The Structure and Population Genetics of the Breakpoints Associated With the Cosmopolitan Chromosomal Inversion In(3R)Payne in Drosophila melanogaster.
ABSTRACT: We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene. Functional copies of all three genes are found at the opposite breakpoints. We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2-kb portion of the tok gene, as well as the same 2 kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes. The functional tok gene associated with In(3R)P shows little polymorphism. Numerous single-base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.
Project description:We report here the breakpoint structure and sequences of the Drosophila melanogaster cosmopolitan chromosomal inversion In(3R)P. Combining in situ hybridization to polytene chromosomes and long-range PCR, we have identified and sequenced the distal and proximal breakpoints. The breakpoints are not simple cut-and-paste structures; gene fragments and small duplications of DNA are associated with both breaks. The distal breakpoint breaks the tolkin (tok) gene and the proximal breakpoint breaks CG31279 and the tolloid (tld) gene. Functional copies of all three genes are found at the opposite breakpoints. We sequenced a representative sample of standard (St) and In(3R)P karyotypes for a 2-kb portion of the tok gene, as well as the same 2 kb from the pseudogene tok fragment found at the distal breakpoint of In(3R)P chromosomes. The tok gene in St arrangements possesses levels of polymorphism typical of D. melanogaster genes. The functional tok gene associated with In(3R)P shows little polymorphism. Numerous single-base changes, as well as deletions and duplications, are associated with the truncated copy of tok. The overall pattern of polymorphism is consistent with a recent origin of In(3R)P, on the order of Ne generations. The identification of these breakpoint sequences permits a simple PCR-based screen for In(3R)P.
Project description:Chromosomal polymorphism is widespread in the Drosophila genus, with extensive evidence supporting its adaptive character in diverse species. Moreover, inversions are the major contributors to the genus chromosomal evolution. The molecular characterization of a reduced number of polymorphic inversion breakpoints in Drosophila melanogaster and Drosophila subobscura supports that their inversions would have mostly originated through a mechanism that generates duplications -staggered double-strand breaks- and has thus the potential to contribute to their adaptive character. There is also evidence for inversion breakpoint reuse at different time scales. Here, we have characterized the breakpoints of two inversions of D. subobscura -O4 and O8- involved in complex arrangements that are frequent in the warm parts of the species distribution area. The duplications detected at their breakpoints are consistent with their origin through the staggered-break mechanism, which further supports it as the prevalent mechanism in D. subobscura. The comparative analysis of inversions breakpoint regions across the Drosophila genus has revealed several genes affected by multiple disruptions due not only to inversions but also to single-gene transpositions and duplications.
Project description:That closely related species often differ by chromosomal inversions was discovered by Sturtevant and Plunkett in 1926. Our knowledge of how these inversions originate is still very limited, although a prevailing view is that they are facilitated by ectopic recombination events between inverted repetitive sequences. The availability of genome sequences of related species now allows us to study in detail the mechanisms that generate interspecific inversions. We have analyzed the breakpoint regions of the 29 inversions that differentiate the chromosomes of Drosophila melanogaster and two closely related species, D. simulans and D. yakuba, and reconstructed the molecular events that underlie their origin. Experimental and computational analysis revealed that the breakpoint regions of 59% of the inversions (17/29) are associated with inverted duplications of genes or other nonrepetitive sequences. In only two cases do we find evidence for inverted repetitive sequences in inversion breakpoints. We propose that the presence of inverted duplications associated with inversion breakpoint regions is the result of staggered breaks, either isochromatid or chromatid, and that this, rather than ectopic exchange between inverted repetitive sequences, is the prevalent mechanism for the generation of inversions in the melanogaster species group. Outgroup analysis also revealed evidence for widespread breakpoint recycling. Lastly, we have found that expression domains in D. melanogaster may be disrupted in D. yakuba, bringing into question their potential adaptive significance.
