TRF1和TRF2结合端粒是由核小体组织调制

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection.

• http://nar.oxfordjournals.org/cgi/content/short/gkv507v1?rss=1

[详细]

的转录终止因子ρ结核分枝杆菌ATP依赖的运动活动

The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (_MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that _MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the _MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of _MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.

• http://nar.oxfordjournals.org/cgi/content/short/gkv505v1?rss=1

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甲基膦酸二甲酯立体专一性抑制活性位点突变体取代基揭示了位点特异性的DNA重组过程中的立体化学

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The S_P preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or R_P-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

• http://nar.oxfordjournals.org/cgi/content/short/gkv513v1?rss=1

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人类疱疹病毒6 / B U94 ATP酶，解旋酶和DNA结合蛋白的表征，核酸外切酶

Human herpesvirus-6A (HHV-6A) and HHV-6B integrate their genomes into the telomeres of human chromosomes, however, the mechanisms leading to integration remain unknown. HHV-6A/B encode a protein that has been proposed to be involved in integration termed U94, an ortholog of adeno-associated virus type 2 (AAV-2) Rep68 integrase. In this report, we addressed whether purified recombinant maltose-binding protein (MBP)-U94 fusion proteins of HHV-6A/B possess biological functions compatible with viral integration. We could demonstrate that MBP-U94 efficiently binds both dsDNA and ssDNA containing telomeric repeats using gel shift assay and surface plasmon resonance. MBP-U94 is also able to hydrolyze adenosine triphosphate (ATP) to ADP, providing the energy for further catalytic activities. In addition, U94 displays a 3' to 5' exonuclease activity on dsDNA with a preference for 3'-recessed ends. Once the DNA strand reaches 8–10 nt in length, the enzyme dissociates it from the complementary strand. Lastly, MBP-U94 compromises the integrity of a synthetic telomeric D-loop through exonuclease attack at the 3' end of the invading strand. The preferential DNA binding of MBP-U94 to telomeric sequences, its ability to hydrolyze ATP and its exonuclease/helicase activities suggest that U94 possesses all functions required for HHV-6A/B chromosomal integration.

• http://nar.oxfordjournals.org/cgi/content/short/gkv503v1?rss=1

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DNA-PKcs的磷酸化hnrnp-a1便于复制后rpa-to-pot1开关和端粒封盖

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3' overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3' overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations.

• http://nar.oxfordjournals.org/cgi/content/short/gkv539v1?rss=1

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基因调节的新机制：结核分枝杆菌蛋白的RNA聚合酶rv1222锚定到DNA抑制转录

We propose a novel mechanism of gene regulation in Mycobacterium tuberculosis where the protein Rv1222 inhibits transcription by anchoring RNA polymerase (RNAP) onto DNA. In contrast to our existing knowledge that transcriptional repressors function either by binding to DNA at specific sequences or by binding to RNAP, we show that Rv1222-mediated transcription inhibition requires simultaneous binding of the protein to both RNAP and DNA. We demonstrate that the positively charged C-terminus tail of Rv1222 is responsible for anchoring RNAP on DNA, hence the protein slows down the movement of RNAP along the DNA during transcription elongation. The interaction between Rv1222 and DNA is electrostatic, thus the protein could inhibit transcription from any gene. As Rv1222 slows down the RNA synthesis, upon expression of the protein in Mycobacterium smegmatis or Escherichia coli, the growth rate of the bacteria is severely impaired. The protein does not possess any significant affinity for DNA polymerase, thus, is unable to inhibit DNA synthesis. The proposed mechanism by which Rv1222 inhibits transcription reveals a new repertoire of prokaryotic gene regulation.

