Loss Of DNA Research Articles

Abstract 3670: Development of cfDNA-based methylation assay for early cancer detections

Abstract Introduction: DNA methylation is the essential epigenetic mechanism of cell regulation, and one of the earliest signatures in cancer development. Blood-based DNA methylation has been tested for cancer early detection. Several methods have been used for DNA methylation sequencing, which include whole genome bisulfite sequencing (WGBS) and antibody-dependent DNA immunoprecipitation (MeDIP). However, WGBS has known issues of DNA loss and fragmentations, and MeDIP only captures the methylated DNA regions and are biased. Therefore, methods that can accurately quantify DNA methylation status while keeping DNA less damaged and loss are highly needed, especially for liquid biopsy at early cancer stages (due to its low cfDNA amount). Methods: We developed a probe capture-based cfDNA methylation assay using proprietary in-house developed liquid biopsy NGS platform and DeepSEA software algorithm. Analytic assay validation has been conducted using reference materials with known methylation profiles. Both cell line gDNA with known methylation profiles and plasma cfDNA from patients were used for this methylation study. Methylation profiles, DNA damages, and losses were evaluated and compared. Fragment distribution and copy number variations (CNV) were compared between untreated and treated samples. Results: In this study, we developed a cfDNA-based methylation assay that measures DNA methylation status and genomic profiling simultaneously. Using both cell line gDNA and plasma cfDNA, the assay detects both methylation change and genomic alterations, including fragment size distribution and CNV in a single test. Discussion: The successful development of cfDNA methylation assay could help identify cancer at its early stage. It also enables the detection and monitoring of minimal residual disease (MRD) and in obtaining information on therapeutic targets and resistance mechanisms relevant to the management of personalized cancer care. Citation Format: Binggang Xiang, Zhixin Zhao, Maya Stevens, Chao Dai, Feng Xie, Xiaohong Wang, Kemin Zhou, Shidong Jia, Pan Du, Jianjun Yu. Development of cfDNA-based methylation assay for early cancer detections [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3670.

3D Printing-Enabled DNA Extraction for Long-Read Genomics.

Long-read genomics technologies such as nanopore sequencing and genome mapping in nanochannels extract genomic information in the kilobase to megabase pair range from single DNA molecules, thereby overcoming read-length limitations in next-generation DNA sequencing. Long-read technologies start with long DNA molecules as the input and thus benefit from universal sample preparation methods that are fast and shear-free and present a scope of automation and direct upstream integration. We describe a 3D printing-assisted poly(dimethylysiloxane)-based DNA sample preparation device, where diffusive chemical lysis followed by electrophoresis produces circa 100 ng of long DNA directly from cells with less than 5 min of labor. Assessment of the product DNA by confinement in nanochannels reveals that the DNA sizes are commensurate with the requirements for long-read single-molecule technologies. Microfluidics not only expedites sample preparation, but also offers the opportunity for integration with genomics technologies to eliminate DNA fragmentation and loss during transfer to the genomic device.

Exclusive use of digital PCR allows an absolute assay of heat-killed Lactobacilli in foods targeting multiple copies of 16S rDNA

The real-time PCR (qPCR) and digital PCR (dPCR) to amplify a single-copy of house-keeping genes (i.e., hsp60, pheS or tuf) are used for the assay of limited microbial species. In general, with a single-copy gene, there are obviously varied DNA sequences for even the same microbial species, which could cause difficulties with design of primers and probes for PCR when targeting various single copy genes. In general, for identification by dPCR (as a representative case: Lactobacillus paracasei), accumulated DNA sequence information of 16S rDNA, which is much more frequently used, should be targeted. In contrast, next-generation sequencing revealed that there are five copies of 16S rDNA in a live L. paracasei MCC1849. Therefore, we aimed to reveal, if heat-killed L. paracasei supplemented in nutritional foods that aid the host immune system have the relevant five copies per chromosomal DNA, and if the relevant copies remain unchanged on the same chromosomal DNA or remain to be different chromosomal DNA fragments. So, we revealed the actual distribution of the potential original five copies of 16S rDNA using our innovative dPCR, in which both 16S rDNA and hsp60 genes were simultaneously elongated. The molecular ratios of 16S rDNA/hsp60 dispersed in the dPCR chip were then estimated. The 16S rDNA/hsp60 molecular ratios of the heat-killed L. paracasei in foods, resultantly ranged from 5.0 to 7.2, being the same or higher than that of the five copies determined by next-generation sequencing. The 16S rDNA copy number/ratio indicated the chromosomal DNA molecular number and the associated cell number. As significance, different nutritional foods could potentially cause the loss of chromosomal DNA of supplemented beneficial microbes to a much greater degree. Our absolute dPCR does not require standard correlative samples for the estimation of final products. The estimation principle of the ratio of 16S rDNA/a house-keeping single-copy gene by our absolute dPCR could lead to a useful and accurate assay for various nutritional foods.

