Genome-Wide Characterization of Tetrahymena thermophila Chromosome Breakage Sites. II. Physical and Genetic MappingSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY653004, AY653005, AY653006, AY653007, AY653008, AY653009, AY653010, AY653011, AY653012, AY653013, AY653014, AY653015, AY653016, AY653017, AY653018, AY653019, AY653020, AY653021, AY653022, AY653023, AY653024, AY653025, AY653026, AY653027, AY653028, AY653029.

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, directed by a conserved chromosome breakage sequence (Cbs element). An accompanying article in this issue reports the development of a successful scheme for the genome-wide cloning and identification of functional chromosome breakage sites. This article reports the physical and genetic characterization of 30 functional chromosome breakage junctions. Unique sequence tags and physical sizes were obtained for the pair of macronuclear chromosomes generated by fragmentation at each Cbs. Cbs-associated polymorphisms were used to genetically map 11 junctions to micronuclear linkage groups and macronuclear coassortment groups. Two pairs of junctions showed statistically significant similarity of the sequences flanking the Cbs, suggestive of relatively recent duplications of entire Cbs junctions during Tetrahymena genome evolution. Two macronuclear chromosomes that lose at least one end in an age-related manner were also identified. The whole-genome shotgun sequencing of the Tetrahymena macronucleus has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from natural ends of macronuclear chromosomes, Cbs junctions will provide useful sequence tags for relating macro-and micronuclear genetic, physical, and whole-genome sequence maps.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

“…Several reports document duplications of Cbs and surrounding sequences in T. thermophila (Cassidy-Hanley et al, 2005; Hamilton et al, 2006b; Yao et al, 1987). The pilot study described above revealed four Cbs duplications among the 22 sequenced sites in three other Tetrahymena species (Figure 4B).…”

MRE11 and COM1/SAE2 are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena

“…For a similar procedure see Cassidy-Hanley et al (2005). In short, 4 × 10 6 cells (∼20 ml of cell culture) were centrifuged for 3 min at 1000 × g. The pellet was resuspended in 50 µl ice-cold 0.05 M Na 2 EDTA, 0.01 M Tris-Cl (pH 7.5).…”

“…In the absence of known recombination hotspots in Tetrahymena, we adopted a method by which DSBs can be detected as a smear during the separation of chromosomesized DNA molecules by PFGE (Game et al 1989;Loidl et al 1998). In Tetrahymena, the region of the gel that is expected to display a DSB-generated smear is densely covered with an excess of MAC-derived chromosome fragments (Cassidy-Hanley et al 2005 and Supplement S5). Therefore, we probed the gel with a MIC-specific DNA sequence.…”

Programmed Minichromosome Elimination as a Mechanism for Somatic Genome Reduction in Tetrahymena thermophila

“…One study has shown that a single MAC minichromosome end that was retained at or before 30 fissions after conjugation was lost after 100 fissions. This age-related loss could suggest the loss of the entire minichromosome and hinted at the loss of additional minichromosomes during ageing [50]. However, whether it plays a significant role in programmed DNA elimination, as it does in nematodes, is unclear.…”