• http://nar.oxfordjournals.org/cgi/content/short/gkv516v1?rss=1

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内含入侵痕迹藻类形态和揭示几乎相同的北极和南极Micromonas种群

Spliceosomal introns are a hallmark of eukaryotic genes that are hypothesized to play important roles in genome evolution but have poorly understood origins. Although most introns lack sequence homology to each other, recently new families of spliceosomal introns that are repeated hundreds of times in individual genomes have been discovered in a few organisms. The prevalence and conservation of these introner elements (IEs) or introner-like elements (ILEs) in other taxa, as well as their evolutionary relationships to regular spliceosomal introns, are still unknown. Here, we systematically investigate introns in the widespread marine green alga Micromonas and report new families of IEs, numerous intron presence-absence polymorphisms, and potential intron insertion hot-spots. The new families enabled identification of conserved IE secondary structure features and establishment of a novel general model for repetitive intron proliferation across genomes. Despite shared secondary structure, the IE families from each Micromonas lineage bear no obvious sequence similarity to those in the other lineages, suggesting their appearance is intimately linked with the process of speciation. Two of the new IE families come from an Arctic culture (Micromonas Clade E2) isolated from a polar region where this alga is increasing in abundance due to climate change. The same two families were detected in metagenomic data from Antarctica – a system where Micromonas has never before been reported. Strikingly high identity between the Arctic isolate and Antarctic coding sequences that flank the IEs suggests connectivity between populations in the two polar systems that we postulate occurs through deep-sea currents. Recovery of Clade E2 sequences in North Atlantic Deep Waters beneath the Gulf Stream supports this hypothesis. Our work illuminates the dynamic relationships between an unusual class of repetitive introns, genome evolution, speciation and global distribution of this sentinel marine alga.

• http://mbe.oxfordjournals.org/cgi/content/short/msv122v1?rss=1

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有害突变的积累作为一个归化和向日葵等菊科作物改良的后果

For populations to maintain optimal fitness, harmful mutations must be efficiently purged from the genome. Yet, under circumstances that diminish the effectiveness of natural selection, such as the process of plant and animal domestication, deleterious mutations are predicted to accumulate. Here, we compared the load of deleterious mutations in 21 accessions from natural populations and 19 domesticated accessions of the common sunflower using whole-transcriptome single nucleotide polymorphism data. Although we find that genetic diversity has been greatly reduced during domestication, the remaining mutations were disproportionally biased toward nonsynonymous substitutions. Bioinformatically predicted deleterious mutations affecting protein function were especially strongly over-represented. We also identify similar patterns in two other domesticated species of the sunflower family (globe artichoke and cardoon), indicating that this phenomenon is not due to idiosyncrasies of sunflower domestication or the sunflower genome. Finally, we provide unequivocal evidence that deleterious mutations accumulate in low recombining regions of the genome, due to the reduced efficacy of purifying selection. These results represent a conundrum for crop improvement efforts. Although the elimination of harmful mutations should be a long-term goal of plant and animal breeding programs, it will be difficult to weed them out because of limited recombination.

• http://mbe.oxfordjournals.org/cgi/content/short/msv106v2?rss=1

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无模型的RNA序列和结构对齐了解形状探测揭示了16S rRNA保守交替二级结构

by Christopher A. Lavender, Ronny Lorenz, Ge Zhang, Rita Tamayo, Ivo L. Hofacker, Kevin M. Weeks

Discovery and characterization of functional RNA structures remains challenging due to deficiencies in de novo secondary structure modeling. Here we describe a dynamic programming approach for model-free sequence comparison that incorporates high-throughput chemical probing data. Based on SHAPE probing data alone, ribosomal RNAs (rRNAs) from three diverse organisms – the eubacteria E. coli and C. difficile and the archeon H. volcanii – could be aligned with accuracies comparable to alignments based on actual sequence identity. When both base sequence identity and chemical probing reactivities were considered together, accuracies improved further. Derived sequence alignments and chemical probing data from protein-free RNAs were then used as pseudo-free energy constraints to model consensus secondary structures for the 16S and 23S rRNAs. There are critical differences between these experimentally-informed models and currently accepted models, including in the functionally important neck and decoding regions of the 16S rRNA. We infer that the 16S rRNA has evolved to undergo large-scale changes in base pairing as part of ribosome function. As high-quality RNA probing data become widely available, structurally-informed sequence alignment will become broadly useful for de novo motif and function discovery.