Factors affecting DNA recovery from cartridge cases

Cartridge cases are often the sole items left behind after a shooting incident and DNA traces from these can identify persons connected to the shooting. However, the chance of retrieving usable DNA profiles from cartridge cases is limited, due to the low amounts of deposited DNA and subsequent DNA loss associated with the firing process. In the current study, we set out to increase the DNA recovery from cartridge cases and cartridges by evaluating different swab types and detergents used for trace collection. A protocol applying nylon-flocked swabs instead of cotton swabs was implemented in casework at the Swedish National Forensic Centre (NFC), increasing DNA yield. The number of samples providing a DNA concentration ≥ 0.001 ng/μL (the in-house cut-off for processing low-template samples) increased from 11.1 to 28.6 % for cartridge cases and from 16.0 to 43.3 % for cartridges. There was also a substantial increase in mixed STR profiles, too complex to use for comparisons. Thus, it was not possible to take the full advantage of the elevated DNA yield provided by nylon-flocked swabs. The number of usable STR profiles increased from 5.0 to 8.0 % for cartridge cases and remained unchanged for cartridges. Controlled studies were performed to assess the impact on the DNA recovery from different persons handling the ammunition, different material and size of the cartridge cases, and the type of firearm used. These studies reflected an ideal situation, where all cartridges were extensively handled and loaded without gloves, thus providing a higher expected DNA yield compared to most casework samples. The total peak height differed by up to a factor of ∼50 when 20 different persons handled cartridges prior to shooting. By evaluating eleven combinations of different firearms and ammunition, it was found that the casing material and type of firearm also have a substantial impact on DNA yield.

Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae.

Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8–99.3%; R2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicability of our protocol for field samples, we analyzed three different Hong Kong coral samples. Six Symbiodiniaceae genera were detected in Acropora valida, Oulastrea crispata, and Platygyra acuta, with Breviolum, Effrenium, Fugacium, and Gerakladium sp. being reported for the first time. Our results suggest that aggressively disrupting cells to ensure thorough cell lysis, estimating cell loss and DNA loss, and validating qPCR assays are critical for success. The number of species examined here is limited, but the primers are potentially applicable to most species in respective genera, and the protocol and the approach to develop it provide a base and template toward a standardized procedure for quantitatively characterizing Symbiodiniaceae communities in corals.

The accessory subunit of human DNA polymerase γ is required for mitochondrial DNA maintenance and is able to stabilize the catalytic subunit

Human DNA polymerase γ (POLG) is a mitochondria-specific replicative DNA polymerase consisting of a single catalytic subunit, POLGα, and a dimeric accessory subunit, POLGβ. To gain a deeper understanding of the role of POLGβ, we knocked out this protein in cultured human cybrid cells and established numerous knockout clones. POLGβ-knockout clones presented a clear phenotype of mitochondrial DNA loss, indicating that POLGβ is necessary for mitochondrial DNA replication. Moreover, POLGβ-knockout cells showed a severe decrease in POLGα levels and acute suppression of POLGβ expression efficiently down-regulated POLGα levels. These results suggest that, in addition to its role as the processivity factor of POLG, POLGβ acts as a POLGα stabilizer, an important role for POLGβ in mitochondrial DNA maintenance.

Six Multiplex TaqManTM-qPCR Assays for Quantitative Diagnostics of Pseudomonas Species Causative of Bacterial Blotch Diseases of Mushrooms.

