Duplicated DNA Sequences Research Articles

The contribution of copy number variants to psychiatric symptoms and cognitive ability.

Copy number variants (CNVs) are deletions and duplications of DNA sequence. The most frequently studied CNVs, which are described in this review, are recurrent CNVs that occur in the same locations on the genome. These CNVs have been strongly implicated in neurodevelopmental disorders, namely autism spectrum disorder (ASD), intellectual disability (ID), and developmental delay (DD), but also in schizophrenia. More recent work has also shown that CNVs increase risk for other psychiatric disorders, namely, depression, bipolar disorder, and post-traumatic stress disorder. Many of the same CNVs are implicated across all of these disorders, and these neuropsychiatric CNVs are also associated with cognitive ability in the general population, as well as with structural and functional brain alterations. Neuropsychiatric CNVs also show incomplete penetrance, such that carriers do not always develop any psychiatric disorder, and may show only mild symptoms, if any. Variable expressivity, whereby the same CNVs are associated with many different phenotypes of varied severity, also points to highly complex mechanisms underlying disease risk in CNV carriers. Comprehensive and longitudinal phenotyping studies of individual CNVs have provided initial insights into these mechanisms. However, more work is needed to estimate and predict the effect of non-recurrent, ultra-rare CNVs, which also contribute to psychiatric and cognitive outcomes. Moreover, delineating the broader phenotypic landscape of neuropsychiatric CNVs in both clinical and general population cohorts may also offer important mechanistic insights.

A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.

Trapped topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast

Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5' end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) the product of which generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that overexpress the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.

Inverted duplicate DNA sequences increase translocation rates through sequencing nanopores resulting in reduced base calling accuracy.

Inverted duplicated DNA sequences are a common feature of structural variants (SVs) and copy number variants (CNVs). Analysis of CNVs containing inverted duplicated DNA sequences using nanopore sequencing identified recurrent aberrant behavior characterized by low confidence, incorrect and missed base calls. Inverted duplicate DNA sequences in both yeast and human samples were observed to have systematic elevation in the electrical current detected at the nanopore, increased translocation rates and decreased sampling rates. The coincidence of inverted duplicated DNA sequences with dramatically reduced sequencing accuracy and an increased translocation rate suggests that secondary DNA structures may interfere with the dynamics of transit of the DNA through the nanopore.

Genome Synteny Has Been Conserved Among the Octoploid Progenitors of Cultivated Strawberry Over Millions of Years of Evolution.

Allo-octoploid cultivated strawberry (Fragaria × ananassa) originated through a combination of polyploid and homoploid hybridization, domestication of an interspecific hybrid lineage, and continued admixture of wild species over the last 300 years. While genes appear to flow freely between the octoploid progenitors, the genome structures and diversity of the octoploid species remain poorly understood. The complexity and absence of an octoploid genome frustrated early efforts to study chromosome evolution, resolve subgenomic structure, and develop a single coherent linkage group nomenclature. Here, we show that octoploid Fragaria species harbor millions of subgenome-specific DNA variants. Their diversity was sufficient to distinguish duplicated (homoeologous and paralogous) DNA sequences and develop 50K and 850K SNP genotyping arrays populated with co-dominant, disomic SNP markers distributed throughout the octoploid genome. Whole-genome shotgun genotyping of an interspecific segregating population yielded 1.9M genetically mapped subgenome variants in 5,521 haploblocks spanning 3,394 cM in F. chiloensis subsp. lucida, and 1.6M genetically mapped subgenome variants in 3,179 haploblocks spanning 2,017 cM in F. × ananassa. These studies provide a dense genomic framework of subgenome-specific DNA markers for seamlessly cross-referencing genetic and physical mapping information and unifying existing chromosome nomenclatures. Using comparative genomics, we show that geographically diverse wild octoploids are effectively diploidized, nearly completely collinear, and retain strong macro-synteny with diploid progenitor species. The preservation of genome structure among allo-octoploid taxa is a critical factor in the unique history of garden strawberry, where unimpeded gene flow supported its origin and domestication through repeated cycles of interspecific hybridization.