• http://feeds.plos.org/~r/ploscompbiol/NewArticles/~3/zICeiWIlSog/info%3Adoi%2F10.1371%2Fjournal.pcbi.1004126

[详细]

在随机分散的景观的集合种群的持久性

by Jacopo Grilli, György Barabás, Stefano Allesina

Habitat destruction and land use change are making the world in which natural populations live increasingly fragmented, often leading to local extinctions. Although local populations might undergo extinction, a metapopulation may still be viable as long as patches of suitable habitat are connected by dispersal, so that empty patches can be recolonized. Thus far, metapopulations models have either taken a mean-field approach, or have modeled empirically-based, realistic landscapes. Here we show that an intermediate level of complexity between these two extremes is to consider random landscapes, in which the patches of suitable habitat are randomly arranged in an area (or volume). Using methods borrowed from the mathematics of Random Geometric Graphs and Euclidean Random Matrices, we derive a simple, analytic criterion for the persistence of the metapopulation in random fragmented landscapes. Our results show how the density of patches, the variability in their value, the shape of the dispersal kernel, and the dimensionality of the landscape all contribute to determining the fate of the metapopulation. Using this framework, we derive sufficient conditions for the population to be spatially localized, such that spatially confined clusters of patches act as a source of dispersal for the whole landscape. Finally, we show that a regular arrangement of the patches is always detrimental for persistence, compared to the random arrangement of the patches. Given the strong parallel between metapopulation models and contact processes, our results are also applicable to models of disease spread on spatial networks.

• http://feeds.plos.org/~r/ploscompbiol/NewArticles/~3/b1pTwYKfSLA/info%3Adoi%2F10.1371%2Fjournal.pcbi.1004251

[详细]

基于结构的RNA基因组和三HIV相关共识的二级结构

by Christopher A. Lavender, Robert J. Gorelick, Kevin M. Weeks

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1_NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.

• http://feeds.plos.org/~r/ploscompbiol/NewArticles/~3/5Iryh2B4WEA/info%3Adoi%2F10.1371%2Fjournal.pcbi.1004230

[详细]

拓扑关联域及其远程触点在G1早期的复制定时程序[研究]建立一致的建立

Mammalian genomes are partitioned into domains that replicate in a defined temporal order. These domains can replicate at similar times in all cell types (constitutive) or at cell type specific times (developmental). Genome-wide chromatin conformation capture (Hi-C) has revealed sub-megabase topologically-associating domains (TADs), which are the structural counterparts of replication domains. Hi-C also segregates inter-TAD contacts into defined 3D spatial compartments that align precisely to genome-wide replication timing profiles. Determinants of replication timing program are re-established during early G1 phase of each cell cycle and lost in G2 phase, but it is not known when TAD structure and inter-TAD contacts are re-established after their elimination during mitosis. Here we use multiplexed 4C-seq to study dynamic changes in chromatin organization during early G1. We find that both establishment of TADs and their compartmentalization occur during early G1, within the same time frame as the establishment of the replication-timing program. Once established, this 3D organization is preserved either after withdrawal into quiescence or for the remainder of interphase including G2 phase, implying 3D structure is not sufficient to maintain replication timing. Finally, we find that developmental domains are less well compartmentalized than constitutive domains and display chromatin properties that distinguish them from early and late constitutive domains. Overall, this study uncovers a strong connection between chromatin re-organization during G1, establishment of replication timing and its developmental control.

