We have explored in detail the sequence composition of chromosomal regions that were broken, inverted, and re-combined in the lineage leading to Drosophila mojavensis
after its split from D. arizonae
. Our results failed to obtain direct support for either a canonical mechanism of transposon- or other repetitive-element-mediated chromosomal rearrangements (e.g., Finnegan 1989
) or an alternative model of chromosomal re-modeling proposed by Ranz et al. (2007)
. We cannot completely exclude these possibilities, though, since, for example, a repetitive element may have been lost from a breakpoint region in the D. arizonae
lineage. However, this seems unlikely based on the very low level of sequence divergence between these species. We do find, however, additional sequence features near to the chromosomal breakpoints that may contribute to local chromosomal instability in this region, predisposing it to accelerated breakages and rearrangements.
In the recent model proposed by Ranz et al. (2007)
, inversions are initiated by two pairs of staggered single-strand breaks in the chromatin. This process does not require common sequence between the two breakpoints in the ancestral chromosome. Rather, nearby nicks in the chromosome backbone cause base-pairing to fail between these breaks and the chromosome regions to separate. Overhanging ends generated by this process can be fixed, either by a loss of 5′overhanging nucleotides, or by filling in the missing strand. Finally, non-homologous end-joining can recombine the chromosomes in a new orientation. Inversions generated in this way may leave clear signatures of the process in the descendant’s genomic sequence. Fixing both 5′ overhangs of the same breakpoint in the same manner results in either inverted duplications of the regions between the nick-points or reciprocal deletions of these regions in the descendant’s chromosome. If the two overhangs of a breakpoint are fixed in opposite ways, though, no DNA is gained or lost, and no evidence of this process will be apparent in the genomic sequence. Hence, although we failed to find evidence for this process, we cannot exclude its operation in this instance.
While our sequence data cannot adequately distinguish between one outcome of the staggered double-strand break model and a model in which clean double-stranded breaks separated out this chromosomal region, the local genome features around the Xe breakpoints suggest possible explanations for why the breakpoints occurred at these locations. Ranz et al. (2007)
suggest that certain regions may be predisposed to chromatin breaks and are more likely to be involved in chromosome rearrangements. In the D. mojavensis
genome, we observed two key features in the vicinity of the inversion breakpoints on the X-chromosome. The proximal breakpoint is associated with a repeat element that is duplicated in high numbers across the genome. The distal breakpoint is near to a local gene duplication event. Since the sequence homology between the two copies of CG2056 breaks down immediately at the end of the coding sequences (suggesting a fairly ancient duplication), it is impossible to tell if this inversion-duplication event also used this breakpoint. But, its location does seem suggestive that a possible inherent weakness of this genomic region caused both events. Alternatively, base-pairing between the paralogous copies would cause a fairly tight hairpin loop in the chromatin, which may induce local chromosomal instability. Similarly, while the repeat element spanning the proximal breakpoint is not associated with the distal breakpoint, it seems possible that the high-copy number of this region may have increased its susceptibility to chromosome breaks.
Non-repetitive element-associated double-strand breaks in chromatin have been proposed to explain inversions in other Drosophila
species. Cirera et al. (1995)
suggest such a mechanism for an inversion fixed between D. melanogaster
and D. subobscura
. They note that alternating purine-pyrimidine sequences (RY repeats) may predispose certain regions towards chromosomal breakages due to topoisomerase II activity. Interestingly, the two Xe breakpoints we identify are centered within short RY repeat sequences (6 and 5 unit repeats for the proximal and distal breakpoint, respectively). This mechanism may be worthy of further study.
Our analysis of the evolution of the duplicated copies of the gene CG2056 in D. mojavensis
and D. arizonae
provides an interesting aside to the story of the Xe inversion. We have demonstrated that this duplication event predated the inversion in the D. replete
group ancestor. Since that point, the function of this gene appears to have been taken over by the derived copy at the expense of the ancestral copy, as evidenced by the expression of CG2056_G1 in D. mojavensis
, the amino-acid level conservation of CG2056_G1 in both species, the high level of sequence evolution in both CG2056_G2 genes and the early stop codon in Dmoj_CG2056_G2. Yet, it seems remarkable that Dmoj_CG2056_G1 remains functional despite this inversion event. Much of the 5′ UTR region of the gene was replaced by the rearrangement, which is the region where most regulatory sequences of genes are thought to reside. The nearer breakpoint is located ~500 bases from the start of exon B, but as few as 91 bases from the hypothesized start codons in exon A (Gnomon prediction, Gilbert 2007
). It may be that neither exon A nor this 5′UTR are accurately annotated and the entire regulatory region is located within the first 500 bases of exon B, or elsewhere 3′ of this gene, but this result seems worthy of future studies.
The generation of genome-sequences from groups of related organisms is beginning to promote a new level of understanding of the processes underlying genome-evolution. For decades, chromosomal rearrangements have been identified as of great importance in processes of adaptation and speciation, but until recently, models that explain their occurrence have been not been tested against actual genomic data. New results from Drosophila
and other taxonomic groups (e.g. Bailey et al. 2004
; Ranz et al. 2007
) have shown that chromosomes do appear to show pockets of instability, increasing the likelihood of rearrangements. Thus, identifying sequence features that increase this brittleness is of great utility to any studies of genome-evolution and speciation in general. We present here a case study of a recent chromosomal rearrangement that shows two types of sequence features (repetitive elements and adjacent inverted duplicated sequence) that each may be important for predicting chromosomal instability.