Heterochromatin Bands and rDNA Sites Evolution in Polyploidization Events in Cynodon Rich. (Poaceae)

Cynodon is a genus with a wide distribution in tropical and subtropical areas. Ploidy levels in Cynodon range from diploid to hexaploid; hence, polyploidy is the most important driver of chromosome number variation in this genus. After polyploidization, structural rearrangements such as deletions, insertion, or duplications of DNA sequences frequently occur, allowing variation of chromosome morphology, size, and number. These events might result in a wide diversity of karyotypes, contributing to reproductive isolation, and consequently to speciation. In this study, we investigate the karyotype variation of Cynodon based on comparative cytogenetic analyses. We conducted chromosome counts, DNA quantification, CMA/DAPI double staining, and FISH mapping of 5S and 35S rDNA sites. Cytomolecular data were analyzed in a phylogenetic framework, in order to trace the evolutionary history of the karyotype variation considering polyploidy events in this group. Our results indicate that the most recent common ancestor of Cynodon had two 35S and 5S rDNA sites, two CMA bands, nine DAPI+ bands on the long arm, five DAPI+ bands on the short arm, and three DAPI+ bands in the pericentromeric region. During polyploidization events, there were losses and gains of heterochromatic sequences mainly on the short arms and centromeric regions.

The Neurospora crassa Standard Oak Ridge Background Exhibits Atypically Efficient Meiotic Silencing by Unpaired DNA.

Meiotic silencing by unpaired DNA (MSUD), an RNAi-mediated gene silencing process, is efficient in crosses made in the Neurospora crassa standard Oak Ridge (OR) genetic background. However, MSUD was decidedly less efficient when the OR-derived MSUD testers were crossed with many wild-isolated strains (W), suggesting that either sequence heterozygosity in tester x W crosses suppresses MSUD, or that OR represents the MSUD-conducive extreme in the range of genetic variation in MSUD efficiency. Our results support the latter model. MSUD was less efficient in near-isogenic crosses made in the novel N. crassa B/S1 genetic background, and in N. tetrasperma strain 85. Possibly, in B/S1 and 85, additional regulatory cues, absent from OR, calibrate the MSUD response. A locus in distal chromosome 1R appears to underlie the OR vs. B/S1 difference. Repeat-induced point mutation (RIP) destroys duplicated genes by G:C to A:T mutation of duplicated DNA sequences. Chromosome segment duplications (Dps) dominantly suppress RIP, possibly by titrating out the RIP machinery. In Dp x N crosses, the Dp–borne genes cannot pair properly, hence efficient MSUD, as in OR, silences them and renders the crosses barren. We speculate that the increased productivity engendered by inefficient MSUD enables small duplications to escape RIP.

Stalled replication forks generate a distinct mutational signature in yeast

Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication-associated genome rearrangements are poorly understood, largely due to methodological difficulties in analyzing specific replication forks in vivo. To provide an insight into this process, we analyzed the mutagenic consequences of replication fork stalling at a single, site-specific replication barrier (the Escherichia coli Tus/Ter complex) engineered into the yeast genome. We demonstrate that transient stalling at this barrier induces a distinct pattern of genome rearrangements in the newly replicated region behind the stalled fork, which primarily consist of localized losses and duplications of DNA sequences. These genetic alterations arise through the aberrant repair of a single-stranded DNA gap, in a process that is dependent on Exo1- and Shu1-dependent homologous recombination repair (HRR). Furthermore, aberrant processing of HRR intermediates, and elevated HRR-associated mutagenesis, is detectable in a yeast model of the human cancer predisposition disorder, Bloom's syndrome. Our data reveal a mechanism by which cellular responses to stalled replication forks can actively generate genomic alterations and genetic diversity in normal proliferating cells.

Characterization of a spermine/spermidine transport system reveals a novel DNA sequence duplication in Neisseria gonorrhoeae.