• http://genome.cshlp.org/cgi/reprint/gr.183699.114v1?rss=1

[详细]

GC有偏见的基因转换在人类基因组中[方法]量化

Many evidences indicate that GC-biased gene conversion (gBGC) has a major impact on the evolution of mammalian genomes. However, a detailed quantification of the process is still lacking. The strength of gBGC can be measured from the analysis of derived allele frequency spectra (DAF) but this approach is sensitive to a number of confounding factors. In particular, we show by simulations that the inference is pervasively affected by polymorphism polarization errors and by spatial heterogeneity in gBGC strength. We propose a new general method to quantify gBGC from DAF spectra, incorporating polarization errors, taking spatial heterogeneity into account, and jointly estimating mutation bias. Applying it to human polymorphism data from the 1000 Genomes Project we show that the strength of gBGC does not differ between hypermutable CpG sites and non-CpG sites, suggesting that in humans gBGC is not caused by the base-excision repair machinery. Genome-wide, the intensity of gBGC is in the nearly neutral area. However, given that recombination occurs primarily within recombination hotspots, 1 to 2% of the human genome is subject to strong gBGC. On average, gBGC is stronger in African than in non-African populations, reflecting differences in effective population sizes. However, due to more heterogeneous recombination landscapes, the fraction of the genome affected by strong gBGC is larger in non-African than in African populations. Given that the location of recombination hotspots evolves very rapidly, our analysis predicts that in the long term, a large fraction of the genome is affected by short episodes of strong gBGC.

• http://genome.cshlp.org/cgi/reprint/gr.185488.114v1?rss=1

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agplus:聚合块快速和灵活的工具

Summary: Aggregation plots are frequently used to evaluate signal distributions at user-interested points in ChIP-Seq data analysis. agplus, a new and simple command-line tool, enables rapid and flexible generation of text tables tailored for aggregation plots from which users can easily design multiple groups based on user-definitions such as regulatory regions or transcription initiation sites.

Availability and Implementation: This software is implemented in Ruby, supported on Linux and Mac OSX, and freely available at http://github.com/kazumits/agplus

Contact: yohkawa@epigenetics.med.kyushu-u.ac.jp

• http://bioinformatics.oxfordjournals.org/cgi/content/short/btv322v1?rss=1

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ABC SNVs差分工具:(A allele-specific限于ChIP-Seq experiments缔结具有约束力的标音

Motivation: Detection of allelic imbalances in ChIP-Seq reads is a powerful approach to identify functional non-coding single nucleotide variants (SNVs), either polymorphisms or mutations, which modulate the affinity of transcription factors for chromatin. We present ABC, a computational tool that identifies allele specific binding of transcription factors from aligned ChIP-Seq reads at heterozygous SNVs. ABC controls for potential false positives resulting from biases introduced by the use of short sequencing reads in ChIP-Seq and can efficiently process a large number of heterozygous SNVs.

Results: ABC successfully identifies previously characterized functional SNVs, such as the rs4784227 breast cancer risk associated SNP that modulates the affinity of FOXA1 for the chromatin.

Implementation: ABC is written in PERL and can be run on any platform with both PERL (≥5.18.1) and R (≥3.1.1) installed. The script requires the PERL Statistics::R module.

Availability: The code is open-source under an Artistic-2.0 license and versioned on GitHub (https://github.com/mlupien/ABC/).

Supplementary Information: Supplementary information can be found at Bioinformatics online

Contact: mlupien@uhnres.utoronto.ca

• http://bioinformatics.oxfordjournals.org/cgi/content/short/btv321v1?rss=1

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基于计算机视觉的自动选择应用于蛋白质核磁共振光谱峰值

Motivation: A detailed analysis of multidimensional NMR spectra of macromolecules requires the identification of individual resonances (peaks). This task can be tedious and time-consuming and often requires support by experienced users. Automated peak picking algorithms were introduced more than 25 years ago, but there are still major deficiencies/flaws that often prevent complete and error free peak picking of biological macromolecule spectra. The major challenges of automated peak picking algorithms is both the distinction of artifacts from real peaks particularly from those with irregular shapes and also picking peaks in spectral regions with overlapping resonances which are very hard to resolve by existing computer algorithms. In both of these cases a visual inspection approach could be more effective than a "blind" algorithm.

