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. 2006 Apr;5(4):771-80.
doi: 10.1128/EC.5.4.771-780.2006.

The highly conserved family of Tetrahymena thermophila chromosome breakage elements contains an invariant 10-base-pair core

Eileen P Hamilton  1 , Sondra Williamson, Sandra Dunn, Virginia Merriam, Cindy Lin, Linh Vong, Jessica Russell-Colantonio, Eduardo Orias
Affiliations

The highly conserved family of Tetrahymena thermophila chromosome breakage elements contains an invariant 10-base-pair core

Eileen P Hamilton et al. Eukaryot Cell. 2006 Apr.

Abstract

As a typical ciliate, Tetrahymena thermophila is a unicellular eukaryote that exhibits nuclear dimorphism: each cell contains a diploid, germ line micronucleus (MICN) and a polyploid, somatic macronucleus (MACN). During conjugation, when a new MACN differentiates from a mitotic descendant of the diploid fertilization nucleus, the five MICN chromosomes are site-specifically fragmented into 250 to 300 MACN chromosomes. The classic chromosome breakage sequence (CBS) is a 15-bp element (TAAACCAACCTCTTT) reported to be necessary and sufficient for chromosome breakage. To determine whether a CBS is present at every site of chromosome fragmentation and to assess the range of sequence variation tolerated, 31 CBSs were isolated without preconception as to the sequence of the chromosome breakage element. Additional CBS-related sequences were identified in the whole-genome sequence by their similarities to the classic CBS. Forty CBS elements behaved as authentic chromosome breakage sites. The CBS nucleotide sequence is more diverse than previously thought: nearly half of the CBS elements identified by unbiased methods have a variant of the classic CBS. Only an internal 10-bp core is completely conserved, but the entire 15-bp chromosome breakage sequence shows significant sequence conservation. Our results suggest that any one member of the CBS family provides a necessary and sufficient cis element for chromosome breakage. No chromosome breakage element totally unrelated to the classic CBS element was found; such elements, if they exist at all, must be rare.

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Figures

FIG. 1.
FIG. 1.
Locations and nomenclature of PCR primers used for IPCR walks and CBS PCR tests. Open bar, MACN-destined DNA; vertically hatched bar, telomere sequence; solid bar, CBS element; stippled (including solid) bar, BES; arrows, primer locations and their 5′-to-3′ orientations. (A) Divergent specific primers for IPCR walks, designed using a Tel clone sequence (top line). Divergent proximal primers (DVP) and divergent distal primers (DVD) are relative to the telomere (in MACN DNA) or the CBS (in MICN DNA). RE, restriction enzyme sites. Note that a PCR product is obtained only when the RE-bounded MICN segment (bottom line) has been circularized. (B) Convergent specific PCR primers to test chromosome breakage function, designed using a BES-adjacent sequence. For CBS junctions isolated by IPCR, Tel CVD (convergent distal) is derived from Tel clone sequence and Tel CBS is derived from sequence on the opposite side of the BES, obtained after the IPCR walk. For CBS junctions available as a previously sequenced CBS clone, CBS C and CBS G were designed on opposite sides of the junction; see the work of Hamilton et al. (16) for more details. Note that the use of the two convergent specific primers generates products exclusively templated from MICN DNA (top line); thus, no product is seen when the chromosome arm containing the CBS is missing in the MICN of a nullisomic strain. Using a convergent specific primer with the Tel primer generates the MACN-templated PCR product only when a telomere is added nearby after complete (or conceivably partial) chromosome breakage.
FIG. 2.
FIG. 2.
Nucleotide conservation measurements across the 15-bp CBS-related and flanking sequences. (A) Conservation measurements in 62 functional CBSs. (B) Conservation measurements in 44 nonfunctional CBS-related elements (controls). (C) Nucleotide frequencies in the 62 functional CBSs shown in panel A. The CBS element occupies positions 21 to 35 in all panels, emphasized by the vertical lines in panels B and C. Nucleotide conservation was calculated at every position, as described in Materials and Methods, using aligned C-strand nucleotide sequences. At any given position in the logo plot, 2 bits represent maximum conservation (i.e., the same nucleotide occupies that position in every CBS junction), and 0 bits corresponds to no conservation (i.e., maximum diversity; all 4 nucleotides are equally frequent at that position). Note that (i) in panel B, positions corresponding to the completely conserved CBS core (22 to 30 and 32) have been marked with dots because variation at those positions was forbidden by the method used for the identification of nonfunctional CBS and (ii) the average conservation of flanking region positions for nonfunctional CBS-like elements (B) is significantly lower than that observed for regions flanking functional CBS (A). This likely reflects a bias for higher GC content in the flanking sequence introduced by the selection for the GC-rich (50%) CBS conserved core sequence. For example, 20 of the 44 CBS-like elements and surrounding regions match genes in the predicted proteome, which statistically is GC richer than the intron or intergenic region (36).
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