Genome-wide characterization of tetrahymena thermophila chromosome breakage sites. I. Cloning and identification of functional sites

Abstract

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.

Figure 1.—

Specific PCR amplification products templated by MIC or MAC DNA in a Cbs region. SP-C and SP-G are specific PCR primers designed from the DNA sequence of a Cbs insert. GCbs and CCbs are the single strands of the canonical Cbs element 5′-AAAGAGGTTGGTTTA-3′ (GCbs) and its inverse complement (CCbs). Tel is an inwardly oriented PCR primer derived from the telomere sequence 5′-CCCCAACCCCAACC-3′. (Top) PCR amplification products templated exclusively by MIC DNA: MIC-FL is the full-length product, while MIC-C and MIC-G are the two “half-products.” (Bottom) PCR amplification “half-products” templated exclusively by MAC DNA.

Figure 2.—

Nullisomic mapping of the Cbs 1L-5 junction. DNA from inbred strain B and C3 (controls) and from nullisomic strains missing both copies of the indicated chromosomes or chromosome arms was PCR amplified using the Cbs 1L-5G and 1L-5C primers. The amplification pattern of the 0.6-kb band (arrow) allows assignment of this junction to the left arm of MIC chromosome 1 (1L).

Figure 3.—

Specific PCR amplification of MIC-limited or MAC-destined segments flanking a functional Cbs element results in products of similar length. Amplification products from the Cbs 1R-1 junction of inbred strain B are illustrated here. Primer nomenclature and sequences are as in Figure 1 and Table 2, respectively. Arrows point to pairs of related PCR products from either side of the junction.

Figure 4.—

Genetic and developmental DNA polymorphisms associated with Cbs 1R-4G. DNAs from mass cultures of young B and C3 homozygotes (<30 fissions old) and from B/C3 heterozygotes passaged for at least 500 fissions (panel of terminal assortants; see materials and methods) were PCR amplified using the Tel and Cbs 1R-4G primers. The SB prefix has been omitted from the names of the terminal assortants (1805–1821). The major size difference (compare B and C3 lanes) is determined by the germline-encoded allele difference; the allele assorted to purity is indicated under each lane. The minor, within-allele size variation (e.g., compare assortants 1810–1812) is developmental and results from telomere addition site microheterogeneity associated with chromosome breakage during MAC differentiation (see text).

Figure 5.—

Sequence analysis of developmental microheterogeneity in the macronuclear telomere addition site at the 3L-2 Cbs junction. The telomere-adjacent regions of 11 terminal assortants were PCR amplified using the Tel and 3L-2G primers. The sequences of the products are shown in 5′–3′ orientation. Telomeric repeats are shown in boldface type, beginning at the first position where the MAC and MIC sequence (bottom line) diverge. Sequences corresponding to the Tel primer are underlined. When a partial telomere repeat unit in the MIC (italicized) just precedes the site of MIC/MAC sequence divergence, it is unclear which was the first nucleotide added by telomerase. PCR products templated from whole-cell DNA from mass cultures of inbred B and C3 progeny (analogous to lanes B and C3 in Figure 4) showed a major sequence component identical in size to that of assortant SB1806. No genetic polymorphism is associated with this Cbs.