Project description:Balancers are rearranged chromosomes used in Drosophila melanogaster to maintain deleterious mutations in stable populations, preserve sets of linked genetic elements and construct complex experimental stocks. Here, we assess the phenotypes associated with breakpoint-induced mutations on commonly used third chromosome balancers and show remarkably few deleterious effects. We demonstrate that a breakpoint in p53 causes loss of radiation-induced apoptosis and a breakpoint in Fucosyltransferase A causes loss of fucosylation in nervous and intestinal tissue-the latter study providing new markers for intestinal cell identity and challenging previous conclusions about the regulation of fucosylation. We also describe thousands of potentially harmful mutations shared among X or third chromosome balancers, or unique to specific balancers, including an Ankyrin 2 mutation present on most TM3 balancers, and reiterate the risks of using balancers as experimental controls. We used long-read sequencing to confirm or refine the positions of two inversions with breakpoints lying in repetitive sequences and provide evidence that one of the inversions, In(2L)Cy, arose by ectopic recombination between foldback transposon insertions and the other, In(3R)C, cleanly separates subtelomeric and telomeric sequences and moves the subtelomeric sequences to an internal chromosome position. In addition, our characterization of In(3R)C shows that balancers may be polymorphic for terminal deletions. Finally, we present evidence that extremely distal mutations on balancers can add to the stability of stocks whose purpose is to maintain homologous chromosomes carrying mutations in distal genes. Overall, these studies add to our understanding of the structure, diversity and effectiveness of balancer chromosomes.
Project description:Chromosomal rearrangements constitute a significant feature of genome evolution, and inversion polymorphisms in Drosophila have been studied intensely for decades. Population geneticists have long recognized that the sequence features associated with inversion breakpoints would reveal much about the mutational origin, uniqueness, and genealogical history of individual inversion polymorphisms, but the cloning of breakpoint sequences is not trivial. With the aid of a method for rapid recovery of DNA clones spanning rearrangement breakpoints, we recover and examine the DNA sequences spanning the breakpoints of the cosmopolitan inversion In(3L)Payne in Drosophila melanogaster. By examining the sequence diversity associated with six standard and seven inverted chromosomes from natural populations, we find that the inversion is monophyletic in origin, the sequences are genetically isolated from recombination at the breakpoints, and there is no association with features such as transposable elements. The inverted sequences show 17-fold less nucleotide polymorphism, but there are eight fixed differences in the region spanning both breakpoints. This suggests that this inversion is not recently derived. Finally, Northern analysis and transcript mapping find that the distal breakpoint has disrupted three transcripts that are normally expressed in the standard arrangement. Incidentally, the method introduced here can be used to isolate breakpoint sequences of arrangements associated with many human diseases.
Project description:Although Drosophila melanogaster has been the subject of intensive analysis of polymorphism and divergence, little is known about the distribution of variation at the most distal regions of chromosomes arms. Here we report a survey of genetic variation on the tip of 3L in D. melanogaster and D. simulans. Levels of single nucleotide polymorphism in the most distal euchromatic sequence are approximately one order of magnitude less than that typically observed in genomic regions of normal crossing over, consistent with what might be expected under models of linked selection in regions of low crossing over. However, despite this reduced level of nucleotide variation, we found abundant deletion polymorphism. These deletions create at least three gene presence/absence polymorphisms within D. melanogaster: the putative G-protein coupled receptor mthl-8 (which is the most distal known or predicted gene on 3L) and the unannotated mRNAs AY060886 and BT006009. Strikingly, D. simulans is also segregating deletions that cause mthl8 presence/absence polymorphism. Breakpoint sequencing and tests of correlations with segregating SNPs in D. melanogaster suggest that each deletion is unique. Cloned breakpoint sequences revealed the presence of Het-A elements just distal to unique, canonical euchromatic sequences. This pattern suggests a model in which repeated telomeric deficiencies cause deletions of euchromatic sequence followed by subsequent "healing" by retrotranposition of Het-A elements. These data reveal the dominance of telomeric dynamics on the evolution of closely linked sequences in Drosophila.
Project description:Chromosomal breaks and rearrangements have been observed in conjunction with autism and autistic spectrum disorders. A chromosomal inversion has been previously reported in autistic siblings, spanning the region from approximately 7q22.1 to 7q31. This family is distinguished by having multiple individuals with autism and associated disabilities. The region containing the inversion has been strongly implicated in autism by multiple linkage studies, and has been particularly associated with language defects in autism as well as in other disorders with language components. Mapping of the inversion breakpoints by FISH has localized the inversion to the region spanning approximately 99-108.75 Mb of chromosome 7. The proximal breakpoint has the potential to disrupt either the coding sequence or regulatory regions of a number of cytochrome P450 genes while the distal region falls in a relative gene desert. Copy number variant analysis of the breakpoint regions detected no duplication or deletion that could clearly be associated with disease status. Association analysis in our autism data set using single nucleotide polymorphisms located near the breakpoints showed no significant association with proximal breakpoint markers, but has identified markers near the distal breakpoint ( approximately 108-110 Mb) with significant associations to autism. The chromosomal abnormality in this family strengthens the case for an autism susceptibility gene in the chromosome 7q22-31 region and targets a candidate region for further investigation.