Bacterial blotch is a group of economically important diseases of the common button mushroom (Agaricus bisporus). Once the pathogens are introduced to a farm, mesophilic growing conditions (that are optimum for mushroom production) result in severe and widespread secondary infections. Efficient, timely and quantitative detection of the pathogens is hence critical for the design of localized control strategies and prediction of disease risk. This study describes the development of real-time TaqManTM assays that allow molecular diagnosis of three currently prevalent bacterial blotch pathogens: “Pseudomonas gingeri,” Pseudomonas tolaasii and (as yet uncharacterized) Pseudomonas strains (belonging to Pseudomonas salomonii and Pseudomonas edaphica). For each pathogen, assays targeting specific DNA markers on two different loci, were developed for primary detection and secondary verification. All six developed assays showed high diagnostic specificity and sensitivity when tested against a panel of 63 Pseudomonas strains and 40 other plant pathogenic bacteria. The assays demonstrated good analytical performance indicated by linearity across calibration curve (>0.95), amplification efficiency (>90%) and magnitude of amplification signal (>2.1). The limits of detection were optimized for efficient quantification in bacterial cultures, symptomatic tissue, infected casing soil and water samples from mushroom farms. Each target assay was multiplexed with two additional assays. Xanthomonas campestris was detected as an extraction control, to account for loss of DNA during sample processing. And the total Pseudomonas population was detected, to quantify the proportion of pathogenic to beneficial Pseudomonas in the soil. This ratio is speculated to be an indicator for blotch outbreaks. The multiplexed assays were successfully validated and applied by routine testing of diseased mushrooms, peat sources, casing soils, and water from commercial production units.

Optimization of Soil DNA Extraction Protocol Using Na2EDTA, SDS, Heating, Vortexing and CaCl2 and Its Validation for Metagenomic Studies

Aim: Several methods described previously for isolation and purification of soil DNA. Most of these protocols use combination of techniques or methods but the role and contribution of each individual method or component used is not clearly discussed. This study aims at analysing the effect of individual components used in extraction of DNA from soil and finally to optimize soil DNA isolation protocol and its validation by using 16SrDNA sequence analysis.
 Methods and Results: The soil was washed with anionic buffers before lysis step to reduce humic substances and release microbial cells from soil matrix, then the cells were lysed using combination of SDS, heating and vortexing and finally humic substances were removed using chemical flocculation. Pre-lysis washing of soil with 100 mmol l-1 Na2EDTA proved good for releasing microbial cells from soil matrix. Heating the soil sample at 75°C yielded good quantity (15.73 µg g-1 soil) DNA followed by 2% SDS (10.28 µg g-1 soil) and vortexing at 1400 rpm (8.94 µg g-1 soil). Combination of heating, SDS and vortexing yielded 25 µg DNA per gram of soil. Different concentrations of chemical flocculants like AlNH4(SO4)2, FeCl3, CaCl2 and MgCl2 were used to reduce humic substances. Flocculation with 100 mmol l-1 CaCl2 removed 5.2 mg humic substances without significant loss of DNA. 16S rDNA sequence analysis of DNA extracted from soil reveals presence of all the common soil bacterial species indicating the protocol is unbiased.
 Conclusion: Combination of chemical (SDS) and physical (heating and vortexing) methods yield good DNA whereas addition of enzyme (lysozyme) did not show significant effect on cell lysis. The digestion of isolated DNA with restriction enzyme and amplification of 16S rDNA using Taq DNA polymerase indicates the isolated DNA is pure enough for metagnomic analysis. 16Sr DNA sequencing of soil DNA indicates that this protocol can extract good quality and quantity DNA from range of bacteria present in soil varying in their cell wall composition. The optimised protocol is unbiased, very simple, does not need special equipments and many samples can be processed simultaneously.

Integrative genomics approach identifies molecular features associated with early-stage ovarian carcinoma histotypes

Ovarian cancer comprises multiple subtypes (clear-cell (CCC), endometrioid (EC), high-grade serous (HGSC), low-grade serous (LGSC), and mucinous carcinomas (MC)) with differing molecular and clinical behavior. However, robust histotype-specific biomarkers for clinical use have yet to be identified. Here, we utilized a multi-omics approach to identify novel histotype-specific genetic markers associated with ovarian carcinoma histotypes (CCC, EC, HGSC, and MC) using DNA methylation, DNA copy number alteration and RNA sequencing data for 96 primary invasive early-stage (stage I and II) ovarian carcinomas. More specifically, the DNA methylation analysis revealed hypermethylation for CCC in comparison with the other histotypes. Moreover, copy number imbalances and novel chromothripsis-like rearrangements (n = 64) were identified in ovarian carcinoma, with the highest number of chromothripsis-like patterns in HGSC. For the 1000 most variable transcripts, underexpression was most prominent for all histotypes in comparison with normal ovarian samples. Overall, the integrative approach identified 46 putative oncogenes (overexpressed, hypomethylated and DNA gain) and three putative tumor suppressor genes (underexpressed, hypermethylated and DNA loss) when comparing the different histotypes. In conclusion, the current study provides novel insights into molecular features associated with early-stage ovarian carcinoma that may improve patient stratification and subclassification of the histotypes.