During infection, Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, comes into contact with numerous host compounds including polyamines (e.g. spermine and spermidine). Here, we show that spermine and spermidine concentrations in the growth medium decrease to undetectable levels in the presence of gonococci over time, but not when proteins of the putative polyamine transport system are lost due to mutation. We propose that gonococci have a functional and sole polyamine transport system (PotFGHI) that specifically imports spermine and spermidine. Bioinformatics and molecular analyses showed that the transporter's potGHI genes are organized as an operon while the gene encoding the necessary cognate periplasmic polyamine-binding protein (PotF) is located elsewhere on the chromosome. Interestingly, within the potGHI locus, we identified a novel duplicated sequence, which we term the Pot-Gene-Associated-Duplication-Element, present in variable copy numbers in different gonococcal strains that was likely formed from the 5(') and 3(') ends of the coding sequences of the tandemly linked potH and potG genes, respectively.

New histone supply regulates replication fork speed and PCNA unloading

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.

Biochemical Characterization of Novel Retroviral Integrase Proteins

Integrase is an essential retroviral enzyme, catalyzing the stable integration of reverse transcribed DNA into cellular DNA. Several aspects of the integration mechanism, including the length of host DNA sequence duplication flanking the integrated provirus, which can be from 4 to 6 bp, and the nucleotide preferences at the site of integration, are thought to cluster among the different retroviral genera. To date only the spumavirus prototype foamy virus integrase has provided diffractable crystals of integrase-DNA complexes, revealing unprecedented details on the molecular mechanisms of DNA integration. Here, we characterize five previously unstudied integrase proteins, including those derived from the alpharetrovirus lymphoproliferative disease virus (LPDV), betaretroviruses Jaagsiekte sheep retrovirus (JSRV), and mouse mammary tumor virus (MMTV), epsilonretrovirus walleye dermal sarcoma virus (WDSV), and gammaretrovirus reticuloendotheliosis virus strain A (Rev-A) to identify potential novel structural biology candidates. Integrase expressed in bacterial cells was analyzed for solubility, stability during purification, and, once purified, 3′ processing and DNA strand transfer activities in vitro. We show that while we were unable to extract or purify accountable amounts of WDSV, JRSV, or LPDV integrase, purified MMTV and Rev-A integrase each preferentially support the concerted integration of two viral DNA ends into target DNA. The sequencing of concerted Rev-A integration products indicates high fidelity cleavage of target DNA strands separated by 5 bp during integration, which contrasts with the 4 bp duplication generated by a separate gammaretrovirus, the Moloney murine leukemia virus (MLV). By comparing Rev-A in vitro integration sites to those generated by MLV in cells, we concordantly conclude that the spacing of target DNA cleavage is more evolutionarily flexible than are the target DNA base contacts made by integrase during integration. Given their desirable concerted DNA integration profiles, Rev-A and MMTV integrase proteins have been earmarked for structural biology studies.

Clusters of Ancestrally Related Genes That Show Paralogy in Whole or in Part Are a Major Feature of the Genomes of Humans and Other Species

Arrangements of genes along chromosomes are a product of evolutionary processes, and we can expect that preferable arrangements will prevail over the span of evolutionary time, often being reflected in the non-random clustering of structurally and/or functionally related genes. Such non-random arrangements can arise by two distinct evolutionary processes: duplications of DNA sequences that give rise to clusters of genes sharing both sequence similarity and common sequence features and the migration together of genes related by function, but not by common descent [1], [2], [3]. To provide a background for distinguishing between the two, which is important for future efforts to unravel the evolutionary processes involved, we here provide a description of the extent to which ancestrally related genes are found in proximity.Towards this purpose, we combined information from five genomic datasets, InterPro, SCOP, PANTHER, Ensembl protein families, and Ensembl gene paralogs. The results are provided in publicly available datasets (http://cgd.jax.org/datasets/clustering/paraclustering.shtml) describing the extent to which ancestrally related genes are in proximity beyond what is expected by chance (i.e. form paraclusters) in the human and nine other vertebrate genomes, as well as the D. melanogaster, C. elegans, A. thaliana, and S. cerevisiae genomes.With the exception of Saccharomyces, paraclusters are a common feature of the genomes we examined. In the human genome they are estimated to include at least 22% of all protein coding genes. Paraclusters are far more prevalent among some gene families than others, are highly species or clade specific and can evolve rapidly, sometimes in response to environmental cues. Altogether, they account for a large portion of the functional clustering previously reported in several genomes.