Project description:We have used the inversion system of Drosophila pseudoobscura to investigate how genetic flux occurs among the gene arrangements. The patterns of nucleotide polymorphism at seven loci were used to infer gene conversion events between pairs of different gene arrangements. We estimate that the average gene conversion tract length is 205 bp and that the average conversion rate is 3.4 x 10(-6), which is 2 orders of magnitude greater than the mutation rate. We did not detect gene conversion events between all combinations of gene arrangements even though there was sufficient nucleotide variation for detection and sufficient opportunity for exchanges to occur. Genetic flux across the inverted chromosome resulted in higher levels of differentiation within 0.1 Mb of inversion breakpoints, but a slightly lower level of differentiation in central inverted regions. No gene conversion events were detected within 17 kb of an inversion breakpoint suggesting that the formation of double-strand breaks is reduced near rearrangement breakpoints in heterozygotes. At least one case where selection rather than proximity to an inversion breakpoint is responsible for reduction in polymorphism was identified.
Project description:We describe genomic structures of 59 X-chromosome segmental duplications that include the proteolipid protein 1 gene (PLP1) in patients with Pelizaeus-Merzbacher disease. We provide the first report of 13 junction sequences, which gives insight into underlying mechanisms. Although proximal breakpoints were highly variable, distal breakpoints tended to cluster around low-copy repeats (LCRs) (50% of distal breakpoints), and each duplication event appeared to be unique (100 kb to 4.6 Mb in size). Sequence analysis of the junctions revealed no large homologous regions between proximal and distal breakpoints. Most junctions had microhomology of 1-6 bases, and one had a 2-base insertion. Boundaries between single-copy and duplicated DNA were identical to the reference genomic sequence in all patients investigated. Taken together, these data suggest that the tandem duplications are formed by a coupled homologous and nonhomologous recombination mechanism. We suggest repair of a double-stranded break (DSB) by one-sided homologous strand invasion of a sister chromatid, followed by DNA synthesis and nonhomologous end joining with the other end of the break. This is in contrast to other genomic disorders that have recurrent rearrangements formed by nonallelic homologous recombination between LCRs. Interspersed repetitive elements (Alu elements, long interspersed nuclear elements, and long terminal repeats) were found at 18 of the 26 breakpoint sequences studied. No specific motif that may predispose to DSBs was revealed, but single or alternating tracts of purines and pyrimidines that may cause secondary structures were common. Analysis of the 2-Mb region susceptible to duplications identified proximal-specific repeats and distal LCRs in addition to the previously reported ones, suggesting that the unique genomic architecture may have a role in nonrecurrent rearrangements by promoting instability.
Project description:Chromosome rearrangements are a significant cause of intellectual disability and birth defects. Subtelomeric rearrangements, including deletions, duplications and translocations of chromosome ends, were first discovered over 40 years ago and are now recognized as being responsible for several genetic syndromes. Unlike the deletions and duplications that cause some genomic disorders, subtelomeric rearrangements do not typically have recurrent breakpoints and involve many different chromosome ends. To capture the molecular mechanisms responsible for this heterogeneous class of chromosome abnormality, we coupled high-resolution array CGH with breakpoint junction sequencing of a diverse collection of subtelomeric rearrangements. We analyzed 102 breakpoints corresponding to 78 rearrangements involving 28 chromosome ends. Sequencing 21 breakpoint junctions revealed signatures of non-homologous end-joining, non-allelic homologous recombination between interspersed repeats and DNA replication processes. Thus, subtelomeric rearrangements arise from diverse mutational mechanisms. In addition, we find hotspots of subtelomeric breakage at the end of chromosomes 9q and 22q; these sites may correspond to genomic regions that are particularly susceptible to double-strand breaks. Finally, fine-mapping the smallest subtelomeric rearrangements has narrowed the critical regions for some chromosomal disorders.