Discordant evolution of mitochondrial and nuclear yeast genomes at population level

BackgroundMitochondria are essential organelles partially regulated by their own genomes. The mitochondrial genome maintenance and inheritance differ from the nuclear genome, potentially uncoupling their evolutionary trajectories. Here, we analysed mitochondrial sequences obtained from the 1011 Saccharomyces cerevisiae strain collection and identified pronounced differences with their nuclear genome counterparts.ResultsIn contrast with pre-whole genome duplication fungal species, S. cerevisiae mitochondrial genomes show higher genetic diversity compared to the nuclear genomes. Strikingly, mitochondrial genomes appear to be highly admixed, resulting in a complex interconnected phylogeny with a weak grouping of isolates, whereas interspecies introgressions are very rare. Complete genome assemblies revealed that structural rearrangements are nearly absent with rare inversions detected. We tracked intron variation in COX1 and COB to infer gain and loss events throughout the species evolutionary history. Mitochondrial genome copy number is connected with the nuclear genome and linearly scale up with ploidy. We observed rare cases of naturally occurring mitochondrial DNA loss, petite, with a subset of them that do not suffer the expected growth defect in fermentable rich media.ConclusionsOverall, our results illustrate how differences in the biology of two genomes coexisting in the same cells can lead to discordant evolutionary histories.

Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR.

Despite the interest in characterizing genomic variation, the presence of large repeats at the breakpoints hinders the analysis of many structural variants. This is especially problematic for inversions, since there is typically no gain or loss of DNA. Here, we tested novel linkage-based droplet digital PCR (ddPCR) assays to study 20 inversions ranging from 3.1 to 742 kb flanked by inverted repeats (IRs) up to 134 kb long. Of those, we validated 13 inversions predicted by different genome-wide techniques. In addition, we obtained new experimental human population information across 95 African, European, and East Asian individuals for 16 inversions, including four already validated variants without high-throughput genotyping methods. Through comparison with previous data, independent replicates and both inversion breakpoints, we demonstrate that the technique is highly accurate and reproducible. Most studied inversions are widespread across continents, and their frequency is negatively correlated with genetic length. Moreover, all except two show clear signs of being recurrent, and we could better define the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, the generated genotypes have allowed us to check inversion functional effects, validating gene expression differences reported before for two inversions and finding new candidate associations. Therefore, the developed methodology makes it possible to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

Effect of High-Dose Topical Minoxidil on Erythrocyte Quality in SKH1 Hairless Mice.

Simple SummaryIn any animal species, involuntary exposure to unknown agents may increase genetic material damage. This genetic damage can also induce the appearance of diseases such as cancer or other pathologies, including problems that can be passed on to the offspring of the damaged individual. For instance, living organisms may be affected due to the use of medications or exposure to certain chemical, physical, or biological agents which cause cell failure. This impaired function acts as an indicator that helps identify and evaluate damage in order to avoid or minimize it. In this work, excessive doses of a cosmetic drug for topical use in dermatological treatments, known as minoxidil, produced defects in the blood of hairless mice, particularly in red cells, indicating loss of DNA, a situation that may compromise life or the offspring by causing damage to their genetic material. It is important to consider that compounds may be tissue- or species-specific, although we cannot rule out the possibility that similar damage could occur in other animal species. Thus, excessive exposure to this compound should be prevented.SKH1 hairless mice are widely used in carcinogenesis and dermatology research due to their bare skin, as exposure to different agents is facilitated. Minoxidil is a cosmetic drug that is recognized as a mitogenic agent, and mitogens are suggested to have carcinogenic and mutagenic potential by inducing cell division and increasing the possibility of perpetuating DNA damage. Therefore, we hypothesized that the application of high doses of minoxidil to the skin of hairless mice would increase the number of micronucleated erythrocytes (MNEs) in peripheral blood. The objective of this study was to evaluate the topical administration of high doses of minoxidil on peripheral blood erythrocytes of SKH1 mice by means of micronucleus assay. Minoxidil was administered on the entire body surface of mice every 12 or 24 h. Minoxidil dosing every 24 h increased the number of micronucleated polychromatic erythrocytes (MNPCEs), and dosing every 12 h increased the number of MNEs and MNPCEs, as compared to baseline and the negative control group. No decrease in polychromatic erythrocyte frequencies was observed in the minoxidil groups. Therefore, topical application of high minoxidil doses to mice can produce DNA damage, as observed through an increase in the number of MNEs, without producing cytotoxicity, possibly due to its mitogenic effect.