Patterns of Repeat-Induced Point Mutation in Transposable Elements of Basidiomycete Fungi

Transposable elements (TEs) are ubiquitous genomic parasites that have prompted the evolution of genome defense systems that restrict their activity. Repeat-induced point mutation (RIP) is a homology-dependent genome defense that introduces C-to-T transition mutations in duplicated DNA sequences and is thought to control the proliferation of selfish repetitive DNA. Here, we determine the taxonomic distribution of hypermutation patterns indicative of RIP among basidiomycetes. We quantify C-to-T transition mutations in particular di- and trinucleotide target sites for TE-like sequences from nine fungal genomes. We find evidence of RIP-like patterns of hypermutation at TpCpG trinucleotide sites in repetitive sequences from all species of the Pucciniomycotina subphylum of the Basidiomycota, Microbotryum lychnidis-dioicae, Puccinia graminis, Melampsora laricis-populina, and Rhodotorula graminis. In contrast, we do not find evidence for RIP-like hypermutation in four species of the Agaricomycotina and Ustilaginomycotina subphyla of the Basidiomycota. Our results suggest that a RIP-like process and the specific nucleotide context for mutations are conserved within the Pucciniomycotina subphylum. These findings imply that coevolutionary interactions between TEs and a hypermutating genome defense are stable over long evolutionary timescales.

A factor in a wild isolated Neurospora crassa strain enables a chromosome segment duplication to suppress repeat-induced point mutation

Repeat-induced point mutation (RIP) is a sexual stage-specific mutational process of Neurospora crassa and other fungi that alters duplicated DNA sequences. Previous studies from our laboratory showed that chromosome segment duplications (Dps) longer than (approx.) 300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps less than 200 kbp did not individually suppress RIP, they could do so in homozygous and multiply heterozygous crosses, provided the sum of the duplicated DNA exceeds (approx.) 300 kbp. Here we demonstrate suppression of RIP in a subset of progeny carrying the normally sub-threshold 154 kbp Dp(R2394) from a cross of T(R2394) to the wild isolated Carrefour Mme. Gras strain (CMG). Thus, the CMG strain contains a factor that together with Dp(R2394) produces a synthetic RIP suppressor phenotype. It is possible that the factor is a cryptic Dp that together with Dp(R2394) can exceed the size threshold for titration of the RIP machinery and thereby causes RIP suppression.

Copy Number Variants and Schizophrenia

Approximately 12% of the human genome consists of relatively common copy number variations (CNVs) — i.e., insertions or duplications of DNA sequences

Titration of repeat-induced point mutation (RIP) by chromosome segment duplications in Neurospora crassa

Repeat-induced point mutation (RIP) is a hypermutational process that alters duplicated DNA sequences in Neurospora crassa. In previous studies, five of six large ( > 100 kb) chromosome segment duplications (Dp's) examined were shown to dominantly suppress RIP in smaller (< 5 kb) duplications. The suppressor duplications were > 270 kb, whereas the lone non-suppressor duplication was approximately 117 kb. We have now screened another 33 duplications and found 29 more suppressors and four more non-suppressors. All 22 suppressor duplications whose size could be estimated were > 270 kb, whereas two newly identified non-suppressor duplications examined were 140-154 kb. RIP was suppressed in a subset of crosses heterozygous for more than one ordinarily non-suppressor duplication. These results strengthen the hypothesis that large duplications titrate out the RIP machinery and suggest the "equivalence point" for the titration is close to 300 kb.

Mutations in mating-type genes greatly decrease repeat-induced point mutation process in the fungus Podospora anserina

RIP (Repeat-Induced point Mutation) and PR (Premeiotic Recombination) are two developmentally regulated processes in filamentous ascomycetes. RIP detects and mutates duplicated DNA sequences, while PR results in deletion of the interstitial sequence between cis-duplicated DNA sequences. These two silencing processes take place between fertilization and premeiotic replication, a period during which the mating-type genes play an active role in several developmental processes. Previous studies have shown that mutations in the mating-type genes affect the development of the fruiting body. This study provides evidence that mutations in the mating-type genes reduce the frequency of RIP and PR. It establishes that alleles which have the more stringent effect on fruiting-body development, have also the strongest effect on RIP and PR frequencies. We propose two models for the relation between mating-type genes and RIP and PR, one based on the direct control of RIP and PR by mating-type regulatory proteins, the other based on an indirect effect through the control of a development step during which RIP and PR take place.