Results: We present a novel approach using computer vision (CV) methodology which could be better adapted to the problem of peak recognition. After suitable "training" we successfully applied the CV algorithm to spectra of medium sized soluble proteins up to molecular weights of 26 kDa and to a 130 kDa complex of a tetrameric membrane protein in detergent micelles. Our CV approach outperforms commonly used programs. With suitable training data sets the application of presented method can be extended to automated peak picking in multidimensional spectra of nucleic acids or carbohydrates and adapted to solid state NMR spectra.

Availability: CV-Peak Picker is available upon request from the authors.

• http://bioinformatics.oxfordjournals.org/cgi/content/short/btv318v1?rss=1

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Trans-species学习细胞信号网络系统与双峰很深的信仰

Motivation: Model organisms play critical roles in biomedical research of human diseases and drug development. An imperative task is to translate information/knowledge acquired from model organisms to humans. In this study, we address a trans-species learning problem: predicting human cell responses to diverse stimuli, based on the responses of rat cells treated with the same stimuli.

Results: We hypothesized that rat and human cells share a common signal-encoding mechanism but employ different proteins to transmit signals, and we developed a bimodal deep belief network (bDBN) and a semi-restricted bimodal deep belief network (sbDBN) to represent the common encoding mechanism and perform trans-species learning. These "deep learning" models include hierarchically organized latent variables capable of capturing the statistical structures in the observed proteomic data in a distributed fashion. The results show that the models significantly outperform two current state-of-the-art classification algorithms. Our study demonstrated the potential of using deep hierarchical models to simulate cellular signaling systems.

Availability: The software is available at the following URL: http://pubreview.dbmi.pitt.edu/TransSpeciesDeepLearning/. The data are available through SBV IMPROVER website, https://www.sbvimprover.com/challenge-2/overview, upon publication of the report by the organizers.

• http://bioinformatics.oxfordjournals.org/cgi/content/short/btv315v1?rss=1

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发现着丝粒蛋白：从冷白的手的A，B，C CENPs

The kinetochore is a complex molecular machine that directs chromosome segregation during mitosis. It is one of the most elaborate subcellular protein structures in eukaryotes, comprising more than 100 different proteins. Inner kinetochore proteins associate with specialized centromeric chromatin containing the histone H3 variant centromere

• http://feeds.nature.com/~r/nrm/rss/current/~3/YkcO4ZH36L0/nrm4001

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一个有条件的自我消除HAC基因载体通过一ttavp64表达盒掺入代

Human artificial chromosome (HAC)-based vectors represent an alternative technology for gene delivery and expression with a potential to overcome the problems caused by virus-based vectors. The recently developed alphoid^tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, tTA or tTS, to its centromeric tetO sequences. This provides a unique control for phenotypes induced by genes loaded into the HAC. The alphoid^tetO-HAC elimination is highly efficient when a high level of chromatin modifiers as tetR fusion proteins is achieved following transfection of cells by a retrovirus vector. However, such vectors are potentially mutagenic and might want to be avoided under some circumstances. Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. We demonstrated that a single copy of tTA^VP64 carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function. To adopt the alphoid^tetO-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA^VP64 cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid^tetO-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/e57?rss=1

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RNAE：靶向蛋白翻译增强人工非sineb2重复RNA编码的一种有效方法

