8–11 are from one transformed line and those in lanes 5–7 and 12–14 from the other.