Loss of the rpl32 gene from the chloroplast genome and subsequent acquisition of a preexisting transit peptide within the nuclear gene in Populus

Gene transfer events from organelle genomes (mitochondria and chloroplasts in plants) to the nuclear genome are important processes in the evolution of the eukaryotic cell. It is highly likely that the gene transfer event is still an ongoing process in higher plant mitochondria and chloroplasts. The number and order of genes encoded in the chloroplast genome of higher plants are highly conserved. Recently, several exceptional cases of gene loss from the chloroplast genome have been discovered as the number of complete chloroplast genome sequences has increased. The Populus chloroplast genome has lost the rpl32 gene, while the corresponding the chloroplast rpl32 (cp rpl32) gene has been identified in the nuclear genome. Nuclear genes transferred from the chloroplast genome need to gain a sequence that encodes a transit peptide. Here, we revealed that the nuclear cp rpl32 gene has acquired the exon sequence, which is highly homologous to a transit peptide derived from the chloroplast Cu–Zn superoxide dismutase (cp sod-1) gene. The cp rpl32 gene has acquired the sequence that encodes not only for the transit peptide, but also for the conserved N-terminal portion of the mature SOD protein from the cp sod-1 gene, suggesting the occurrence of DNA sequence duplication. Unlike cp SOD-1, cp RPL32 did not show biased localization in the chloroplasts. This difference may be caused by mutations accumulated in the sequence of the SOD domain on the cp rpl32 gene. We provide new insight into the fate of the inherent sequence derived from a transit peptide.

Spontaneous duplications in diploid Saccharomyces cerevisiae cells

The duplication of DNA sequences is a powerful determinant of genomic plasticity and is known to be one of the key factors responsible for evolution. Recent genomic sequence data demonstrate the abundance of duplicated genes in all surveyed organisms. Over the past years, experimental systems were adequately designed to explore the molecular mechanisms involved in their formation in haploid Saccharomyces cerevisiae strains. To obtain a more global and accurate view of the events leading to DNA sequence duplications, we have selected and characterized duplication occurrences in diploid S. cerevisiae cells. The molecular analysis showed that two other predominant ways lead to duplication in this context: formation of extra chimeric chromosomes and non-reciprocal translocation events. Moreover, we demonstrated that these two types of rearrangements are RAD52 independent and therefore that homologous recombination plays no part in their formation. Finally, our results show the multiplicity of mechanisms involved in duplication events and provide the first experimental evidence that these mechanisms might be ploidy dependent.

A common 93-kb duplicated DNA sequence at 1q21.2 in acute lymphoblastic leukemia and Burkitt lymphoma

In three patients with acute lymphoblastic leukemia (ALL) and in another with Burkitt lymphoma (BL), conventional cytogenetics and fluorescence in situ hybridization (FISH), applied singly or in combination, showed 1q duplication in two cases of ALL with hyperdiploid karyotypes, 1q duplication resulting from an unbalanced translocation in a third case of ALL, and inv dup(1)(q) in a patient with BL. Centromeric or telomeric breakpoints and extension of the 1q duplicons varied in each case. FISH defined a minimal, common duplicated region of 93kb at band 1q21.2 corresponding to clone RP11-212K13. In this region three putative oncogenes or tumor suppressor genes have been mapped: SF3B4 (splicing factor 3b, subunit 4), OTUD7B (OTU domain containing 7B), and MTMR11 (myotubularin related protein 11). For the first time, a minimal common 1q21.2 duplicated sequence has been identified in lymphoid malignancies in a region where putative oncogenes or suppressor genes have been mapped. This finding elucidates the genomic background of ALL and BL with 1q duplication and provides the basis for molecular studies investigating which genes are involved in leukemogenesis or disease progression in these cases.