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Chromosomal mechanics - centromeres, telomeres and rearrangements
- The genome of the social amoeba Dictyostelium discoideum

Repeat clusters may serve as centromeres
Centromeres mobilize eukaryotic chromosomes during cell division but vary widely in their structure and organisation19, making them difficult to identify. Each Dictyostelium chromosome carries a single cluster of repeats rich in DIRS (Dictyostelium intermediate repeat sequence) elements20, 21 near one end22, and this sole but striking structural consistency suggests that these clusters may serve as centromeres. Although the repetitive nature of the chromosomal termini impeded their assembly, most of the cluster on Chromosome 1 was assembled (Fig. 3) and shows a complex pattern of DIRS and related Skipper elements, each preferentially associated with others of the same type. Frequent insertions and partial deletions have created a mosaic with little long-range order.
In Dictyostelium cells demonstrating condensed chromosomes characteristic of mitosis, DIRS-element probes hybridise to one end of each chromosome (Fig. SI 4), consistent with the mapping data. DIRS-like elements in other species are more uniformly scattered along the chromosomes23 suggesting that their restricted distribution in Dictyostelium chromosomes is functionally important. Further, the DIRS-containing ends of the chromosomes cluster not only during mitosis, but also during interphase (Fig. SI 4), as has been observed for centromeres in Schizosaccharomyces pombe,24.

rDNA sequences appear to act as telomeres
No G+T-rich telomere-like motifs were identified in the sequence. However, earlier findings22 suggested that the chromosomes terminate in the same G+A-rich repeat motif which caps the extrachromosomal rDNA element. We therefore surveyed all shotgun sequence to identify reads containing a junction between complex repetitive elements and rDNA-like sequence. Only 556 such reads were identified, of which 221 could be built into 13 contigs which we refer to as 'C/R (complex-repeat/rDNA) junctions'.

Of the 13 junctions, two represented already-known regions lying internally in the chromosomal assemblies. Of the remaining 11, one had twice the sequence coverage of the others, suggesting that it represents two distinct but identical portions of the genome (a possibility supported by the fact that another two of the junctions differed from each other by only two bases). Hence, we infer that the 11 remaining contigs represent 12 distinct junctions between repetitive elements and rDNA-like sequences - potentially one for every chromosomal end.

Based on their content of sequence-reads from each of the whole-chromosome libraries, we assigned two of the C/R contigs to each of the chromosomes. Chromosomes 4 and 5 cannot be distinguished in this way but three junctions, including the one believed to be present as two copies, are assigned to this chromosome pair. The point in the rDNA palindrome which is represented differs from one junction to the next (Fig. SI 5), but several junctions fall at common parts of the palindrome. This may reflect a preference in the mechanism which forms or maintains the junctions, or may result from an homogenizing recombination between them or with other rDNA sequences. Certainly the low frequency of differences between the rDNA components of the junction fragments and the extrachromosomal rDNA element argues for some process that limits or rectifies mutation. At each junction, we see only the rDNA sequence that immediately adjoins the complex repeat, since further assembly is precluded by the multicopy nature of rDNA. Therefore we cannot tell whether each junctional rDNA sequence extends to the telomere-repeat-carrying tip of the rDNA palindrome sequence, nor whether other sequences lie beyond the rDNA components.

HAPPY mapping of markers derived from six of these C/R junctions confirmed not only the chromosomal assignments which had been made based on the origins of their component sequences, but also their locations at the termini of the mapped regions of the chromosomes. For the other junctions, the absence of unique sequence features precluded such mapping. Taken as a whole, this evidence strongly suggests that rDNA-like elements form part of the telomere structure in D. discoideum and that common mechanisms stabilise both the extrachromosomal rDNA element and the chromosomal termini.

Chromosome 2 duplication - aftermath of a 'breakage-fusion-bridge' event?
Chromosome 2 of D. discoideum AX4 carries a perfect inverted 1.51Mb duplication (Fig. 2; references 9, 25, and this work). This duplication, containing 608 genes, is known25 to be absent from the wild-type isolate NC4 and from one of its direct descendents (AX2), but present in another (AX3); AX4 in turn is derived from AX3. The sequences adjoining the right-hand end of the duplication - a partial copy of a DIRS element (and a partial DDT-A element) and a region identical to part of the rDNA palindrome, both at about 3.74Mb (Fig. 2) – have been implicated in centromeric and telomeric functions, respectively, elsewhere in the genome.

We propose that this duplication arose from a 'breakage-fusion-bridge' cycle as first described in maize26, and since observed in many genomes. The nearby DIRS and rDNA components, in this view, represent abortive attempts to stabilize the halves of the broken chromosome by establishing new telomeres and centromeres, followed by re-fusion of the pieces to create a restored and enlarged chromosome (Fig. SI 6).

Chromosome 2 (the largest of the chromosomes, even discounting the duplication in AX4) may be prone to breakage: in the Bonner isolate of NC4, maintained in vegetative growth for 50 years, chromosome 2 is represented by two smaller fragments27. Comparison with more recent data22 indicates that the break-point in NC4-Bonner lies in the same region as the duplication in AX4, suggesting that NC4-Bonner underwent the early stages of this process, but that the chromosome fragments were stabilized and maintained after the initial breakage. Preliminary results (not shown) from HAPPY mapping also suggest that, whilst wild-type isolates V12M2 and NC4 both lack the duplication seen in AX4, NC4 may carry a duplication of ~300kb near the opposite end of chromosome 2.

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