The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication.

ABSTRACTIt is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.

Drug Targeting of Genomic Instability in Multiple Myeloma.

Genomic instability can be observed at both chromosomal and chromatin levels. Instability at the macro level includes centrosome abnormalities (CA) resulting in numerical as well as structural chromosomal changes, whereas instability at the micro level is characterized by defects in DNA repair pathways resulting in microsatellite instability (MIN) or mutations. Genomic instability occurs during carcinogenesis without impairing survival and growth, though the precise mechanisms remain unclear. Solid tumors arising from most cells of epithelial origin are characterized by genomic instability which renders them resistant to chemotherapy and radiotherapy. This instability is also observed in 25% of myeloma patients and has been shown to be highly prognostic, independently of the international staging system (ISS). However, a biomarker of aberrant DNA repair and loss of heterozygosity (LOH), was only observed at a frequency of 5% in newly diagnosed patients. Several new molecules targeting the pathways involved in genomic instability are under development and some have already entered clinical trials. Poly(ADP-ribose) polymerase-1 (PARP) inhibitors have been FDA-approved for the treatment of breast cancer type 1 susceptibility protein (BRCA1)-mutated metastatic breast cancer, as well as ovarian and lung cancer. Topoisomerase inhibitors and epigenetic histone modification-targeting inhibitors, such as HDAC (Histone Deacetylase) inhibitors which are novel agents that can target genomic instability. Several of the small molecule inhibitors targeting chromosomal level instability such as PARP, Akt, Aurora kinase, cyclin dependent kinase or spindle kinase inhibitors have been tested in mouse models and early phase I/II trials. ATM, ATR kinase inhibitors and DNA helicase inhibitors are also promising novel agents. However, most of these drugs are not effective as single agents but appear to act synergistically with DNA damaging agents such as radiotherapy, platinum derivatives, immunomodulators, and proteasome inhibitors. In this review, new drugs targeting genomic instability and their mechanisms of action will be discussed.

Tumor cells suppress radiation-induced immunity by hijacking caspase 9 signaling.

High-dose radiation activates caspases in tumor cells to produce abundant DNA fragments for DNA sensing in antigen-presenting cells, but the intrinsic DNA sensing in tumor cells after radiation is rather limited. Here we demonstrate that irradiated tumor cells hijack caspase 9 signaling to suppress intrinsic DNA sensing. Instead of apoptotic genomic DNA, tumor-derived mitochondrial DNA triggers intrinsic DNA sensing. Specifically, loss of mitochondrial DNA sensing in Casp9-/- tumors abolishes the enhanced therapeutic effect of radiation. We demonstrated that combining emricasan, a pan-caspase inhibitor, with radiation generates synergistic therapeutic effects. Moreover, loss of CASP9 signaling in tumor cells led to adaptive resistance by upregulating programmed death-ligand 1 (PD-L1) and resulted in tumor relapse. Additional anti-PD-L1 blockade can further overcome this acquired immune resistance. Therefore, combining radiation with a caspase inhibitor and anti-PD-L1 can effectively control tumors by sequentially blocking both intrinsic and extrinsic inhibitory signaling.

Rapid loss of endogenous DNA in pig bone buried in five different environments

The rate at which endogenous DNA from differently prepared (butchered, boiled and baked) compact pig bones degrades in five different Danish terrestrial and marine environments over 12 months was investigated. Although > 70% of the estimated endogenous mtDNA is lost after just four weeks of exposure, no cytosine deamination of DNA was recognised. A correlation between the presence of oxygen and the amount of preserved DNA was observed. The results provide valuable information on the interaction between the endogenous DNA and the depositional environment in the early stages of bone diagenesis, which can be a support in the interpretation of the initial diagenetic pathways of archaeological bone.