In this study, a universal protein expression enhancement RNA tool, termed RNAe, was developed by modifying a recently discovered natural long non-coding RNA. At the moment, RNAe is the only technology for gene expression enhancement, as opposed to silencing, at the post-transcriptional level. With this technology, an expression enhancement of 50–1000% is achievable, with more than 200% enhancement achieved in most cases. This work identified the sufficient and necessary element for RNAe function, which was found to be merely 300 nucleotides long and was named minRNAe. It contains a 72-nt 5' pairing sequence which determines the specificity, a 167-nt short non-pairing interspersed nuclear element (SINE) B2 sequence which enhances ribosome recruitment to the target mRNA, and a poly(A) tail, provided together on a plasmid bearing the appropriate sequences. Cellular delivery of RNAe was achieved using routine transfection. The RNAe platform was validated in several widely-used mammalian cell lines. It was proven to be efficient and flexible in specifically enhancing the expression of various endogenous and exogenous proteins of diverse functions in a dose-dependent manner. Compared to the expression-inhibitory tool RNAi, the RNAe tool has a comparable effect size, with an enhancing as opposed to inhibitory effect. One may predict that this brand new technology for enhancing the production of proteins will find wide applications in both research and biopharmaceutical production.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/e58?rss=1

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快速、灵敏的检测缺失诱导的精确基因打靶

The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/e59?rss=1

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SC3序列：一种高度并行的、定量的单细胞基因表达的检测

Single-cell mRNA sequencing (RNA-seq) methods have undergone rapid development in recent years, and transcriptome analysis of relevant cell populations at single-cell resolution has become a key research area of biomedical sciences. We here present single-cell mRNA 3-prime end sequencing (SC3-seq), a practical methodology based on PCR amplification followed by 3-prime-end enrichment for highly quantitative, parallel and cost-effective measurement of gene expression in single cells. The SC3-seq allows excellent quantitative measurement of mRNAs ranging from the 10,000-cell to 1-cell level, and accordingly, allows an accurate estimate of the transcript levels by a regression of the read counts of spike-in RNAs with defined copy numbers. The SC3-seq has clear advantages over other typical single-cell RNA-seq methodologies for the quantitative measurement of transcript levels and at a sequence depth required for the saturation of transcript detection. The SC3-seq distinguishes four distinct cell types in the peri-implantation mouse blastocysts. Furthermore, the SC3-seq reveals the heterogeneity in human-induced pluripotent stem cells (hiPSCs) cultured under on-feeder as well as feeder-free conditions, demonstrating a more homogeneous property of the feeder-free hiPSCs. We propose that SC3-seq might be used as a powerful strategy for single-cell transcriptome analysis in a broad range of investigations in biomedical sciences.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/e60?rss=1

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multisnv：提高从多个相关的肿瘤样本体点突变的检测概率的方法

Somatic variant analysis of a tumour sample and its matched normal has been widely used in cancer research to distinguish germline polymorphisms from somatic mutations. However, due to the extensive intratumour heterogeneity of cancer, sequencing data from a single tumour sample may greatly underestimate the overall mutational landscape. In recent studies, multiple spatially or temporally separated tumour samples from the same patient were sequenced to identify the regional distribution of somatic mutations and study intratumour heterogeneity. There are a number of tools to perform somatic variant calling from matched tumour-normal next-generation sequencing (NGS) data; however none of these allow joint analysis of multiple same-patient samples. We discuss the benefits and challenges of multisample somatic variant calling and present multiSNV, a software package for calling single nucleotide variants (SNVs) using NGS data from multiple same-patient samples. Instead of performing multiple pairwise analyses of a single tumour sample and a matched normal, multiSNV jointly considers all available samples under a Bayesian framework to increase sensitivity of calling shared SNVs. By leveraging information from all available samples, multiSNV is able to detect rare mutations with variant allele frequencies down to 3% from whole-exome sequencing experiments.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/e61?rss=1

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通过对NEIL1蛋白质丰度Rad9调控高效碱基切除修复是很重要的

RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9^–/– relative to Rad9^+/+ embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9^–/– mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.

• http://nar.oxfordjournals.org/cgi/content/short/43/9/4531?rss=1

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