Hox genes reveal variations in the genomic DNA of allotetraploid hybrids derived from Carassius auratus red var. (female) \xd7 Cyprinus carpio L. (male)

BackgroundHox transcription factors are master regulators of animal development. Although highly conserved, they can contribute to the formation of novel biological characteristics when modified, such as during the generation of hybrid species, thus potentially serving as species-specific molecular markers. Here, we systematically studied the evolution of genomic sequences of Hox loci in an artificial allotetraploid lineage (4nAT, 4n = 200) derived from a red crucian carp (♀, RCC, 2n = 100) × common carp (♂, CC, 2n = 100) cross and its parents (RCC and CC).ResultsPCR amplification yielded 23 distinct Hox gene fragments from 160 clones in 4nAT, 22 fragments from 90 clones in RCC, and 19 fragments from 90 clones in CC. Sequence alignment of the HoxA3a and HoxC10a genes indicated both the inheritance and loss of paternal genomic DNA in 4nAT. The HoxA5a gene from 4nAT consisted of two subtypes from RCC and two subtypes from CC, indicating that homologous recombination occurred in the 4nAT hybrid genome. Moreover, 4nAT carried genomic pseudogenization in the HoxA10b and HoxC13a loci. Interestingly, a new type of HoxC9a gene was found in 4nAT as a hybrid sequence of CC and RCC by recombination in the intronic region.ConclusionThe results revealed the influence of Hox genes during polyploidization in hybrid fish. The data provided insight into the evolution of vertebrate genomes and might be benefit for artificial breeding programs.

Evolution and Diversity of the Wild Rice Oryza officinalis Complex, across Continents, Genome Types, and Ploidy Levels.

The Oryza officinalis complex is the largest species group in Oryza, with more than nine species from four continents, and is a tertiary gene pool that can be exploited in breeding programs for the improvement of cultivated rice. Most diploid and tetraploid members of this group have a C genome. Using a new reference C genome for the diploid species O. officinalis, and draft genomes for two other C genome diploid species Oryza eichingeri and Oryza rhizomatis, we examine the influence of transposable elements on genome structure and provide a detailed phylogeny and evolutionary history of the Oryza C genomes. The O. officinalis genome is 1.6 times larger than the A genome of cultivated Oryza sativa, mostly due to proliferation of Gypsy type long-terminal repeat transposable elements, but overall syntenic relationships are maintained with other Oryza genomes (A, B, and F). Draft genome assemblies of the two other C genome diploid species, Oryza eichingeri and Oryza rhizomatis, and short-read resequencing of a series of other C genome species and accessions reveal that after the divergence of the C genome progenitor, there was still a substantial degree of variation within the C genome species through proliferation and loss of both DNA and long-terminal repeat transposable elements. We provide a detailed phylogeny and evolutionary history of the Oryza C genomes and a genomic resource for the exploitation of the Oryza tertiary gene pool.

Cohesin Is Out for Stalled Replication Fork Restart.

Replication stress underlies many genomic alterations in cancer cells. In this issue of Developmental Cell, Benedict etal. show that WAPL-dependent cohesin removal is needed to restart DNA synthesis at stalled forks and promote survival following replication stress, uncovering an unexpected link between stress and sister chromatid cohesion loss.

Locus-Specific Regulation of Xist Expression Using the CRISPR-Cas9-Based System.

DNA methylation inhibitor or loss and gain of function of DNA methylation key players were widely used to investigate the regulation of X inactive-specific transcript (Xist) expression by DNA methylation, which results in global change of DNA methylation. Here, we reported a novel method for regulation of Xist using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. First, Xist expression was increased in 5-aza-2'-deoxycytidine-treated female goat fibroblast cells. Second, three single-guide RNAs (sgRNAs) that target the Xist differential methylation region (DMR) were inserted to deactivated Cas9 (dCas9) nuclease and the catalytic domain of the DNA methyltransferase Dnmt3a coexpression plasmid. Bisulfite PCR analysis and quantitative real-time PCR revealed that the methylation level of the DMR was significantly increased, while the expression of Xist was downregulated in all three sgRNAs, compared with the mock-transfected cells. Third, the methylation activity at the sites of 37 bp from the protospacer-adjacent motif sequence showed the strong change relative to the mock-transfected cells. Furthermore, genome-wide DNA methylation and expression of the DNA methylation key players were not statistically changed in all three sgRNAs. Therefore, we confirmed that Xist expression was regulated by DNA methylation, and directed DNA methylation of Xist DMR at locus-specific solution decreased Xist expression.